21 research outputs found

    Automated wounding machine to create standardized cartilage defects in an osteochondral model

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    Non-invasive raman spectroscopy and quantitative real-time PCR distinguish among undifferentiated human mesenchymal stem cells and redifferentiated nucleus pulposus cells and chondrocytes In Vitro

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    Background: The most common cause of lower back pain is the pathological degeneration of the nucleus pulposus (NP). Promising NP regeneration strategies involving human mesenchymal stem cells (hMSCs) would require specific markers to confirm successful differentiation into the NP lineage and to distinguish the articular cartilage (AC). Objective: We sought specific NP mRNA markers that are upregulated in native NP cells but not in dedifferentiated NP cells, undifferentiated hMSCs or chondrocytes. We also considered the suitability of non-invasive Raman spectroscopy to distinguish among these classes of cells. Method: We used quantitative real-time PCR and Raman spectroscopy to analyse undifferentiated hMSCs in monolayers and embedded in hydrogels, and compared the results with dedifferentiated and redifferentiated human NP and AC cells. Results: The redifferentiation of NP cells induced the expression of annexin A3 (ANXA3), collagen type II (COL2) and proteoglycan mRNAs , whereas the redifferentiation of AC cells only induced proteoglycan expression. Redifferentiated NP cells expressed higher levels of ANXA3, COL2, paired box 1 (PAX1) and OCT4 mRNA than redifferentiated AC cells. Redifferentiated NP cells and undifferentiated hMSC-TERT cells expressed similar amount of OCT4 mRNA, indicating that only ANXA3, COL2 and PAX1 are promising markers for redifferentiated NP cells. Raman spectra clearly differed among the three cell types and highlighted their differentiation status. Conclusion: We recommend ANXA3, COL2 and PAX1 as markers to determine the success of hMSC-based differentiation to regenerate NP cells. Raman spectroscopy can be used to determine cell type and differentiation status especially in the context of clinical trials

    Identifying chondrogenesis strategies for tissue engineering of articular cartilage

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    A key step in the tissue engineering of articular cartilage is the chondrogenic differentiation of mesenchymal stem cells (MSCs) into chondrocytes (native cartilage cells). Chondrogenesis is regulated by transforming growth factor-β (TGF-β), a short-lived cytokine whose effect is prolonged by storage in the extracellular matrix. Tissue engineering applications aim to maximise the yield of differentiated MSCs. Recent experiments involve seeding a hydrogel construct with a layer of MSCs lying below a layer of chondrocytes, stimulating the seeded cells in the construct from above with exogenous TGF-β and then culturing it in vitro. To investigate the efficacy of this strategy, we develop a mathematical model to describe the interactions between MSCs, chondrocytes and TGF-β. Using this model, we investigate the effect of varying the initial concentration of TGF-β, the initial densities of the MSCs and chondrocytes, and the relative depths of the two layers on the long-time composition of the tissue construct

    Osteogenesis and mineralization of mesenchymal stem cells in collagen type I-based recombinant peptide scaffolds

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    Recombinant peptides have the power to harness the inherent biocompatibility of natural macromolecules, while maintaining a defined chemistry for use in tissue engineering. Creating scaffolds from peptides requires stabilization via crosslinking, a process known to alter both mechanics and density of adhesion ligands. The chemistry and mechanics of linear scaffolds from a recombinant peptide based on human collagen type I (RCP) was investigated after crosslinking. Three treatments were compared: dehydrothermal treatment (DHT), hexamethylene diisocyanate (HMDIC), and genipin. With crosslinking, mechanical properties were not significantly altered, ranging from 1.9 to 2.7 kPa. However, the chemistry of the scaffolds was changed, affecting properties such as water uptake, and initial adhesion of human mesenchymal stem cells (hMSCs). Genipin crosslinking supported the lowest adhesion, especially during osteoblastic differentiation. While significantly altered, RCP scaffold chemistry did not affect osteoblastic differentiation of hMSCs. After four weeks in vitro, all scaffolds showed excellent cellular infiltration, with up-regulated osteogenic markers (RUNX2, Osteocalcin, Collagen type I) and mineralization, regardless of the crosslinker. Thus, it appears that, without significant changes to mechanical properties, crosslinking chemistry did not regulate hMSC differentiation on scaffolds from recombinant peptides, a growing class of materials with the ability to expand the horizons of regenerative medicine

    Inducing chondrogenesis in MSC/chondrocyte co-cultures using exogenous TGF-β: a mathematical model

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    The differentiation of mesenchymal stem cells (MSCs) into chondrocytes (native cartilage cells), or chondrogenesis, is a key step in the tissue engineering of articular cartilage, where the motility and high proliferation rate of MSCs used as seed cells are exploited. Chondrogenesis is regulated by transforming growth factor-beta (TGF-β), a short-lived cytokine whose effect is prolonged by storage in the extracellular matrix. Tissue engineering applications require the complete differentiation of an initial population of MSCs, and two common strategies used to achieve this in vitro are (1) co-culture the MSCs with chondrocytes, which constitutively produce TGF-β; or (2) add exogenous TGF-β. To investigate these strategies we develop an ordinary differential equation model of the interactions between TGF-β, MSCs and chondrocyte. Here the dynamics of TGF-β are much faster than those of the cell processes; this difference in time-scales is exploited to simplify subsequent model analysis. Using our model we demonstrate that under strategy 1 complete chondrogenesis will be induced if the initial proportion of chondrocytes exceeds a critical value. Similarly, under strategy 2 we find that there is a critical concentration of exogenous TGF-β above which all MSCs will ultimately differentiate. Finally, we use the model to demonstrate the potential advantages of adopting a hybrid strategy where exogenous TGF-β is added to a co-culture of MSCs and chondrocytes, as compared to using either strategy 1 or 2 in isolation

    A mathematical model for cell infiltration and proliferation in a chondral defect

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    We develop a mathematical model to describe the regeneration of a hydrogel inserted into an ex vivo osteochondral explant. Specifically we use partial differential equations to describe the evolution of two populations of cells that migrate from the tissue surrounding the defect, proliferate, and compete for space and resources within the hydrogel. The two cell populations are chondrocytes and cells that infiltrate from the subchondral bone. Model simulations are used to investigate how different seeding strategies and growth factor placement within the hydrogel affect the spatial distribution of both cell types. Since chondrocyte migration is extremely slow, we conclude that the hydrogel should be seeded with chondrocytes prior to culture in order to obtain zonal chondrocyte distributions typical of those associated with healthy cartilage

    Ex vivo cartilage defect model: Evaluation of cartilage hydrogels

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    Inducing chondrogenesis in MSC/chondrocyte co-cultures using exogenous TGF-beta: a mathematical model

    No full text
    The differentiation of mesenchymal stem cells (MSCs) into chondrocytes (native cartilage cells), or chondrogenesis, is a key step in the tissue engineering of articular cartilage, where the motility and high proliferation rate of MSCs used as seed cells are exploited. Chondrogenesis is regulated by transforming growth factor-beta (TGF-β), a short-lived cytokine whose effect is prolonged by storage in the extracellular matrix. Tissue engineering applications require the complete differentiation of an initial population of MSCs, and two common strategies used to achieve this in vitro are (1) co-culture the MSCs with chondrocytes, which constitutively produce TGF-β; or (2) add exogenous TGF-β. To investigate these strategies we develop an ordinary differential equation model of the interactions between TGF-β, MSCs and chondrocyte. Here the dynamics of TGF-β are much faster than those of the cell processes; this difference in time-scales is exploited to simplify subsequent model analysis. Using our model we demonstrate that under strategy 1 complete chondrogenesis will be induced if the initial proportion of chondrocytes exceeds a critical value. Similarly, under strategy 2 we find that there is a critical concentration of exogenous TGF-β above which all MSCs will ultimately differentiate. Finally, we use the model to demonstrate the potential advantages of adopting a hybrid strategy where exogenous TGF-β is added to a co-culture of MSCs and chondrocytes, as compared to using either strategy 1 or 2 in isolation.Michael J. Chen, Jonathan P. Whiteley, Colin P. Please, Andrea Schwab, Franziska Ehlicke, Sarah L. Waters, Helen M. Byrn
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