Differential effects of tyrosine-rich amelogenin peptide on chondrogenic and osteogenic differentiation of adult chondrocytes.

H D Amin,C R Ethier

doi:10.1007/s00441-015-2292-7

Abstract

Current approaches to treat osteoarthritis (OA) are insufficient. Autologous chondrocyte implantation (ACI) has been used for the past decade to treat patients with OA or focal cartilage defects. However, a number of complications have been reported post-ACI, including athrofibrosis and symptomatic hypertrophy. Thus, a long-term ACI strategy should ideally incorporate methods to ‘prime’ autologous chondrocytes to form a cartilage-specific matrix and suppress hypertrophic mineralization. The objective of this study is to examine the effects of tyrosine-rich amelogenin peptide (TRAP; an isoform of the developmental protein amelogenin) on human articular cartilage cell (HAC) chondrogenic differentiation and hypertrophic mineralization in vitro. Effects of chemically synthesized TRAP on HAC chondrogenic differentiation were determined by assessing: (1) sGAG production; (2) Alcian blue staining for proteoglycans; (3) collagen type II immunostaining; and (4) expression of the chondrogenic genes SOX9, ACAN and COL2A1. Hypertrophic mineralization was assayed by: (1) ALP expression; (2) Alizarin red staining for Ca+2-rich bone nodules; (3) OC immunostaining; and (4) expression of the osteogenic/hypertrophic genes Ihh and BSP. Chemically synthesized TRAP was found to suppress terminal osteogenic differentiation of HACs cultured in hypertrophic mineralization-like conditions, an effect mediated via down-regulation of the Ihh gene. Moreover, TRAP was found to augment chondrogenic differentiation of HACs via induction of SOX9 gene expression when cells were cultured in pro-chondrogenic media. The results obtained from this proof-of-concept study motivate further studies on the use of TRAP as part of a preconditioning regimen in autologous chondrocyte implantation procedures for OA patients and patients suffering from focal cartilage defects.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-015-2292-7) contains supplementary material, which is available to authorized users.

Highlights

Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage (Felson 2006)
In vitro functional outcomes: Effects of tyrosine-rich amelogenin peptide (TRAP) on differentiation of articular cartilage cells In TRAP-treated cells cultured in chondrogenic media (CM), a large dose-dependent increase in sulfated glycosaminoglycan (sGAG) levels was observed (Table 1); 10 and 50 μg/ml TRAP increased sGAG levels by 353 and 344 %, respectively, compared with control conditions of human articular cartilage cell (HAC) cultured in CM alone (p
For determining the effects of TRAP on hypertrophic mineralization, HACs were first preconditioned for 2 weeks in CM and cultured for 3 weeks in hypertrophic media (HTM) or HTM+TRAP, as a simplified in vitro model of conditions changing from chondrogenic to osteogenic in a process of endochondral ossification in osteoarthritic joints

Summary

Introduction

Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage (Felson 2006). It was recently identified as the second most common cause of disability and the major cause for disability allowance in the United States (Felson 2006). Physiotherapy, exercise and analgesics are currently used for early stage management of OA, whereas in later stages, surgical interventions are typically required (Bartha, et al 2006; Felson 2006). Existing therapies do not reverse the development of OA and achieve cartilage regeneration (Bartha et al 2006; Felson 2006). Current efforts to develop regenerative medicine-based therapeutic interventions for OA or focal cartilage defect

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