Systemically Administered Mesenchymal Stromal Cells Transduced with Insulin-like Growth Factor-I Localize to a Fracture Site and Potentiate Healing

Journal of Orthopaedic Trauma. Oct, 2002  |  Pubmed ID: 12368646

To determine to the ability of systemically administered pluripotential mesenchymal stromal cells to localize to a fracture site and whether transduction with a therapeutic gene, insulin-like growth factor-I (D1-IGF-I), could potentiate healing.

Transforming Growth Factor-beta in Calcium Alginate Beads for the Treatment of Articular Cartilage Defects in the Rabbit

Arthroscopy : the Journal of Arthroscopic & Related Surgery : Official Publication of the Arthroscopy Association of North America and the International Arthroscopy Association. Oct, 2002  |  Pubmed ID: 12368788

Articular cartilage has only limited capability for intrinsic repair. The use of growth factors has been suggested to improve the repair of cartilage after injury. Reliable delivery systems for these agents are needed. In this study we tested calcium alginate for the delivery of TGF-beta in the treatment of osteochondral defects in the rabbit knee.

Transplanted Bone Marrow Cells Localize to Fracture Callus in a Mouse Model

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Nov, 2002  |  Pubmed ID: 12472234

Bone marrow contains many cellular elements that may contribute to fracture repair. We used a pluripotential stromal cell in a mouse model to demonstrate the presence of transplanted cells in fracture hematoma and subsequently in maturing fracture callus. Cells were transduced with traceable genes (lac Z and neomycin resistance) and traced in vivo after intravenous injection into syngeneic mice. These transduced cells home to bone marrow, suggesting that they might be detected in fracture callus. Cells were injected intravenously into mice and stabilized femoral shaft fractures were induced. Control mice received intravenous lactated-Ringer's solution prior to fracture. Callus tissue and marrow were examined histologically from I to 10 weeks after fracture to detect transplanted cells. Transplanted cells were detected in fracture callus in areas, and at times, of most active bone formation. Control specimens showed minimal staining of the callus tissue. Levels of the traceable gene in fracture callus increased, reached a peak between 3 and 4 weeks after fracture, then diminished and disappeared by 10 weeks post-fracture as woven bone at the fracture site was replaced by lamellar bone with cells from the host mouse. The results show that pluripotent bone marrow cells home to the marrow after systemic injection and localize in fracture callus.

Repair of Full-thickness Cartilage Defects in Rabbit Knees with Free Periosteal Graft Preincubated with Transforming Growth Factor

Orthopedics. Apr, 2003  |  Pubmed ID: 12722910

This study compared different concentrations of transforming growth factor-beta 1 (TGF-beta1) used for short-term preincubation in vitro of large periosteal explants to determine the effect of chondrogenesis and the fate of repair cartilage over time. Eighty-two rabbits were divided into four groups: group A, non-grafted; group B, non-incubated grafted; group C, 100 ng/mL recombinant human (rh) TGF-beta1 preincubated grafted; and group D, 20 ng/mL rhTGF-beta1 preincubated grafted. Rabbits from each group were sacrificed at intervals between 2 and 24 weeks. Histologic slides were stained with safranin O and were scored based on a subjective scoring system. Group A healed with non-cartilaginous material. Group B healed with hyaline cartilage-like material with progressive thinning of this regenerated layer; at 24 weeks, this layer was fibrous tissue. Group C enhanced repair with hyaline cartilage-like material but accelerated early degeneration and osteophyte formation; the cartilage became fibrous tissue at 24 weeks. Group D did not enhance cartilaginous repair. At 24 weeks, all groups had the same result. The 100 ng/mL rhTGF-beta1 preincubation in vitro with periosteum enhanced early osteochondral repair but did not show prolonged durability. Clinical application of this growth factor necessitates further study.

Tracing Transduced Cells in Osteochondral Defects

Journal of Pediatric Orthopedics. Jul-Aug, 2003  |  Pubmed ID: 12826937

In this study the authors explored the feasibility of using transduced cells for gene therapy to induce healing of osteochondral defects. Both a mouse mesenchymal cell line and mixed rabbit adherent stromal cells were transduced with either liposomal transfection or retroviral transduction using a traceable gene. Transduction efficiency was more than 95% with the retroviral construct and expression was maintained for over 6 months of passage. The liposomal transfection led to a transient expression with an efficiency of 50%. The expression of osteochondral genes was diminished but preserved after transduction in vitro. Transduced rabbit cells were transplanted into osteochondral defects in rabbit femoral condyles. Cells transplanted in vivo could be detected for 4 weeks in the repair tissue. The authors' data demonstrate that mesenchymal cells from bone marrow, stably transduced with a traceable gene product, retain the bone and cartilage phenotype and can be followed in vivo after transplantation into cartilage defects.

Chondrocyte Transplantation into Articular Cartilage Defects with Use of Calcium Alginate: the Fate of the Cells

The Journal of Bone and Joint Surgery. American Volume. Sep, 2003  |  Pubmed ID: 12954835

The fate of transplanted chondrocytes used to elicit the repair of osteochondral defects is unknown. The objective of this study was to examine the fate and the expression of cartilage-specific genes in chondrocytes when the chondrocyte phenotype was maintained preoperatively by alginate suspension culture, the cells were labeled with enhanced green fluorescent protein, and the chondrocytes in alginate were then implanted into full-thickness osteochondral defects in rabbits.

Lovastatin Inhibits Adipogenic and Stimulates Osteogenic Differentiation by Suppressing PPARgamma2 and Increasing Cbfa1/Runx2 Expression in Bone Marrow Mesenchymal Cell Cultures

Bone. Oct, 2003  |  Pubmed ID: 14555271

The mechanism whereby lovastatin can counteract steroid-induced osteonecrosis and osteoporosis is poorly understood. We assessed the effect of lovastatin on a multipotential cell line, D1, which is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPARgamma2, respectively, were determined. 422aP2 gene expression was analyzed. Osteocalcin promoter activity was measured by cotransfecting the cells with the phOC-luc and pSV beta-Gal plasmids. Lovastatin enhanced osteoblast differentiation as assessed by a 1.8x increase in expression of Cbfa1/Runx2 and by a 5x increase in osteocalcin promoter activity. Expression of PPARgamma2 was decreased by 60%. By enhancing osteoblast gene expression and by inhibiting adipogenesis, lovastatin may shunt uncommitted osteoprogenitor cells in marrow from the adipocytic to the osteoblastic differentiation pathway. Future evaluation of lovastatin and other lipid-lowering drugs will help determine their potential as therapeutic agents for osteonecrosis and osteoporosis.

A Fibronectin Fragment Stimulates Intervertebral Disc Degeneration in Vivo

Spine. Oct, 2003  |  Pubmed ID: 14560080

A radiographic, histologic, biochemical, and gene expression study was conducted in vivo in a rabbit model to determine the effect of injection of the N-terminal 30 kDa fibronectin fragment (Fn-f) into the intervertebral disc along with various control substances.

Cotargeting Tumor and Stroma in a Novel Chimeric Tumor Model Involving the Growth of Both Human Prostate Cancer and Bone Stromal Cells

Cancer Gene Therapy. Feb, 2004  |  Pubmed ID: 14695756

Stromal-epithelial interaction contributes to local prostate tumor growth, androgen-independent progression and distant metastasis. We have established in vitro coculture and in vivo chimeric tumor models to evaluate the roles of stromal cells isolated from either osteosarcoma or normal bone, a site where prostate cancer cells frequently metastasize, in contributing to the growth and survival of human prostate cancer cells. We have evaluated extensively the effects of toxic gene therapy using luciferase-tagged chimeric human prostate cancer models both in vitro and in vivo. In the in vitro cocultured cell model, we assessed cancer cell growth and residual cellular proteins after targeting either prostate cancer epithelial cells alone or both prostate cancer and bone stromal cells. In the in vivo animal model, we measured tumor volume and serum prostate-specific antigen (PSA) in mice bearing chimeric prostate tumors comprised of human prostate tumor cells and normal bone stromal cells. Our results demonstrated that: (1) The rate of human prostate cancer cell growth in vitro is accelerated by coculturing with human and rat osteosarcoma or normal mouse bone marrow stromal cell lines. No growth stimulation was noted when cocultured with a human prostate epithelial cell line. (2) Disabling the growth of normal bone stromal cells using transgenic targeting with a bystander gene, herpes simplex virus thymidine kinase (hsv-TK), plus the pro-drug ganciclovir (GCV) or acyclovir markedly depressed the growth of cocultured human prostate cancer cells in vitro and human prostate cancer-mouse normal bone stroma chimeric tumors in vivo. (3) By cotargeting both human prostate cancer and normal mouse bone stromal cells in vitro with an adenoviral construct, Ad-hOC-TK (a replication-defective Ad5 vector with the bystander transgene hsv-TK under the control of a human osteocalcin (hOC) promoter) plus GCV4, we observed greater inhibition of tumor cell growth than by targeting a single cell compartment with Ad-PSA-TK (a vector construct similar to Ad-hOC-TK except that the transgene expression is under regulation by a full-length human PSA promoter). These results, taken together, established a basic principle that cotargeting both tumor and its supporting stroma is more efficacious than targeting a single cell compartment in the treatment of human prostate cancer bone metastasis. This principle can be applied to other clinical conditions of cancer growth where stroma contribute to the overall growth and survival potential of the cancer.

In Vivo Bioluminescent Imaging of Virus-mediated Gene Transfer and Transduced Cell Transplantation in the Intervertebral Disc

Spine. Apr, 2004  |  Pubmed ID: 15082981

Work presented here used a small animal model to demonstrate the feasibility and usefulness of in vivo bioluminescent imaging to studying degenerative disc disease.

Semitendinosus Regrowth: Biochemical, Ultrastructural, and Physiological Characterization of the Regenerate Tendon

The American Journal of Sports Medicine. Jul-Aug, 2004  |  Pubmed ID: 15262639

Previous studies have suggested that hamstring tendons can regenerate following harvesting for anterior cruciate ligament reconstruction.

Collagen and Proteoglycan Abnormalities in the GDF-5-deficient Mice and Molecular Changes when Treating Disk Cells with Recombinant Growth Factor

Spine. Oct, 2004  |  Pubmed ID: 15480133

A magnetic resonance image, histologic, biochemical, and gene expression study was conducted to characterize the effects of growth and development factor-5 (GDF-5) deficiency on the health of the intervertebral disc.

A Fibronectin Fragment Alters the Metabolism by Rabbit Intervertebral Disc Cells in Vitro

Spine. Jun, 2005  |  Pubmed ID: 15928546

A biochemical and gene expression study was conducted to determine the effects of the 30-kDa N-terminal fibronectin fragment (Fn-f) on the glycosaminoglycan content of nucleus pulposus (NP) explant cultures, and on the gene expression profile of NP cells in alginate culture.

A New Method of Selecting Schwann Cells from Adult Mouse Sciatic Nerve

Journal of Neuroscience Methods. Nov, 2005  |  Pubmed ID: 15970332

We describe a method of using laminin for the selection and purification of Schwann cells in vitro. We also studied the viability of the selected cells suspended in alginate beads both in vitro and in vivo. We observed that the homogeneity of the Schwann cell culture increased with each round of laminin selection and reached 85-90% after five passages. The viability of cells after incubation within an alginate bead in vivo was between 73 and 76% compared with greater than 90% viability for cells that were maintained in monolayer culture. This new method of serial selection using laminin-coated surfaces has optimized the purification of a Schwann cell culture expanded from cells harvested from the adult sciatic nerve of a mouse. This method has the advantage of being technically easier than other methods described and results in a Schwann cell culture that is 80-90% homogenous.

Modulation of Chondrocytic Properties of Fat-derived Mesenchymal Cells in Co-cultures with Nucleus Pulposus

Connective Tissue Research. 2005  |  Pubmed ID: 16019417

Human subcutaneous fat-derived mesenchymal cells recently have been shown to have the potential to differentiate in vitro into a variety of cell types, including adipocytes, osteoblasts, chondrocytes, and myoblasts. This effect suggests that fat tissue may serve as an abundant and easily acquired source of multipotent cells for tissue engineering. The multipotential characteristics of fat-derived mesenchymal cells from the inguinial fat pad of rabbit have not been clearly defined. In this study we have isolated a population of mesenchymal cells from inguinal fat from adult New Zealand white rabbits. The cells that were maintained under various differentiation conditions were shown to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes; this differentiation was demonstrated using gene expression for tissue-specific proteins. We also co-cultured the cells with intervertebral disk tissue from the nucleus pulpous or from the annulus fibrosus. The fat-derived cells co-cultured with nucleus pulposus showed an increase in expression of type II collagen and aggrecan genes, compared with cells in alginate alone and cells co-cultured with annulus fibrosus. The data suggest that the fat-derived mesenchymal cells responded to soluble mediators from the disk. Future studies on intervertebral disk reconstruction could be based on our findings with fat-derived multipotential cells from the inguinal region of the rabbit that were co-cultured with disk tissue and may prove useful in tissue engineering strategies.

BMP-14 Deficiency Inhibits Long Bone Fracture Healing: a Biochemical, Histologic, and Radiographic Assessment

Journal of Orthopaedic Trauma. Oct, 2005  |  Pubmed ID: 16247308

Bone morphogenetic proteins (BMPs) represent a distinct subset of the transforming growth factor-beta family best known for their role in joint formation and bone growth, and several recent clinical trials have begun to look at their efficacy in the augmentation of fracture healing. The goal of this research is to examine the effect of BMP-14, also known as growth differentiation factor-5 and cartilage- derived morphogenetic protein-1 (GDF-5, CDMP-1) on fracture healing by studying the long bone repair process in mice with a deficiency in this signaling peptide.

Steroid Effects on Osteogenesis Through Mesenchymal Cell Gene Expression

Osteoporosis International : a Journal Established As Result of Cooperation Between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. Jan, 2005  |  Pubmed ID: 15205891

We have studied the mechanism of steroid-induced osteonecrosis by examining the effect of dexamethasone on a multipotential cell line, D1, which is derived from bone marrow and is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPARgamma2, were determined. Osteocalcin promoter activity was measured by co-transfecting the cells with the phOC-luc and pSV beta-Gal plasmids. Dexamethasone increased PPARgamma2 gene expression 2-fold, while Cbfa1/Runx2 gene expression and osteocalcin promoter activity decreased by 50-60%, and VEGF protein, measured by ELISA, decreased by 55%. These changes indicate enhanced adipogenesis and decreased osteogenesis by mesenchymal cells in vitro, together with a decrease in VEGF, a potent angiogeneic factor, suggesting that dexamethasone may shunt uncommitted osteoprogenitor cells in marrow from osteoblastic differentiation into the adipocytic pathway, leading to diminished vascularization and eventual osteonecrosis.

Demineralized Bone Matrix Gelatin As Scaffold for Osteochondral Tissue Engineering

Biomaterials. Apr, 2006  |  Pubmed ID: 16343611

To develop a single-unit osteochondral tissue with demineralized bone matrix gelatin (BMG), rabbit chondrocytes were cultured on demineralized bone matrix gelatin for 6 weeks. The engineered osteochondral tissue was characterized with histology, immunolocalization, TEM, SEM, biochemical assay, and gene expression analysis. About 1.3mm viable neo-cartilage was produced on demineralized BMG. RT-PCR, immunohistochemistry, TEM, biochemical assay, and histology revealed hyaline-like cartilage with zonal layers, intense type II collagen expression, and abundant proteoglycan content formed upon BMG compared with normal cartilage. But hydroxyproline content and type I collagen gene and protein expressions were significantly lower. We consider engineering cartilage tissue with chondrocytes cultured on allogenic demineralized BMG is a good approach for osteochondral tissue engineering.

Tendon Tissue Engineering and Gene Transfer: the Future of Surgical Treatment

The Journal of Hand Surgery. May-Jun, 2006  |  Pubmed ID: 16713828

Technologic improvements in the field of tissue engineering are leading to new potential developments in the currently used approaches to treat tendon injuries including difficult clinical scenarios such as zone II flexor tendon injuries of the hand and the mutilated hand with extensive tendon defects. A combination of mesenchymal (adult stem) cells, growth factors, and bioresorbable polymers can provide a solution for the treatment of difficult tendon injuries. Extensive research is needed to show that the extracellular matrix produced in response to the cell/growth factor/polymer composites in vivo is effective and functional as a regenerate tissue. Further exciting advances are foreseen in cell-based genetic engineering with the transfer of DNA to the site of tendon lacerations. These treatment modalities require improved safety precautions to reduce the risks and enhance the benefits of gene therapy.

Alcohol-induced Adipogenesis in a Cloned Bone-marrow Stem Cell

The Journal of Bone and Joint Surgery. American Volume. Nov, 2006  |  Pubmed ID: 17079381

Alcohol has been shown to be associated with osteoporosis and osteonecrosis in patients and in animal models. Recent studies have demonstrated that alcohol contributes to abnormal lipid metabolism in the stromal cells of bone marrow, but the mechanisms have not been defined. The purpose of this study was to evaluate the effects of alcohol on the differentiation of a stem cell that was cloned from bone marrow.

Use of Genetically Engineered Bone-marrow Stem Cells to Treat Femoral Defects: an Experimental Study

The Journal of Bone and Joint Surgery. American Volume. Nov, 2006  |  Pubmed ID: 17079384

Treatment of osteonecrosis continues to be a challenging problem. The replacement of necrotic bone with graft materials that promote osteogenesis and angiogenesis may provide better outcomes for early stage disease. In this study, genetically engineered bone-marrow stem cells were used to enhance repair of a defect in the distal aspect of the femur.

Osteogenic Differentiation of Adipose-derived Stromal Cells Treated with GDF-5 Cultured on a Novel Three-dimensional Sintered Microsphere Matrix

The Spine Journal : Official Journal of the North American Spine Society. Nov-Dec, 2006  |  Pubmed ID: 17088192

It is well known that under the proper conditions multipotential bone marrow stromal cells are capable of osteogenic differentiation. Recently studies have demonstrated that an analogous subpopulation of cells exist within adipose tissue. Although early studies characterizing these adipose-derived stromal (ADS) cells in culture exist, investigations exploring the characteristics and viability of these cells cultured on a three-dimensional sintered microsphere matrix are absent.

Recombinant Growth/differentiation Factor-5 Stimulates Osteogenic Differentiation of Fat-derived Stromal Cells in Vitro

Connective Tissue Research. 2006  |  Pubmed ID: 17118748

Fat-derived stromal cells can differentiate into various skeletal tissues. Currently the mechanism that determines whether stromal cells differentiate into osteoblasts is unclear and the role of growth/differentiation factor (GDF)-5 in differentiation of fat-derived stromal cells is not fully understood. It appears that the differentiation of stromal cells is greatly enhanced by GDF-5 that plays a role in a variety of musculoskeletal processes such as joint formation, tendon maintenance, and bone formation. Our study showed that GDF-5 promotes the differentiation of rat fat-derived stromal cells into osteogenic lineages in vitro. Furthermore, these findings were confirmed by histology, biochemical assay for alkaline phosphatase activity, and analysis of gene expression. The ability to preferentially stimulate fat-derived stromal cells down the osteogenic pathway holds significance in a variety of clinical scenarios.

Novel Biodegradable Poly(1,8-octanediol Malate) for Annulus Fibrosus Regeneration

Macromolecular Bioscience. Nov, 2007  |  Pubmed ID: 17683110

A novel elastic scaffold that simulates the deformability of annulus fibrosus (AF) and has good biocompatibility was developed. The scaffold was formed of a malic acid-based polyester poly(1,8-octanediol malate) (POM), which was synthesized by direct polycondensation. The tensile strength of POM gradually increased with the extension of the polymerization time, while the degradation rate decreased. Rat AF cells proliferated on the POM films and maintained their phenotype. The 3D scaffold also supported the growth of the AF cells, as confirmed by Safranin-O and type II collagen staining. POM also demonstrated a good biocompatibility in an in vivo foreign body response assay, an important prerequisite for tissue engineering applications. This study suggests that elastic POM scaffold may be an ideal candidate for AF tissue engineering.

Growth and Differentiation Factor-5 (GDF-5) Stimulates Osteogenic Differentiation and Increases Vascular Endothelial Growth Factor (VEGF) Levels in Fat-derived Stromal Cells in Vitro

Bone. Feb, 2007  |  Pubmed ID: 17070126

Fat-derived adult mesenchymal stem cells can differentiate into different phenotypes reflecting their potential to regenerate various skeletal tissues. These properties together with the association of adipose with skeletal tissues formed the basis of our study to establish an experimental model for using fat-derived stromal cells to undergo osteogenic differentiation in vitro under the influence of either growth and differentiation factor-5 (GDF-5) or bone morphogenetic protein-2 (BMP-2). Members of the BMP/GDF family of proteins are known for their ability to elicit skeletal morphogenesis, but little is known about the mechanism whereby these morphogens exert their effect on the osteogenic differentiation of fat-derived stromal cells. We compared the effects of GDF-5 and BMP-2 in their recombinant forms to qualitatively and quantitatively determine their influence on the osteogenic differentiation of fat derived stromal cells by examining the effects on mineralization, extracellular matrix, cell proliferation, biochemistry, and gene expression. We identified that GDF-5 not only promotes osteogenic differentiation of rat fat-derived stromal cells, but also may promote angiogenic activity of stromal cells by increasing vascular endothelial growth factor (VEGF) gene expression in vitro. These data suggest that several distinct regulatory mechanisms may exist in association with osteogenic differentiation.

Mouse Growth and Differentiation Factor-5 Protein and DNA Therapy Potentiates Intervertebral Disc Cell Aggregation and Chondrogenic Gene Expression

The Spine Journal : Official Journal of the North American Spine Society. Mar-Apr, 2008  |  Pubmed ID: 17974491

Growth and differentiation factor-5 (GDF-5)-deficient mice showed abnormalities in intervertebral disc (IVD) structure and extracellular matrix. Adenovirus-mediated GDF-5 delivery can promote the growth of rabbit disc cells.

Tendon: Biology, Biomechanics, Repair, Growth Factors, and Evolving Treatment Options

The Journal of Hand Surgery. Jan, 2008  |  Pubmed ID: 18261674

Surgical treatment of tendon ruptures and lacerations is currently the most common therapeutic modality. Tendon repair in the hand involves a slow repair process, which results in inferior repair tissue and often a failure to obtain full active range of motion. The initial stages of repair include the formation of functionally weak tissue that is not capable of supporting tensile forces that allow early active range of motion. Immobilization of the digit or limb will promote faster healing but inevitably results in the formation of adhesions between the tendon and tendon sheath, which leads to friction and reduced gliding. Loading during the healing phase is critical to avoid these adhesions but involves increased risk of rupture of the repaired tendon. Understanding the biology and organization of the native tendon and the process of morphogenesis of tendon tissue is necessary to improve current treatment modalities. Screening the genes expressed during tendon morphogenesis and determining the growth factors most crucial for tendon development will likely lead to treatment options that result in superior repair tissue and ultimately improved functional outcomes.

Adenovirus-mediated Expression of Growth and Differentiation Factor-5 Promotes Chondrogenesis of Adipose Stem Cells

Growth Factors (Chur, Switzerland). Jun, 2008  |  Pubmed ID: 18569021

The repair of articular cartilage injuries is impeded by the avascular and non-innervated nature of cartilage. Transplantation of autologous chondrocytes has a limited ability to augment the repair process due to the highly differentiated state of chondrocytes and the risks of donor-site morbidity. Mesenchymal stem cells can undergo chondrogenesis in the presence of growth factors for cartilage defect repair. Growth and differentiation factor-5 (GDF5) plays an important role in chondrogenesis. In this study, we examined the effects of GDF5 on chondrogenesis of adipose-derived stem cells (ADSCs) and evaluate the chondrogenic potentials of GDF5 genetically engineered ADSCs using an in vitro pellet culture model. Rat ADSCs were grown as pellet cultures and treated with chondrogenic media (CM). Induction of GDF5 by an adenovirus (Ad-GDF5) was compared with exogenous supplementation of GDF5 (100 ng/ml) and transforming growth factor-beta (TGF-beta1; 10 ng/ml). The ADSCs underwent chondrogenic differentiation in response to GDF5 exposure as demonstrated by production of proteoglycan, and up-regulation of collagen II and aggrecan at the protein and mRNA level. The chondrogenic potential of a one-time infection with Ad-GDF5 was weaker than exogenous GDF5, but equal to that of TGF-beta1. Stimulation with growth factors or CM alone induced transient expression of the mRNA for collagen X, indicating a need for optimization of the CM. Our findings indicate that GDF5 is a potent inducer of chondrogenesis in ADSCs, and that ADSCs genetically engineered to express prochondrogenic growth factors, such as GDF5, may be a promising therapeutic cell source for cartilage tissue engineering.

Receptor-mediated Gene Delivery Using PAMAM Dendrimers Conjugated with Peptides Recognized by Mesenchymal Stem Cells

Molecular Pharmaceutics. Jun, 2010  |  Pubmed ID: 20230026

As mesenchymal stem cells (MSCs) can differentiate into multiple cell types, the delivery of exogenous genes to this type of cell can be an important tool in tissue regeneration and engineering. However transfection of MSCs using nonviral gene delivery vectors is difficult, the development of more efficient and safe DNA vehicles being necessary. Moreover, specific transfection of MSCs may be required to avoid unwanted side effects in other tissues. In this study, a novel family of gene delivery vectors based on poly(amidoamine) (PAMAM) dendrimers functionalized with peptides displaying high affinity toward MSCs was prepared. The vectors were characterized with respect to their ability to neutralize, bind and compact plasmid DNA (pDNA). The complexes formed between the vectors and pDNA were analyzed concerning their size, zeta-potential, capacity of being internalized by cells and transfection efficiency. These new vectors exhibited low cytotoxicity, receptor-mediated gene delivery into MSCs and transfection efficiencies superior to those presented by native dendrimers and by partially degraded dendrimers.

VEGF and BMP-6 Enhance Bone Formation Mediated by Cloned Mouse Osteoprogenitor Cells

Growth Factors (Chur, Switzerland). Oct, 2010  |  Pubmed ID: 20497064

New strategies such as combined utilization of growth factors may provide a better treatment for difficult fractures. We have demonstrated enhanced angiogenesis and osteogenesis through the actions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) on the osteogenic differentiation of a cloned mouse osteoprogenitor cell in vitro and ectopic bone formation in vivo. Human VEGF and BMP-6 genes expressed together produced a significant increase in alkaline phosphatase activity, expression of the RunX2 and osteocalcin genes and mineralization. Microcomputed tomographic analysis of subcutaneous implants consisting of cells transfected with VEGF and BMP-6 cDNA and delivered on a 3D poly (lactic-co-glycolic acid) scaffold confirmed the additive effects between VEGF and BMP-6. Ectopic bone formation in the VEGF plus BMP-6 group was greatest compared to that in either VEGF or BMP-6 alone. This is the first study that demonstrates osteogenesis in vitro and in vivo through the additive effects of VEGF and BMP-6.

Adipose-derived Mesenchymal Stem Cells Treated with Growth Differentiation Factor-5 Express Tendon-specific Markers

Tissue Engineering. Part A. Sep, 2010  |  Pubmed ID: 20575691

Adipose-derived mesenchymal stem cells (ADMSCs) are a unique population of stem cells with therapeutic potential in the treatment of connective tissue injuries. Growth differentiation factor-5 (GDF)-5 is known to play a role in tendon repair and maintenance. The aim of this study was to investigate the effects of GDF-5 on proliferation and tendonogenic gene expression of rat ADMSCs.

Use of a Bioactive Scaffold for the Repair of Bone Defects in a Novel Reproducible Vertebral Body Defect Model

Bone. Aug, 2010  |  Pubmed ID: 20580872

Bone defects in vertebral bodies (VB) usually occur after the reduction of fractures or are caused by bone disease. Besides the treatment of original disease, repair of the bone defect can restore the structure of VB and improve stabilization of the spine to protect the spinal cord nerves. To aid studies of the efficacy of bioengineering techniques for repair of VB, we developed a rat model with a critical size bone defect in VB. Air-motivated burrs were used to create two sizes of bone defect (2 x 3 x 1.5 mm; 2 x 3 x 3 mm) in the anterior part of VB in 6-month-old Fischer 344 rats. Quantitative CT analyses and histological assays demonstrated that neither defects self-repaired by 8 weeks post surgery. Moreover, the tendency of bone formation was monitored in the same animal by serial CT image evaluations, allowing us to demonstrate that there was significant bone growth during the 4- to 6-week period after the creation of the bone defect. We then implanted sintered poly(lactic-co-glycolic acid) (PLGA) microsphere scaffolds loaded with Matrigel with or without recombinant human bone morphogenetic protein 2 (rhBMP2; 2.0 microg rhBMP2/10 microL Matrigel/scaffold) into the bone defect (2 x 3 x 3 mm) in the VB. Bone formation was detected by quantitative analyses of serial CT images, which demonstrated bone growth in rats that received the rhBMP2 implant, in both surrounding areas and inside area of the scaffold. In addition to a rapid increase within 2 weeks of the operation, another significant bone formation period was found between 4 and 8 weeks after the implantation. By contrast, the control group that received the implant without rhBMP2 did not show similar bone formation tendencies. The results of CT analyses were confirmed by histological studies. This study suggests that a critical size bone defect of the anterior VB can be developed in a rat model. Characterization of this model demonstrated that 4 to 6 weeks after creation of the defect was a significant bone growth period for VB bone repair in rats. This animal model has further utility for the study of different biomaterials for VB bone repair. Implantation of a bioactive PLGA scaffold carrying rhBMP2 allowed more successful repair of the VB defect. Although further characterization studies are needed, the bioactive PLGA scaffold developed in this study will likely adapt easily to other in vivo osteogenesis applications.

Growth Differentiation Factor-5 Regulation of Extracellular Matrix Gene Expression in Murine Tendon Fibroblasts

Journal of Tissue Engineering and Regenerative Medicine. Mar, 2011  |  Pubmed ID: 20653042

The synthesis and organization of extracellular matrix (ECM) of tendon, in resting and states of repair, are governed by fibroblasts. Growth differentiation factor-5 (GDF-5) may enhance the cellular response to tendon injury, thus improving the structural outcome of the regenerative tissue. This study was an attempt to identify potential mechanisms controlling the response of fibroblasts to injury and GDF-5, in the pursuit of improved tissue regeneration. There were two sets of experiments. Isolated mice Achilles tendon fibroblasts were treated with different concentrations of rGDF-5 (0-100 ng/ml) for 0-12 days in cell culture. The temporal effect of rGDF-5 on ECM gene expression was analysed for type I collagen and aggrecan expression. Microarray and gene expression analysis were performed on cells treated with 100 ng/ml for 4 days. Forty-five mice underwent bilateral mid-substance Achilles tendon tenotomy and suture repair. Repair sites were injected with 10 µg rGDF-5 or saline. Tendons were assessed histologically at 2, 4 and 6 weeks. Expression of ECM genes procollagen IX, aggrecan, matrix metalloproteinase 9 and fibromodulin were upregulated. Proinflammatory reaction genes were downregulated. rGDF-5 led to an increase in total DNA, glycosaminoglycan (GAG) and hydroxyproline (OHP). The OHP:DNA ratio of fibroblast cultures was increased over all time points, with increased GAG:DNA at day 12. rGDF-5 treatment showed improved collagen organization over controls. The results delineate the mode of action of rGDF-5 at the cellular and gene level. rGDF-5 could play a role in tendon repair and be used for future therapies that promote tendon healing.

Combined VEGF and LMP-1 Delivery Enhances Osteoprogenitor Cell Differentiation and Ectopic Bone Formation

Growth Factors (Chur, Switzerland). Feb, 2011  |  Pubmed ID: 21222516

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

Growth/differentiation Factor-5 Modulates the Synthesis and Expression of Extracellular Matrix and Cell-adhesion-related Molecules of Rat Achilles Tendon Fibroblasts

Connective Tissue Research. 2011  |  Pubmed ID: 21250863

This study was designed to examine the cellular and molecular response of tendon fibroblasts to growth/differentiation factor-5 (GDF-5). Rat Achilles tendon fibroblasts (ATFs) were treated in culture with varying concentrations of GDF-5 (0-1000 ng/ml) over varying periods of time (0-12 days). Cell proliferation, evaluated through use of a standard MTT colorimetric assay, confirmed that GDF-5 stimulates ATF proliferation in a concentration- and time-dependent fashion. Temporal and concentration analysis revealed that GDF-5 increases total DNA, glycosaminoglycan (GAG), and hydroxyproline (HYP) content. Ratios of HYP/DNA and GAG/DNA increased with increasing concentrations of GDF-5 (0-1000 ng/ml). Expression of the following 12 extracellular matrix (ECM) and cell-adhesion-related genes was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR): collagen I (col I), collagen III (col III), matrix metalloproteinases (MMP)-3 and -13, aggrecan, tissue inhibitor of matrix metalloproteinase (TIMP)-2, syndecan-4, N-cadherin, tenascin-C, biglycan, versican, and decorin. RT-PCR data revealed an increase in the expression of col I, col III, MMP-3, MMP-13, TIMP-2, syndecan-4, N-cadherin, tenascin-C, and aggrecan genes by day 6. A statistically significant decrease in TIMP-2 and MMP-13 was observed on day 12. Decorin expression was depressed at all time points in cells treated with GDF-5. There was no significant change in biglycan expression in ATFs supplemented with GDF-5. These findings suggest that GDF-5 induces cellular proliferation and ECM synthesis as well as expression of ECM and cell-adhesion-related genes in ATFs. This study further defines the influence of GDF-5 on rat ATFs through its action on the expression of genes that are associated with tendon ECM.

Enhancement of Matrix Production and Cell Proliferation in Human Annulus Cells Under Bioreactor Culture

Tissue Engineering. Part A. Jun, 2011  |  Pubmed ID: 21303231

Tissue engineering is a promising approach for treatment of disc degeneration. Herein, we evaluated effects of rotating bioreactor culture on the extracellular matrix production and proliferation of human annulus fibrosus (AF) cells. AF cells were embedded into alginate beads, and then cultured up to 3 weeks in a rotating wall vessel bioreactor or a static vessel. By real-time reverse transcription-polymerase chain reaction, expression of aggrecan, collagen type I and type II, and collagen prolyl 4-hydroxylase II was remarkably elevated, whereas expression of matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 5 was significantly decreased under bioreactor. Biochemical analysis revealed that the levels of the whole cell-associated proteoglycan and collagen were approximately five- and twofolds in rotating bioreactor, respectively, compared to those in static culture. Moreover, AF cell proliferation was augmented in rotating bioreactor. DNA contents were threefolds higher in rotating bioreactor than that in static culture. Expression of the proliferating cell nuclear antigen was robustly enhanced in rotating bioreactor as early as 1 week. Our findings suggested that rotating bioreactor culture would be an effective technique for expansion of human annulus cells for tissue engineering driven treatment of disc degeneration.

The Reaction of Bone to Tumor Growth from Human Breast Cancer Cells in a Rat Spine Single Metastasis Model

Spine. Apr, 2011  |  Pubmed ID: 21422981

In vivo experiments to develop a rat spine single metastasis model by using human breast cancer cells.

A Message from the Editor-in-Chief

Connective Tissue Research. Dec, 2011  |  Pubmed ID: 22185576