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Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production.

ABSTRACT: Regenerative medicine and tissue engineering studies are actively developing novel means to repair adult articular cartilage defects using biological approaches. One such approach is the harnessing of adult human therapeutic cells such as those referred to as mesenchymal stem cells. Upon exposure to chondrogenic signals, these cells differentiate and initiate the production of a complex and voluminous cartilaginous matrix that is crucial to both the structure and function of cartilage. Furthermore, this complexity requires the time-sensitive activation of a large number of genes to produce the components of this matrix. The current study analyzed the kinetics of matrix production in an aggregate culture model where adult human mesenchymal stem cells were induced to differentiate as chondrocytes. The results indicate the existence of a biphasic mode of differentiation and maturation during which matrix genes and molecules are differentially activated and secreted. These results have important implications for developing novel approaches for the creation of tissue engineered articular cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1757-1766, 2018.

SUBMITTER: Sorrell JM

PROVIDER: S-EPMC5976510 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production.

Sorrell J Michael JM Somoza Rodrigo A RA Caplan Arnold I AI

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20171222 6

Regenerative medicine and tissue engineering studies are actively developing novel means to repair adult articular cartilage defects using biological approaches. One such approach is the harnessing of adult human therapeutic cells such as those referred to as mesenchymal stem cells. Upon exposure to chondrogenic signals, these cells differentiate and initiate the production of a complex and voluminous cartilaginous matrix that is crucial to both the structure and function of cartilage. Furthermo ...[more]

PMID: 29194731

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Project description:INTRODUCTION: In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a "tri-culture" model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants. METHODS: Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures). RESULTS: In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young's and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1?), IL-6, and IL-8. Stimulation of monocultures with IL-1? and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only. CONCLUSIONS: Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1? and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs' chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies.

Project description:Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo.Statement of significanceCurrent cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.

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Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production.

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Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production.

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