Gene Therapy and Tissue Engineering in Orthopaedic Surgery

The Journal of the American Academy of Orthopaedic Surgeons. Jan-Feb, 2002  |  Pubmed ID: 11809046

A new biologic era of orthopaedic surgery has been initiated by basic scientific advances that have resulted in the development of gene therapy and tissue engineering approaches for treating musculoskeletal disorders. The terminology, fundamental concepts, and current research in this burgeoning field must be understood by practicing orthopaedic surgeons. Different gene therapy approaches, multiple gene vectors, a multitude of cytokines, a growing list of potential scaffolds, and putative stem cells are being studied. Gene therapy and tissue engineering applications for bone healing, articular disorders, intervertebral disk pathology, and skeletal muscle injuries are being explored. Innovative methodologies that ensure patient safety can potentially lead to many new treatment strategies for musculoskeletal conditions.

Genetic Engineering of Meniscal Allografts

Tissue Engineering. Feb, 2002  |  Pubmed ID: 11886659

Allograft meniscal transplantation represents one of the few available treatment options after menisectomy. Despite acceptable early results, a considerable controversy exists with regard to poor graft regeneration, shrinkage and biomechanical failure of transplanted menisci. Transfer of specific growth factor genes may improve the regeneration process of meniscal allografts. The aim of this study was to investigate the feasibility of gene transfer in meniscal allografts in rabbits. Four different viral vectors encoding marker genes, including lacZ, luciferase, and green fluorescence protein were used to investigate viral transduction in 50 lapine menisci for 4 weeks in vitro. Subsequently, 16 unilateral meniscus replacements were performed with ex vivo retrovirally transduced meniscal allografts, and the expression of the lacZ gene was examined histologically at 2, 4, 6, and 8 weeks after transplantation. Gene expression in the superficial cell layers of the menisci can be detected for up to 4 weeks in vitro, but the level of gene transfer declined over time. The transduction with retrovirus showed better persistence and deep penetration of the menisci with infected cells. In vivo, declining numbers of beta-galactosidase-positive cells were also detected in retrovirally transduced allografts up to 8 weeks. Consistently, transduced cells were found at the menisco-synovial junction of the transplants and in deeper layers of the menisci. There was no evidence of cellular immune response in the transduced transplants. This investigation showed a prospective for growth factor delivery in auto- and allografts. In further experiments, vectors expressing therapeutic proteins such as growth factors will be investigated to assess their potential to improve remodeling and healing of meniscal allografts.

Muscle Injuries and Repair: Current Trends in Research

The Journal of Bone and Joint Surgery. American Volume. May, 2002  |  Pubmed ID: 12004029

The Use of Ex Vivo Gene Transfer Based on Muscle-derived Stem Cells for Cardiovascular Medicine

Trends in Cardiovascular Medicine. Apr, 2002  |  Pubmed ID: 12007736

Cell transplantation is a potential therapy for patients suffering from congestive heart failure. Many cell types have been experimentally tested for their ability to improve cardiac function. In this review, we discuss the potential of cell transplantation into the heart using various cell sources and introduce an attractive new cell source: Muscle-derived stem cells (MDSCs) are capable of delivering therapeutic genes and potentially differentiating toward a cardiomyocyte lineage within an injected heart. MDSCs are an attractive, alternate cell source because in addition to being multipotent (i.e., capable of differentiating into various lineages), they are easily accessible via simple biopsy of the patient's own muscle. This review will describe the isolation and unique characteristics of MDSCs and outline their potential use in regenerative medicine.

Identification of a Novel Population of Muscle Stem Cells in Mice: Potential for Muscle Regeneration

The Journal of Cell Biology. May, 2002  |  Pubmed ID: 12021255

Three populations of myogenic cells were isolated from normal mouse skeletal muscle based on their adhesion characteristics and proliferation behaviors. Although two of these populations displayed satellite cell characteristics, a third population of long-time proliferating cells expressing hematopoietic stem cell markers was also identified. This third population comprises cells that retain their phenotype for more than 30 passages with normal karyotype and can differentiate into muscle, neural, and endothelial lineages both in vitro and in vivo. In contrast to the other two populations of myogenic cells, the transplantation of the long-time proliferating cells improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. The long-time proliferating cells' ability to proliferate in vivo for an extended period of time, combined with their strong capacity for self-renewal, their multipotent differentiation, and their immune-privileged behavior, reveals, at least in part, the basis for the improvement of cell transplantation. Our results suggest that this novel population of muscle-derived stem cells will significantly improve muscle cell-mediated therapies.

Enhancement of Tendon-bone Integration of Anterior Cruciate Ligament Grafts with Bone Morphogenetic Protein-2 Gene Transfer: a Histological and Biomechanical Study

The Journal of Bone and Joint Surgery. American Volume. Jul, 2002  |  Pubmed ID: 12107310

The integration of tendon grafts used for replacement of the anterior cruciate ligament is still sometimes unsatisfactory and may be associated with postoperative anterior-posterior laxity. The goal of this study was to examine the capacity of bone morphogenetic protein-2 (BMP-2) gene transfer to improve the integration of semitendinosus tendon grafts at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits.

Insulin Growth Factor-1 Decreases Muscle Atrophy Following Denervation

Microsurgery. 2002  |  Pubmed ID: 12111979

Despite modern microsurgical techniques for nerve repair, functional outcome following proximal injury is often unsatisfactory because irreversible muscle atrophy may develop before reinnervation occurs. Because insulin growth factor-1 (IGF-1) has been shown to improve muscle regeneration after injury, and may have a role in muscle preservation following denervation, the purpose of this investigation was to evaluate the histological, immunohistochemical, and electrophysiological differences between normal, denervated, and IGF-1-injected denervated muscle over an 8-week period. Denervated mice gastrocnemius muscles demonstrated a decrease in muscle weight, a decrease in myofiber diameter, an absence of muscle regeneration, an early increase in the number of neuromuscular junctions (NMJs), and a decrease in fast-twitch and maximum tetanic strength as compared to normal muscle up to 8 weeks following denervation. IGF-1-injected denervated muscle, on the other hand, sustained muscle diameter and muscle weight, maintained a smaller number of NMJs, and relatively sustained fast-twitch and maximum tetanic strength as compared to normal muscle over 8 weeks. These data suggest that IGF-1 may help prevent muscle atrophy and secondary functional compromise after denervation.

Adenovirus Mediated Gene Transfer to Skeletal Muscle

Microscopy Research and Technique. Jul, 2002  |  Pubmed ID: 12112422

Transfer of therapeutic genes into muscle tissue has promise for the treatment of a variety of muscular dystrophies. Various vectors have been used to deliver genes to skeletal muscle but their application has faced several major limitations including: (1) the lack of transgene persistence caused by the immune rejection of transduced myofibers and/or vector toxicity, and (2) the maturation dependence of viral transduction. While the immunorejection and/or cytotoxic problems are being overcome with the development of new vectors, maturation-dependent viral transduction is still a major hurdle in gene transfer to skeletal muscle. Poor adenoviral transduction in mature myofibers has been attributed to: (1) the extracellular matrix of mature myofibers may form a physical barrier and prevent the passage of large viral particles; (2) viral receptors are down-regulated with muscle maturation; and (3) loss of myoblasts with muscle maturation, which serve as intermediaries in the viral transduction. In this review, we will focus on recent developments in overcoming those hurdles of gene therapy in skeletal muscle, especially to adenovirus (Ad), including: (1) new mutant vectors lacking all viral genes to decrease immunogenicity, and hence, improve persistence of transgene expression in muscle in vivo; (2) using tissue specific promoters to evade immunorejection; (3) permeabilization of the extracellular matrix; (4) modifying the viral receptors in mature myofibers; and (5) myoblast or muscle stem cell mediated ex vivo gene transfer.

Enhancement of Bone Healing Based on Ex Vivo Gene Therapy Using Human Muscle-derived Cells Expressing Bone Morphogenetic Protein 2

Human Gene Therapy. Jul, 2002  |  Pubmed ID: 12133273

Molecular biological advances have allowed the use of gene therapy in a clinical setting. In addition, numerous reports have indicated the existence of inducible osteoprogenitor cells in skeletal muscle. Because of this, we hypothesized that skeletal muscle cells might be ideal vehicles for delivery of bone-inductive factors. Using ex vivo gene transfer methods, we genetically engineered freshly isolated human skeletal muscle cells with adenovirus and retrovirus to express human bone morphogenetic protein 2 (BMP-2). These cells were then implanted into nonhealing bone defects (skull defects) in severe combined immune deficiency (SCID) mice. The closure of the defect was monitored grossly and histologically. Mice that received BMP-2-producing human muscle-derived cells experienced a full closure of the defect by 4 to 8 weeks posttransplantation. Remodeling of the newly formed bone was evident histologically during the 4- to 8-week period. When analyzed by fluorescence in situ hybridization, a small fraction of the transplanted human muscle-derived cells was found within the newly formed bone, where osteocytes normally reside. These results indicate that genetically engineered human muscle-derived cells enhance bone healing primarily by delivering BMP-2, while a small fraction of the cells seems to differentiate into osteogenic cells.

BMP4-expressing Muscle-derived Stem Cells Differentiate into Osteogenic Lineage and Improve Bone Healing in Immunocompetent Mice

Molecular Therapy : the Journal of the American Society of Gene Therapy. Aug, 2002  |  Pubmed ID: 12161183

Recent advances in molecular biology have led the way for novel approaches to improve bone healing. The ideal growth factor, vector, and delivery systems for producing bone in an immune competent animal model, however, have yet to be identified. Using a retrovirus encoding BMP4 and recently isolated muscle-derived stem cells (MDSCs), we demonstrated the following: MDSCs undergo osteogenic differentiation in response to BMP4 in a dose-dependent manner; retrovirus encoding BMP4 can efficiently transduce MDSCs, both enhancing osteogenic differentiation and inhibiting myogenic differentiation; transduced MDSCs can produce high levels of functional BMP4 as they differentiate toward an osteogenic lineage; allogeneic transduced MDSCs can induce robust de novo bone formation in immunocompetent mice despite the presence of an immune reaction, demonstrating the ability of this retroviral-BMP4-muscle construct to provide sufficient stimuli for osteoinduction in vivo; MDSCs appear to deliver BMP4, respond to the human BMP4 in an autocrine manner, and actively participate in bone formation, thus serving both osteoinductive and osteoproductive roles; and the BMP4-expressing MDSCs can induce bone formation and improve bone healing in a critical-sized skull defect in immunocompetent mice. Therefore, we believe that technology based on the MDSCs and vector system has great potential for promoting bone healing in a variety of musculoskeletal conditions.

Muscle-based Gene Therapy and Tissue Engineering for Treatment of Growth Plate Injuries

Journal of Pediatric Orthopedics. Sep-Oct, 2002  |  Pubmed ID: 12198455

Growth plate injuries may lead to a progressive angular deformity or longitudinal growth disturbance. The authors investigated the feasibility of gene therapy and tissue engineering based on autologous muscle-and adenoviral-mediated gene transfer of insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) to treat tibial physeal defects in rabbits. The medial half of the left proximal tibial growth plate was completely excised in 44 6-week-old New Zealand white rabbits. Four experimental groups were created: no treatment (I), autologous muscle interposition (II), autologous muscle interposition injected with adIGF-1 (III), and autologous muscle interposition injected with adBMP-2 (IV). Radiographic and histologic assessments were obtained postoperatively. Significant tibial shortening and a compact osseous bridge were observed in groups I and IV. Growth plates remained open in groups II and III. This experiment demonstrates that IGF-1 had a supportive effect on physeal chondrocytes, while BMP-2 caused increased osteogenic activity in the injured growth plates.

Differentiation of Muscle-derived Cells into Myofibroblasts in Injured Skeletal Muscle

The American Journal of Pathology. Sep, 2002  |  Pubmed ID: 12213718

Injured muscle can initiate regeneration promptly by activating myogenic cells that proliferate and differentiate into myotubes and myofibers. However, the recovery of the injured skeletal muscle often is hindered by the development of fibrosis. We hypothesized that the early-appearing myogenic cells in the injured area differentiate into myofibroblasts and eventually contribute to the development of fibrosis. To investigate this, we transplanted a genetically engineered clonal population of muscle-derived stem cells (MC13 cells) into the skeletal muscle of immunodeficient SCID mice, which were lacerated 4 weeks after transplantation. The MC13 cells regenerated numerous myofibers in the nonlacerated muscle and these myogenic cells were gradually replaced by myofibroblastic cells in the injured muscle. Our results suggest that the release of local environmental stimuli after muscle injury triggers the differentiation of myogenic cells (including MC13 cells) into fibrotic cells. These results demonstrate the potential of muscle-derived stem cells to differentiate into different lineages and illustrate the importance of controlling the local environment within the injured tissue to optimize tissue regeneration via the transplantation of stem cells.

Synergistic Enhancement of Bone Formation and Healing by Stem Cell-expressed VEGF and Bone Morphogenetic Protein-4

The Journal of Clinical Investigation. Sep, 2002  |  Pubmed ID: 12235106

We investigated the interaction between angiogenic and osteogenic factors in bone formation and bone healing with ex vivo gene therapy using muscle-derived stem cells genetically engineered to express human bone morphogenetic protein-4 (BMP4), VEGF, or VEGF-specific antagonist (soluble Flt1). Our results show that although VEGF alone did not improve bone regeneration, it acted synergistically with BMP4 to increase recruitment of mesenchymal stem cells, to enhance cell survival, and to augment cartilage formation in the early stages of endochondral bone formation. These early effects, coupled with accelerated cartilage resorption, eventually led to a significant enhancement of bone formation and bone healing. The beneficial effect of VEGF on bone healing elicited by BMP4 depends critically on the ratio of VEGF to BMP4, with an improper ratio leading to detrimental effects on bone healing. Finally, we show that soluble Flt1 inhibits bone formation elicited by BMP4. Thus, VEGF plays an important role in bone formation elicited by BMP4, and it can significantly enhance BMP4-elicited bone formation and regeneration through multiple mechanisms. This study has important implications for the formulation of new strategies to improve bone healing through increasing mesenchymal stem cell recruitment and survival, in combination with muscle-derived stem cell-based gene therapy.

Effect of Cryoinjury on the Contractile Parameters of Bladder Strips: a Model of Impaired Detrusor Contractility

Brain Research Bulletin. Oct, 2002  |  Pubmed ID: 12372544

In anesthetized Sprague-Dawley rats, the bladder was exposed and cryoinjury was induced by abruptly freezing the serosal side of the bladder wall with a chilled aluminum rod previously placed on dry ice (-40 degrees C). Five days later, the rats were euthanized, and strips were prepared from the area adjacent to the injury. Neurally and alpha,beta methylene-ATP (alpha,beta m-ATP; 50 microM)-evoked contractions were measured in bladder strips from cryoinjured or intact bladders prepared from sham-operated rats. Cryoinjured bladder strips produced significantly lower contractile forces than intact strips to electrical stimulation at higher (10-40 Hz) frequencies. The maximal rate of the neurally evoked contractions was slower in the cryoinjured bladders. The contractile response to alpha,beta m-ATP was smaller in the cryoinjured preparations indicating that the changes may have also occurred at the postjunctional site. In addition, atropine was more effective at inhibiting the neurally evoked contractions in the cryoinjured bladder strips suggesting that a cholinergic dominance occurs after cryoinjury. It is concluded that cryoinjury is a viable method of causing a defined, reproducible injury to the urinary bladder resulting in impaired function of both the cholinergic transmission and the smooth muscle. The bladder cryoinjury can be used as a model for studying impaired bladder compliance and detrusor contractility as well as treatments that may improve bladder function such as tissue engineering.

Gene Therapy and Tissue Engineering Based on Muscle-derived Stem Cells

Current Opinion in Molecular Therapeutics. Aug, 2002  |  Pubmed ID: 12222876

Skeletal muscle represents a convenient source of stem cells for cell-based tissue and genetic engineering. Muscle-derived stem cells (MDSCs) exhibit both multipotentiality and self-renewal capabilities, and are considered to be distinct from the well-studied satellite cell, another type of muscle stem cell that is capable of self-renewal and myogenic lineage differentiation. The MDSC appears to have less restricted differentiation capabilities as compared with the satellite cell, and may be a precursor of the satellite cell. This review considers the evidence for the existence of MDSCs as well as their origin. We will discuss recent investigations highlighting the potential of stem cell transplantation for the treatment of skeletal, cardiac and smooth muscle injuries and disease. We will highlight challenges in bridging the gap between understanding basic stem cell biology and clinical utilization for cell therapy.

Muscle Derived, Cell Based Ex Vivo Gene Therapy for Treatment of Full Thickness Articular Cartilage Defects

The Journal of Rheumatology. Sep, 2002  |  Pubmed ID: 12233887

To evaluate the effectiveness of transplanted allogeneic muscle derived cells (MDC) embedded in collagen gels for the treatment of full thickness articular cartilage defects, to compare the results to those from chondrocyte transplantation, and to evaluate the feasibility of MDC based ex vivo gene therapy for cartilage repair.

The Role of CD34 Expression and Cellular Fusion in the Regeneration Capacity of Myogenic Progenitor Cells

Journal of Cell Science. Nov, 2002  |  Pubmed ID: 12376567

Characterization of myogenic subpopulations has traditionally been performed independently of their functional performance following transplantation. Using the preplate technique, which separates cells based on their variable adhesion characteristics, we investigated the use of cell surface proteins to potentially identify progenitors with enhanced regeneration capabilities. Based on previous studies, we used cell sorting to investigate stem cell antigen-1 (Sca-1) and CD34 expression on myogenic populations with late adhesion characteristics. We compared the regeneration efficiency of these sorted progenitors, as well as those displaying early adhesion characteristics, by quantifying their ability to regenerate skeletal muscle and restore dystrophin following transplantation into allogenic dystrophic host muscle. Identification and utilization of late adhering populations based on CD34 expression led to differential regeneration, with CD34-positive populations exhibiting significant improvements in dystrophin restoration compared with both their CD34-negative counterparts and early adhering cell populations. Regenerative capacity was found to correspond to the level of myogenic commitment, defined by myogenic regulatory factor expression, and the rate and degree of induced cell differentiation and fusion. These results demonstrate the ability to separate definable subpopulations of myogenic progenitors based on CD34 expression and reveal the potential implications of defining myogenic cell behavioral and phenotypic characteristics in relation to their regenerative capacity in vivo.

Muscle Derived Cell Mediated Ex Vivo Gene Transfer to the Lower Urinary Tract: Comparison of Viral Vectors

Urological Research. Oct, 2002  |  Pubmed ID: 12389119

Gene therapy is a novel form of molecular medicine that may have a major impact on the future of human health care. We explored the efficacy of skeletal muscle derived cells (MDC) transduced with four viruses for ex vivo gene transfer into the lower urinary tract. Primary MDC were isolated from normal neonatal rats and transduced with: (1). adenovirus, (2). herpes simplex virus type-1 (HSV-1), (3). retrovirus or (4). adeno-associated virus (AAV), all of which express the beta-galactosidase reporter gene. Adult Sprague Dawley rats ( n=4 each group-time) were used. The MDC were injected into the right and left lateral bladder walls. The number of injected MDC ranged from 1 to 1.5 x 10(6). The tissues were harvested after 1, 4, 7, and 15 days, sectioned and assayed for beta-galactosidase expression. In the bladder wall, we noted cells expressing beta-galactosidase for each viral group. Adenoviral and HSV-1 transduced cells showed strong expression at 1 and 4 days post-injection, but the expression decreased gradually and was not detectable at 15 days post-injection. Retroviral transduced cells were detected at each time point with a strong expression persisting for 15 days but decreasing gradually over time. Although expression of the AAV transduced cells was initially weak, the later time points exhibited a much stronger expression, especially at day 7 post-injection. This expression persisted for at least 15 days post-injection. In conclusion, successful MDC mediated ex vivo gene transfer into the lower urinary tract was achieved with all four viral vectors. Our results suggest that the ex vivo approach may lead to an efficient and persistent viral gene delivery to the lower urinary tract while minimizing exposure of the host to virus.

Muscle-based Gene Therapy and Tissue Engineering to Improve Bone Healing

Clinical Orthopaedics and Related Research. Oct, 2002  |  Pubmed ID: 12394474

Failed fracture healing is a significant problem in orthopaedics, often seen in patients with scaphoid fractures, high-energy injuries, and osteoporosis. Current treatments often result in poor outcomes and donor site morbidity. Gene therapy has been the focus of much recent research to improve bone healing. In the current review, the authors specifically evaluate the use of muscle-derived cells as a gene delivery vehicle and inducible osteoprogenitor cell that can enhance bone regeneration. Muscle-derived cells have been used to deliver bone morphogenetic protein-2 and produce ectopic bone. These cells express osteocalcin and have been found within newly generated bone in locations normally occupied by osteoblasts and osteocytes. Finally, it is shown that muscle-derived cells coupled with ex vivo gene therapy can heal critical-sized calvarial defects.

Gene Therapy and Tissue Engineering for Sports Medicine

The Journal of Gene Medicine. Feb, 2003  |  Pubmed ID: 12539148

Sports injuries usually involve tissues that display a limited capacity for healing. The treatment of sports injuries has improved over the past 10 to 20 years through sophisticated rehabilitation programs, novel operative techniques, and advances in the field of biomechanical research. Despite this considerable progress, no optimal solution has been found for treatment of various sports-related injuries, including muscle injuries, ligament and tendon ruptures, central meniscal tears, cartilage lesions, and delayed bone fracture healing. New biological approaches focus on the treatment of these injuries with growth factors to stimulate and hasten the healing process. Gene therapy using the transfer of defined genes encoding therapeutic proteins represents a promising way to efficiently deliver suitable growth factors into the injured tissue. Tissue engineering, which may eventually be combined with gene therapy, may potentially result in the creation of tissues or scaffolds for regeneration of tissue defects following trauma. In this article we will discuss why gene therapy and tissue engineering are becoming increasingly important in modern orthopaedic sports medicine practice. We then will review recent research achievements in the area of gene therapy and tissue engineering for sports-related injuries, and highlight the potential clinical applications of this technology in the treatment of patients with musculoskeletal problems following sports-related injuries.

Gene Therapy in Orthopaedic Surgery

Instructional Course Lectures. 2003  |  Pubmed ID: 12690900

Gene therapy has the potential to revolutionize the treatment of a variety of musculoskeletal disorders. In the past decade, more than 4,000 patients have been enrolled in clinical trials involving gene therapy. Gene therapy is becoming increasingly important in modern orthopaedic clinical practice. It is important to discuss the issues that physicians and scientists face when designing an experiment or clinical trial involving gene therapy, along with the potential clinical applications for gene therapy in the treatment of patients with musculoskeletal problems.

Antifibrotic Effects of Suramin in Injured Skeletal Muscle After Laceration

Journal of Applied Physiology (Bethesda, Md. : 1985). Aug, 2003  |  Pubmed ID: 12730151

Muscle injuries are very common in traumatology and sports medicine. Although muscle tissue can regenerate postinjury, the healing process is slow and often incomplete; complete recovery after skeletal muscle injury is hindered by fibrosis. Our studies have shown that decreased fibrosis could improve muscle healing. Suramin has been found to inhibit transforming growth factor (TGF)-beta1 expression by competitively binding to the growth factor receptor. We conducted a series of tests to determine the antifibrotic effects of suramin on muscle laceration injuries. Our results demonstrate that suramin (50 microg/ml) can effectively decrease fibroblast proliferation and fibrotic-protein expression (alpha-smooth muscle actin) in vitro. In vivo, direct injection of suramin (2.5 mg) into injured murine muscle resulted in effective inhibition of muscle fibrosis and enhanced muscle regeneration, which led to efficient functional muscle recovery. These results support our hypothesis that prevention of fibrosis could enhance muscle regeneration, thereby facilitating more efficient muscle healing. This study could significantly contribute to the development of strategies to promote efficient muscle healing and functional recovery.

Muscle Stem Cells Differentiate into Haematopoietic Lineages but Retain Myogenic Potential

Nature Cell Biology. Jul, 2003  |  Pubmed ID: 12792651

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.

Stem Cells in the Treatment of Muscle and Connective Tissue Diseases

Current Opinion in Pharmacology. Jun, 2003  |  Pubmed ID: 12810201

Current data indicate the existence of two types of postnatal stem cells. Tissue non-specific stem cells are haematopoietic in origin and can differentiate into different blood lineages. In contrast, tissue-specific stem cells preferentially differentiate into cells of the residing tissue, although they also possess a limited ability to turn into other lineages. In terms of therapeutic potential, unmodified muscle-derived stem cells have been proven capable of regenerating dystrophic muscle. Furthermore, when genetically modified to express growth factors, these cells are versatile in promoting bone healing. This also occurs with mesenchymal stem cells, which have been used in an attempt to repair defects of cartilage and ligaments. Thus, stem-cell-based therapy--particularly genetically engineered therapy--holds great potential for the treatment of a variety of disorders and conditions affecting the muscle and connective tissue.

Gamma Interferon As an Antifibrosis Agent in Skeletal Muscle

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Sep, 2003  |  Pubmed ID: 12919866

Muscle injuries are a common problem in sports medicine. Skeletal muscle can regenerate itself, but the process is both slow and incomplete. Previously we and others have used growth factors to improve the regeneration of muscle, but the muscle healing was impeded by scar tissue formation. However, when we blocked the fibrosis process with decorin, an antifibrosis agent, we improved the muscle healing. Here we show that gammainterferon (gammaINF)--a cytokine that inhibits the signaling of transforming growth factor beta1 (TGFbeta1), a fibrotic stimulator--reduces fibrosis formation and improves the healing of lacerated skeletal muscle. With gammaINF treatment, the growth rate of muscle-derived fibroblasts was reduced and the level of fibrotic protein expression induced by TGFbeta1 (including TGFbeta1, vimentin, and alpha-smooth muscle actin) was down-regulated in vitro. In a mouse laceration model, the area of fibrosis decreased when gammaINF was injected at either 1 or 2 weeks after injury. More importantly, the injection of gammaINF at either 1 or 2 weeks post-injury was found to improve muscle function in terms of both fast-twitch and tetanic strength. This study demonstrates that gammaINF is a potent antifibrosis agent that can improve muscle healing after laceration injury.

Improvement of Muscle Healing Through Enhancement of Muscle Regeneration and Prevention of Fibrosis

Muscle & Nerve. Sep, 2003  |  Pubmed ID: 12929198

Skeletal muscle is able to repair itself through regeneration. However, an injured muscle often does not fully recover its strength because complete muscle regeneration is hindered by the development of fibrosis. Biological approaches to improve muscle healing by enhancing muscle regeneration and reducing the formation of fibrosis are being investigated. Previously, we have determined that insulin-like growth factor-1 (IGF-1) can improve muscle regeneration in injured muscle. We also have investigated the use of an antifibrotic agent, decorin, to reduce muscle fibrosis following injury. The aim of this study was to combine these two therapeutic methods in an attempt to develop a new biological approach to promote efficient healing and recovery of strength after muscle injuries. Our findings indicate that further improvement in the healing of muscle lacerations is attained histologically by the combined administration of IGF-1 to enhance muscle regeneration and decorin to reduce the formation of fibrosis. This improvement was not associated with improved responses to physiological testing, at least at the time-points tested in this study.

Gene Therapy to Improve Osteogenesis in Bone Lesions with Severe Soft Tissue Damage

Langenbeck's Archives of Surgery / Deutsche Gesellschaft Für Chirurgie. Oct, 2003  |  Pubmed ID: 14504931

Ex vivo gene therapy can induce bone formation when delivery cells carrying the bone morphogenetic protein (BMP) gene are used. The hypothesis for this study was that the cell-mediated gene therapy could improve the healing of bony lesions with severe soft tissue damage.

Dystrophin Delivery in Dystrophin-deficient DMDmdx Skeletal Muscle by Isogenic Muscle-derived Stem Cell Transplantation

Human Gene Therapy. Nov, 2003  |  Pubmed ID: 14577915

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.

The Role of Cell Type in Bone Healing Mediated by Ex Vivo Gene Therapy

Langenbeck's Archives of Surgery / Deutsche Gesellschaft Für Chirurgie. Oct, 2003  |  Pubmed ID: 14534793

The ideal cellular vehicle for use in cell-mediated gene therapy to enhance bone healing has not yet been identified. The purpose of this study was to compare the capacity of two types of cells transduced with retro-bone morphogenetic protein 4 (BMP4)-muscle-derived cells (MDCs) and unfractioned bone marrow stromal cells (BMSCs).

Improved Sphincter Contractility After Allogenic Muscle-derived Progenitor Cell Injection into the Denervated Rat Urethra

Urology. Nov, 2003  |  Pubmed ID: 14624934

To study the physiologic outcome of allogenic transplant of muscle-derived progenitor cells (MDPCs) in the denervated female rat urethra.

Muscle-derived Stem Cells: Potential for Muscle Regeneration

Birth Defects Research. Part C, Embryo Today : Reviews. Aug, 2003  |  Pubmed ID: 14671776

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness caused by the lack of dystrophin expression at the sarcolemma of muscle fibers. Although various approaches to delivering dystrophin in dystrophic muscle have been investigated extensively (e.g., cell and gene therapy), there is still no treatment that alleviates the muscle weakness in this common inherited muscle disease. The transplantation of myoblasts can enable transient delivery of dystrophin and improve the strength of injected dystrophic muscle, but this approach has various limitations, including immune rejection, poor cellular survival rates, and the limited spread of the injected cells. The isolation of muscle cells that can overcome these limitations would enhance the success of myoblast transplantation significantly. The efficiency of cell transplantation might be improved through the use of stem cells, which display unique features, including (1) self-renewal with production of progeny, (2) appearance early in development and persistence throughout life, and (3) long-term proliferation and multipotency. For these reasons, the development of muscle stem cells for use in transplantation or gene transfer (ex vivo approach) as treatment for patients with muscle disorders has become more attractive in the past few years. In this paper, we review the current knowledge regarding the isolation and characterization of stem cells isolated from skeletal muscle by highlighting their biological features and their relationship to satellite cells as well as other populations of stem cells derived from other tissues. We also describe the remarkable ability of stem cells to regenerate skeletal muscle and their potential use to alleviate the muscle weakness associated with DMD.

Muscle-derived Stem Cells Seeded into Acellular Scaffolds Develop Calcium-dependent Contractile Activity That is Modulated by Nicotinic Receptors

Urology. Jun, 2003  |  Pubmed ID: 12809930

To explore the contractile activity and physiologic properties of muscle-derived stem cells (MDSCs) incorporated into small intestinal submucosa (SIS) scaffolds.

Development of an Experimental System for the Study of Urethral Biomechanical Function

American Journal of Physiology. Renal Physiology. Feb, 2004  |  Pubmed ID: 14506075

Despite its principal mechanical function in the storage and release of urine, the biomechanical properties of the urethra have remained largely unexplored. The purpose of this study was to develop and validate an experimental model that can be used for evaluating whole urethral tissue in such a manner. Bladder-urethral specimens were excised from halothane-anesthetized female rats and mounted at in vivo length within the experimental apparatus consisting of a tissue perfusion chamber, an adjustable fluid column, and a laser micrometer. Outer diameter measurements were made at proximal, mid, and distal axial locations in response to increases in intraluminal pressure and after addition of various muscle-responsive agents. Basal smooth muscle tone and regional variations in compliance were detected through pressure-diameter responses. Chemically evoked contractile responses were measured and correspond to regional compositions of intrinsic smooth and striated muscle components. The results presented illustrate the utility of this system, which should permit a more thorough characterization of structure-function relationships and urethral biomechanical function in relation to normal and dysfunctional tissue states.

Retroviral Delivery of Noggin Inhibits the Formation of Heterotopic Ossification Induced by BMP-4, Demineralized Bone Matrix, and Trauma in an Animal Model

The Journal of Bone and Joint Surgery. American Volume. Jan, 2004  |  Pubmed ID: 14711949

The heterotopic ossification of muscles, tendons, and ligaments is a common problem faced by orthopaedic surgeons. We investigated the ability of Noggin (a BMP [bone morphogenetic protein] antagonist) to inhibit heterotopic ossification.

Muscle-derived Stem Cells

Cell Cycle (Georgetown, Tex.). Feb, 2004  |  Pubmed ID: 14712064

Researchers have identified 2 types of stem cells in skeletal muscle: satellite cells and multipotent stem cells (MPSCs). The latter category includes different cell populations isolated by various researchers using several techniques. The methods used to isolate these cells appear to influence the stem cell characteristics of the MPSCs. Although MPSCs and satellite cells could represent different stages of maturation of the same progenitor cells, they also could represent distinct populations of stem cells that exist in skeletal muscle. This article summarizes the recent developments in muscle-derived stem cell research.

Noninvasive Monitoring of Stem Cell Transfer for Muscle Disorders

Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. Feb, 2004  |  Pubmed ID: 14755651

In this study the ability of magnetodendrimers to efficiently label cultured muscle stem cells and allow for subsequent in vivo cell detection was determined. Magnetodendrimer-labeled cells exhibited normal growth rates in culture, and retained their capacity to undergo proliferation and form normal myotubes. Labeled stem cells possessed high in vivo proton relaxivities that enhanced MRI contrast properties and enabled us to noninvasively monitor the stem cells' incorporation into dystrophic muscle. Well defined regions of decreased signal intensity were observed in both T2- and T1-weighted image sequences. MRI was used to longitudinally follow stem cell dynamics in dystrophic muscle with in-plane resolutions on the order of a single muscle fiber (22 x 43 microm2). Regions of decreased signal intensity were well correlated with iron accumulation and other histochemical markers of stem cell incorporation. We concluded that this technique may be useful for continuous noninvasive readouts of stem cell transfer, replacing sequential muscle biopsies and tissue harvesting.

Establishing Reliable Criteria for Isolating Myogenic Cell Fractions with Stem Cell Properties and Enhanced Regenerative Capacity

Blood Cells, Molecules & Diseases. Jan-Feb, 2004  |  Pubmed ID: 14757409

Despite a focused effort within the myogenic cell transplantation community, little progress has been made toward the reliable identification and isolation of progenitors that are capable of tolerating the initial posttransplantation environment and effectively regenerating clinically relevant quantities of muscle. The future success of myogenic-based treatment modalities requires an enhanced understanding of the highly heterogeneous nature of the myogenic progenitor cell pool, which has been previously documented by numerous researchers. Further, for translation of experimental animal results to clinical application, reliable in vitro selection criteria must be established and must be translatable across species. While research into the utility of surface markers is ongoing, as an alternative we have investigated in vitro cell behavioral characteristics under imposed conditions which challenge the propensity of myogenic progenitors to choose between various cell fates (i.e., proliferation, quiescence, or differentiation). Previous observations in the mouse suggest an enhanced in vivo regenerative capacity of myogenic populations with respect to their in vitro ability to maintain a proliferative and undifferentiated state [J. Cell Sci. 115 (2002) 4361]. From these observations it is thus proposed that such behavior may represent an a priori indicator of regenerative capacity following transplantation. To challenge this proposition, a rat cell isolation and transplantation model was evaluated in an identical manner. In agreement with the results obtained from the mouse, a significant correlation between regenerative capacity and induction of differentiation was observed. These results contribute to the growing body of scientific evidence documenting the underlying behavioral differences that exist between various myogenic progenitors while also, importantly, providing evidence that such differences may significantly impact the functional capabilities of these cells posttransplantation. This information further implies that from a therapeutic standpoint isolation strategies aimed toward obtaining efficient myogenic progenitors should, in the absence of a reliable surface marker(s), focus on identifying populations displaying desirable in vitro behavior (i.e., high proliferative capacity and low induced differentiation). Incorporating such criteria into cell isolation and/or purification schemes may yield significant returns in the clinical myogenic transplantation setting.

Gene Transfer to Skeletal Muscle Using Herpes Simplex Virus-based Vectors

Methods in Molecular Biology (Clifton, N.J.). 2004  |  Pubmed ID: 14970600

Type 1 herpes simplex virus (HSV-1)-based vectors, which are naturally capable of carrying large DNA fragments like the 14 kb dystrophin cDNA, have been studied for their ability to transduce muscle cells. These vectors can persist in the host cell in a nonintegrated state and can be prepared at adequately high titers (10(7)-10(9) PFU/mL). They also infect myoblasts, myotubes, and immature myofibers efficiently. The major disadvantage of the first-generation HSV vectors is their relatively high cytotoxicity, which hampers long-term transgene expression. Second-generation mutants defective for multiple immediate early (IE) genes (e.g., ICP4, ICP22, and ICP27) display substantially reduced cytotoxicity in vitro, which improves the duration of transgene expression (6-11). In this chapter, we describe a new method of gene delivery using second-generation HSV-1 vectors. This procedure should enable an investigator to transduce normal mouse muscle cells, both in vitro and in vivo. We explain the conditions for muscle cell isolation, transduction in vitro and in vivo, and the technique for evaluating transduction efficiency (beta-galactosidase; beta-gal) using histology or the beta-gal assay (ONPG) method.

Ex Vivo Gene Transfer to Mature Skeletal Muscle by Using Adenovirus Helper Cells

The Journal of Gene Medicine. Feb, 2004  |  Pubmed ID: 14978769

Adenoviral gene transfer to adult skeletal muscle is hindered by several major limitations, including host immune responses and maturation-dependent loss of myofiber infectivity. Ex vivo gene delivery is more efficient than direct viral injection in surmounting maturation-dependent adenoviral transduction. Here we investigated the use of helper cells to improve the efficiency of ex vivo gene transfer to adult mouse skeletal muscle.

Transforming Growth Factor-beta1 Induces the Differentiation of Myogenic Cells into Fibrotic Cells in Injured Skeletal Muscle: a Key Event in Muscle Fibrogenesis

The American Journal of Pathology. Mar, 2004  |  Pubmed ID: 14982854

Transforming growth factor-beta1 (TGF-beta1) is thought to play a crucial role in fibrotic diseases. This study demonstrates for the first time that TGF-beta1 stimulation can induce myoblasts (C2C12 cells) to express TGF-beta1 in an autocrine manner, down-regulate the expression of myogenic proteins, and initiate the production of fibrosis-related proteins in vitro. Direct injection of human recombinant TGF-beta1 into skeletal muscle in vivo stimulated myogenic cells, including myofibers, to express TGF-beta1 and induced scar tissue formation within the injected area. We also observed the local expression of this growth factor by myogenic cells, including regenerating myofibers, in injured skeletal muscle. Finally, we demonstrated that TGF-beta1 gene-transfected myoblasts (CT cells) can differentiate into myofibroblastic cells after intramuscular transplantation, but that decorin, an anti-fibrosis agent, prevents this differentiation process by blocking TGF-beta1. In summary, these findings indicate that TGF-beta1 is a major stimulator that plays a significant role in both the initiation of fibrotic cascades in skeletal muscle and the induction of myogenic cells to differentiate into myofibroblastic cells in injured muscle.

Converse Relationship Between in Vitro Osteogenic Differentiation and in Vivo Bone Healing Elicited by Different Populations of Muscle-derived Cells Genetically Engineered to Express BMP4

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Apr, 2004  |  Pubmed ID: 15005851

In this study, we compared the use of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve bone healing to that of muscle-derived non-osteoprogenitor cells (PP1 cells). Surprisingly, the use of PP1 cells resulted in an improved outcome because of the lack of adverse responses to BMP4 involving cell differentiation, proliferation, and apoptosis.

Intraurethral Muscle-derived Cell Injections Increase Leak Point Pressure in a Rat Model of Intrinsic Sphincter Deficiency

Urology. Apr, 2004  |  Pubmed ID: 15072911

To determine whether allogenic muscle-derived cells (MDCs) could restore sphincter function in rats with intrinsic sphincter deficiency (ISD). ISD denotes a malfunction of the urethral sphincter.

Muscle-derived Stem Cells for Musculoskeletal Tissue Regeneration and Repair

Transplant Immunology. Apr, 2004  |  Pubmed ID: 15157924

Muscle recently has been identified as a good source of adult stem cells that can differentiate into cells of different lineages. The most well-known muscle progenitor cells are satellite cells, which not only contribute to the replenishment of the myogenic cell pool but also can become osteoblasts, adipocytes and chondrocytes. Other populations of stem cells that appear to be distinct from satellite cells also have been discovered recently. Muscle-derived stem cells (MDSCs) can be divided into two major categories based on these cells' varied abilities to differentiate into myogenic lineages. Interestingly, MDSCs that can differentiate readily into myogenic cells are usually CD45-. In contrast, MDSCs with less myogenic potential are CD45+. Various lines of evidence suggest that different populations of MDSCs are closely related. Furthermore, MDSCs appear to be closely related to endothelial cells or pericytes of the capillaries surrounding myofibers. When used in tissue engineering applications, MDSCs--particularly those genetically engineered to express growth factors--have been demonstrated to possess great potential for the regeneration and repair of muscle, bone and cartilage. Further research is necessary to delineate the relationship between different populations of MDSCs and between MDSCs and other adult stem cells, to investigate their developmental origin, and to determine the regulatory pathways and factors that control stem cell self-renewal, proliferation and differentiation. This knowledge could greatly enhance the usefulness of muscle-derived stem cells, as well as other adult stem cells, for tissue repair and regeneration applications.

Ex Vivo Gene Therapy-induced Endochondral Bone Formation: Comparison of Muscle-derived Stem Cells and Different Subpopulations of Primary Muscle-derived Cells

Bone. Jun, 2004  |  Pubmed ID: 15193544

Muscle-based gene therapy and tissue engineering hold great promise for improving bone healing. However, the relative advantage of muscle-derived stem cells (MDSCs) or primary muscle-derived cells (MDCs) remains to be defined. We compared the ability of MDSCs and different subpopulations of MDCs (PP1 and PP3) to induce bone formation via ex vivo gene therapy. We were able to efficiently transduce the MDSCs and all the other evaluated populations of MDCs (efficiency of transduction = approximately 80%) by using a retroviral vector expressing human bone morphogenetic protein 4 (BMP4). All the transduced cell populations secreted high levels of BMP4 (140-300 ng/10(6) cells/24 h), but the MDSCs differentiated toward the osteogenic lineage more effectively than did the other muscle cell populations, as indicated by the expression of alkaline phosphatase, an early osteogenic marker. von Kossa staining indicated that mineralized bone formed as early as 7 days after implantation of any of the BMP4-expressing cell populations into immunocompetent syngeneic mice; however, MDSCs expressing BMP4 produced significantly more bone than did the other MDC populations, as evidenced by both histomorphometry and biochemical analysis. Further investigation revealed that MDSCs expressing BMP4 persisted for a significantly longer period of time at the bone forming sites than did the other BMP4-expressing MDC populations. Additionally, MDSCs expressing BMP4 triggered a smaller infiltration of CD4 lymphocytes within the bone forming areas than did the other MDC populations expressing BMP4. Finally, we demonstrated that MDSCs expressing BMP4 can heal a critical-sized skull bone defect in immunocompetent mice. In summary, this study shows that MDSCs are better than primary MDCs for use as cellular vehicles in BMP4-based ex vivo gene therapy to improve bone healing. The advantage of MDSCs may be attributable, at least in part, to their lower immunogenicity and higher capacity for in vivo survival.

Development of a Self-inactivating Tet-on Retroviral Vector Expressing Bone Morphogenetic Protein 4 to Achieve Regulated Bone Formation

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2004  |  Pubmed ID: 15194055

The aims of this study were to explore the possibility of improving the design of self-inactivating (SI) retroviral vectors and to develop an SI vector that would allow optimal tet-on-regulated therapeutic gene expression. To minimize any interference between the viral promoter and the inducible promoter, we deleted different regulatory elements in the 3'LTR and examined their effects on transgene expression in transfected or transduced cells. In transfected cells, such deletions reduced the transgene expression. The insertion of a polyadenylation sequence could not completely compensate for this effect. We observed three patterns of transgene expression in cells transduced with these tet-on retroviral vectors: (1) high levels of both basal and inducible expression, (2) low levels of both basal and inducible expression, and (3) low levels of basal and high levels of inducible expression. After using the optimal vector to transduce muscle-derived stem cells, we were able to regulate the strong in vitro expression of transgenes-including enhanced green fluorescent protein and bone morphogenetic protein 4-via the addition or withdrawal of doxycycline (Dox). Implantation of the transduced cells and subsequent Dox-dependent induction of gene expression resulted in bone formation in vivo. Thus, we have developed an optimal SI retroviral vector that maintains a high titer, efficiently transduces muscle-derived stem cells, and enables both high levels of inducible gene expression in vitro and robust regulated bone formation in vivo.

Structural and Functional Healing of Critical-size Segmental Bone Defects by Transduced Muscle-derived Cells Expressing BMP4

The Journal of Gene Medicine. Sep, 2004  |  Pubmed ID: 15352071

Our previous studies have shown that muscle-derived cells, including a population of muscle stem cells, transduced with a retroviral vector expressing bone morphogenetic protein 4 (BMP4) can improve the healing of critical-size calvarial defects. However, we did not evaluate the functionality of the healed bone. The purpose of this study was to determine whether primary muscle-derived cells transduced with retroBMP4 can heal a long bone defect both structurally and functionally.

Tissue Engineering with Muscle-derived Stem Cells

Current Opinion in Biotechnology. Oct, 2004  |  Pubmed ID: 15464371

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Researchers continue to investigate stem cells in mature tissues and demonstrate the potential ability of organ-specific cells to differentiate into multiple lineages. One stem cell that displays such promise is the muscle-derived stem cell (MDSC). Data supporting the existence of MDSCs have emerged as part of investigations to improve myoblast cell transplantation for the treatment of muscular dystrophies. As these efforts continue, the potential for MDSC-based therapy for other musculoskeletal injuries, as well as for cardiac and smooth muscle injuries, is currently being explored.

Noninvasive Monitoring and Tracking of Muscle Stem Cell Transplants

Transplantation. Dec, 2004  |  Pubmed ID: 15591951

Efficient techniques to noninvasively monitor stem cell transplants will accelerate the development of stem cell therapies. Magnetic resonance (MR) imaging of labeled stem cells is a noninvasive approach that can provide images with high spatial resolution. In this report, we have evaluated the ability of a commercially available, FDA-approved contrast agent to allow for the monitoring of therapeutic stem cell transplants in murine dystrophic muscle.

The Use of Suramin, an Antifibrotic Agent, to Improve Muscle Recovery After Strain Injury

The American Journal of Sports Medicine. Jan, 2005  |  Pubmed ID: 15610998

Muscle strain injuries are extremely common in sports medicine. Muscle healing often is hindered by scar tissue formation after injury.

The Vascular Wall As a Source of Stem Cells

Annals of the New York Academy of Sciences. Jun, 2005  |  Pubmed ID: 15958696

We have characterized the emerging hematopoietic system in the human embryo and fetus. Two embryonic organs, the yolk sac and aorta, support the primary emergence of hematopoietic stem cells (HSCs), but only the latter contributes lymphomyeloid stem cells for definitive, adult-type hematopoiesis. A common feature of intra- and extraembryonic hematopoiesis is that in both locations hematopoietic cells emerge in close vicinity to vascular endothelial cells. We have provided evidence that a population of angiohematopoietic mesodermal stem cells, marked by the expression of flk-1 and the novel BB9/ACE antigen, migrate from the paraaortic splanchnopleura into the ventral part of the aorta, where they give rise to hemogenic endothelial cells and, in turn, hematopoietic cells. HSCs also appear to develop from endothelium in the embryonic liver and fetal bone marrow, albeit at a much lower frequency. This would imply that the organism does not function during its whole life on a stock of hematopoietic stem cells established in the early embryo, as is usually accepted. We next examined whether the vessel wall can contribute stem cells for other cell lineages, primarily in the model of adult skeletal muscle regeneration. By immunohistochemistry and flow cytometry, we documented the existence in skeletal muscle, besides genuine endothelial and myogenic cells, of a subset of satellite cells that coexpress endothelial cell markers. This suggested the existence of a continuum of differentiation from vascular cells to endothelial cells that was confirmed in long-term culture. The regenerating capacity of these cells expressing both myogenic and endothelial markers is being investigated in skeletal and cardiac muscle, and the results are being compared with those generated by satellite cells. Altogether, these results point to a generalized progenitor potential of a subset of endothelial, or endothelium-like, cells in blood vessel walls, in pre- and postnatal life.

Noggin Improves Bone Healing Elicited by Muscle Stem Cells Expressing Inducible BMP4

Molecular Therapy : the Journal of the American Society of Gene Therapy. Aug, 2005  |  Pubmed ID: 16043095

The aim of this study was to test the hypothesis that a specific antagonist may enhance the efficacy of its corresponding growth factor in a regulated tissue engineering strategy. Our prior research has led to the development of a retroviral vector that enables optimal regulated bone morphogenetic protein 4 (BMP4) expression in vitro and regulated bone formation in vivo with transduced muscle stem cells. However, when implanted in critical-sized calvarial defects, these cells led to residual bone formation without induction or bone overgrowth with induction, even at reduced cell doses. We thus co-implanted the aforementioned cells with stem cells engineered to express Noggin, a specific BMP antagonist. This approach, while preserving our ability to regulate bone regeneration closely, prevented both the basal level bone regeneration and the bone overgrowth and, more importantly, led to the regeneration of bone that more closely resembled normal bone. We believe that this regulatable tissue engineering strategy, enhanced by utilizing a specific antagonist, constitutes a new paradigm for tissue engineering and regenerative medicine.

Differential Effect of BMP4 on NIH/3T3 and C2C12 Cells: Implications for Endochondral Bone Formation

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Sep, 2005  |  Pubmed ID: 16059633

After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage.

Initial Failure in Myoblast Transplantation Therapy Has Led the Way Toward the Isolation of Muscle Stem Cells: Potential for Tissue Regeneration

Current Topics in Developmental Biology. 2005  |  Pubmed ID: 16125002

Myoblast transfer therapy can restore dystrophin expressing myofibers in mdx mice and patients with Duchenne muscular dystrophy (DMD). However, the effectiveness of this technique is hindered by numerous limitations, including minimal distribution of cells after injection, immune rejection, and poor cell survival. Initial studies revealed that only a small population of cells was responsible for muscle regeneration. Compared with myoblast transplantation, the injection of a population of myogenic cells purified with the pre-plate technique results in a superior regeneration of dystrophin-expressing myofibers. These postnatal muscle-derived stem cells (MDSC) undergo self-renewal, display long-term proliferation, and differentiate into multiple lineages. This review examines the initial obstacles encountered in myoblast transplantation, the regenerative properties of MDSC, and the potential use of these stem cells not only for DMD therapy but also for multiple applications, including bone repair and blood reconstitution.

Differential Myocardial Infarct Repair with Muscle Stem Cells Compared to Myoblasts

Molecular Therapy : the Journal of the American Society of Gene Therapy. Dec, 2005  |  Pubmed ID: 16125468

Myoblast transplantation for cardiac repair has generated beneficial results in both animals and humans; however, poor viability and poor engraftment of myoblasts after implantation in vivo limit their regeneration capacity. We and others have identified and isolated a subpopulation of skeletal muscle-derived stem cells (MDSCs) that regenerate skeletal muscle more effectively than myoblasts. Here we report that in comparison with a myoblast population, MDSCs implanted into infarcted hearts displayed greater and more persistent engraftment, induced more neoangiogenesis through graft expression of vascular endothelial growth factor, prevented cardiac remodeling, and elicited significant improvements in cardiac function. MDSCs also exhibited a greater ability to resist oxidative stress-induced apoptosis compared to myoblasts, which may partially explain the improved engraftment of MDSCs. These findings indicate that MDSCs constitute an alternative to other myogenic cells for use in cardiac repair applications.

Biaxial Mechanical Properties of Muscle-derived Cell Seeded Small Intestinal Submucosa for Bladder Wall Reconstitution

Biomaterials. Feb, 2005  |  Pubmed ID: 15275818

Bladder wall replacement remains a challenging problem for urological surgery due to leakage, infection, stone formation, and extensive time needed for tissue regeneration. To explore the feasibility of producing a more functional biomaterial for bladder reconstitution, we incorporated muscle-derived cells (MDC) into small intestinal submucosa (SIS) scaffolds. MDC were harvested from mice hindleg muscle, transfected with a plasmid encoding for beta-galactosidase, and placed into single-layer SIS cell culture inserts. Twenty-five MDC and/or SIS specimens were incubated at 37 degrees C for either 10 or 20 days. After harvesting, mechanical properties were characterized using biaxial testing, and the areal strain under 1 MPa peak stress used to quantify tissue compliance. Histological results indicated that MDC migrated throughout the SIS after 20 days. The mean (+/-SE) areal strain of the 0 day control group was 0.182 +/- 0.027 (n=5). After 10 days incubation, the mean (+/-SE) areal strain in MDC/SIS was 0.247 +/- 0.014 (n=5) compared to 10 day control SIS 0.200 +/- 0.024 (n=6). After 20 days incubation, the mean areal strain of MDC/SIS was 0.255 +/- 0.019 (n=5) compared to control SIS 0.170 +/- 0.025 (n=5). Both 10 and 20 days seeded groups were significantly different (p=0.027) than that of incubated SIS alone, but were not different from each other. These results suggest that MDC growth was supported by SIS and that initial remodeling of the SIS ECM had occurred within the first 10 days of incubation, but may have slowed once the MDC had grown to confluence within the SIS.

Cutting-edge Muscle Recovery: Using Antifibrosis Agents to Improve Healing

The Physician and Sportsmedicine. May, 2005  |  Pubmed ID: 20086363

Muscle injuries, the most frequent type of sports-related injury, present challenging problems in traumatology and sports medicine. Injured skeletal muscle can repair itself via spontaneous regeneration, but extracellular matrix overgrowth and collagen deposition can lead to fibrosis, resulting in incomplete functional recovery and a propensity for injury recurrence. Physicians may be able to improve skeletal muscle healing after injury when researchers understand more about the mechanisms involved in scar-tissue development. Techniques may be refined to prevent muscle fibrosis-specifically via the inactivation of transforming growth factor-beta-1-and, ultimately, improve muscle healing after injuries.

The Effect of Relaxin Treatment on Skeletal Muscle Injuries

The American Journal of Sports Medicine. Dec, 2005  |  Pubmed ID: 16157846

Injured skeletal muscle can repair itself via spontaneous regeneration; however, the overproduction of extracellular matrix and excessive collagen deposition lead to fibrosis. Neutralization of the effect of transforming growth factor-beta 1, a key fibrotic cytokine, on myogenic cell differentiation after muscle injury can prevent fibrosis, enhance muscle regeneration, and thereby improve the functional recovery of injured muscle.

NS-398, a Cyclooxygenase-2-specific Inhibitor, Delays Skeletal Muscle Healing by Decreasing Regeneration and Promoting Fibrosis

The American Journal of Pathology. Oct, 2005  |  Pubmed ID: 16192645

Nonsteroidal anti-inflammatory drugs are often prescribed after muscle injury. However, the effect of nonsteroidal anti-inflammatory drugs on muscle healing remains primarily controversial. To further examine the validity of using these drugs after muscle injury, we investigated the working mechanism of NS-398, a cyclooxygenase-2-specific inhibitor. In vitro experiments showed that NS-398 inhibited the proliferation and maturation of differentiated myogenic precursor cells, suggesting a detrimental effect on skeletal muscle healing. Using a mouse laceration model, we analyzed the in vivo effect of NS-398 on skeletal muscle healing at time points up to 4 weeks after injury. The in vivo results revealed delayed muscle regeneration at early time points after injury in the NS-398-treated mice. Compared to controls, lacerated muscles treated with NS-398 expressed higher levels of transforming growth factor-beta1, which corresponded with increased fibrosis. In addition, transforming growth factor-beta1 co-localized with myostatin, a negative regulator of skeletal muscle growth. We also found reduced neutrophil and macrophage infiltration in treated muscles, indicating that the delayed skeletal muscle healing observed after NS-398 treatment could be influenced by the anti-inflammatory effect of NS-398. Our findings suggest that the use of cyclooxygenase-2-specific inhibitors to treat skeletal muscle injuries warrants caution because they may interfere with muscle healing.

VEGF Improves, Whereas SFlt1 Inhibits, BMP2-induced Bone Formation and Bone Healing Through Modulation of Angiogenesis

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Nov, 2005  |  Pubmed ID: 16234975

We studied the interaction between VEGF and BMP2 during bone formation and bone healing. Results indicate that VEGF antagonist inhibited BMP2-elicited bone formation, whereas the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing through modulation of angiogenesis.

Influence of BMPs on the Formation of Osteoblastic Lesions in Metastatic Prostate Cancer

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Dec, 2005  |  Pubmed ID: 16294272

The purpose of this study was to evaluate the role of BMPs on the formation of metastatic prostate cancer lesions to bone. Our results show that BMPs influence the development and progression of osteoblastic lesions and suggest that therapies that inhibit BMP activity may reduce the formation and progression of osteoblastic lesions.

Musculoskeletal Gene Therapy and Its Potential Use in the Treatment of Complicated Musculoskeletal Infection

Infectious Disease Clinics of North America. Dec, 2005  |  Pubmed ID: 16297745

Tissue repair is a major issue in orthopedics. Many musculoskeletal tissues, including cartilage, meniscus, and the anterior cruciate ligament, heal poorly after injury. Recent studies have led to the identification of numerous growth factors and other gene products that can promote the regeneration of damaged musculoskeletal tissues. In the last century, the discovery and evolving use of antibiotics has significantly decreased the prevalence and severity of infectious diseases. In many orthopedic scenarios, however, treatment of infections can be difficult, and often involves a prolonged course of antibiotics with concomitant surgical interventions and loss of tissue. Although studies have demonstrated the successful transfer of target genes and the associated manipulation of the musculoskeletal tissue environment, researchers have made few attempts designed to use gene therapy to treat infectious musculoskeletal diseases in animal models. Before it is possible to use gene-based approaches to treat such diseases effectively, researchers must perform more studies to investigate the potential problems that may arise when using gene therapy in an infectious environment.

Cell Therapy for Muscle Regeneration and Repair

Physical Medicine and Rehabilitation Clinics of North America. Nov, 2005  |  Pubmed ID: 16214050

Biomechanical Characterization of the Urethral Musculature

American Journal of Physiology. Renal Physiology. May, 2006  |  Pubmed ID: 16368741

Rigorous study of the associations between urethral structural anatomy and biomechanical function is necessary to advance the understanding of the development, progression, and treatment of urethral pathologies. An ex vivo model was utilized to define the relative biomechanical contributions of the active (muscle) elements of the female urethra relative to its passive (noncontractile) elements. Whole urethras from female, adult rats were tested under a range of applied intraluminal pressures (0 to 20 mmHg) as a laser micrometer simultaneously measured midurethral outer diameter. Active tissue characterization was performed during induced contraction of either smooth muscle alone (N(omega)-nitro-l-arginine, phenylephrine), striated muscle alone (sodium nitroprusside, atropine, hexamethonium, acetylcholine), or during collective activation of both muscles (N(omega)-nitro-l-arginine, phenylephrine, acetylcholine). The subsequent collection of paired passive biomechanical responses permitted the determination of parameters related to intrinsic muscle contractile function. Activation of each muscle layer significantly influenced the biomechanical responses of the tissue. Measures of muscle responsiveness over a wide range of sustained opposing pressures indicated that an activated striated muscle component was approximately one-third as effective as activated smooth muscle in resisting tissue deformation. The maximum circumferential stress generated by the striated muscle component under these conditions was also determined to be approximately one-third of that generated by the smooth muscle (748 +/- 379 vs. 2,229 +/- 409 N/m(2)). The experiments quantitatively reveal the relative influence of the intrinsic urethral smooth and striated muscle layers with regard to their effect on the mechanical properties and maximum functional responses of the urethra to applied intralumenal stresses in the complete absence of extrinsic influences.

Cartilage Repair Using Bone Morphogenetic Protein 4 and Muscle-derived Stem Cells

Arthritis and Rheumatism. Feb, 2006  |  Pubmed ID: 16447218

Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle exhibit long-time proliferation, high self-renewal, and multipotent differentiation. This study was undertaken to investigate the ability of MDSCs that were retrovirally transduced to express bone morphogenetic protein 4 (BMP-4) to differentiate into chondrocytes in vitro and in vivo and enhance articular cartilage repair.

Nerve Growth Factor Improves the Muscle Regeneration Capacity of Muscle Stem Cells in Dystrophic Muscle

Human Gene Therapy. Feb, 2006  |  Pubmed ID: 16454652

Researchers have attempted to use gene- and cell-based therapies to restore dystrophin and alleviate the muscle weakness that results from Duchenne muscular dystrophy (DMD). Our research group has isolated populations of muscle-derived stem cells (MDSCs) from the postnatal skeletal muscle of mice. In comparison with satellite cells, MDSCs display an improved transplantation capacity in dystrophic mdx muscle that we attribute to their ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages. Here we tested whether the use of nerve growth factor (NGF) improves the transplantation efficiency of MDSCs. We used two methods of in vitro NGF stimulation: retroviral transduction of MDSCs with a CL-NGF vector and direct stimulation of MDSCs with NGF protein. Neither method of NGF treatment changed the marker profile or proliferation behavior of the MDSCs, but direct stimulation with NGF protein significantly reduced the in vitro differentiation ability of the cells. NGF stimulation also significantly enhanced the engraftment efficiency of MDSCs transplanted within the dystrophic muscle of mdx mice, resulting in the regeneration of numerous dystrophin-positive muscle fibers. These findings highlight the importance of NGF as a modulatory molecule, the study of which will broaden our understanding of its biologic role in the regeneration and repair of skeletal muscle by musclederived cells.

Inhibited Skeletal Muscle Healing in Cyclooxygenase-2 Gene-deficient Mice: the Role of PGE2 and PGF2alpha

Journal of Applied Physiology (Bethesda, Md. : 1985). Oct, 2006  |  Pubmed ID: 16778000

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat skeletal muscle injury. However, studies have shown that NSAIDs may be detrimental to the healing process. Mediated by prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)), the cycloxygenase-2 (COX-2) pathway plays an important role in muscle healing. We hypothesize that the COX-2 pathway is important for the fusion of muscle cells and the regeneration of injured muscle. For the in vitro experiments, we isolated myogenic precursor cells from wild-type (Wt) and COX-2 gene-deficient (COX-2(-/-)) mice and examined the effect of PGE(2) and PGF(2alpha) on cell fusion. For the in vivo experiments, we created laceration injury on the tibialis anterior (TA) muscles of Wt and COX-2(-/-) mice. Five and 14 days after injury, we examined the TA muscles histologically and functionally. We found that the secondary fusion between nascent myotubes and myogenic precursor cells isolated from COX-2(-/-) mice was severely compromised compared with that of Wt controls but was restored by the addition of PGF(2alpha) or, to a lesser extent, PGE(2) to the culture. Histological and functional assessments of the TA muscles in COX-2(-/-) mice revealed decreased regeneration relative to that observed in the Wt mice. The findings reported here demonstrate that the COX-2 pathway plays an important role in muscle healing and that prostaglandins are key mediators of the COX-2 pathway. It suggests that the decision to use NSAIDs to treat muscle injuries warrants critical evaluation because NSAIDs might impair muscle healing by inhibiting the fusion of myogenic precursor cells.

Mouse Adipose-derived Stem Cells Undergo Multilineage Differentiation in Vitro but Primarily Osteogenic and Chondrogenic Differentiation in Vivo

Tissue Engineering. Jul, 2006  |  Pubmed ID: 16889519

Human, rat, and mouse studies have demonstrated the existence of a population of adipose-derived adult stem (ADAS) cells that can undergo multilineage differentiation in vitro. However, it remains unclear whether these cells maintain their multilineage potential in vivo. The aim of this study was to examine the in vitro and in vivo characteristics and behavior of a potential population of murine ADAS (muADAS) cells isolated from the visceral fat of the abdominal cavity of C57BL/10J mice. We used flow cytometry to examine the cells' expression of CD29, CD31, CD45, CD34, CD44, CD144, CD146, Flk1, and Sca-1. The isolated cell population was CD45 negative, which precludes contamination by hematopoietic cells, but was partially positive for Sca-1 and CD34: 2 stem-cell markers. After induction in conditioned medium, the muADAS cells gained the ability to undergo adipogenic, osteogenic, chondrogenic, myogenic, and hematopoietic differentiation in vitro. The muADAS cells readily differentiated to form bone and cartilage in vivo for up to 24 weeks, but their ability to regenerate muscle or reconstitute bone marrow was found to be limited.

Periurethral Cellular Injection: Comparison of Muscle-derived Progenitor Cells and Fibroblasts with Regard to Efficacy and Tissue Contractility in an Animal Model of Stress Urinary Incontinence

Urology. Aug, 2006  |  Pubmed ID: 16904482

To compare muscle-derived cells (MDCs) and fibroblasts with regard to their potential for restoration of urethral function on injection in a previously established animal model of stress urinary incontinence.

VEGF and BMP Expression in Mouse Osteosarcoma Cells

Clinical Orthopaedics and Related Research. Sep, 2006  |  Pubmed ID: 16906080

Osteosarcoma is the most common primary bone malignancy. Despite improvements in therapy, approximately 30% of patients experience pulmonary metastasis. Expression of several growth factors, including VEGF and BMPs, has been implicated in tumor progression and metastatic potential. We hypothesized increased metastatic potential of mouse osteosarcoma cells positively correlates with the expression of VEGF and BMPs. We studied the expression patterns of these growth factors in two murine osteosarcoma cell lines with varying degrees of metastatic potential: K7M2 (highly metastatic) and K12 (minimally metastatic). Expression of VEGF and BMP2 were higher in the metastatic K7M2 cell line. We also investigated the effects of the BMP antagonist noggin on osteosarcoma growth characteristics in vitro. We noted decreased motility, altered morphology, and increased cell death in the highly metastatic K7M2 cell line. Less metastatic K12 cells showed substantial cell death without clear alteration of motility or morphology. These data suggest BMP2 expression may be an important factor in osteosarcoma metastasis and noggin administration theoretically could block its actions. Inhibition of BMPs and VEGF should be investigated further as a possible strategy for decreasing the incidence of pulmonary metastases in osteosarcoma.

Tissue Engineering and Developmental Biology: Going Biomimetic

Tissue Engineering. Dec, 2006  |  Pubmed ID: 17518669

This article contains the collective views expressed at the first session of the workshop "Tissue Engineering--The Next Generation," which was devoted to the interactions between developmental biology and tissue engineering. Donald Ingber discussed the chasms between developmental biology and tissue engineering from the perspective of a cell biologist who has had interest in tissue engineering since its early days. Van C. Mow shared a historical perspective on the development of tissue engineering as one of the first engineers involved in the field. David Butler offered an assessment of functional tissue engineering, a new area he helped establish and promote. Laura Niklason discussed how to be more effective in developing cellular therapies for large numbers of patients. Johnny Huard described his approach to tissue engineering, based on the use of muscle-derived cells. Jeremy Mao focused on cell homing and cell density in the context of native development and relevance to tissue engineering. Ioannis Yannas proposed a set of "rules" in organ regeneration. Collectively, the faculty expressed a remarkable level of enthusiasm for bridging the gaps between developmental biology and tissue engineering and offered new ideas on how to facilitate the interaction between the two fields.

Overexpression of Noggin Inhibits BMP-mediated Growth of Osteolytic Prostate Cancer Lesions

Bone. Feb, 2006  |  Pubmed ID: 16126463

Although a majority of metastatic prostate cancer lesions are osteoblastic in nature, some are mixed or lytic; and, osteoblastic lesions require osteolytic activity in order to progress. The role of BMPs in the formation of prostate cancer metastases to bone remains unknown. We hypothesized that BMPs influence the development and progression of osteolytic prostate cancer lesions.

Gene Therapy and Tissue Engineering in Orthopaedic Surgery

Instructional Course Lectures. 2006  |  Pubmed ID: 16958493

Despite setbacks in other fields, gene therapy in orthopaedic surgery continues to serve as the basis for novel treatments of various musculoskeletal disorders. Even in the brief time since the last review of scientific progress in this area, another orthopaedic-related disease has joined the ranks of those studied in gene therapy clinical trials. Armed with new techniques and new reagents, and committed to the increased use of tissue engineering, physicians and scientists continue to work together to accelerate tissue repair and reverse the course of chronic debilitating diseases.

Mixed Metastatic Lung Cancer Lesions in Bone Are Inhibited by Noggin Overexpression and Rank:Fc Administration

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Oct, 2006  |  Pubmed ID: 16995812

Lung cancer metastases to bone produce a primarily mixed osteolytic/osteoblastic lesion. The purpose of this study was to determine if blockade of both pathways would inhibit the formation these lesions in bone. Inhibition of the osteoblastic lesion with noggin and the osteolytic lesion with RANK:Fc was a successful treatment strategy to inhibit progression of mixed lung cancer lesions in bone.

Stem and Progenitor Cells in Skeletal Muscle Development, Maintenance, and Therapy

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2007  |  Pubmed ID: 17387336

Satellite cells are dormant progenitors located at the periphery of skeletal myofibers that can be triggered to proliferate for both self-renewal and differentiation into myogenic cells. In addition to anatomic location, satellite cells are typified by markers such as M-cadherin, Pax7, Myf5, and neural cell adhesion molecule-1. The Pax3 and Pax7 transcription factors play essential roles in the early specification, migration, and myogenic differentiation of satellite cells. In addition to muscle-committed satellite cells, multi-lineage stem cells encountered in embryonic, as well as adult, tissues exhibit myogenic potential in experimental conditions. These multi-lineage stem cells include side-population cells, muscle-derived stem cells (MDSCs), and mesoangioblasts. Although the ontogenic derivation, identity, and localization of these non-conventional myogenic cells remain elusive, recent results suggest their ultimate origin in blood vessel walls. Indeed, purified pericytes and endothelium-related cells demonstrate high myogenic potential in culture and in vivo. Allogeneic myoblasts transplanted into Duchenne muscular dystrophy (DMD) patients have been, in early trials, largely inefficient owing to immune rejection, rapid death, and limited intramuscular migration--all obstacles that are now being alleviated, at least in part, by more efficient immunosuppression and escalated cell doses. As an alternative to myoblast transplantation, stem cells such as mesoangioblasts and CD133+ progenitors administered through blood circulation have recently shown great potential to regenerate dystrophic muscle.

Thermosensitive Hydrogel PEG-PLGA-PEG Enhances Engraftment of Muscle-derived Stem Cells and Promotes Healing in Diabetic Wound

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2007  |  Pubmed ID: 17406344

Regenerating new tissue using cell transplantation has relied on successful cell engraftment in the host; however, cell engraftment into the diabetic skin wound is not as successful as in many other tissues. We used a biodegradable and biocompatible triblock co-polymer poly(ethylene glycol-b-[DL-lactic acid-co-glycolic acid]-b-ethylene glycol) (PEG-PLGA-PEG), which forms a thermosensitive hydrogel, as a wound dressing and scaffold. We found that the thermosensitive hydrogel increased the engraftment of muscle-derived stem cells (MDSCs) by 20- to 30-fold until day 20, when the wound was completely closed in a db/db genetically diabetic mouse model. At day 9, 30% of the transplanted MDSCs were found to remain, and 15% remained at day 20 after transplantation. The increased engraftment resulted in enhanced wound healing, as indicated by the wound closure rate, epithelium migration, and collagen deposition. Using MDSCs stably expressing beta-gal and immunofluorescence, we found that 25% of MDSCs differentiated into fibroblasts, 10% into myofibroblasts, and 10% into endothelial cells. We conclude that using the thermosensitive hydrogel as a scaffold increased the engraftment of MDSCs, which leads to improved diabetic wound healing, possibly by retaining the cells at the wound site for longer.

A Role for Cell Sex in Stem Cell-mediated Skeletal Muscle Regeneration: Female Cells Have Higher Muscle Regeneration Efficiency

The Journal of Cell Biology. Apr, 2007  |  Pubmed ID: 17420291

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.

Noggin Inhibits Postoperative Resynostosis in Craniosynostotic Rabbits

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Jul, 2007  |  Pubmed ID: 17437358

Inhibition of bone formation after surgery to correct craniosynostosis would alleviate the need for secondary surgeries and decrease morbidity and mortality. This study used a single dose of Noggin protein to prevent resynostosis and improve postoperative outcomes in a rabbit model of craniosynostosis.

Matrix Metalloproteinase-1 Therapy Improves Muscle Healing

Journal of Applied Physiology (Bethesda, Md. : 1985). Jun, 2007  |  Pubmed ID: 17551103

Muscle undergoes time-dependent phases of healing after injury, which ultimately results in residual fibrosis in the injured area. The use of exogenous matrix metalloproteinases (MMPs) may improve recovery after muscle injury by promoting the digestion of existing fibrous tissue and releasing local growth factors. In the current experiment, bilateral gastrocnemius (GM) lacerations were created in severe combined immunodeficient mice. Twenty-five days after injury (peak posttraumatic fibrosis), C2C12 cells (myoblasts) transduced with the LacZ reporter gene were injected with exogenous MMP-1 into the right GMs at the site of injury; the cells were also injected along with PBS (control) at the site of injury in the left GMs. The muscle tissues were examined histologically via X-gal, hemotoxylin and eosin, and Masson's trichrome staining. The MMP-treated limbs contained more regenerating myofibers than did the control limbs (MMP 170+/-96 fibers, control 62+/-51 fibers; P<0.001). Less fibrous tissue was observed within MMP-treated muscles (MMP: 24+/-11%, control: 35+/-15%; P<0.01). These results suggest that the direct injection of MMP-1 into the zone of injury during fibrosis can enhance muscle regeneration by increasing the number of myofibers and decreasing the amount of fibrous tissue.

Regenerative Medicine in Orthopaedic Surgery

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Oct, 2007  |  Pubmed ID: 17551972

Regenerative medicine holds great promise for orthopaedic surgery. As surgeons continue to face challenges regarding the healing of diseased or injured musculoskeletal tissues, regenerative medicine aims to develop novel therapies that will replace, repair, or promote tissue regeneration. This review article will provide an overview of the different research areas involved in regenerative medicine, such as stem cells, bioinductive factors, and scaffolds. The potential use of stem cells for orthopaedic tissue engineering will be addressed by presenting the current progress with skeletal muscle-derived stem cells. As well, the development of a revascularized massive allograft will be described and will serve as a prototypic model of orthopaedic tissue engineering. Lastly, we will describe current approaches used to design cell instructive materials and how they can be used to promote and regulate the formation of bony tissue.

Malignant Transformation of Multipotent Muscle-derived Cells by Concurrent Differentiation Signals

Stem Cells (Dayton, Ohio). Sep, 2007  |  Pubmed ID: 17569791

Recent studies have shown that germ-line determination occurs early in development and that extracellular signaling can alter this fate. This denial of a cell's fate by counteracting its intrinsic signaling pathways through extrinsic stimulation is believed to be associated with oncogenesis. Using specific populations of multipotent skeletal muscle-derived stem cells (MDSCs), we have been able to generate tumors by subjecting cells with specific lineage predilections to concomitant differentiation signals. More specifically, when a stem cell that had a predilection toward osteogenesis was implanted into a skeletal muscle, tumors formed in 25% of implanted mice. When cells predilected to undergo myogenesis were pretreated with bone morphogenetic protein 4 (BMP4) for 4 days prior to implantation, they formed tumors in 25% of mice. These same myogenic predilected cells, when transduced to express BMP4 and implanted into either a long-bone or cranial defect, formed bone, but they formed tumors in 100% of mice when implanted into the skeletal muscle. The tumors generated in this latter study were serially transplantable as long as they retained BMP4 expression. Furthermore, when we impeded the ability of the cells to undergo myogenic differentiation using small interfering RNA to the myogenic regulator MyoD1, we stopped transformation. Based on our findings, we postulate that specific MDSC populations can undergo concomitant signal-induced transformation and that the initial stages of transformation may be due to changes in the balance between the inherent nature of the cell and extrinsic signaling pathways. This theory represents a potential link between somatic stem cells and cancer and suggests an involvement of the niche/environment in transformation.

Skeletal Muscle Fiber Type Conversion During the Repair of Mouse Soleus: Potential Implications for Muscle Healing After Injury

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Nov, 2007  |  Pubmed ID: 17593537

We used a mouse model of cardiotoxin injury to examine fiber type conversion during muscle repair. We evaluated the soleus muscles of 37 wild-type mice at 2, 4, 8, and 12 weeks after injury. We also used antibodies (fMHC and sMHC) against fast and slow myosin heavy chain to classify the myofibers into three categories: fast-, slow-, and mixed (hybrid)-type myofibers (myofibers expressing both fMHC and sMHC). Our results revealed an increase in the percentage of slow-type myofibers and a decrease in the percentage of fast-type myofibers during the repair process. The percentage of hybrid-type myofibers increased 2 weeks after injury, then gradually decreased over the following 6 weeks. Similarly, our analysis of centronucleated myofibers showed an increase in the percentage of slow-type myofibers and decreases in the percentages of fast- and hybrid-type myofibers. We also investigated the relationship between myofiber type conversion and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha). The expression of both PGC-1alpha protein, which is expressed in both the nucleus and the cytoplasm of regenerating myofibers, and sMHC protein increased with time after cardiotoxin injection, but we observed no significant differential expression of fMHC protein in regenerating muscle fibers during muscle repair. PGC-1alpha-positive myofibers underwent fast to slow myofiber type conversion during the repair process. These results suggest that PGC-1alpha contributes to myofiber type conversion after muscle injury and that this phenomenon could influence the recovery of the injured muscle.

Relationships Between Transforming Growth Factor-beta1, Myostatin, and Decorin: Implications for Skeletal Muscle Fibrosis

The Journal of Biological Chemistry. Aug, 2007  |  Pubmed ID: 17597062

Recent studies have shown that myostatin, first identified as a negative regulator of skeletal muscle growth, may also be involved in the formation of fibrosis within skeletal muscle. In this study, we further explored the potential role of myostatin in skeletal muscle fibrosis, as well as its interaction with both transforming growth factor-beta1 and decorin. We discovered that myostatin stimulated fibroblast proliferation in vitro and induced its differentiation into myofibroblasts. We further found that transforming growth factor-beta1 stimulated myostatin expression, and conversely, myostatin stimulated transforming growth factor-beta1 secretion in C2C12 myoblasts. Decorin, a small leucine-rich proteoglycan, was found to neutralize the effects of myostatin in both fibroblasts and myoblasts. Moreover, decorin up-regulated the expression of follistatin, an antagonist of myostatin. The results of in vivo experiments showed that myostatin knock-out mice developed significantly less fibrosis and displayed better skeletal muscle regeneration when compared with wild-type mice at 2 and 4 weeks following gastrocnemius muscle laceration injury. In wild-type mice, we found that transforming growth factor-beta1 and myostatin co-localize in myofibers in the early stages of injury. Recombinant myostatin protein stimulated myofibers to express transforming growth factor-beta1 in skeletal muscles at early time points following injection. In summary, these findings define a fibrogenic property of myostatin and suggest the existence of co-regulatory relationships between transforming growth factor-beta1, myostatin, and decorin.

Osteogenic Potential of Postnatal Skeletal Muscle-derived Stem Cells is Influenced by Donor Sex

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research. Oct, 2007  |  Pubmed ID: 17605633

This study compared the osteogenic differentiation of F-MDSCs and M-MDSCs. Interestingly, M-MDSCs expressed osteogenic markers and underwent mineralization more readily than F-MDSCs; a characteristic likely caused by more osteoprogenitor cells within the M-MDSCs than the F-MDSCs and/or an accelerated osteogenic differentiation of M-MDSCs.

Decorin Gene Transfer Promotes Muscle Cell Differentiation and Muscle Regeneration

Molecular Therapy : the Journal of the American Society of Gene Therapy. Sep, 2007  |  Pubmed ID: 17609657

We have shown that decorin, a small leucine-rich proteoglycan, can inhibit transforming growth factor (TGF)-beta1 to prevent fibrous scar formation and improve muscle healing after injury. In the decorin-treated muscle, an enhancement of muscle regeneration is observed through histological examination. In this article, we report our determination of whether decorin has a direct effect on myogenic cells' differentiation. Our results indicate that myoblasts genetically engineered to express decorin (CD cells) differentiated into myotubes at a significantly higher rate than did control myoblasts (C2C12). This enhanced differentiation led to the up-regulation of myogenic genes (Myf5, Myf6, MyoD, and myogenin) in CD cells in vitro. We speculate that the higher rate of differentiation exhibited by the CD cells is due to the up-regulation of follistatin, peroxisome-proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha), p21, and the myogenic genes, and the down-regulation of TGF-beta1 and myostatin. Decorin gene transfer in vivo promoted skeletal muscle regeneration and accelerated muscle healing after injury. These results suggest that decorin not only prevents fibrosis but also improves muscle regeneration and repair.

Prospective Identification of Myogenic Endothelial Cells in Human Skeletal Muscle

Nature Biotechnology. Sep, 2007  |  Pubmed ID: 17767154

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.

Muscle-derived Stem Cells for Tissue Engineering and Regenerative Therapy

Biomaterials. Dec, 2007  |  Pubmed ID: 17915311

Skeletal muscle has been recognized as an essential source of progenitor or satellite cells, which are primarily responsible for muscle regeneration. Recently, muscle has also been identified as a valuable source of postnatal stem cells that appear to be distinct from satellite cells and possess the ability to differentiate into other cell lineages. These cells, named muscle-derived stem cells, possess a high myogenic capacity and effectively regenerate both skeletal and cardiac muscle. Remarkably, when genetically modified ex vivo to express growth factors, these cells can differentiate into osteogenic and chondrogenic lineages and have been shown to promote the repair of bone and cartilage. Muscle stem cell-based regenerative therapy and tissue engineering using ex vivo gene therapy, are promising approaches for the treatment of various musculoskeletal, cardiovascular, and urological disorders.

[The Sex of Muscle Stem Cells: Must It Be Taken into Account?]

Médecine Sciences : M/S. Oct, 2007  |  Pubmed ID: 17937884

A Relationship Between Vascular Endothelial Growth Factor, Angiogenesis, and Cardiac Repair After Muscle Stem Cell Transplantation into Ischemic Hearts

Journal of the American College of Cardiology. Oct, 2007  |  Pubmed ID: 17950150

We investigated whether vascular endothelial growth factor (VEGF) was associated with the angiogenic and therapeutic effects induced after transplantation of skeletal muscle-derived stem cells (MDSCs) into a myocardial infarction (MI).

In Vitro Interaction Between Muscle-derived Stem Cells and Nucleus Pulposus Cells

The Spine Journal : Official Journal of the North American Spine Society. Sep-Oct, 2008  |  Pubmed ID: 18023623

Current therapies for intervertebral disc degeneration (IDD) are aimed at treating the clinical symptoms arising from IDD rather than directly treating the underlying problem. Pathophysiology of IDD is characterized by a progressive decrease in proteoglycan content and cell density in the nucleus pulposus (NP). A cell-based therapy is a promising concept that uses various cell types to repopulate the disc in an attempt to slow, stop, or reverse the progressive loss of proteoglycans. Stem cells appear to be an excellent candidate for this purpose, based on their ability to differentiate into various connective tissue lineages. The muscle tissue could serve as a good source of adult stem cells because of its vast abundance through out the human body.

Development of a Tissue-engineered Vascular Graft Combining a Biodegradable Scaffold, Muscle-derived Stem Cells and a Rotational Vacuum Seeding Technique

Biomaterials. Mar, 2008  |  Pubmed ID: 18035412

There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77+/-20% vs. 66+/-6% at day 0) while CD34 expression was reduced (19+/-12% vs. 61+/-10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127+/-900 mm Hg and suture retention strength was 1.3+/-0.3N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.

Regenerative Medicine for the Musculoskeletal System Based on Muscle-derived Stem Cells

The Journal of the American Academy of Orthopaedic Surgeons. Feb, 2008  |  Pubmed ID: 18252837

The identification and characterization of stem cells is introducing a paradigm shift in the field of orthopaedic surgery. Whereas in the past, diseased tissue was replaced with allograft material, current trends in research revolve around regenerating damaged tissue. Muscle-derived stem cells have an application in regeneration of articular cartilage, bone, and skeletal muscle. These postnatal (ie, adult) stem cells can be readily isolated via muscle biopsy. They can display long-term proliferation, high self-renewal, and multipotent differentiation. They also can be genetically modified to secrete growth factors important to tissue healing, thereby functioning as implantable, long-lasting reservoirs for these molecules. Taken together, this evidence suggests that muscle-derived stem cells are well suited for gene therapy and tissue engineering applications for the musculoskeletal system. Effective implementation of even just a few applications of muscle-derived stem cell-based tissue engineering has the potential to revolutionize the way certain musculoskeletal diseases are managed.

Sex Differences in Muscle-derived Stem Cells and Skeletal Muscle

Critical Reviews in Eukaryotic Gene Expression. 2008  |  Pubmed ID: 18304031

Sex is well known to influence life expectancy and disposition to disease. Stem and progenitor cells are believed to persist throughout life, and they contribute to the repair and healthy maintenance of tissue; consequently, sex-related differences demonstrated by stem cells may provide insight to sex-related differences in aging, disease, and healing. However, cell sex is an often overlooked variable in stem cell biology.

Dominant Negative Bmp5 Mutation Reveals Key Role of BMPs in Skeletal Response to Mechanical Stimulation

BMC Developmental Biology. 2008  |  Pubmed ID: 18380899

Over a hundred years ago, Wolff originally observed that bone growth and remodeling are exquisitely sensitive to mechanical forces acting on the skeleton. Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse. Although the processes of mechanotransduction and functional response of bone to mechanical strain have been extensively studied, the molecular signaling mechanisms that mediate the response of bone cells to mechanical stimulation remain unclear.

Matrix Metalloproteinase-1 Treatment of Muscle Fibrosis

Acta Biomaterialia. Sep, 2008  |  Pubmed ID: 18440885

The onset of scarring after injury may impede the regeneration and functional recovery of skeletal muscle. Matrix metalloproteinase-1 (MMP-1) hydrolyzes type I collagen and thus may improve muscle regeneration by resolving fibrotic tissue. We examined the effect of recombinant human MMP-1 on fibrosis in the lacerated gastrocnemius muscle of NOD/scid mice, allowing treatment potential to be ascertained in isolation from immune response. The efficacy of proMMP-1 and active MMP-1 were compared with or without poly(ethylene glycol) (PEG) modification, which was intended to increase the enzyme's stability. Active MMP-1 was most effective in reducing fibrosis, although treatment with proMMP-1 was also beneficial relative to controls. PEG-modified MMP-1 had minimal activity in vivo, despite retaining activity towards a thioester substrate. Moreover, the modified enzyme was inactivated by trypsin and subtilisin at rates comparable to that of native MMP-1. These results and those of computational structural studies suggest that modification occurs at the C-terminal hemopexin domain of MMP-1, which plays a critical role in collagen turnover. Site-specific modifications that spares catalytic and substrate binding sites while protecting susceptible proteolytic digestion sites may be beneficial. We conclude that active MMP-1 can effectively reduce muscle scarring and that its activity is related to the ability of the enzyme to digest collagen, thereby facilitating remodeling of the injured muscle.

Purification and Long-term Culture of Multipotent Progenitor Cells Affiliated with the Walls of Human Blood Vessels: Myoendothelial Cells and Pericytes

Methods in Cell Biology. 2008  |  Pubmed ID: 18442653

We have identified with molecular markers and purified by flow cytometry two populations of cells that are developmentally and anatomically related to blood vessel walls in human tissues: myoendothelial cells, found in skeletal muscle and coexpressing markers of endothelial and myogenic cells, and pericytes--aka mural cells--which surround endothelial cells in capillaries and microvessels. Purified myoendothelial cells and pericytes exhibit multilineage developmental potential and differentiate, in culture and in vivo, into skeletal myofibers, bone, cartilage, and adipocytes. Myoendothelial cells and pericytes can be cultured on the long term with sustained marker expression and differentiation potential and clonal populations thereof have been derived. Yet, these blood vessel wall-derived progenitors exhibit no tendency to malignant transformation upon extended culture. Our results suggest that multipotent progenitor cells, such as mesenchymal stem cells, previously isolated retrospectively from diverse cultured adult tissues are derived from a subset of perivascular cells. We present in this chapter the main strategies and tactics used to purify, culture on the long term, and phenotypically characterize these novel multipotent cells.

Angiotensin II Receptor Blockade Administered After Injury Improves Muscle Regeneration and Decreases Fibrosis in Normal Skeletal Muscle

The American Journal of Sports Medicine. Aug, 2008  |  Pubmed ID: 18550776

Several therapeutic agents have been shown to inhibit fibrosis and improve regeneration after injury in skeletal muscle by antagonizing transforming growth factor-beta1. Angiotensin receptor blockers have been shown to have a similar effect on transforming growth factor-beta1 in a variety of tissues.

Improved Muscle Healing After Contusion Injury by the Inhibitory Effect of Suramin on Myostatin, a Negative Regulator of Muscle Growth

The American Journal of Sports Medicine. Dec, 2008  |  Pubmed ID: 18725651

Muscle contusions are the most common muscle injuries in sports medicine. Although these injuries are capable of healing, incomplete functional recovery often occurs.

Purification and Culture of Human Blood Vessel-associated Progenitor Cells

Current Protocols in Stem Cell Biology. Mar, 2008  |  Pubmed ID: 18770640

Multilineage progenitor cells, diversely designated as MSC, MAPC, or MDSC, have been previously extracted from long-term cultures of fetal and adult organs (e.g., bone marrow, brain, lung, pancreas, muscle, adipose tissue, and several others). The identity and location, within native tissues, of these elusive stem cells are described here. Subsets of endothelial cells and pericytes, which participate in the architecture of human blood vessels, exhibit, following purification to homogeneity, developmental multipotency. The selection from human tissues, by flow cytometry using combinations of positive and negative cell surface markers, of endothelial and perivascular cells is described here. In addition, a rare subset of myoendothelial cells that express markers of both endothelial and myogenic cell lineages and exhibit dramatic myogenic and cardiomyogenic potential has been identified and purified from skeletal muscle. The culture conditions amenable to the long-term proliferation of these blood vessel-associated stem cells in vitro are also described.

Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle- and Bone-like Subpopulations

Stem Cells (Dayton, Ohio). Jan, 2008  |  Pubmed ID: 17901398

In vivo, growth factors exist both as soluble and as solid-phase molecules, immobilized to cell surfaces and within the extracellular matrix. We used this rationale to develop more biologically relevant approaches to study stem cell behaviors. We engineered stem cell microenvironments using inkjet bioprinting technology to create spatially defined patterns of immobilized growth factors. Using this approach, we engineered cell fate toward the osteogenic lineage in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary muscle-derived stem cells (MDSCs) isolated from adult mice. This patterning approach was conducive to patterning the MDSCs into subpopulations of osteogenic or myogenic cells simultaneously on the same chip. When cells were cultured under myogenic conditions on BMP-2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time-lapse microscopy was used to visualize the formation of multinucleated myotubes, and immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off BMP-2 pattern. This work provides proof-of-concept for engineering spatially controlled multilineage differentiation of stem cells using patterns of immobilized growth factors. This approach may be useful for understanding cell behaviors to immobilized biological patterns and could have potential applications for regenerative medicine.

Interaction Between Macrophages, TGF-beta1, and the COX-2 Pathway During the Inflammatory Phase of Skeletal Muscle Healing After Injury

Journal of Cellular Physiology. Feb, 2008  |  Pubmed ID: 17657727

Inflammation, an important phase of skeletal muscle healing, largely involves macrophages, TGF-beta1, and the COX-2 pathway. To improve our understanding of how these molecules interact during all phases of muscle healing, we examined their roles in muscle cells in vitro and in vivo. Initially, we found that depletion of macrophages in muscle tissue led to reduced muscle regeneration. Macrophages may influence healing by inducing the production of TGF-beta1 and PGE2 in different muscle cell types. We then found that the addition of TGF-beta1 induced PGE2 production in muscle cells, an effect probably mediated by COX-2 enzyme. It was also found that TGF-beta1 enhanced macrophage infiltration in wild-type mice after muscle injury. However, this effect was not observed in COX-2(-/-) mice, suggesting that the effect of TGF-beta1 on macrophage infiltration is mediated by the COX-2 pathway. Furthermore, we found that PGE2 can inhibit the expression of TGF-beta1. PGE2 and TGF-beta1 may be involved in a negative feedback loop balancing the level of fibrosis formation during skeletal muscle healing. In conclusion, our results suggest a complex regulatory mechanism of skeletal muscle healing. Macrophages, TGF-beta1, and the COX-2 pathway products may regulate one another's levels and have profound influence on the whole muscle healing process.

The Influence of Sex on the Chondrogenic Potential of Muscle-derived Stem Cells: Implications for Cartilage Regeneration and Repair

Arthritis and Rheumatism. Dec, 2008  |  Pubmed ID: 19035511

To explore possible differences in muscle-derived stem cell (MDSC) chondrogenic differentiation in vitro and articular cartilage regeneration in vivo between murine male MDSCs (M-MDSCs) and female MDSCs (F-MDSCs).

Myogenic Endothelial Cells Purified from Human Skeletal Muscle Improve Cardiac Function After Transplantation into Infarcted Myocardium

Journal of the American College of Cardiology. Dec, 2008  |  Pubmed ID: 19038685

The aim of this study was to evaluate the therapeutic potential of human skeletal muscle-derived myoendothelial cells for myocardial infarct repair.

Isolation of a Slowly Adhering Cell Fraction Containing Stem Cells from Murine Skeletal Muscle by the Preplate Technique

Nature Protocols. 2008  |  Pubmed ID: 18772878

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

A Perivascular Origin for Mesenchymal Stem Cells in Multiple Human Organs

Cell Stem Cell. Sep, 2008  |  Pubmed ID: 18786417

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

Re: Angiotensin II Receptor Blockade Administered After Injury Improves Muscle Regeneration and Decreases Fibrosis in Normal Skeletal Muscle

The American Journal of Sports Medicine. Dec, 2008  |  Pubmed ID: 19074403

Blocking Vascular Endothelial Growth Factor with Soluble Flt-1 Improves the Chondrogenic Potential of Mouse Skeletal Muscle-derived Stem Cells

Arthritis and Rheumatism. Jan, 2009  |  Pubmed ID: 19116905

To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt-1 (sFlt-1), on chondrogenesis, using muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle.

Functional Overloading of Dystrophic Mice Enhances Muscle-derived Stem Cell Contribution to Muscle Contractile Capacity

Archives of Physical Medicine and Rehabilitation. Jan, 2009  |  Pubmed ID: 19154831

To evaluate the effect of functional overloading on the transplantation of muscle derived stem cells (MDSCs) into dystrophic muscle and the ability of transplanted cells to increase dystrophic muscle's ability to resist overloading-induced weakness.

Bone Regeneration Mediated by BMP4-expressing Muscle-derived Stem Cells is Affected by Delivery System

Tissue Engineering. Part A. Feb, 2009  |  Pubmed ID: 19061430

This study investigated the delivery of bone morphogenetic protein (BMP)4-secreting muscle-derived stem cells (MDSC-B4) capable of inducing bone formation in mice using collagen gel (CG), fibrin sealant (FS), and gelatin sponge carriers. After implanting these various cell-loaded scaffolds intramuscularly or into critical-size skull defects, we measured the extent of heterotopic ossification and calvarial defect healing over a 6-week period via radiographic, radiomorphometric, histological, and micro-computed tomography analyses. As expected, in the absence of MDSC-B4, there was no ectopic ossification and only minimal calvarial regeneration using each type of scaffold. Although CG and gelatin sponges loaded with BMP4-secreting cells produced the most ectopic bone, FS constructs produced bone with comparably less mineralization. In the mouse calvaria, we observed MDSC-B4-loaded scaffolds able to promote bone defect healing to a variable degree, but there were differences between these implants in the volume, shape, and morphology of regenerated bone. MDSC-B4 delivery in a gelatin sponge produced hypertrophic bone, whereas delivery in a CG and FS healed the defect with bone that closely resembled the quantity and configuration of native calvarium. In summary, hydrogels are suitable carriers for osteocompetent MDSCs in promoting bone regeneration, especially at craniofacial injury sites.

Stem Cells for the Treatment of Skeletal Muscle Injury

Clinics in Sports Medicine. Jan, 2009  |  Pubmed ID: 19064161

Skeletal muscle injuries are extremely common, accounting for up to 35%-55% of all sports injuries and quite possibly affecting all musculoskeletal traumas. These injuries result in the formation of fibrosis, which may lead to the development of painful contractures, increases patients' risk for repeat injuries, and limits their ability to return to a baseline or pre-injury level of function. The development of successful therapies for these injuries must consider the pathophysiology of these musculoskeletal conditions. We discuss the direct use of muscle-derived stem cells and some key cell population dynamics as well as the use of clinically applicable modalities that may enhance the local supply of stem cells to the zone of injury by promoting angiogenesis.

The Effect of Muscle Loading on Skeletal Muscle Regenerative Potential: an Update of Current Research Findings Relating to Aging and Neuromuscular Pathology

American Journal of Physical Medicine & Rehabilitation / Association of Academic Physiatrists. Feb, 2009  |  Pubmed ID: 19169178

Skeletal muscle is a dynamic tissue with a remarkable ability to continuously respond to environmental stimuli. Among its adaptive responses is the widely investigated ability of skeletal muscle to regenerate after loading or injury or both. Although significant basic science efforts have been dedicated to better understand the underlying mechanism controlling skeletal muscle regeneration, there has been relatively little impact in the clinical approaches used to treat skeletal muscle injuries and wasting. The purpose of this review article is to provide an overview of the basic biology of satellite cell function in response to muscle loading and to relate these findings in the context of aging and neuromuscular pathology for the rehabilitation medicine specialist.

Ex Vivo Noggin Gene Therapy Inhibits Bone Formation in a Mouse Model of Postoperative Resynostosis

Plastic and Reconstructive Surgery. Feb, 2009  |  Pubmed ID: 19182668

Resynostosis following surgical correction of primary craniosynostosis necessitates further surgical intervention, thereby increasing morbidity and mortality. Bone morphogenetic proteins are known to be expressed during normal bone healing. This study tested the hypothesis that treatment of suturectomy sites with Noggin, an extracellular antagonist of bone morphogenetic proteins, would inhibit postoperative resynostosis in a mouse suturectomy model.

The Dose of Growth Factors Influences the Synergistic Effect of Vascular Endothelial Growth Factor on Bone Morphogenetic Protein 4-induced Ectopic Bone Formation

Tissue Engineering. Part A. Aug, 2009  |  Pubmed ID: 19215221

Although vascular endothelial growth factor (VEGF) has been shown to act synergistically with bone morphogenetic protein (BMP)2 and BMP4 to promote ectopic endochondral bone formation via cell-based BMP gene therapy, the optimal ratio of VEGF to either of the BMPs required to obtain this beneficial effect remains unclear. In the current study, two cell types (C2C12, NIH/3T3) were retrovirally transduced to express BMP4 only or both BMP4 and VEGF. The resulting groups of cells were tested for their cellular proliferation, in vitro mineralization capacity, survival potential, and ability to undergo ectopic bone formation when implanted into a gluteofemoral muscle pocket created in severe combined immunodeficient mice. Results showed that VEGF inhibited the in vitro calcification of C2C12 and NIH/3T3 cells transduced to express BMP4. In vivo, C2C12 and NIH/3T3 cells expressing BMP4 and VEGF displayed significantly less bone formation than the same cells expressing only BMP4. In vivo, our results indicated that, when the ratio of VEGF to BMP4 is high, a detrimental effect on ectopic bone formation is observed; however, when the ratio is kept low and constant over time, the detrimental effect that VEGF has on ectopic bone formation is lost. Our studies revealed that VEGF's synergistic role in BMP4 induced ectopic bone formation is dose and cell-type dependent, which is an important consideration for cell-based gene therapy and tissue engineering for bone healing.

Cartilage Repair in a Rat Model of Osteoarthritis Through Intraarticular Transplantation of Muscle-derived Stem Cells Expressing Bone Morphogenetic Protein 4 and Soluble Flt-1

Arthritis and Rheumatism. May, 2009  |  Pubmed ID: 19404941

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt-1 (sFlt-1), on the chondrogenesis of skeletal muscle-derived stem cells (MDSCs) in a rat model of osteoarthritis (OA).

Sex of Muscle Stem Cells Does Not Influence Potency for Cardiac Cell Therapy

Cell Transplantation. 2009  |  Pubmed ID: 19523348

We have previously shown that populations of skeletal muscle-derived stem cells (MDSCs) exhibit sex-based differences for skeletal muscle and bone repair, with female cells demonstrating superior engrafting abilities to males in skeletal muscle while male cells differentiating more robustly toward the osteogenic and chondrogenic lineages. In this study, we tested the hypothesis that the therapeutic capacity of MDSCs transplanted into myocardium is influenced by sex of donor MDSCs or recipient. Male and female MDSCs isolated from the skeletal muscle of 3-week-old mice were transplanted into recipient male or female dystrophin-deficient (mdx) hearts or into the hearts of male SCID mice following acute myocardial infarction. In the mdx model, no difference was seen in engraftment or blood vessel formation based on donor cell or recipient sex. In the infarction model, MDSC-transplanted hearts showed higher postinfarction angiogenesis, less myocardial scar formation, and improved cardiac function compared to vehicle controls. However, sex of donor MDSCs had no significant effects on engraftment, angiogenesis, and cardiac function. VEGF expression, a potent angiogenic factor, was similar between male and female MDSCs. Our results suggest that donor MDSC or recipient sex has no significant effect on the efficiency of MDSC-triggered myocardial engraftment or regeneration following cardiac injury. The ability of the MDSCs to improve cardiac regeneration and repair through promotion of angiogenesis without differentiation into the cardiac lineage may have contributed to the lack of sex difference observed in these models.

Perivascular Multi-lineage Progenitor Cells in Human Organs: Regenerative Units, Cytokine Sources or Both?

Cytokine & Growth Factor Reviews. Oct-Dec, 2009  |  Pubmed ID: 19926515

Multi-lineage progenitors, e.g. mesenchymal stem cells, persist in adult developed organs, making a windfall for the cell therapist but an enigma for stem cell biologists. Recent results from our own and other laboratories show that the ancestor of these elusive adult stem cells is likely to be found in the perivascular area, explaining the ubiquitous distribution of these cells in the body. We have prospectively identified and purified vascular pericytes in multiple human organs and shown that these cells are potent mesodermal progenitors that give rise to genuine mesenchymal stem cells in culture. Pericytes can differentiate into diverse cell lineages, but also secrete multiple paracrine growth factors/cytokines, which likely explains in part their robust regenerative potential.

Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle

Molecular Therapy : the Journal of the American Society of Gene Therapy. Oct, 2009  |  Pubmed ID: 19603004

We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)-mediated angiogenesis in MDSC transplantation-based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.

Antioxidant Levels Represent a Major Determinant in the Regenerative Capacity of Muscle Stem Cells

Molecular Biology of the Cell. Jan, 2009  |  Pubmed ID: 19005220

Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation.

The Synergistic Effect of Treadmill Running on Stem-cell Transplantation to Heal Injured Skeletal Muscle

Tissue Engineering. Part A. Mar, 2010  |  Pubmed ID: 19788347

Muscle-derived stem-cell (MDSC) transplantation presents a promising method for the treatment of muscle injuries. This study investigated the ability of exercise to enhance MDSC transplantation into the injured muscle. Mice were divided into four groups: contusion + phosphate-buffered saline (C + PBS; n = 14 muscles), C + MDSC transplantation (n = 12 muscles), C + PBS + treadmill running (C + PBS + TM; n = 17 muscles), and C + MDSC + TM (n = 13 muscles). One day after injury, the TM groups began running for 1 or 5 weeks. Two days after injury, muscles of C + MDSC and C + MDSC + TM groups were injected with MDSCs. One or 5 weeks later, the number and differentiation of transplanted MDSCs, myofiber regeneration, collagen I formation, and vascularity were assessed histologically. In vitro, MDSCs were subjected to mechanical stimulation, and growth kinetics were quantified. In vitro, mechanical stimulation decreased the MDSC population doubling time (18.6 +/- 1.6 h) and cell division time (10.9 +/- 0.7 h), compared with the controls (population doubling time: 23.0 +/- 3.4 h; cell division time: 13.3 +/- 1.1 h) (p = 0.01 and 0.03, respectively). In vivo, 5 weeks of TM increased the myogenic contribution of transplanted MDSCs, compared with the controls (p = 0.02). C + MDSC, C + PBS + TM, and C + MDSC + TM demonstrated decreased fibrosis at 5 weeks, compared with the C + PBS controls (p = 0.00, p = 0.03, and p = 0.02, respectively). Results suggest that the mechanical stimulation favors MDSC proliferation, both in vitro and in vivo, and that exercise enhances MDSC transplantation after injury.

In Vivo Assessment of a Tissue-engineered Vascular Graft Combining a Biodegradable Elastomeric Scaffold and Muscle-derived Stem Cells in a Rat Model

Tissue Engineering. Part A. Apr, 2010  |  Pubmed ID: 19895206

Limited autologous vascular graft availability and poor patency rates of synthetic grafts for bypass or replacement of small-diameter arteries remain a concern in the surgical community. These limitations could potentially be improved by a tissue engineering approach. We report here our progress in the development and in vivo testing of a stem-cell-based tissue-engineered vascular graft for arterial applications. Poly(ester urethane)urea scaffolds (length = 10 mm; inner diameter = 1.2 mm) were created by thermally induced phase separation (TIPS). Compound scaffolds were generated by reinforcing TIPS scaffolds with an outer electrospun layer of the same biomaterial (ES-TIPS). Both TIPS and ES-TIPS scaffolds were bulk-seeded with 10 x 10(6) allogeneic, LacZ-transfected, muscle-derived stem cells (MDSCs), and then placed in spinner flask culture for 48 h. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks. Angiograms and histological assessment were performed at the time of explant. Cell-seeded constructs showed a higher patency rate than the unseeded controls: 65% (ES-TIPS) and 53% (TIPS) versus 10% (acellular TIPS). TIPS scaffolds had a 50% mechanical failure rate with aneurysmal formation, whereas no dilation was observed in the hybrid scaffolds. A smooth-muscle-like layer of cells was observed near the luminal surface of the constructs that stained positive for smooth muscle alpha-actin and calponin. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of von Willebrand Factor-positive cells was observed in the lumen of MDSC-seeded constructs, whereas acellular controls showed platelet and fibrin deposition. This is the first evidence that MDSCs improve patency and contribute to the remodeling of a tissue-engineered vascular graft for arterial applications.

Inkjet-based Biopatterning of Bone Morphogenetic Protein-2 to Spatially Control Calvarial Bone Formation

Tissue Engineering. Part A. May, 2010  |  Pubmed ID: 20028232

The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitro and bone formation in vivo using inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or (125)I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivo were comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitro and tissue formation in vivo in register to printed patterns.

Platelets in Regeneration

Seminars in Thrombosis and Hemostasis. Mar, 2010  |  Pubmed ID: 20414833

Platelets, as the first cellular response after disruption of vascular and/or tissue integrity, cover any existing injury within our body. But is the regenerative potential of platelets limited to providing a cellular patch for wounds? This review highlights the recent advance in our understanding of platelets being distinctly regulated and regulating cells that contribute immensely to the healing process from the very initial stage to the late events of tissue regeneration. For instance, the intrinsic actions of platelets as a regenerative cell, the participation of platelets in angiogenic processes, and the interplay of platelets and circulating stem and progenitor cells, as well as potential therapeutic implications, are addressed. Although we are starting to understand the underlying mechanisms connecting platelets to the components of tissue regeneration just mentioned, many aspects remain to be elucidated. The demand to invest research in this area is underscored by the fact that platelets or platelet-derived molecules are already applied in clinical contexts such as connective tissue regeneration, whereas other research fields have largely neglected platelet effects going beyond their participation in the coagulation cascade. Understanding the mechanisms connecting platelets to tissue regeneration, however, will inevitably open novel options in regenerative medicine.

Testing the Critical Size in Calvarial Bone Defects: Revisiting the Concept of a Critical-size Defect

Plastic and Reconstructive Surgery. Jun, 2010  |  Pubmed ID: 20517092

There is a clinical need for bone replacement strategies because of the shortfalls endemic to autologous bone grafting, especially in the pediatric patient population. For the past 25 years, the animal model that has been used to test bone replacement strategies has been the calvarial critical-size defect, based on the initial size of the bone defect. This study was undertaken to test the concept of the critical size in several different models. A review of the theoretical and scientific bases for the critical-size defect was also undertaken.

Effect of Phosphatidyl Inositol 3-kinase, Extracellular Signal-regulated Kinases 1/2, and P38 Mitogen-activated Protein Kinase Inhibition on Osteogenic Differentiation of Muscle-derived Stem Cells

Tissue Engineering. Part A. Dec, 2010  |  Pubmed ID: 20617875

Skeletal muscle-derived stem cells (MDSCs) can undergo osteogenesis when treated with bone morphogenetic proteins (BMPs), making them a potential cell source for bone tissue engineering. The signaling pathways that regulate BMP4-induced osteogenesis in MDSCs are not well understood, although they may provide a means to better regulate differentiation during bone regeneration. The objective of this study was to characterize the signaling pathways involved in the BMP4-induced osteogenesis of MDSCs. Cells were treated with BMP4 and specific inhibitors to the extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and phosphatidyl inositol 3-kinase (PI3K) pathways (PD98059, SB203580, and Ly294002, respectively). Cellular proliferation, expression of osteoblast-related genes, alkaline phosphatase (ALP) activity, and tissue mineralization were measured to determine the role of each pathway in the osteogenic differentiation of MDSCs. Inhibition of the ERK1/2 pathway increased ALP activity and mineralization, whereas inhibition of the p38 MAPK pathway decreased osteogenesis, suggesting opposing roles of these pathways in the BMP4-induced osteogenesis of MDSCs. Inhibition of the PI3K pathway significantly increased mineralization by MDSCs. These findings highlight the involvement of the ERK1/2, p38 MAPK, and PI3K pathways in opposing capacities in MDSC differentiation and warrant further investigation, as it may identify novel therapeutic targets for the development of stem cell-based therapies for bone tissue engineering.

Cellular Antioxidant Levels Influence Muscle Stem Cell Therapy

Molecular Therapy : the Journal of the American Society of Gene Therapy. Oct, 2010  |  Pubmed ID: 20664528

Although cellular transplantation has been shown to promote improvements in cardiac function following injury, poor cell survival following transplantation continues to limit the efficacy of this therapy. We have previously observed that transplantation of muscle-derived stem cells (MDSCs) improves cardiac function in an acute murine model of myocardial infarction to a greater extent than myoblasts. This improved regenerative capacity of MDSCs is linked to their increased level of antioxidants such as glutathione (GSH) and superoxide dismutase. In the current study, we demonstrated the pivotal role of antioxidant levels on MDSCs survival and cardiac functional recovery by either reducing the antioxidant levels with diethyl maleate or increasing antioxidant levels with N-acetylcysteine (NAC). Both the anti- and pro-oxidant treatments dramatically influenced the survival of the MDSCs in vitro. When NAC-treated MDSCs were transplanted into infarcted myocardium, we observed significantly improved cardiac function, decreased scar tissue formation, and increased numbers of CD31(+) endothelial cell structures, compared to the injection of untreated and diethyl maleate-treated cells. These results indicate that elevating the levels of antioxidants in MDSCs with NAC can significantly influence their tissue regeneration capacity.

Differential Efficacy of Gels Derived from Small Intestinal Submucosa As an Injectable Biomaterial for Myocardial Infarct Repair

Biomaterials. Oct, 2010  |  Pubmed ID: 20674011

Injectable biomaterials have been recently investigated as a therapeutic approach for cardiac repair. Porcine-derived small intestinal submucosa (SIS) material is currently used in the clinic to promote accelerated wound healing for a variety of disorders. In this study, we hypothesized that gels derived from SIS extracellular matrix would be advantageous as an injectable material for cardiac repair. We evaluated 2 forms of SIS gel, types B (SIS-B) and C (SIS-C), for their ability to provide a therapeutic effect when injected directly into ischemic myocardium using a murine model of an acute myocardial infarction. Echocardiography analysis at both 2 and 6 weeks after infarction demonstrated preservation of end-systolic left ventricular geometry and improvement of cardiac contractility in the hearts injected with SIS-B when compared with control hearts injected with saline. However, the SIS-C gel provided no functional efficacy in comparison with control. Histological analysis revealed that SIS-B reduced infarct size and induced angiogenesis relative to control, whereas injection of SIS-C had minimal effect on these histological parameters. Characterization of both gels revealed differential growth factor content with SIS-B exhibiting higher levels of basic fibroblast growth factor than SIS-C, which may explain, at least in part, the differential histological and functional results. This study suggests that SIS gel offers therapeutic potential as an injectable material for the repair of ischemic myocardium. Further understanding of SIS gel characteristics, such as biological and physical properties, that are critical determinants of efficacy would be important for optimization of this biomaterial for cardiac repair.

Pericyte-based Human Tissue Engineered Vascular Grafts

Biomaterials. Nov, 2010  |  Pubmed ID: 20684982

The success of small-diameter tissue engineered vascular grafts (TEVGs) greatly relies on an appropriate cell source and an efficient cellular delivery and carrier system. Pericytes have recently been shown to express mesenchymal stem cell features. Their relative availability and multipotentiality make them a promising candidate for TEVG applications. The objective of this study was to incorporate pericytes into a biodegradable scaffold rapidly, densely and efficiently, and to assess the efficacy of the pericyte-seeded scaffold in vivo. Bi-layered elastomeric poly(ester-urethane)urea scaffolds (length = 10 mm; inner diameter = 1.3 mm) were bulk seeded with 3 x 10(6) pericytes using a customized rotational vacuum seeding device in less than 2 min (seeding efficiency > 90%). The seeded scaffolds were cultured in spinner flasks for 2 days and then implanted into Lewis rats as aortic interposition grafts for 8 weeks. Results showed pericytes populated the porous layer of the scaffolds evenly and maintained their original phenotype after the dynamic culture. After implantation, pericyte-seeded TEVGs showed a significant higher patency rate than the unseeded control: 100% versus 38% (p < 0.05). Patent pericyte-seeded TEVGs revealed extensive tissue remodeling with collagen and elastin present. The remodeled tissue consisted of multiple layers of alpha-smooth muscle actin- and calponin-positive cells, and a von Willebrand factor-positive monolayer in the lumen. These results demonstrate the feasibility of a pericyte-based TEVG and suggest that the pericytes play a role in maintaining patency of the TEVG as an arterial conduit.

Skeletal Muscle-derived Stem Cells Differentiate into Hepatocyte-like Cells and Aid in Liver Regeneration

International Journal of Clinical and Experimental Pathology. 2010  |  Pubmed ID: 20830239

The liver is unique for its ability to regenerate after injury, however, critical injuries or disease cause it to lose this quality. Stem cells have been explored as a possibility to restore the function of seriously damaged livers, based on their self-renewability and multiple differentiation capacity. These experiments examine the ability of muscle derived stem cells (MDSCs) to differentiate into hepatocyte-like cells in vitro and acquire functional liver attributes for repairing damaged livers. In vitro experiments were performed using MDSCs from postnatal mice and mouse hepatocyte cell lines. Our data revealed that MDSCs differentiated into hepatocyte-like cells and expressed liver cell markers, albumin, hepatocyte nuclear factor 4 α, and alpha feto-protein, both at the RNA and protein level. Additionally, in vivo studies showed successful engraftment of MDSCs into hepatectomized mouse livers of mice. These results provide evidence suggesting that MDSCs have the capacity to differentiate into liver cell-like cells and may serve as potential candidates to aid in liver regeneration.

A Three-dimensional Gel Bioreactor for Assessment of Cardiomyocyte Induction in Skeletal Muscle-derived Stem Cells

Tissue Engineering. Part C, Methods. Jun, 2010  |  Pubmed ID: 19601695

Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.

AAV-directed Muscular Dystrophy Gene Therapy

Expert Opinion on Biological Therapy. Mar, 2010  |  Pubmed ID: 20132060

Muscle-directed gene therapy for genetic muscle diseases can be performed by the recombinant adeno-associated viral (rAAV) vector delivery system to achieve long-term therapeutic gene transfer in all affected muscles.

Accelerated Aging of Intervertebral Discs in a Mouse Model of Progeria

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Dec, 2010  |  Pubmed ID: 20973062

Intervertebral disc degeneration (IDD) is a common and debilitating disorder that results in reduced flexibility of the spine, pain, and reduced mobility. Risk factors for IDD include age, genetic predisposition, injury, and other environmental factors such as smoking. Loss of proteoglycans (PGs) contributes to IDD with advancing age. Currently there is a lack of a model for rapid investigation of disc aging and evaluation of therapeutic interventions. Here we examined progression of disc aging in a murine model of a human progeroid syndrome caused by deficiency of the DNA repair endonuclease, ERCC1-XPF (Ercc1(-/Δ) mice). The ERCC1-deficient mice showed loss of disc height and degenerative structural changes in their vertebral bodies similar to those reported for old rodents. Compared to their wild-type littermates, Ercc1(-/Δ) mice also exhibit other age-related IDD characteristics, including premature loss of disc PG, reduced matrix PG synthesis, and enhanced apoptosis and cell senescence. Finally, the onset of age-associated disc pathologies was further accelerated in Ercc1(-/Δ) mice following chronic treatment with the chemotherapeutic agent mechlorethamine. These results demonstrate that Ercc1(-/Δ) mice represent an accurate and rapid model of disc aging and provide novel evidence that DNA damage negatively impacts PG synthesis.

IOC Consensus Paper on the Use of Platelet-rich Plasma in Sports Medicine

British Journal of Sports Medicine. Dec, 2010  |  Pubmed ID: 21106774

Study of Muscle Cell Dedifferentiation After Skeletal Muscle Injury of Mice with a Cre-Lox System

PloS One. 2011  |  Pubmed ID: 21304901

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied.

Use of an Ultrasonic Blade Facilitates Muscle Repair After Incision Injury

The Journal of Surgical Research. May, 2011  |  Pubmed ID: 21324491

The ultrasonic Harmonic Blade cuts and coagulates soft tissue at temperatures lower than conventional electrosurgery. This study investigated whether improved hemostatic control and reduced collateral damage in skeletal muscle incisions translates into improved myofiber regeneration, reduced fibrosis and faster muscle recovery.

Follistatin Improves Skeletal Muscle Healing After Injury and Disease Through an Interaction with Muscle Regeneration, Angiogenesis, and Fibrosis

The American Journal of Pathology. Aug, 2011  |  Pubmed ID: 21689628

Recovery from skeletal muscle injury is often incomplete because of the formation of fibrosis and inadequate myofiber regeneration; therefore, injured muscle could benefit significantly from therapies that both stimulate muscle regeneration and inhibit fibrosis. To this end, we focused on blocking myostatin, a member of the transforming growth factor-β superfamily and a negative regulator of muscle regeneration, with the myostatin antagonist follistatin. In vivo, follistatin-overexpressing transgenic mice underwent significantly greater myofiber regeneration and had less fibrosis formation compared with wild-type mice after skeletal muscle injury. Follistatin's mode of action is likely due to its ability to block myostatin and enhance neovacularization. Furthermore, muscle progenitor cells isolated from follistatin-overexpressing mice were significantly superior to muscle progenitors isolated from wild-type mice at regenerating dystrophin-positive myofibers when transplanted into the skeletal muscle of dystrophic mdx/severe combined immunodeficiency mice. In vitro, follistatin stimulated myoblasts to express MyoD, Myf5, and myogenin, which are myogenic transcription factors that promote myogenic differentiation. Moreover, follistatin's ability to enhance muscle differentiation is at least partially due to its ability to block myostatin, activin A, and transforming growth factor-β1, all of which are negative regulators of muscle cell differentiation. The findings of this study suggest that follistatin is a promising agent for improving skeletal muscle healing after injury and muscle diseases, such as the muscular dystrophies.

Identification and Characterization of Chondrogenic Progenitor Cells in the Fascia of Postnatal Skeletal Muscle

Journal of Molecular Cell Biology. Dec, 2011  |  Pubmed ID: 21729867

Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation. A chondrogenic phase is typically observed in this process, which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle. Two prospective cell populations were isolated from rat skeletal muscle: fascia-derived cells (FDCs), extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body. Both populations were investigated for their cell surface marker profiles (flowcytometry analysis), proliferation rates as well as their myogenic and chondrogenic potentials. The majority of FDCs expressed mesenchymal stromal cell markers but not endothelial cell markers. FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro, but not myogenic differentiation. Although MDCs showed chondrogenic potential, they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however, the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium. To clarify the role of the muscle-derived myogenic cells in chondrogenesis, mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential. Our results indicated that the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis. Taken together, our results suggest that non-myogenic cells residing in the fascia of skeletal muscle have a strong chondrogenic potential and may represent a novel donor cell source for cartilage regeneration and repair.

Isolation and Characterization of Human Anterior Cruciate Ligament-Derived Vascular Stem Cells

Stem Cells and Development. Aug, 2011  |  Pubmed ID: 21732814

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture.

Terminal Differentiation is Not a Major Determinant for the Success of Stem Cell Therapy - Cross-talk Between Muscle-derived Stem Cells and Host Cells

Stem Cell Research & Therapy. 2011  |  Pubmed ID: 21745421

We have found that when muscle-derived stem cells (MDSCs) are implanted into a variety of tissues only a small fraction of the donor cells can be found within the regenerated tissues and the vast majority of cells are host derived. This observation has also been documented by other investigators using a variety of different stem cell types. It is speculated that the transplanted stem cells release factors that modulate repair indirectly by mobilizing the host's cells and attracting them to the injury site in a paracrine manner. This process is loosely called a 'paracrine mechanism', but its effects are not necessarily restricted to the injury site. In support of this speculation, it has been reported that increasing angiogenesis leads to an improvement of cardiac function, while inhibiting angiogenesis reduces the regeneration capacity of the stem cells in the injured vascularized tissues. This observation supports the finding that most of the cells that contribute to the repair process are indeed chemo-attracted to the injury site, potentially through host neo-angiogenesis. Since it has recently been observed that cells residing within the walls of blood vessels (endothelial cells and pericytes) appear to represent an origin for post-natal stem cells, it is tempting to hypothesize that the promotion of tissue repair, via neo-angiogenesis, involves these blood vessel-derived stem cells. For non-vascularized tissues, such as articular cartilage, the regenerative property of the injected stem cells still promotes a paracrine, or bystander, effect, which involves the resident cells found within the injured microenvironment, albeit not through the promotion of angiogenesis. In this paper, we review the current knowledge of post-natal stem cell therapy and demonstrate the influence that implanted stem cells have on the tissue regeneration and repair process. We argue that the terminal differentiation capacity of implanted stem cells is not the major determinant of the cells regenerative potential and that the paracrine effect imparted by the transplanted cells plays a greater role in the regeneration process.

Injectable Fibroblast Growth Factor-2 Coacervate for Persistent Angiogenesis

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2011  |  Pubmed ID: 21808045

Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform.

Intramuscular Transplantation of Muscle-derived Stem Cells Accelerates Skeletal Muscle Healing After Contusion Injury Via Enhancement of Angiogenesis

The American Journal of Sports Medicine. Sep, 2011  |  Pubmed ID: 21828363

Muscle contusions are common muscle injuries. Although these injuries are capable of healing, incomplete functional recovery often occurs. Muscle-derived stem cells (MDSCs) are likely derived from blood vessel cells and have a multilineage differentiation potential.

Placental Perivascular Cells for Human Muscle Regeneration

Stem Cells and Development. Mar, 2011  |  Pubmed ID: 20923371

Perivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin. In vitro-cultured placental villi released rapidly adhering and migratory CD146+CD34⁻CD45⁻CD56⁻ cells of putative perivascular origin that expressed mesenchymal stem cell markers. CD146+CD34⁻CD45⁻CD56⁻ perivascular cells isolated and purified from the placental villi by flow cytometry were indeed highly myogenic in culture, and generated dystrophin-positive myofibers, and they promoted angiogenesis after transplantation into SCID/mdx mouse muscles. These observations confirm the existence of mesenchymal progenitor cells within the walls of human blood vessels, and suggest that the richly vascularized human placenta is an abundant source of perivascular myogenic cells able to migrate within dystrophic muscle and regenerate myofibers.

Skeletal Muscle-derived Stem Cells: Implications for Cell-mediated Therapies

Medicina (Kaunas, Lithuania). 2011  |  Pubmed ID: 22156603

Current advances in stem cell research and innovative biological approaches in the field of tissue engineering and regenerative medicine could eventually translate into prospective clinical applications. Various adult organs and tissues harbor stem and progenitor cells that could potentially be used to repair, regenerate, and restore a variety of different tissues following acute injury or tissue destructive diseases. Skeletal muscle is a very convenient and plentiful source of somatic stem cells. It contains several distinct populations of myogenic stem cells including satellite cells that are mainly responsible for muscle growth and regeneration, and multipotent muscle-derived stem cells (MDSCs). Although both cell populations share some phenotypic similarities, MDSCs display a much greater differentiation potential in vitro and are capable of regenerating various tissues in vivo. Furthermore, these cells not only participate in the regeneration process by differentiating into tissue-specific cell types, but also promote endogenous tissue repair by secreting a multitude of trophic factors. In this article, we describe the biological aspects of MDSC isolation and characterization and provide an overview of potential therapeutic application of these cells for the treatment of cardiac and skeletal muscle injuries and diseases, urological dysfunction, and bone and cartilage defects. We also discuss major challenges and limitations currently faced by MDSC-based therapies that await resolution before these techniques can be applied clinically.

NF-κB Negatively Impacts the Myogenic Potential of Muscle-derived Stem Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Dec, 2011  |  Pubmed ID: 22158056

Inhibition of the inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) pathway enhances muscle regeneration in injured and diseased skeletal muscle, but it is unclear exactly how this pathway contributes to the regeneration process. In this study, we examined the role of NF-κB in regulating the proliferation and differentiation of muscle-derived stem cells (MDSCs). MDSCs isolated from the skeletal muscles of p65(+/-) mice (haploinsufficient for the p65 subunit of NF-κB) had enhanced proliferation and myogenic differentiation compared to MDSCs isolated from wild-type (wt) littermates. In addition, selective pharmacological inhibition of IKKβ, an upstream activator of NF-κB, enhanced wt MDSC differentiation into myotubes in vitro. The p65(+/-) MDSCs also displayed a higher muscle regeneration index than wt MDSCs following implantation into adult mice with muscular dystrophy. Additionally, using a muscle injury model, we observed that p65(+/-) MDSC engraftments were associated with reduced inflammation and necrosis. These results suggest that inhibition of the IKK/NF-κB pathway represents an effective approach to improve the myogenic regenerative potential of MDSCs and possibly other adult stem cell populations. Moreover, our results suggest that the improved muscle regeneration observed following inhibition of IKK/NF-κB, is mediated, at least in part, through enhanced stem cell proliferation and myogenic potential.

Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

PloS One. 2011  |  Pubmed ID: 22195027

Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.

Lentivirus-mediated Wnt11 Gene Transfer Enhances Cardiomyogenic Differentiation of Skeletal Muscle-derived Stem Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Apr, 2011  |  Pubmed ID: 21304494

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Engineering Spatial Control of Multiple Differentiation Fates Within a Stem Cell Population

Biomaterials. May, 2011  |  Pubmed ID: 21316755

The capability to engineer microenvironmental cues to direct a stem cell population toward multiple fates, simultaneously, in spatially defined regions is important for understanding the maintenance and repair of multi-tissue units. We have previously developed an inkjet-based bioprinter to create patterns of solid-phase growth factors (GFs) immobilized to an extracellular matrix (ECM) substrate, and applied this approach to drive muscle-derived stem cells toward osteoblasts 'on-pattern' and myocytes 'off-pattern' simultaneously. Here this technology is extended to spatially control osteoblast, tenocyte and myocyte differentiation simultaneously. Utilizing immunofluorescence staining to identify tendon-promoting GFs, fibroblast growth factor-2 (FGF-2) was shown to upregulate the tendon marker Scleraxis (Scx) in C3H10T1/2 mesenchymal fibroblasts, C2C12 myoblasts and primary muscle-derived stem cells, while downregulating the myofibroblast marker α-smooth muscle actin (α-SMA). Quantitative PCR studies indicated that FGF-2 may direct stem cells toward a tendon fate via the Ets family members of transcription factors such as pea3 and erm. Neighboring patterns of FGF-2 and bone morphogenetic protein-2 (BMP-2) printed onto a single fibrin-coated coverslip upregulated Scx and the osteoblast marker ALP, respectively, while non-printed regions showed spontaneous myotube differentiation. This work illustrates spatial control of multi-phenotype differentiation and may have potential in the regeneration of multi-tissue units.

Venous Graft-derived Cells Participate in Peripheral Nerve Regeneration

PloS One. 2011  |  Pubmed ID: 21966370

Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle.

Human Skeletal Muscle Cells with a Slow Adhesion Rate After Isolation and an Enhanced Stress Resistance Improve Function of Ischemic Hearts

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2012  |  Pubmed ID: 22068427

Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.

Muscle-derived Stem/progenitor Cell Dysfunction Limits Healthspan and Lifespan in a Murine Progeria Model

Nature Communications. 2012  |  Pubmed ID: 22215083

With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs, isolated from young wild-type mice, to progeroid mice confer significant lifespan and healthspan extension. The transplanted MDSPCs improve degenerative changes and vascularization in tissues where donor cells are not detected, suggesting that their therapeutic effect may be mediated by secreted factor(s). Indeed, young wild-type-MDSPCs rescue proliferation and differentiation defects of aged MDSPCs when co-cultured. These results establish that adult stem/progenitor cell dysfunction contributes to ageing-related degeneration and suggests a therapeutic potential of post-natal stem cells to extend health.

Biologic Approaches to Enhance Rotator Cuff Healing After Injury

Journal of Shoulder and Elbow Surgery / American Shoulder and Elbow Surgeons ... [et Al.]. Feb, 2012  |  Pubmed ID: 22244061

Despite the advances in surgical procedures to repair the rotator cuff, there is a high incidence of failure. Biologic approaches, such as growth factor delivery and stem cell and gene therapy, are potential targets for optimization to improve the outcome of rotator cuff therapies and reduce rates of reinjury. This article outlines the current evidence for growth factor and stem cell therapy in tendon healing and the augmentation of rotator cuff repair.

Role of Angiogenesis After Muscle Derived Stem Cell Transplantation in Injured Medial Collateral Ligament

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Apr, 2012  |  Pubmed ID: 21913220

We performed this study to investigate the therapeutic role of vascular endothelial growth factor (VEGF) in medial collateral ligament (MCL) healing. Murine muscle derived stem cells (MDSCs) obtained via the preplate technique were retrovirally transduced to express: (1) VEGF and nLacZ (MDSC-VEGF), (2) soluble fms-like tyrosine kinase-1 (sFLT1, a VEGF-specific antagonist) and nLacZ (MDSC-sFLT1), and (3) nLacZ (MDSC-nLacZ). After transecting the MCL of immunodeficient rats, 5 × 10(5)  cells of each of the transduction groups list above were transplanted into the MCL injury site. A control group was injected with phosphate-buffered saline (PBS) only. Immunohistochemical staining demonstrated that there were more Isolectin B4 and β-galactosidase double positive cells in the rats transplanted with MDSC-VEGF transduced cells than the other groups at week 1. Capillary density was significantly higher in the MDSC-VEGF group than the other groups at week 2; however, there were no significant differences in the biomechanical assessment between the MDSC-VEGF and MDSC-nLacZ groups. On the other hand, the MDSC-sFLT1 group revealed a lower capillary density than the other two groups and the functional ligament healing of the MDSC-sFLT1 group was significantly decreased compared to the other groups when assessed biomechanically. The findings of the present study suggest that angiogenesis plays a critical role in the healing process of injured MCL. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:627-633, 2012.

Mechanical Loading of Stem Cells for Improvement of Transplantation Outcome: The Role of Loading History

Tissue Engineering. Part A. Jan, 2012  |  Pubmed ID: 22280442

Stem cell therapy for tissue repair is a rapidly evolving field and the factors which dictate the physiological responsiveness of stem cells remain under intense investigation. In this study we hypothesized that the mechanical loading history of muscle derived stem cells (MDSCs) would significantly impact MDSC survival, host tissue angiogenesis, and myocardial function following MDSC transplantation into acutely infarcted myocardium. Mice with acute myocardial infarction by permanent left coronary artery ligation were injected with either non-stimulated (NS) or mechanically stimulated (MS) MDSCs. Mechanical stimulation consisted of stretching the cells with equibiaxial stretch with a magnitude of 10% and frequency of 0.5 Hz. MS cell transplanted hearts showed improved cardiac contractility, increased numbers of host CD31 positive cells and decreased fibrosis, in the peri-infarct region, compared to the hearts treated with NS MDSCs. MS MDSCs displayed higher vascular endothelial growth factor (VEGF) expression than NS cells in vitro. These findings highlight an important role for cyclic mechanical loading preconditioning of donor MDSCs in optimizing MDSC transplantation for myocardial repair.