Synergistic effects of biological stimuli and flexion induce microcavities promote hypertrophy and inhibit chondrogenesis during in vitro culture of human mesenchymal stem cell aggregates

Interzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis-inducing [CIM] and hypertrophy-inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin-O and hematoxylin & eosin (H&E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high-stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.