Introduction

Mesenchymal stem cells (MSCs) are a potential source of cells for the repair of articular cartilage and osteochondral defects (OCD) in the ankle. Synovial tissue has been shown to be a rich source of MSCs with the ability to undergo chondrogenic differentiation. Although these cells represent a heterogenous population, clonal populations have not been previously studied.

INTRODUCTION

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Introduction

Mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied.

INTRODUCTION

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Introduction

Mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied.

Introduction: Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Material and Methods: Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis.

Results: Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased.

Discussion: Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells.

Introduction: There is an ever-increasing clinical need for the regeneration and replacement of tissue to replace soft tissue lost due to trauma, disease and cosmetic surgery. A potential alternative to the current treatment modalities is the use of tissue engineering applications using mesenchymal stem cells that have been identified in many tissues including the fat pad. In this study, stem cells isolated from the fat pad were characterised and their differentiation potential assessed.

Materials and Methods: The infrapatellar fat pad was obtained from total knee replacement for osteoarthritis. Cells were isolated, expanded and stained for a number of stem cell markers. For adipogenic differentiation, cells were cultured in adipogenic inducing medium (10ug/ml insulin, 1uM dexamthasone, 100uM indomethacin and 500uM 3-isobutyl-1-methyl xanthine). Gene expression analyses and Oil red O staining was performed to assess adipogenesis.

Results: Cells at passage 2 stained strongly for CD13, CD29, CD44, CD90 and CD105 (mesenchymal stem cell markers). The cells stained sparsely for 3G5 (peri-cyte marker). On gene expression analyses, the cells cultured under adipogenic conditions had almost a 1,000 fold increase in expression of peroxisome proliferator-activated receptor gamma-2 (PPAR gamma-2) and 1,000,000 fold increase in expression of lipoprotein lipase (LPL). Oil red O staining revealed triglyceride accumulation within typical adipogenic morphology, confirming the adipogenic nature of the observed vacuoles, and showed failure of staining in control cells.

Discussion: Fat pad derived stem cells expressed a cell surface epitope profile of mesenchymal stem cells, and exhibited the potential to undergo adipogenic differentiation. Our results show that the human fat pad is a viable potential autogeneic source for mesenchymal stem cells capable of adipogenic differentiation as well as previously documented ostegenic and chondrogenic differentiation. This cell source has potential use in tissue engineering applications.

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis.

Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased.

Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells.

There is an ever-increasing clinical need for the regeneration and replacement of tissue to replace soft tissue lost due to trauma, disease and cosmetic surgery. A potential alternative to the current treatment modalities is the use of tissue engineering applications using mesenchymal stem cells that have been identified in many tissue including the infrapatellar fat pad. In this study, stem cells isolated from the infrapatellar fat pad were characterised to ascertain their origin, and allowed to undergo adipogenic differentiation to confirm multilineage differentiation potential.

The infrapatellar fat pad was obtained from total knee replacement for osteoarthritis. Cells were isolated and expanded in monolayer culture. Cells at passage 2 stained strongly for CD13, CD29, CD44, CD90 and CD105 (mesenchymal stem cell markers). The cells stained poorly for LNGFR and STRO1 (markers for freshly isolated bone marrow derived stem cells), and sparsely for 3G5 (pericyte marker). Staining for CD34 (haematopoetic marker) and CD56 (neural and myogenic lineage marker) was negative. {BR}For adipogenic differentiation, cells were cultured in adipogenic inducing medium consisting of basic medium with 10ug/ml insulin, 1uM dexamthasone, 100uM indomethacin and 500uM 3-isobutyl-1-methyl xanthine. By day 16, many cells had lipid vacuoles occupying most of the cytoplasm. On gene expression analyses, the cells cultured under adipogenic conditions had almost a 1,000 fold increase in expression of peroxisome proliferator-activated receptor gamma-2 (PPAR gamma-2) and 1,000,000 fold increase in expression of lipoprotein lipase (LPL). Oil red O staining confirmed the adipogenic nature of the observed vacuoles and showed failure of staining in control cells.

Our results show that the human infrapatellar fat pad is a viable potential autogeneic source for mesenchymal stem cells capable of adipogenic differentiation as well as previously documented ostegenic and chondrogenic differentiation. This cell source has potential use in tissue engineering applications.

Introduction: Autologous chondrocytes harvested from articular cartilage are being used for the repair of focal cartilage defects. The procedure involves injury to the cartilage and alternative sources of stem cells for use in repair are being explored. Stem cells have been found in many tissue including bone marrow and the infrapa-tellar fat pad. Infrapatellar fat pad derived stem cells present a viable and easily accessible source of stem cells for the repair of cartilage defects and tissue engineering applications.

Hypoxia has been shown to improve chondrogenesis in stem cells derived from the bone marrow. We explore the hypothesis that this effect would also apply to stem cells derived from the infrapatellar fat pad.

Materials and methods: Cell aggregates from early passage stem cells isolated from the infrapatellar fat pad were placed in chondrogenic media for 14 days either in a normoxic (20% oxygen) or hypoxic (5% oxygen) environment. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemi-cal studies were performed on the aggregates to assess chondrogenesis.

Results: Cells grown under hypoxic conditions showed significantly improved chondrogenesis as determined by relatively higher gene expression of proteoglycans, collagens and SOX genes. The cell aggregates also had a higher glycosoaminoglycan content and glycosoamino-glycan content per DNA. Immunohistochemical studies confirm enhanced production of collagen types I and II and aggrecan.

Discussion: These findings confirm the previously documented effects of hypoxic culture conditions on stem cells and extend the findings to include infrapatellar fat pad derived stem cells. Our findings suggest that oxygen tension has a role in regulating the function of stem cells as they undergo chondrogenesis. In culture these cells appear to function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension. This has important implications in future tissue engineering applications of these cells.

Introduction: In this study infrapatellar fat pad (IPFP) derived stem cells were expanded with and without Fibroblast Growth Factor-2 (FGF-2) supplementation and were compared with regards to their ability to proliferate and differentiate into chondrocytes.

Materials and Methods: Cells were isolated from the IPFP tissue and expanded in monolayer culture with and without rhFGF-2 supplementation (final concentration 10ng/ ml). Cell aggregates were placed in chondrogenic media for two weeks. Gene expression studies were carried out using quantitative real time PCR. Immunohistochemical labelling was performed with antibody localisation determined by an immunoperoxidase procedure. The pellets were also weighed and digested in papain for DNA and glycosoaminoglycan (GAG) analysis.

Results: Cells expanded in FGF-2 supplemented media were smaller and proliferated more rapidly. The FGF-2 supplemented cell aggregates also showed 100 times higher expression of collagen type II (COL2A1). Immunohistochemical studies showed that pellets made from FGF-2 treated cells stained more strongly for collagen II and more weakly for collagen I. Pellets made with FGF-2 treated cells were larger, continued with enhanced proliferation and contained more proteoglycan.

Conclusion: Our findings show enhanced proliferation and chondrogenic differentiation in IPFP derived stem cells expanded in FGF-2 supplemented media.