Abstract: BACKGROUND: The vicious cycle of osteoarthritis initiation and progression is driven by the combined effects of mechanical microenvironment disruption and collapse of osmotic pressure homeostasis. Sustained abnormal osmotic pressure disrupts chondrocyte homeostasis and markedly impairs the ability of bone marrow mesenchymal stem cells to differentiate into chondrocytes. Consequently, this compromises the regenerative capacity of cartilage and accelerates the degeneration of articular cartilage.
OBJECTIVE: To develop a pathological osmotic pressure model for use in osmotic intervention experiments, in order to investigate the effects of osmotic pressure on chondrogenic differentiation of bone marrow mesenchymal stem cells and chondrocyte matrix metabolism, and to explore the role of imbalanced osmotic pressure within the joint cavity in the pathogenesis of osteoarthritis.
METHODS: Bone marrow mesenchymal stem cells were isolated from 6- to 8-week-old rats and cultured to the third passage. Third-passage rat chondrocytes were revived and performed expansion culture. Physiological or pathological osmotic pressure regulating solutions were prepared by adding NaCl to the culture medium, and their biocompatibility was assessed via cell counting kit-8 assays. Bone marrow mesenchymal stem cells were treated with different osmotic pressure regulating solutions in chondrogenic induction medium for 7 or 14 days. Safranin O staining was used to identify the secretion of glycosaminoglycan (a cartilage marker). qRT-PCR was performed to detect expression of genes related to cartilage synthesis. The effects of physiological or pathological osmotic pressure regulating solution on chondrocyte anabolism and catabolism were further evaluated after interleukin-1β inflammation induction. Furthermore, RNA-seq was employed to identify differentially expressed genes between the physiological and pathological osmotic pressure groups and an enrichment analysis was performed.
RESULTS AND CONCLUSION: (1) Third-passage bone marrow mesenchymal stem cells were successfully isolated and cultured, and third-passage chondrocytes were successfully revived and expanded. (2) Cell counting kit-8 assay demonstrated that these physiological and pathological osmotic pressure regulating solutions exhibited good biocompatibility. (3) Safranin O staining indicated that the chondrogenic capacity of the physiological osmotic pressure group was significantly enhanced compared to the pathological osmotic pressure group on both day 7 and day 14. (4) qRT-PCR further confirmed that compared with the interleukin 1β group and the pathological osmotic pressure group, the physiological osmotic pressure group could significantly upregulate the expression of cartilage synthesis-related genes aggrecan and collagen type II α1 chain, while downregulating the catabolism-related genes matrix metalloproteinase 13 and matrix metalloproteinase 3. (5) RNA sequencing results revealed that under physiological osmotic conditions, the expression of multiple molecules and signaling pathways associated with osteoarthritis pathogenesis was markedly suppressed. 

Key words: osteoarthritis, osmotic pressure of joint fluid, bone marrow mesenchymal stem cells, chondrogenic induction, chondrocytes, physiological osmotic pressure, pathological osmotic pressure