Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients

Southmead Hospital, and University of Bristol, Bristol, UK.
Arthritis & Rheumatology (Impact Factor: 7.76). 01/2007; 56(1):177-87. DOI: 10.1002/art.22285
Source: PubMed


To determine whether it is possible to engineer 3-dimensional hyaline cartilage using mesenchymal stem cells derived from the bone marrow (BMSCs) of patients with osteoarthritis (OA).
Expanded BMSCs derived from patients with hip OA were seeded onto polyglycolic acid scaffolds and differentiated using transforming growth factor beta3 in the presence or absence of parathyroid hormone-related protein (PTHrP) to regulate hypertrophy. Micromass pellet cultures were established using the same cells for comparison. At the end of culture, the constructs or pellets were processed for messenger RNA (mRNA) analysis by quantitative real-time reverse transcription-polymerase chain reaction. Matrix proteins were analyzed using specific assays.
Cartilage constructs engineered from BMSCs were at least 5 times the weight of equivalent pellet cultures. Histologic, mRNA, and biochemical analyses of the constructs showed extensive synthesis of proteoglycan and type II collagen but only low levels of type I collagen. The protein content was almost identical to that of cartilage engineered from bovine nasal chondrocytes. Analysis of type X collagen mRNA revealed a high level of mRNA in chondrogenic constructs compared with that in undifferentiated BMSCs, indicating an increased risk of hypertrophy in the tissue-engineered cells. However, the inclusion of PTHrP at a dose of 1 microM or 10 microM during the culture period resulted in significant suppression of type X collagen mRNA expression and a significant decrease in alkaline phosphatase activity, without any loss of the cartilage-specific matrix proteins.
Three-dimensional hyaline cartilage can be engineered using BMSCs from patients with OA. This method could thus be used for the repair of cartilage lesions.

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Available from: Trevor J Sims, Oct 14, 2014
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    • "Taken together, the results of our study indicate that PTHrP could be used clinically to inhibit undesirable hypertrophic chondrocyte differentiation. Accordingly , in vitro work has already demonstrated that PTHrP successfully inhibited hypertrophic differentiation of articular chondrocytes (Wang et al., 2011; Zhang et al., 2013) and cartilage constructs engineered from bone marrow-derived mesenchymal stem cells (BMSCs), without losing cartilagespecific matrix proteins (US patent 20080318859, Kafienah et al., 2007). Moreover, in vivo, intra-articular PTHrP injection together with collagen-silk scaffold implantation (4–6 weeks post-injury) inhibited terminal differentiation and enhanced chondrogenesis in induced osteochondral defects in rabbits (Zhang et al., 2013). "
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    ABSTRACT: The endocrine feedback loop between vitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10−8 M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Dec 2014 · Journal of Cellular Physiology
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    • "Many pathways are involved in the regulation of chondrocyte hypertrophy, including parathyroid hormone-related protein (PTHrP)/Indian hedgehog (IHH), wingless/int (WNT)/β-catenin, and TGF-β/sma and mad-related family (SMAD) pathways, converging on runt-related transcription factor 2 (RUNX2) and myocyte enhancer factor 2C (MEF2C) to drive expression of hypertrophic genes. Modulation of these pathways to suppress hypertrophy of MSC-derived cartilage-like tissues has been explored, either directly for example by treatment with PTHrP55–57, or indirectly through factors including hypoxia, co-culture with articular chondrocytes, epigenetic modulation, and biomaterial composition58. Such studies have demonstrated that modulation of chondrogenic hypertrophy is feasible, at least in vitro. "
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    ABSTRACT: Repair of lesions of the articular cartilage lining the joints remains a major clinical challenge. Surgical interventions include osteochondral autograft transfer and microfracture. They can provide some relief of symptoms to patients, but generally fail to durably repair the cartilage. Autologous chondrocyte implantation has thus far shown the most promise for the durable repair of cartilage, with long-term follow-up studies indicating improved structural and functional outcomes. However, disadvantages of this technique include the need for additional surgery, availability of sufficient chondrocytes for implantation, and maintenance of their phenotype during culture-expansion. Mesenchymal stem cells offer an attractive alternative cell-source for cartilage repair, due to their ease of isolation and amenability to ex vivo expansion while retaining stem cell properties. Preclinical and clinical studies have demonstrated the potential of mesenchymal stem cells to promote articular cartilage repair, but have also highlighted several key challenges. Most notably, the quality and durability of the repair tissue, its resistance to endochondral ossification, and its effective integration with the surrounding host tissue. In addition, challenges exist related to the heterogeneity of mesenchymal stem cell preparations and their quality-control, as well as optimising the delivery method. Finally, as our knowledge of the cellular and molecular mechanisms underlying articular cartilage repair increases, promising studies are emerging employing bioactive scaffolds or therapeutics that elicit an effective tissue repair response through activation and mobilisation of endogenous stem and progenitor cells.
    Full-text · Article · Jul 2013 · Osteoarthritis and Cartilage
    • "However, as well as inducing chondrogenic factors TGF-ß also causes type X collagen expression when given to cells in vitro (Kafienah et al., 2007). Type X collagen is a marker of hypertrophy, and when given to MSCs in vitro (but not chondrocytes) TGF-ß causes hypertrophy, this prevents the cells from being therapeutically useful (Kafienah et al., 2007). It may be that other soluble factors that aid and influence TGF-ßs action such as IGF or PTHrP this may allow for the artificial prevention of hypertrophy in MSCs induced into chondrogenesis by TGF-ß. "
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    ABSTRACT: Despite its remarkable ability to resist mechanical loading, articular cartilage is not capable of mounting a useful reparative reaction in response to damage caused by trauma or disease. As a result numerous surgical and medical approaches have been developed to aid the healing of articular cartilage. Despite the success of surgical techniques such as microfracture, recently attentions have been turned to cell based therapies such as autologous chondrocyte implantation (ACI). ACI has produced encouraging results, however better results may be achievable through an evolution of this surgical approach. Since the first generation of ACI techniques changes have been made in the technique e.g. the introduction of collagen membranes instead of periosteal flaps, and more recently the use of collagen scaffolds for cellular delivery. The procedure has also moved on from being performed as an open operation and can now be performed arthroscopically. Despite these advances the procedure still uses chondrocytes harvested from the joint being repaired. These cells are vulnerable to dedifferentiation during the required in vitro expansion, and as a result may not be capable of producing repair tissue once implanted back into the joint. Mesenchymal stem cells (MSCs) may provide a dedifferentiation resistant alternative to chondrocytes. MSCs would also allow for the use of one arthroscopic operation on the affected joint, as opposed to the two operations that are currently required for ACI.
    No preview · Article · Jan 2013 · Histology and histopathology
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