In JoVE (1)

Other Publications (20)

Articles by Lauren E. Flynn in JoVE

 JoVE Bioengineering

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

1Biomedical Engineering Graduate Program, The University of Western Ontario, 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, 3Department of Chemical Engineering, Queen's University, 4Department of Chemical & Biochemical Engineering, Faculty of Engineering, The University of Western Ontario

JoVE 55436

Other articles by Lauren E. Flynn on PubMed

Proliferation and Differentiation of Adipose-derived Stem Cells on Naturally Derived Scaffolds

Biomaterials. Apr, 2008  |  Pubmed ID: 18242690

A tissue-engineered substitute that facilitates large-volume regeneration of the subcutaneous adipose tissue layer is needed for reconstructive plastic surgery. Towards this goal, we describe the in vitro culture of primary human adipose-derived stem cells (ASC) seeded into placental decellular matrix (PDM) and cross-linked hyaluronan (XLHA) scaffolds. Specifically, we evaluated cellular proliferation and adipogenic differentiation in the PDM, XLHA, and PDM combined with XLHA scaffolds. Cellular proliferation, viability, and glucose consumption were determined prior to the induction of differentiation. Adipogenesis within each of the scaffolds was investigated through gene expression analysis using end point and real time reverse transcriptase polymerase chain reaction (RT-PCR). The results indicate that the cell-adhesive PDM scaffolds facilitated proliferation and viability, while differentiation was augmented when the cells were encapsulated in the non-adhesive XLHA gels.

From Competence to Waive Counsel to Competence to Represent Oneself: the Supreme Court Advances Fairness in Edwards

Mental and Physical Disability Law Reporter. Jan-Feb, 2009  |  Pubmed ID: 20698084

The Effect of Serial Passaging on the Proliferation and Differentiation of Bovine Adipose-derived Stem Cells

Cells, Tissues, Organs. 2012  |  Pubmed ID: 21893933

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis.

Design and Characterization of Tissue-specific Extracellular Matrix-derived Microcarriers

Tissue Engineering. Part C, Methods. Mar, 2012  |  Pubmed ID: 21981618

The three-dimensional (3D) extracellular matrix (ECM) environment plays a critical role in mediating normal cellular behavior and tissue organization. While commercially available microcarriers have shown promise, limited research has been conducted on the design of tissue-specific, custom-fabricated microcarriers, engineered to mimic the composition of the native ECM of cells or tissues of interest. Moving toward this goal, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via minimally-cytotoxic protocols. Characterization by microscopy confirmed the production of stable spherical microcarriers, with a microporous surface topography and porous interior. The mean diameter of the DAT microcarriers was 934±51 μm, while the porosity was estimated as 29%±4% using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT microcarriers were effectively stabilized with the photochemical crosslinking agent rose bengal, with total protein release in a simulated physiological environment remaining below 10 μg/mL at all time points. Preliminary cell culture studies with human adipose-derived stem cells (ASCs) in a spinner flask system indicated enhanced cell attachment and proliferation of ASCs on DAT microcarriers over 14 days, as compared with gelatin control microcarriers fabricated using similar methods. Testing confirmed injectability of the DAT microcarriers, further supporting the clinical potential of the approach for localized cell delivery and small volume augmentation in plastic and reconstructive surgery. Overall, tissue-specific microcarriers prepared from solubilized DAT were found to be highly supportive of human ASCs cultured in a 3D dynamic environment.

The Performance of Decellularized Adipose Tissue Microcarriers As an Inductive Substrate for Human Adipose-derived Stem Cells

Biomaterials. Jun, 2012  |  Pubmed ID: 22456084

With the aim of developing a clinically-translatable cell expansion and delivery vehicle for adipose tissue engineering, the adipogenic differentiation of human adipose-derived stem cells (ASCs) was investigated on microcarriers fabricated from human decellularized adipose tissue (DAT). ASCs seeded on the DAT microcarriers and cultured in adipogenic differentiation medium within a low-shear spinner culture system demonstrated high levels of adipogenic differentiation, as measured by the expression of adipogenic genes, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and intracellular lipid accumulation. In contrast, gelatin microcarrier controls did not demonstrate significant adipogenesis, emphasizing the role of the native matrix in mediating ASC differentiation. Interestingly, ASCs cultured on the DAT microcarriers in proliferation medium expressed elevated levels of the adipogenic markers, suggesting that the DAT provided an adipo-inductive substrate for the human ASCs. In vivo testing of the DAT and gelatin microcarriers in a subcutaneous Wistar rat model confirmed injectability and demonstrated stable volume retention over 28 days. Under histological analysis, the DAT microcarriers demonstrated no evidence of immunogenicity or cytotoxicity, with the DAT supporting cellular infiltration and tissue remodeling. Pre-seeding the DAT microcarriers with allogenic rat ASCs enhanced cellularity and angiogenesis within the implant region.

Co-delivery of Adipose-derived Stem Cells and Growth Factor-loaded Microspheres in RGD-grafted N-methacrylate Glycol Chitosan Gels for Focal Chondral Repair

Biomacromolecules. Aug, 2012  |  Pubmed ID: 22746668

The coencapsulation of growth factor-loaded microspheres with adipose-derived stem cells (ASCs) within a hydrogel matrix was studied as a potential means to enhance ASC chondrogenesis in the development of a cell-based therapeutic strategy for the regeneration of partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel used to encapsulate microspheres loaded with bone morphogenetic protein 6 (BMP-6) and transforming growth factor-β3 (TGF-β3) with human ASCs. ASC viability and retention were enhanced when the Young's modulus of the MGC ranged between 225 and 380 kPa. Grafting an RGD-containing peptide onto the MGC backbone (RGD-MGC) improved ASC viability within the gels, remaining at greater than 90% over 14 days in culture. The effects of BMP-6 and TGF-β3 released from the polymer microspheres on ASC chondrogenesis were assessed, and the level of differentiation was compared to ASCs in control gels containing nongrowth factor-loaded microspheres cultured with and without the growth factors supplied in the medium. There was enhanced expression of chondrogenic markers at earlier time points when the ASCs were induced with the sustained and local release of BMP-6 and TGF-β3 from the microspheres. More specifically, the normalized glycosaminoglycan and collagen type II protein expression levels were significantly higher than in the controls. In addition, the ratio of collagen type II to type I was significantly higher in the microsphere delivery group and increased over time. End-point RT-PCR analysis supported that there was a more rapid induction and enhancement of ASC chondrogenesis in the controlled release group. Interestingly, in all of the assays, there was evidence of chondrogenic differentiation when the ASCs were cultured in the gels in the absence of growth factor stimulation. Overall, the co-delivery of growth-factor-loaded microspheres and ASCs in RGD-modified MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair.

Characterization of Biologically Active Insulin-loaded Alginate Microparticles Prepared by Spray Drying

Drug Development and Industrial Pharmacy. Mar, 2013  |  Pubmed ID: 22397581

Spray drying has been used as a means to encapsulate therapeutics in polymeric matrices to improve stability and alter pharmacokinetics. This research aims to characterize alginate microparticles formed by spray drying to encapsulate insulin for therapeutic delivery applications.

Porous Decellularized Adipose Tissue Foams for Soft Tissue Regeneration

Biomaterials. Apr, 2013  |  Pubmed ID: 23384795

To design tissue-specific bioscaffolds with well-defined properties and 3-D architecture, methods were developed for preparing porous foams from enzyme-solubilized human decellularized adipose tissue (DAT). Additionally, a technique was established for fabricating "bead foams" comprised of interconnected networks of porous DAT beads fused through a controlled freeze-thawing and lyophilization procedure. In characterization studies, the foams were stable without the need for chemical crosslinking, with properties that could be tuned by controlling the protein concentration and freezing rate during synthesis. Adipogenic differentiation studies with human adipose-derived stem cells (ASCs) suggested that stiffness influenced ASC adipogenesis on the foams. In support of our previous work with DAT scaffolds and microcarriers, the DAT foams and bead foams strongly supported adipogenesis and were also adipo-inductive, as demonstrated by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, endpoint RT-PCR analysis of adipogenic gene expression, and intracellular lipid accumulation. Adipogenic differentiation was enhanced on the microporous DAT foams, potentially due to increased cell-cell interactions in this group. In vivo assessment in a subcutaneous Wistar rat model demonstrated that the DAT bioscaffolds were well tolerated and integrated into the host tissues, supporting angiogenesis and adipogenesis. The DAT-based foams induced a strong angiogenic response, promoted inflammatory cell migration and gradually resorbed over the course of 12 weeks, demonstrating potential as scaffolds for wound healing and soft tissue regeneration.

Techniques for the Isolation of High-quality RNA from Cells Encapsulated in Chitosan Hydrogels

Tissue Engineering. Part C, Methods. Nov, 2013  |  Pubmed ID: 23448167

Extracting high-quality RNA from hydrogels containing polysaccharide components is challenging, as traditional RNA isolation techniques designed for cells and tissues can have limited yields and purity due to physiochemical interactions between the nucleic acids and the biomaterials. In this study, a comparative analysis of several different RNA isolation methods was performed on human adipose-derived stem cells photo-encapsulated within methacrylated glycol chitosan hydrogels. The results demonstrated that RNA isolation methods with cetyl trimethylammonium bromide (CTAB) buffer followed by purification with an RNeasy® mini kit resulted in low yields of RNA, except when the samples were preminced directly within the buffer. In addition, genomic DNA contamination during reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was observed in the hydrogels processed with the CTAB-based methods. Isolation methods using TRIzol® in combination with one of a Qiaex® gel extraction kit, an RNeasy® mini kit, or an extended solvent purification method extracted RNA suitable for gene amplification, with no evidence of genomic contamination. The latter two methods yielded the best results in terms of yield and amplification efficiency. Predigestion of the scaffolds with lysozyme was investigated as a possible means of enhancing RNA extraction from the polysaccharide gels, with no improvements observed in terms of the purity, yield, or amplification efficiency. Overall, this work highlights the application of a TRIzol®+extended solvent purification method for optimizing RNA extraction that can be applied to obtain reliable and accurate gene expression data in studies investigating cells seeded in chitosan-based scaffolds.

Composite Hydrogel Scaffolds Incorporating Decellularized Adipose Tissue for Soft Tissue Engineering with Adipose-derived Stem Cells

Biomaterials. Feb, 2014  |  Pubmed ID: 24331712

An injectable tissue-engineered adipose substitute that could be used to deliver adipose-derived stem cells (ASCs), filling irregular defects and stimulating natural soft tissue regeneration, would have significant value in plastic and reconstructive surgery. With this focus, the primary aim of the current study was to characterize the response of human ASCs encapsulated within three-dimensional bioscaffolds incorporating decellularized adipose tissue (DAT) as a bioactive matrix within photo-cross-linkable methacrylated glycol chitosan (MGC) or methacrylated chondroitin sulphate (MCS) delivery vehicles. Stable MGC- and MCS-based composite scaffolds were fabricated containing up to 5 wt% cryomilled DAT through initiation with long-wavelength ultraviolet light. The encapsulation strategy allows for tuning of the 3-D microenvironment and provides an effective method of cell delivery with high seeding efficiency and uniformity, which could be adapted as a minimally-invasive in situ approach. Through in vitro cell culture studies, human ASCs were assessed over 14 days in terms of viability, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, adipogenic gene expression and intracellular lipid accumulation. In all of the composites, the DAT functioned as a cell-supportive matrix that enhanced ASC viability, retention and adipogenesis within the gels. The choice of hydrogel also influenced the cell response, with significantly higher viability and adipogenic differentiation observed in the MCS composites containing 5 wt% DAT. In vivo analysis in a subcutaneous Wistar rat model at 1, 4 and 12 weeks showed superior implant integration and adipogenesis in the MCS-based composites, with allogenic ASCs promoting cell infiltration, angiogenesis and ultimately, fat formation.

Comparison of Human Adipose-derived Stem Cells Isolated from Subcutaneous, Omental, and Intrathoracic Adipose Tissue Depots for Regenerative Applications

Stem Cells Translational Medicine. Feb, 2014  |  Pubmed ID: 24361924

Adipose tissue is an abundant source of multipotent progenitor cells that have shown promise in regenerative medicine. In humans, fat is primarily distributed in the subcutaneous and visceral depots, which have varying biochemical and functional properties. In most studies to date, subcutaneous adipose tissue has been investigated as the adipose-derived stem cell (ASC) source. In this study, we sought to develop a broader understanding of the influence of specific adipose tissue depots on the isolated ASC populations through a systematic comparison of donor-matched abdominal subcutaneous fat and omentum, and donor-matched pericardial adipose tissue and thymic remnant samples. We found depot-dependent and donor-dependent variability in the yield, viability, immunophenotype, clonogenic potential, doubling time, and adipogenic and osteogenic differentiation capacities of the ASC populations. More specifically, ASCs isolated from both intrathoracic depots had a longer average doubling time and a significantly higher proportion of CD34(+) cells at passage 2, as compared with cells isolated from subcutaneous fat or the omentum. Furthermore, ASCs from subcutaneous and pericardial adipose tissue demonstrated enhanced adipogenic differentiation capacity, whereas ASCs isolated from the omentum displayed the highest levels of osteogenic markers in culture. Through cell culture analysis under hypoxic (5% O(2)) conditions, oxygen tension was shown to be a key mediator of colony-forming unit-fibroblast number and osteogenesis for all depots. Overall, our results suggest that depot selection is an important factor to consider when applying ASCs in tissue-specific cell-based regenerative therapies, and also highlight pericardial adipose tissue as a potential new ASC source.

Mesenchymal Stem Cell Delivery Strategies to Promote Cardiac Regeneration Following Ischemic Injury

Biomaterials. Apr, 2014  |  Pubmed ID: 24560461

Myocardial infarction (MI) is one of the leading causes of mortality worldwide and is associated with irreversible cardiomyocyte death and pathological remodeling of cardiac tissue. In the past 15 years, several animal models have been developed for pre-clinical testing to assess the potential of stem cells for functional tissue regeneration and the attenuation of left ventricular remodeling. The promising results obtained in terms of improved cardiac function, neo-angiogenesis and reduction in infarct size have motivated the initiation of clinical trials in humans. Despite the potential, the results of these studies have highlighted that the effective delivery and retention of viable cells within the heart remain significant challenges that have limited the therapeutic efficacy of cell-based therapies for treating the ischemic myocardium. In this review, we discuss key elements for designing clinically translatable cell-delivery approaches to promote myocardial regeneration. Key topics addressed include cell selection, with a focus on mesenchymal stem cells derived from the bone marrow (bMSCs) and adipose tissue (ASCs), including a discussion of their potential mechanisms of action. Natural and synthetic biomaterials that have been investigated as injectable cell delivery vehicles for cardiac applications are critically reviewed, including an analysis of the role of the biomaterials themselves in the therapeutic scheme.

Characterization and Assessment of Hyperelastic and Elastic Properties of Decellularized Human Adipose Tissues

Journal of Biomechanics. Nov, 2014  |  Pubmed ID: 25446266

Decellularized adipose tissue (DAT) has shown potential as a regenerative scaffold for plastic and reconstructive surgery to augment or replace damaged or missing adipose tissue (e.g. following lumpectomy or mastectomy). The mechanical properties of soft tissue substitutes are of paramount importance in restoring the natural shape and appearance of the affected tissues, and mechanical mismatching can lead to unpredictable scar tissue formation and poor implant integration. The goal of this work was to assess the linear elastic and hyperelastic properties of decellularized human adipose tissue and compare them to those of normal breast adipose tissue. To assess the influence of the adipose depot source on the mechanical properties of the resultant decellularized scaffolds, we performed indentation tests on DAT samples sourced from adipose tissue isolated from the breast, subcutaneous abdominal region, omentum, pericardial depot and thymic remnant, and their corresponding force-displacement data were acquired. Elastic and hyperelastic parameters were estimated using inverse finite element algorithms. Subsequently, a simulation was conducted in which the estimated hyperelastic parameters were tested in a real human breast model under gravity loading in order to assess the suitability of the scaffolds for implantation. Results of these tests showed that in the human breast, the DAT would show similar deformability to that of native normal tissue. Using the measured hyperelastic parameters, we were able to assess whether DAT derived from different depots exhibited different intrinsic nonlinearities. Results showed that DAT sourced from varying regions of the body exhibited little intrinsic nonlinearity, with no statistically significant differences between the groups.

Multilineage Co-culture of Adipose-derived Stem Cells for Tissue Engineering

Journal of Tissue Engineering and Regenerative Medicine. Jul, 2015  |  Pubmed ID: 23135884

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue-engineering strategies with adipose-derived stem cells (ASCs), the focus of this study was on developing indirect co-culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co-culture with each of the mature cell populations was shown to successfully induce or enhance lineage-specific differentiation of the ASCs. In general, a more homogeneous but lower-level differentiation response was observed in co-culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non-canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF-1 and DKK-1 on multilineage differentiation in co-culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co-culture with the mature cell populations.

Effect of Decellularized Adipose Tissue Particle Size and Cell Density on Adipose-derived Stem Cell Proliferation and Adipogenic Differentiation in Composite Methacrylated Chondroitin Sulphate Hydrogels

Biomedical Materials (Bristol, England). Jul, 2015  |  Pubmed ID: 26225549

An injectable composite scaffold incorporating decellularized adipose tissue (DAT) as a bioactive matrix within a hydrogel phase capable of in situ polymerization would be advantageous for adipose-derived stem cell (ASC) delivery in the filling of small or irregular soft tissue defects. Building on previous work, the current study investigates DAT milling methods and the effects of DAT particle size and cell seeding density on the response of human ASCs encapsulated in photo-cross-linkable methacrylated chondroitin sulphate (MCS)-DAT composite hydrogels. DAT particles were generated by milling lyophilized DAT and the particle size was controlled through the processing conditions with the goal of developing composite scaffolds with a tissue-specific 3D microenvironment tuned to enhance adipogenesis. ASC proliferation and adipogenic differentiation were assessed in vitro in scaffolds incorporating small (average diameter of 38   ±   6 μm) or large (average diameter of 278   ±   3 μm) DAT particles in comparison to MCS controls over a period of up to 21 d. Adipogenic differentiation was enhanced in the composites incorporating the smaller DAT particles and seeded at the higher density of 5   ×   10(5) ASCs/scaffold, as measured by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, semi-quantitative analysis of perilipin expression and oil red O staining of intracellular lipid accumulation. Overall, this study demonstrates that decellularized tissue particle size can impact stem cell differentiation through cell-cell and cell-matrix interactions, providing relevant insight towards the rational design of composite biomaterial scaffolds for adipose tissue engineering.

Adipose-derived Stromal Cells Mediate In vivo Adipogenesis, Angiogenesis and Inflammation in Decellularized Adipose Tissue Bioscaffolds

Biomaterials. Dec, 2015  |  Pubmed ID: 26360790

Decellularized adipose tissue (DAT) has shown promise as an adipogenic bioscaffold for soft tissue augmentation and reconstruction. The objective of the current study was to investigate the effects of allogeneic adipose-derived stem/stromal cells (ASCs) on in vivo fat regeneration in DAT bioscaffolds using an immunocompetent rat model. ASC seeding significantly enhanced angiogenesis and adipogenesis, with cell tracking studies indicating that the newly-forming tissues were host-derived. Incorporating ASCs also mediated the inflammatory response and promoted a more constructive macrophage phenotype. A fraction of the CD163(+) macrophages in the implants expressed adipogenic markers, with higher levels of this "adipocyte-like" phenotype in proximity to the developing adipose tissues. Our results indicate that the combination of ASCs and adipose extracellular matrix (ECM) provides an inductive microenvironment for adipose regeneration mediated by infiltrating host cell populations. The DAT scaffolds are a useful tissue-specific model system for investigating the mechanisms of in vivo adipogenesis that may help to develop a better understanding of this complex process in the context of both regeneration and disease. Overall, combining adipose-derived matrices with ASCs is a highly promising approach for the in situ regeneration of host-derived adipose tissue.

Porous, Ventricular Extracellular Matrix-Derived Foams As a Platform for Cardiac Cell Culture

BioResearch Open Access. 2015  |  Pubmed ID: 26487982

To more closely mimic the native cellular microenvironment, 3D scaffolds derived from the extracellular matrix (ECM) are being developed as alternatives to conventional 2D culture systems. In the present study, we established methods to fabricate nonchemically cross-linked 3D porous foams derived entirely from decellularized porcine left ventricle (DLV) for use as an in vitro cardiac cell culture platform. Furthermore, we explored the effects of physically preprocessing the DLV through mechanical mincing versus cryomilling, as well as varying the ECM concentration on the structure, composition, and physical properties of the foams. Our results indicate that the less highly processed minced foams had a more cohesive and complex network of ECM components, enhanced mechanical properties, and improved stability under simulated culturing conditions. To validate the DLV foams, a proof-of-concept study was conducted to explore the early cardiomyogenic differentiation of pericardial fat adipose-derived stem/stromal cells (pfASCs) on the minced DLV foams relative to purified collagen I gel controls. Differentiation was induced using a modified cardiomyogenic medium (MCM) or through stimulation with 5-azacytidine (5-aza), and cardiomyocyte marker expression was characterized by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction. Our results indicate that early markers of cardiomyogenic differentiation were significantly enhanced on the DLV foams cultured in MCM, suggesting a synergistic effect of the cardiac ECM-derived scaffolds and the culture medium on the induction of pfASC differentiation. Furthermore, in analyzing the response in the noninduced control groups, the foams were observed to provide a mildly inductive microenvironment for pfASC cardiomyogenesis, supporting the rationale for using tissue-specific ECM as a substrate for cardiac cell culture applications.

Comparative Biomechanical Study of Using Decellularized Human Adipose Tissues for Post-mastectomy and Post-lumpectomy Breast Reconstruction

Journal of the Mechanical Behavior of Biomedical Materials. Apr, 2016  |  Pubmed ID: 26735182

Developing suitable biomaterials for post-mastectomy or post-lumpectomy breast reconstruction is highly important. This study is aimed at evaluating biomechanical suitability of decellularized adipose tissue (DAT) for this purpose. The study involves computational experiments for evaluating deformation of the breast reconstructed using DAT under loading conditions pertaining to two common body position changes of prone-to-supine and prone-to-upright. This was conducted using nonlinear finite element models where the breast geometry was obtained from MRI image of a female breast. The experiments were performed using DAT sourced from various adipose tissue depots in comparison to natural adipose tissue. Data obtained from the conducted experiments showed no contour defects with various DAT materials for simulated post-mastectomy or post-lumpectomy breast reconstruction under the loading conditions. They also demonstrated that a breast reconstructed using DAT derived from the breast or subcutaneous abdominal depots exhibit significantly closer deformation, both qualitatively and quantitatively, to that of a normal breast under the same loading conditions. Similarity of DAT deformation to that of natural breast tissue in post-surgery breast reconstruction was assessed using nonlinear finite element analysis. Our results provide evidence that DAT derived from subcutaneous abdominal and breast depots yield more analogous deformation pattern to the natural tissue in post-mastectomy breast reconstruction applications. This is quite encouraging, as breast and subcutaneous adipose tissue can be readily obtained in large quantities from breast or abdominal lipo-reduction surgery procedures. Furthermore, in post-lumpectomy cases all DAT samples used in this research showed similar deformation, and thus are suitable as breast tissue substituents.

Decellularized Adipose Tissue Microcarriers As a Dynamic Culture Platform for Human Adipose-derived Stem/stromal Cell Expansion

Biomaterials. Mar, 2017  |  Pubmed ID: 28038353

With the goal of designing a clinically-relevant expansion strategy for human adipose-derived stem/stromal cells (ASCs), methods were developed to synthesize porous microcarriers derived purely from human decellularized adipose tissue (DAT). An electrospraying approach was applied to generate spherical DAT microcarriers with an average diameter of 428 ± 41 μm, which were soft, compliant, and stable in long-term culture without chemical crosslinking. Human ASCs demonstrated enhanced proliferation on the DAT microcarriers relative to commercially-sourced Cultispher-S microcarriers within a spinner culture system over 1 month. ASC immunophenotype was maintained post expansion, with a trend for reduced expression of the cell adhesion receptors CD73, CD105, and CD29 under dynamic conditions. Upregulation of the early lineage-specific genes PPARγ, LPL, and COMP was observed in the ASCs expanded on the DAT microcarriers, but the cells retained their multilineage differentiation capacity. Comparison of adipogenic and osteogenic differentiation in 2-D cultures prepared with ASCs pre-expanded on the DAT microcarriers or Cultispher-S microcarriers revealed similar adipogenic and enhanced osteogenic marker expression in the DAT microcarrier group, which had undergone a higher population fold change. Further, histological staining results suggested a more homogeneous differentiation response in the ASCs expanded on the DAT microcarriers as compared to either Cultispher-S microcarriers or tissue culture polystyrene. A pilot chondrogenesis study revealed higher levels of chondrogenic gene and protein expression in the ASCs expanded on the DAT microcarriers relative to all other groups, including the baseline controls. Overall, this study demonstrates the promise of applying dynamic culture with tissue-specific DAT microcarriers as a means of deriving regenerative cell populations.

Tough, Semisynthetic Hydrogels for Adipose Derived Stem Cell Delivery for Chondral Defect Repair

Macromolecular Bioscience. Jan, 2017  |  Pubmed ID: 28085994

Cell-based therapies have great potential to regenerate and repair injured articular cartilage, and a range of synthetic and natural polymer-based hydrogels have been used in combination with stem cells and growth factors for this purpose. Although the hydrogel scaffolds developed to date possess many favorable characteristics, achieving the required mechanical properties has remained a challenge. A hydrogel system with tunable mechanical properties, composed of a mixture of natural and synthetic polymers, and its use for the encapsulation of adipose derived stem/stromal cells (ASCs) is described. Solutions of methacrylated chondroitin sulfate (MCS) are mixed with solutions of acrylate-poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate)-acrylate (PEG-(PTMC-A)2 ) in phosphate buffered saline and crosslinked via thermally initiated free radical polymerization. The hydrogel compressive equilibrium moduli and toughness are readily tailored by varying the concentration of the pre-polymers, as well as the molecular weight of the PEG used to prepare the PEG-(PTMC-A)2 . Two peptide sequences, GVOGEA and GGGGRGDS, are individually conjugated to the MCS to facilitate cell binding. The presence of the peptide ligands yields high ASC viability and long term metabolic activity following encapsulation in hydrogels prepared using the thermal initiator system. Overall, these hydrogels show promise as a minimally invasive ASC delivery strategy for chondral defect repair.

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