Summary

While arc-bending strain suppress cell proliferation and stimulate ECM degradation, combined hydrostatic pressure and arc-bending strain overcome these responses, suggesting a key role of compressive loading in maintaining a healthy articular cartilage

Abstract

Introduction

Traumatic knee injuries frequently lead to partial-thickness defects in articular cartilage that can progress to further chondral degeneration and ultimately osteoarthritis. Little is known about progressive degeneration in partial-thickness cartilage defects. This study aimed to characterize cellular behavior in a partial defect model under cyclic hydrostatic pressure (HP) and/or arc-bending strain (BS) to develop novel therapeutic strategies.

Methods

Round articular cartilage discs were harvested from bovine humeral heads using a 6-mm biopsy punch. A partial-thickness defect (2 mm in diameter x 1 mm in depth) was created at the center of each disc with a 2-mm biopsy punch and a drill bit. Discs were randomly allocated to four culture conditions: 1) Unloaded control (Control); 2) cyclic BS at 0-4%, 0.5 Hz; 3) cyclic HP at 0-0.5 MPa, 0.5 Hz; and 4) HP combined with BS (HP/BS). The discs were incubated under the above conditions in culture media at 37°C and 5% CO2 for 9 days. At days 6 and 9, discs were harvested (n=8/group) and cut down the middle. From each sample, a 2-mm thick slice was obtained at the center portion and incubated in Live/Dead staining dye for viability evaluation. The rest of the discs were fixed and embedded in paraffin, and 7-µm sections were stained with safranin-O (Saf-O) to evaluate cartilage matrix and cell shape. To evaluate cell proliferation and degeneration, sections were stained with antibodies against proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-13 (MMP-13), respectively. Percentage of live/dead cells, anucleate cells, PCNA-positive and MMP-13 positive cells were counted within defined areas. Two-way analysis of variance followed by Tukey’s post hoc tests were used for statistical analysis. Statistical significance was set at p < 0.05.

Results

Cell viability remained high in all groups (>80%) over 9 days of culture. Intensity of Saf-O staining was slightly lower under BS compared to other conditions, suggesting a slight reduction in sulfated glycosaminoglycan. Anucleate cells were observed in all conditions; however, at day 6 HP showed significantly fewer anucleate cells compared to BS (P<0.01) or AP (P<0.01). At day 9, AP showed a higher percentage of anucleate cells than BS (P<0.01) or HP alone (P<0.01). The percentage of PCNA-positive cells at day 6 was significantly lower under BS than AP (P<0.01), HP (P<0.01) or HP/BS (P<0.01). At day 9, all conditions showed a reduction in proliferating cells; again, BS presented a lower percentage of PCNA-positive cells in comparison to HP (P<0.01). The percentage of MMP-13-positive cells at day 6 was significantly higher under BS than HP/BS (P< 0.01), HP (P< 0.01), or AP (P <0.01), and higher under HP/BS than AP (P <0.01). At day 9, BS presented a higher percentage of MMP-13-positive cells than HP (P<0.01).

Conclusion

While BS suppressed cell proliferation and stimulated ECM degradation, combined HP/BS overcame these responses, suggesting the key role of compressive loading (HP/BS) in maintaining a healthy articular cartilage. Clinically, BS may mimic non-weight bearing joint movement, whereas HP/BS represents joint movement with weight bearing. This model holds potential in the development of repair strategies and postoperative rehabilitation protocols.