gms | German Medical Science

Objectives: Mesenchymal stem cell (MSC)-based regenerative therapies hold great promise for the repair of articular cartilage defects. However, it is unclear how to generate MSC-derived neocartilage that can adequately respond to mechanical loading, which appears to be critical for their therapeutic success in regenerating articular cartilage lesions in high load-bearing areas. In fact, our previous study demonstrated that a mechanical loading episode that stimulated proteoglycan synthesis rate in articular chondrocytes had opposite effects in MSC-derived chondrocytes. The signals that are responsible for this unfavorable mechanoresponse are not yet fully understood. Parathyroid hormone-related protein (PTHrP) is an interesting candidate cytokine because it is induced by loading in multiple other cell types and regulates extracellular matrix (ECM) synthesis in chondrocytes. The aim of this study was therefore to investigate whether PTHrP could be responsible for the unfavorable loading response in MSC-derived chondrocytes. To test this, we evaluated mechanoinduction of PTHrP and its ability to mimic the effects of load on typical mechanoresponse genes and ECM synthesis rates in the MSC-derived neocartilage. This knowledge may help to identify a factor, that could be targeted to improve the currently limited mechanocompetence of MSC-based engineered cartilage.

Methods: Human bone marrow-derived MSC were seeded in collagen scaffolds, attached toβ-tricalcium phosphate and after 21 days of chondrogenic culture subjected to dynamic unconfined compressive loading (3 h, 25%, 1 Hz) or treated with PTHrP(1-34), cAMP analogue (dbcAMP) or adenylate cyclase inhibitor MDL-12,330A. Cartilage formation was assessed via histology, proteoglycan and collagen synthesis via radiolabeling and cAMP production by ELISA. Mechanoresponse gene expression and hypertrophy markers were assessed by qPCR.

Results and conclusion: PTHrP mRNA and its second messenger cAMP were significantly increased after the loading episode in MSC-derived neocartilage constructs. Interestingly, treatment with exogenous PTHrP peptide increased a subset of mechanoresponse mRNAs (FOS, FOSB, BMP6) and, similar to loading, decreased de novo synthesis rates of proteoglycans and collagen. Importantly, inhibition of adenylate cyclase rescued the load-induced drop in GAG synthesis, indicating involvement of the PTHrP-cAMP signaling axis in the mediation of the unfavourable mechanoresponse. Load and PTHrP treatment also shared the ability to reduce expression of the hypertrophy markers MEF2C and PTH1R when referred to COL2A1. These results indicate, for the first time, PTHrP-cAMP signalling as a mechanotransducer in human MSC-derived neocartilage. PTHrP inhibition should be considered to improve biomechanical properties of MSC-based engineered cartilage.