Effects of simulated muscle exercise on chondrocyte gene expression in a 3D-alginate bead model system

Abstract

Purpose: The joint organ is a complex system composed of bone, muscle, tendon, ligament, synovial and cartilage tissues. Myokines produced and released by muscle cells (myocytes) in response to muscular contractions have autocrine, paracrine and/or endocrine effects. Exercise is one of the recommended core treatments for all osteoarthritis patient; exercise-induced myokines appear to be involved in mediating both systemic as well as local anti-inflammatory effects. We hypothesized that myokines, produced by muscle stimulation mimicking exercise, will have anti-inflammatory and anabolic paracrine effects on chondrocytes. We tested this hypothesis by analyzing the effects of conditioned media from skeletal muscle myobundles (with and without electrical stimulation) on gene expression of chondrocytes in a 3D-alginate culture system. Methods: Electrically responsive, contractile human skeletal muscle myobundles were made from primary myogenic cells isolated from standard muscle biopsies obtained under IRB approval. After four-weeks in culture, when these human myobundles matured, electrical pulse simulation of myobundles was performed to simulate different exercise conditions: 1) Non-stimulated, 2) 1 Hz stimulation (simulated slow-twitch exercise), 3) 10 Hz stimulation (simulated fast-twitch exercise). Conditioned media (CM) from all 3 groups were collected each day for 7 days. Primary chondrocytes, collected as surgical waste, were isolated from normal appearing articular cartilage of the lateral femoral condyle from patients undergoing knee replacement for osteoarthritis (OA). Cartilage was diced, digested in 0.1% (weight/volume) collagenase type II for 16-24 h at 37 °C, and the cells were cultured in F12 media in a T-75 cm2 flask in 5% CO2 until confluent. Passage 1-4 chondrocytes were encapsulated in 1.2% alginate solution (2x10e6 cells/ml) by drop solidification into 102mM CaCl2 solution, then cultured for up to 7 days in DMEM/F12 culture media. Chondrocytes (3 biological replicates) in alginate were cultured for 48 hr in non-stimulated myobundle CM or the stimulated myobundle CM at a ratio of 1:1 to chondrocyte growth media. Quantitative PCR was performed on RNA isolated from alginate bead cultured chondrocytes for analysis of gene expression of MMP13, ADAMTS4, ADAMTS5, COL2A1, COL10A1 and ACAN and YWHAZ (housekeeping control). Results: Compared to non-stimulated CM, stimulated CM tended to reduce chondrocyte gene expression (normalized to the housekeeping control) of ACAN (p=0.086), ADAMTS5 (p=0.081) and MMP13 (p=0.111). Compared to 1 Hz stimulated CM, chondrocytes exposed for 48 hours to 10 Hz stimulated CM exhibited elevated gene expression (normalized to the control and the non-stimulated level of gene expression) of collagens II and X (p=0.045, p=0.01, Figure 1). Conclusions: Our study suggests that myokines produced by muscle stimulation mimicking exercise have anti-catabolic effects. Compared with low frequency stimulation, myokines produced in response to high frequency stimulation induced markers of chondrogenesis (collagen II) and late-stage chondrocyte hypertrophy (collagen X). This pilot study provides evidence for a paracrine effect of muscle on cartilage. Factors from muscle responsible for these effects warrant further investigation and identification to understand the physiologic interactions of muscle and cartilage.Table 1Chondrocyte gene expression in response to muscle conditioned media (CM).GeneChondrocyte gene expression (fold change in stimulated vs non-stimulated muscle CM)P valueChondrocyte gene expression (fold change in 10 Hz vs 1 Hz stimulated muscle CM)P valueCOL21.130.3961.420.045COL100.640.1731.120.010ACAN0.670.0861.040.387ADAMTS40.740.2831.040.442ADAMTS50.630.0811.090.210MMP130.630.1111.050.259 Open table in a new tab