Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface

Objectives

To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis.

Results

hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium.

Conclusions

Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

     

Effects of Fibrinolytic Inhibitors on Chondrogenesis of Bone-marrow Derived Mesenchymal Stem Cells in Fibrin Gels

The objective of this study was to examine the effect of two fibrinolytic inhibitors, aprotinin and aminohexanoic acid, on chondrogenesis of rabbit bone marrow mesenchymal stem cells (BM-MSCs). Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell–fibrin constructs were made by mixing a cell–fibrinogen (10⁷ cells/ml; 40 mg/ml fibrinogen) solution with a thrombin (5 IU/ml) solution and then divided into four groups: aprotinin control, aprotinin + transforming growth factor β (TGF-β), aminohexanoic acid control, and aminohexanoic acid + TGF-β. Each of these groups was further treated with three different concentrations of inhibitors and the TGF-β groups were treated with 10 ng/ml of TGF-β1. The chondrogenic gene expressions, DNA content, and glycosaminoglycan content of samples were analyzed after 14 days of culture. The aprotinin groups exhibited significantly higher levels of aggrecan gene expression and glycosaminoglycan content than the aminohexanoic acid groups. However, inhibitor neither influenced gene expression of type II collagen nor proliferation (i.e., DNA content) of BM-MSCs. These findings suggest that fibrinolytic inhibitors used to control degradation of fibrin clot may influence TGF-β-induced chondrogenesis of BM-MSCs.     

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P < 0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P < 0.05) and that of type I collagen significantly decreased (P < 0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.     

Radiation-Induced Alterations of Osteogenic and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O₂ and 3% O₂ conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O₂ tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O₂ tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.     

Dose-Response of Superparamagnetic Iron Oxide Labeling on Mesenchymal Stem Cells Chondrogenic Differentiation: A Multi-Scale In Vitro Study

Aim

The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues.

Methods

MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity.

Results

No influence of various SPIO concentrations was noted on human bone marow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations.

Conclusions

This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5–25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.     

Evaluation of Metabolomic Changes as a Biomarker of Chondrogenic Differentiation in 3D-cultured Human Mesenchymal Stem Cells Using Proton (1H) Nuclear Magnetic Resonance Spectroscopy

Purpose

The purpose of this study was to evaluate the metabolomic changes in 3D-cultured human mesenchymal stem cells (hMSCs) in alginate beads, so as to identify biomarkers during chondrogenesis using ¹H nuclear magnetic resonance (NMR) spectroscopy.

Materials and Methods

hMSCs (2×10⁶ cells/mL) were seeded into alginate beads, and chondrogenesis was allowed to progress for 15 days. NMR spectra of the chondrogenic hMSCs were obtained at 4, 7, 11, and 15 days using a 14.1-T (600-MHz) NMR with the water suppression sequence, zgpr. Real-Time polymerase chain reaction (PCR) was performed to confirm that that the hMSCs differentiated into chondrocytes and to analyze the metabolomic changes indicated by the NMR spectra.

Results

During chondrogenesis, changes were detected in several metabolomes as hMSC chondrogenesis biomarkers, e.g., fatty acids, alanine, glutamate, and phosphocholine. The metabolomic changes were compared with the Real-Time PCR results, and significant differences were determined using statistical analysis. We found that changes in metabolomes were closely related to biological reactions that occurred during the chondrogenesis of hMSCs.

Conclusions

In this study, we confirm that metabolomic changes detected by ¹H-NMR spectroscopy during chondrogenic differentiation of 3D-cultured hMSCs in alginate beads can be considered as biomarkers of stem cell differentiation.     

Isolation, characterization, and chondrogenic potential of human bone marrow-derived multipotential stromal cells

Multipotential bone marrow stromal cells have the ability to differentiate along multiple connective tissue lineages including cartilage. In this study, we developed an efficient and reproducible procedure for the isolation of stromal cells from bone marrow aspirates of normal human donors based on the expression of endoglin, a type III receptor of the transforming growth factor-beta (TGF-beta) receptor family. We demonstrate that these cells have the ability of multiple lineage differentiation. Stromal cells represented 2-3% of the total mononuclear cells of the marrow. The cells displayed a fibroblastic colony formation in monolayer culture and maintained similar morphology with passage. Expression of cell surface molecules by flow cytometry displayed a stable phenotype with culture expansion. When cocultured with hematopoietic CD34(+) progenitor cells, stromal cells were able to maintain their ability to support hematopoiesis in vitro. Culture expanded stromal cells were placed in a 3-dimensional matrix of alginate beads and cultured in serum-free media in the presence of TGFbeta-3 for chondrogenic lineage progression. Increased expression of type II collagen messenger RNA was observed in the TGFbeta3 treated cultures. Immunohistochemistry performed on sections of alginate beads detected the presence of type II collagen protein. This isolation procedure for stromal cells and the establishment of the alginate culture system for chondrogenic progression will contribute to the understanding of chondrogenesis and cartilage repair.     

The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers

Low back pain is a major public health issue in the Western world, one main cause is believed to be intervertebral disc (IVD) degeneration. To halt/diminish IVD degeneration, cell therapy using different biomaterials e.g. hydrogels as cell carriers has been suggested. In this study, two different hydrogels were examined (in vitro) as potential cell carriers for human mesenchymal stem cells (hMSCs) intended for IVD transplantation. The aim was to investigate cell-survival and chondrogenic differentiation of hMSCs when cultured in hydrogels Puramatrix^® or Hydromatrix^® and potential effects of stimulation with growth hormone (GH). hMSCs/hydrogel cultures were investigated for cell-viability, attachment, gene expression of chondrogenic markers SOX9, COL2A1, ACAN and accumulation of extracellular matrix (ECM). In both hydrogel types, hMSCs were viable for 28 days, expressed integrin β1 which indicates adhesion of hMSCs. Differentiation was observed into chondrocyte-like cells, in a higher extent in hMSCs/Hydromatrix^® cultures when compared to hMSCs/Puramatrix^® hydrogel cultures. Gene expression analyses of chondrogenic markers verified results. hMSCs/hydrogel cultures stimulated with GH displayed no significant effects on chondrogenesis.In conclusion, both hydrogels, especially Hydromatrix^® was demonstrated as a promising cell carrier in vitro for hMSCs, when directed into chondrogenesis. This knowledge could be useful in biological approaches for regeneration of degenerated human IVDs.     

Chondroitin sulfate and dynamic loading alter chondrogenesis of human MSCs in PEG hydrogels

While biochemical and biomechanical cues are known to play important roles in directing stem cell differentiation, there remains little known regarding how these inextricably linked biological cues impact the differentiation fate of human marrow stromal cells (hMSCs). This study investigates the chondrogenic differentiation potential of hMSCs when encapsulated in a three dimensional (3D) hydrogel and exposed to a biochemical cue, chondroitin sulfate (ChS), a biomechanical cue, dynamic loading, and their combination. hMSCs were encapsulated in bioinert poly(ethylene glycol) (PEG) hydrogels only, PEG hydrogels modified with covalently incorporated methacrylated ChS and cultured under free swelling conditions or subjected to delayed intermittent dynamic loading for 2 weeks. The 3D hydrogel environment led to the expression of chondrogenic genes (SOX9) and proteins (aggrecan and collagen II), but also upregulated hypertrophic genes (RUNX2 and Col X mRNA) and proteins (collagen X), while the application of loading generally led to a downregulation in chondrogenic proteins (collagen II). The presence of ChS led to elevated levels of aggrecan, but also collagen I, protein expression and when combined with dynamic loading downregulated, but did not suppress, hypertrophic genes (Col X and RUNX2) and collagen I protein expression. Taken together, this study demonstrates that while the 3D environment induces early terminal differentiation during chondrogenesis of hMSCs, the incorporation of ChS into PEG hydrogels may slow the terminal differentiation process down the hypertrophic lineage particularly when dynamic loading is applied. Biotechnol. Bioeng. 2012; 109: 2671–2682. © 2012 Wiley Periodicals, Inc.     

Prostanoid pattern and iNOS expression during chondrogenic differentiation of human mesenchymal stem cells

Availability of human chondrocytes is a major limiting factor regarding drug discovery projects and tissue replacement therapies. As an alternative human mesenchymal stem cells (hMSCs) from bone marrow are taken into consideration as they can differentiate along the chondrogenic lineage. However, it remains to be shown whether they could form a valid model for primary chondrocytes with regards to inflammatory mediator production, like nitric oxide (NO) and prostanoids. We therefore investigated the production of NO and prostanoids in hMSCs over the course of chondrogenic differentiation and in response to IL-1beta using primary OA chondrocytes as reference. Chondrogenic differentiation was monitored over 28 days using collagen I, collagen II, and collagen X expression levels. Expression levels of inducible nitric oxide synthase (iNOS), levels of NO, and prostanoids were assessed using PCR, Griess assay, and GC/MS/MS, respectively. The hMSCs collagen expression profile during course of differentiation was consistent with a chondrocytic phenotype. Contrary to undifferentiated cells, differentiated hMSCs expressed iNOS and produced NO following stimulation with IL-1beta. Moreover, this induction of iNOS expression was corticosteroid insensitive. The spectrum of prostanoid production in differentiated hMSCs showed similarities to that of OA chondrocytes, with PGE2 as predominant product. We provide the first detailed characterization of NO and prostanoid production in hMSCs in the course of chondrogenic differentiation. Our results suggest that differentiated hMSCs form a valid model for chondrocytes concerning inflammatory mediator production. Furthermore, we propose that IL-1beta stimulation, leading to corticosteroid-insensitive NO synthesis, can be used as a sensitive marker of chondrogenesis.     

Sustained Wnt protein expression in chondral constructs from mesenchymal stem cells

Wnt genes encode a number of secreted glycoproteins which are closely associated with the cell surface and the extracellular matrix. Recently, members of Wnt family have been implicated in regulating chondrocyte differentiation, but their roles in the chondrogenic process are not fully understood. To contribute to an understanding of the roles of Wnts during chondrogenesis, we have analysed the spatiotemporal expression patterns of Wnt using in vitro models for chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSCs). In chondrogenic aggregate culture system, RT-PCR analysis revealed expression of Wnt5a and Wnt4 during late chondrogenesis (days 10 and 15). Immunohistochemical analysis showed widespread distribution of Wnt5a and Wnt4 throughout the aggregates at this late phase of culture (days 14 and 21). In addition, in this aggregate culture system, immunohistochemical staining of Wnt4 and Wnt5a showed similar spatiotemporal expression patterns to that of type II collagen or type X collagen. To confirm the results obtained by immunostaining, the specificity of the anti-Wnt4 or anti-Wnt5a antibody was assessed by Western blot analysis. Of Wnt4 and Wnt5a, only Wnt5a was immunodetectable by Western blot analysis. Western blot analysis showed that Wnt5a was expressed as two different molecular weight forms of 40 and 44 kDa. Treatment with PNGase F, which removes N-linked oligosaccharides, revealed that the mass difference between these two forms could be accounted for by the N-glycosylation status of the protein. When hMSCs were seeded on a porous gelatin sponge, immunolocalization studies showed that type II collagen and type X collagen were detected particularly at the periphery at day 7 of culture. In contrast, Wnt4 and Wnt5a showed even distribution throughout the hMSC/gelatin sponge constructs. Their different spatial expression patterns suggest that Wnt4 and Wnt5a proteins are not functionally linked to type II collagen and type X collagen synthesis in in vitro chondrogenic models of hMSCs.     

Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed a difference in the temporal expression of chondrogenic markers which were up regulated in chondrogenic medium compared to levels in basal medium. Of the three cell types studied, adult chondrocytes offer a more promising cell source for cartilage tissue engineering. This comparative study revealed differences between the microenvironment of all three cell types and provides useful information to inform cell-based therapies for cartilage regeneration.     

Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian‐blue staining, qRT‐PCR, and quantification of alkaline phosphatase (ALP) activity. Pre‐differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)‐β was always required for chondrogenic differentiation and deposition of a collagen‐type‐II‐positive extracellular matrix, while bone morphogenetic protein (BMP)‐2, ‐4, ‐6, ‐7, aFGF, and IGF‐I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF‐β, however, without preventing collagen type X expression. bFGF or parathyroid hormone‐like peptide (PTHrP) inhibited the TGF‐β‐responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up‐regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor‐independent pathway. While TGF‐β was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II⁺/collagen type X^? cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes. J. Cell. Physiol. 223: 84–93, 2010. © 2009 Wiley‐Liss, Inc.     

The role of retinoic acid receptor inhibitor LE135 on the osteochondral differentiation of human bone marrow mesenchymal stem cells

The present study aimed to investigate the role of a retinoic acid receptor-β (RARβ) inhibitor LE135 on TGF-β induced chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs). Pellet culture with exogenous transforming growth factor-β (TGF-β), and a mechanically loaded scaffold system were used to provide two culture models. All samples were cultured for 8 days and changes in early gene expression were determined. Glycosaminoglycan and mRNA expression data showed that LE135 itself did not induce any chondrogenic response in either pellet culture or scaffold culture of hMSCs. LE135 actually inhibited the chondrogenic response caused by exogenous TGF-β, or endogenous TGF-β induced by mechanical load, while the expression of genes normally associated with osteogenesis was not affected. This suggests that the inhibitor LE135 affects the osteochondral differentiation pathway at a different stage, inhibiting chondrogenic gene expression while having no effect on genes normally associated with the osteogenic phenotype. Alternatively, it might be that different cells were proceeding down different lineages. Some cells were undergoing chondrogenesis and this was affected by LE135, while other cells underwent osteogenic differentiation and were not affected by LE135.     

Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs’ physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.     

In VitroDifferentiation Potential of the Periosteal Cells from a Membrane Bone, the Quadratojugal of the Embryonic Chick

The quadratojugal (QJ) is a neural crest-derived membrane bone in the maxillary region of the avian head.In vivoits periosteum undergoes both osteogenesis to form membrane bone and chondrogenesis to form secondary cartilage. This bipotential property, which also exists in some other membrane bones, is poorly understood. The present study used cell culture to investigate the differentiation potential of QJ periosteal cells. Three cell populations were enzymatically released from QJ periostea and plated at different densities. Cell density greatly affected phenotypic expression and differentiation pathways. We found two culture conditions that favored osteogenesis and chondrogenesis, respectively. In micromass culture, the periosteal cells produced a layer of osteogenic cells that expressed alkaline phosphatase (APase) and secreted bony extracellular matrix (ECM). In contrast, low-density monolayer culture elicited chondrogenesis. Cells with pericellular refractile ECM and round shape appeared at 7 to 8 days and formed colonies later. The chondrogenic phenotype of these cells was confirmed by immunolocalization of type II collagen and Alcian blue staining of ECM. This result demonstrated that a fully expressed chondrogenic phenotype can be achieved from membrane bone periosteal cells in primary monolayer culture. Chondrogenesis requires a cell density lower than confluence and cannot be initiated in confluent cultures. Among the three cell populations, those cells from the outer layer have the highest growth rate and require the lowest initial plating density (below 5 × 10³cells/ml) to achieve chondrogenesis. Cells from the inner layer have the slowest growth rate and chondrify at the highest initial density (below 5 × 10⁴cells/ml). Chondrocytes from all populations express distinct phenotypic markers—APase and type I collagen—from initial chondrogenesis, but are not hypertrophic morphologically. Furthermore, the fact that chondrocytes arise within the same colony as APase-positive polygonal cells suggests that chondrocytes may differentiate from precursors related to the osteogenic cell lineage. This cell culture approach mimics secondary cartilage and membrane bone formationin vivo.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.