Summary

Early weight bearing could then be beneficial in promoting the formation of more hyaline-like cartilage repair tissue, as well as the integration between the newly formed tissue and the surrounding hyaline cartilage

Abstract

Introduction

Microfracture is a common procedure to repair local articular defects and consists of the targeted disruption of the subchondral bone to form a clot rich in intramedullary mesenchymal stem cells (MSCs). However, because of the lesser mechanical properties of this fibrocartilaginous repair tissue, in the long term additional treatment is required. In general, early passive motion and limited weight bearing are used in rehabilitation after microfracture, however, it is unclear how these mechanical regimens affect neocartilage tissue formation. There is a need to develop approaches that can improve chondrogenesis after microfracture, to obtain more hyaline-like cartilage repair, as well as to improve mechanical properties and integration with the surrounding hyaline tissue. The purpose of this study was to assess the influence of different mechanical activation regimens simulating partial load bearing or passive motion exercises, on an in vitro mimic of microfracture repair based on fibrin gel scaffolds containing MSCs.

METHODS.
Cylindrical cores in bovine hyaline cartilage plugs were filled with fibrin and bone marrow MSCs from the trabecular bone of juvenile bovine. Controls included: cell-free fibrin filler and a return of the cartilage core (sham). These microfracture models were subject to 3 different loading regimens: free swelling, dynamic compressive loading, rotating shear loading stimulating. To simulate weight bearing (primarily compressive loading), we used the Mechano-Active Tissue Engineering (MATE) system, which allows the application of cyclic compression to tissues with controlled force and displacement. Dynamic compression was applied at 1.5Hz for 2 minutes followed by 2 minutes pause between each sequence with a 9N peak amplitude load force, for a total of 1h a day. To simulate passive motion (primarily shear loading), we used a rotatory cell culture system (RCCS). All samples were cultured in chondrogenic medium with 10ng/ml TGF- ß3. On day 7, 14 and 21, the integration strength between the outer chondral plug and the central core was measured with an electro-force mechanical tester. Then the central core samples were analyzed by real-time RT-PCR, by glycosaminoglycan and PicoGreen assays, and by histology to assess differentiation and integration. Statistical comparisons were done by ANOVA followed by a Tukey post-hoc test with significance of p < 0.05.

Results

In terms of integration strength, for the return plug (sham) group and the fibrin gel scaffold containing BM-MSCs, the MATE (compressive loading) group at day 21 showed significantly higher integration strength than the control, while there were no significant differences in the free swelling and RCCS groups. The GAG/DNA for the MATE group was significantly higher than the FS and RCCS at day 21. Chondrogenic gene expression of the fibrin with BM-MSCs of the MATE condition at day 21, (genes: Sox9, ACAN, ratio of the expressions of Collagen type II / type I) was significantly higher than the FS and the RCCS groups Furthermore, the COL2:COL1 ratio which is an indicator of a less fibrous and more hyaline phenotype for the repair tissue, was most upregulated in MATE group compared to free swelling group at day 21. Furthermore, the catabolic makers MMP-3 and ADAMTS5 were significantly upregulated in the RCCS group at day21.

Discussion

This study supports the concept that chondrogenesis of BM-MSC could be modulated by different mechanical loading. Dynamic compressive loading on an in vitro mimic of microfracture has not only positively affected the generation of more hyaline-like cartilage, but also yielded higher integration strength. Early weight bearing could then be beneficial in promoting the formation of more hyaline-like cartilage repair tissue, as well as the integration between the newly formed tissue and the surrounding hyaline cartilage.