Abstract
Introduction and Objective
Current cartilage repair strategies lack adequate tissue integration capacity and often present mechanical failure at the graft-to-host tissue junction. The design of multilayered osteochondral tissue engineering (TE) constructs is an attractive approach to overcome these problems. However, calcium ion-release from resorbable bone-replacement materials was suggested to compromise chondrogenic differentiation of adjacent cartilage tissue and it is unclear whether articular chondrocytes (AC) or mesenchymal stroma cells (MSC) are more sensitive to such conditions. Aim of the study was to compare how elevated calcium levels affect cartilage matrix production during re-differentiation of AC versus chondrogenic differentiation of MSC. The results of this study will help to identify the ideal cell source for growth of neocartilage adjacent to a calcified bone replacement material for design of multilayered osteochondral TE approaches.
Materials and Methods
Expanded human AC and MSC (6–12 donors per group) were seeded in collagen type I/III scaffolds and cultured under standard chondrogenic conditions at control (1.8mM) or elevated (8.0mM) CaCl2 for 35 days. Proteoglycan and collagen production were assessed via radiolabel-incorporation, ELISA, qPCR and Western blotting. Differences between groups or cell types were calculated using the non-parametric Wilcoxon or Mann-Whitney U test, respectively, with p < 0.05 considered significant.
Results
Elevated calcium significantly reduced GAG synthesis (63% of control, p=0.04) and chondrogenic marker expression of AC, lowering the GAG/DNA content (47% of control, p=0.004) and collagen type II deposition (24% of control, p=0.05) of neocartilage compared to control conditions. Opposite, at elevated calcium levels MSC-derived chondrocytes significantly increased GAG synthesis (130% of control, p=0.02) and collagen type II content (160% of control, p=0.03) of cartilage compared to control tissue. Chondrogenic and hypertrophic marker expression was insensitive to calcium levels in MSC-derived chondrocytes. As a result, maturation under elevated calcium allowed for a significantly higher GAG/DNA content in MSC-derived samples compared to AC constructs, although under control conditions both groups developed similarly.
Conclusions
AC and MSC showed an opposite reaction to elevation of calcium levels regarding cartilage matrix production and we propose MSC as a preferred cell source to grow chondrocytes in vicinity to calcified bone replacement materials. Since MSC remained prone to hypertrophy under elevated calcium, trizonal cartilage TE constructs, where an AC-layer is separated from the bone replacement phase by an intermediate layer of MSC appear as an ideal design for multilayered osteochondral TE with respect to calcium sensitivity of cells and protection of the upper cartilage layer from hypertrophy.
