Abstract

Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-β (TGF-β) and BMP signalling during chondrogenesis. Both TGF-β and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism.

Highlights

  • The healing capacity of cartilage is very limited and various tissue engineering approaches have been investigated to create pheno- and genotypically stable articular cartilage

  • The different steps of endochondral bone development are regulated by a number of signalling molecules including bone morphogenetic proteins (BMPs), transforming growth factor-β (TGF-β), fibroblast growth factors (FGFs), parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), and Wnts (Wingless-related integration sites) [15, 16]

  • Induction of hypertrophy was achieved by addition of T3 and withdrawal of dexamethasone after chondrogenic predifferentiation

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Summary

Introduction

The healing capacity of cartilage is very limited and various tissue engineering approaches have been investigated to create pheno- and genotypically stable articular cartilage. Chondrogenic differentiating MSCs express markers like collagen type X, alkaline phosphatase (ALP), and MMP-13 [6,7,8,9,10,11], indicating hypertrophic conversion. Additional characteristics of terminal differentiation like vascular invasion and matrix calcification have been observed after in vivo transplantation of human chondrogenic MSC pellet cultures into mice [12, 13]. This hypertrophic conversion of chondrogenic differentiating MSCs raises concerns for a tissue engineering application of MSCs in articular cartilage repair. The different steps of endochondral bone development are regulated by a number of signalling molecules including bone morphogenetic proteins (BMPs), transforming growth factor-β (TGF-β), fibroblast growth factors (FGFs), parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), and Wnts (Wingless-related integration sites) [15, 16]

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