Summary

Nasal Chondrocytes are safe and feasible for tissue engineering approaches in articular cartilage repair.

Mesenchymal stem cells (MSC) are suitable candidates for the cell-based cartilage reconstruction and have been isolated from different sources such as bone marrow (BMSC), adipose tissue (ATSC) and synovium (SMSC). The aim of this study was to analyse the tendency of BMSC, ATSC and SMSC to undergo hypertrophy during chondrogenic induction in vitro and to evaluate their in vivo development after ectopic transplantation into SCID mice in order to determine which cell source is most suitable for cartilage regeneration.

Human BMSC, ATSC and SMSC were cultured under chondrogenic conditions for five weeks. Differentiation was evaluated based on histology, gene expression, and analysis of alkaline phosphatase activity (ALP). Pellets were transplanted subcutaneously into SCID mice after chondrogenic induction for 5 weeks and analysed 4 weeks later by histology. Similar COL2A1:COL10A1 mRNA ratios were found in BMSC, ATSC and SMSC. BMSC displayed the highest ALP activities, SMSC had lower and heterogenic ALP activities in vitro which correlated with calcification of spheroids in vivo. Most SMSC transplants specifically lost their collagen type II in vivo or were fully degraded. BMSC and ATSC pellets always underwent vascular invasion and calcification in vivo. Single BMSC samples had the capacity to develop into woven bone or fully developed ossicles with hematopoietic tissue surrounded by a bone capsule.

Neither BMSC nor ATSC or SMSC were able to form stable ectopic cartilage. While BMSC and ATSC underwent developmental processes related to endochondral ossification instead of stable ectopic cartilage formation, SMSC tended to undergo fibrous dedifferentiation or degradation. Besides appropriate induction of chondrogenesis, locking of cells in the desired differentiation state is, thus, a further challenge for adult stem cell-based cartilage repair.

Monolayer expansion of human articular chondrocytes (HAC) is known to result in progressive dedifferentiation and loss of stable cartilage formation capacity in vivo. For optimal outcome of chondrocyte based repair strategies, HAC capable of ectopic cartilage formation may be required. Thus, the aim of this study was to establish appropriate quality control measures capable to predict the ectopic cartilage formation capacity of HAC from culture supernatants. This strategy would avoid the waste of cells for quality control purposes, in order to improve cell therapy and tissue-engineering approaches for the repair of joint surface lesions.

Standardized medium supernatants (n=5) of freshly isolated HAC and chondrocytes expanded for 2 (PD2) or 6 population doublings (PD6) were screened for 15 distinct interleukins, 8 MMPs and 11 miscellaneous soluble factors by a multiplexed immunoassay. Cartilage differentiation markers like COMP and YKL-40 were determined by ELISA. Corresponding HAC were subcutaneously transplanted into SCID-mice and their capacity to form stable ectopic cartilage was examined histologically 4 weeks later.

While freshly isolated chondrocytes generated stable ectopic cartilage positive for collagen type II, none of the PD6 transplants formed cartilaginous matrix. Loss of ectopic stable cartilage formation capacity between PD0 and PD6 correlated with a drop of MMP3 secretion to < 10% of initial levels, while changes for other investigated molecules were not predictive. Chondrocytes from donors with low MMP3 levels (< 10%) at PD2 failed to regenerate ectopic cartilage at PD2, indicating that MMP3 levels of cultured chondrocytes, independent of the number of cell doublings and the time in culture, predicted ectopic cartilage formation.

In conclusion, loss of stable ectopic cartilage formation capacity in the course of HAC dedifferentiation can be predicted by determination of relative MMP3 levels demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.