Biomechanical assessment of osteoarthritic articular cartilage and jaw periosteal cells-based bone constructs

Abstract

Mechanical features influence nearly every aspect of cell biology and function. However, the underlying mechanisms of the role and how mechanical properties and bio-chemical signals are interconnected is not clearly understood. The advent of atomic force microscopy (AFM) provides a powerful tool for quantifying mechanical properties of living cell, typically the elastic modulus (EM). In the present study, a customized AFM approach was applied on load connective tissues to address and investigate two aspects of the musculoskeletal system. The first study investigated the EM changes that occur in the pericellular matrix (PCM) as a function of the cellular pattern reorganization throughout the course of osteoarthritis (OA). The second study investigated the effect of human plate-let lysate (hPL) supplementation on EM and biochemical composition of jaw periosteum derived progenitor cells (JPCs). During osteoarthritis (OA) triggered cartilage degeneration, the chondrocytes in the tissue spatially rearrange from single to double strings, and then to small and finally big clusters. The spatial patterns act as an image-based biomarker for tissue degeneration during OA. In a physiological state, chondrocytes are surrounded by a specialized form of extracellular matrix (ECM) termed the PCM. The PCM, which also dictates the biome-chanical properties of the tissue, is also being progressively degraded throughout the course of OA. The hypothesis of this study was that OA related changes in the cellular organizational patterns (strings, double strings, and clusters) are associated with structural changes of the PCM and with a loss of elastic properties. The biomechanical properties were measured by AFM on specific pattern selected tissue. Biochemical changes of the main components of the PCM (collagen type VI and perlecan) were investigated by pro-tein analysis techniques. The results indicated that there is a significant and stepwise EM decrease alongside each of the cellular pattern rearrangements. At the same time, the ini-tially compact PCM was degraded progressively, losing its structural integrity. The earli-est point with a significant reduction in protein content was at the transition from single strings to small clusters for collagen type VI, and from double strings to small clusters for perlecan. Interestingly, the first significant EM decrease was observed at the transition from single strings to double strings. It must be noted that at this stage, articular cartilage appears macroscopically intact. Both biomechanical properties (EM) as well as biochemi-cal composition (protein content) were the lowest in big clusters. This study is the first to describe the EM as well as structural changes of the PCM in relation to the OA related chondrocyte rearrangement, confirming the role of these patterns as an image-based bi-omarker for early OA events. The hypothesis of the second study was that human plasma lysate (hPL) media en-richment leads to a higher quality of the ECM in JPCs when compared with the standard fetal calf serum (FCS) condition. For this purpose, JPCs cultured with the aforementioned media supplementations were analyzed in two study arms. RAMAN spectroscopy was used for biochemical characterization, and AFM was employed for biomechanical analy-sis. Raman spectroscopic measurements showed significantly higher phosphate to protein ratios and lower carbonate to phosphate ratios under hPL in comparison to FCS culturing. With respect to the ECM collagen maturity, higher ratios of proline to hydroxyproline as well as higher levels of collagen cross-linking were detected in hPL-cultured JPCs. This indicates that hPL induces a higher degree of collagen maturation in JPCs. AFM data showed a significant increase in EM of the ECM under hPL conditions. This study hence demonstrates that hPL media supplementation of JPCs leads to the formation of a higher ECM quality as when compared to the FCS standard settings. In summary, both studies employed AFM-based elasticity measurements to investi-gate biomechanical features of load-bearing tissues. In both studies, the results obtained were significant and provide further insights which may now be fused into existing axi-oms of biochemical processes. Integration of both biomechanical and biochemical features will play a vital role in future scientific endeavors and will serve to establish an in-depth understanding of cellular biology.