Pressure-Regulated Chondrogenesis of BMSCs: Static Negative Pressure Primes Differentiation through Apoptotic Vesicles.

BACKGROUND

It was found that pressure can promote the regeneration and repair of cartilage defects based on bone marrow mesenchymal stem cells (BMSCs). Since the compressive microenvironment of the cartilage in vivo may change with different movement, the mechanobiological effects of different compressive condition on BMSCs, especially the impact for its chondrogenic differentiation and influence on the cartilage microenvironment, is what we concerned about.

METHODS

Rat BMSCs were cultured and subjected to various types of pressure stimulation for 1 h. The Cell Counting Kit-8 (CCK-8) assay was used to analyze cell proliferation, flow cytometry was employed to assess the cell cycle and apoptosis, confocal microscopy was used to observe the cytoskeleton, and transmission electron microscopy was performed to examine the cellular ultrastructure. RT-PCR was used to identify chondrogenic differentiation markers. Apoptotic vesicles derived from BMSCs were isolated by ultracentrifugation, and differentially expressed microRNAs in these vesicles under - 40 kPa compression were identified by transcriptome sequencing.

RESULTS

Specific pressure conditions promoted the proliferation of BMSCs, with dynamic pressure showing a stronger proliferative effect than static pressure. Higher static negative pressure (- 40 kPa) significantly increased the spreading area of BMSCs. Dynamic pressure is stronger than static pressure in promoting cytoskeletal rearrangement, stress fiber formation, and cartilage marker expression in BMSCs. Flow cytometry and transmission electron microscopy results show that both - 40 kPa static and 90 kPa dynamic pressures promote BMSCs apoptosis to some extent. Under - 40 kPa static negative pressure, the differentially expressed microRNAs in BMSCs-derived apoptotic vesicles are involved in stem cell maintenance and chondrogenic proliferation processes.

CONCLUSION

Static negative pressure (- 40 kPa) induces apoptosis in bone marrow mesenchymal stem cells (BMSCs). Notably, compared to chemical induction with staurosporine (STS), BMSCs subjected to - 40 kPa mechanical stimulation display distinct microRNA expression profiles within apoptotic vesicles, specifically enriched in microRNAs implicated in stem cell fate determination and cartilage regeneration. These findings offer valuable insights into biomechanical strategies for optimizing tissue-engineered cartilage repair.