Effects of physiological osmotic pressure on chondrocyte differentiation and extracellular matrix metabolism

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). Quantitative real-time PCR was used to detect the expression of cartilage synthesis-related genes. The effects of physiological or pathological osmotic pressure regulating solutions on chondrocyte anabolic and catabolic metabolism after interleukin-1β inflammation induction were detected. Furthermore, RNA-seq was used to identify differentially expressed genes between the physiological and pathological osmotic pressure groups and perform enrichment analysis. 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) CCK-8 assay results showed that both pathological and physiological osmotic pressure regulating solutions had good biocompatibility. (3) Safranin O staining indicated that at days 7 and 14, the chondrogenic capacity of the physiological osmotic pressure group was significantly enhanced compared with the pathological osmotic pressure group. (4) qRT-PCR results further showed that compared with the interleukin-1β group and the pathological osmotic pressure group, the physiological osmotic pressure group significantly upregulated the expression of cartilage synthesis-related genes aggrecan and collagen type II alpha 1, while downregulating the expression of catabolism-related genes matrix metalloproteinase 13 and matrix metalloproteinase 3. (5) RNA-seq results showed that under physiological osmotic pressure conditions, multiple molecules and signaling pathways related to osteoarthritis pathogenesis were significantly downregulated.

1. Introduction

Osteoarthritis, as the degenerative joint disease with the highest disability rate globally, has become a core public health challenge threatening the health of middle-aged and elderly populations [1-2]. Its pathological features are mainly characterized by progressive degeneration of articular cartilage, subchondral bone sclerosis, and synovial inflammation, ultimately leading to loss of joint function [3]. Although traditional views attribute osteoarthritis to mechanical wear or natural aging [4-5], recent research has revealed that its pathogenesis involves a complex interactive network of mechanical, chemical, and biological factors, among which the disruption of osmotic pressure homeostasis plays a key role.

The pathological process of osteoarthritis is essentially a vicious cycle driven by the combined effects of mechanical microenvironment disruption and collapse of osmotic pressure homeostasis [6]. The osmotic pressure of normal joint fluid is maintained within the range of (404±57) mOsm/kg [7], a property determined by the fixed charge density of proteoglycans in the extracellular matrix [8]. Under physiological conditions, the fixed charge density attracts ions such as Na+ through the Donnan effect to form an osmotic pressure gradient, making the water content of cartilage reach 70%-80%, endowing it with excellent compressive resistance and energy dissipation capacity [9]. However, when abnormal mechanical loads act on cartilage, hyaluronic acid molecules in the extracellular matrix undergo depolymerization due to shear forces, leading to decreased synovial fluid viscosity and reduced osmotic pressure. This hypotonic environment activates chondrocyte volume regulatory responses through ion channels (such as PIEZO1, TRPV4, etc.), forcing cells to take in excess water to maintain osmotic balance, thereby triggering intracellular calcium overload and ferroptosis [10-11].

The pathological coupling effect of abnormal mechanical properties and osmotic pressure changes is particularly significant in the progression of osteoarthritis. Normal joint function depends on uniform biomechanical load distribution, but long-term poor posture or trauma can lead to local pressure abnormalities [12-13], altering the osmotic pressure environment and weakening synovial fluid lubrication, directly exacerbating cartilage damage [14]. In vitro experiments have confirmed that overloaded static pressure activates the integrin β1-FAK pathway abnormally, promoting chondrocytes to secrete matrix metalloproteinase 13 and inhibiting SOX9 transcriptional activity, forming a positive feedback loop of 'mechanical overload → decreased osmotic pressure → extracellular matrix degradation → loss of mechanical buffering capacity' [15-16]. In addition, hypotonic environments weaken the boundary lubrication effect of synovial fluid and stimulate synovial cells to secrete interleukin-6 and prostaglandin E2 [17], these inflammatory factors further aggravate cartilage degradation.