Synergetic Support of Cartilage Homeostasis via Coupled Thermal-Pressure Stimuli: A Transcriptomic Study in Human Cartilage Explants

During daily activities, cartilage encounters complex biophysical cues upon loading. Foremost among these is the coupled stimulation of hydrostatic pressure (HP) and loading-induced temperature increase (T), which encompasses both mechanical and thermal aspects of biophysical stimulations in cartilage. While prior research on this subject has been initiated in our laboratory, the detailed mechanisms of combined HP-T effects on chondrocytes in their natural environment remain largely unexplored. Using a custom bioreactor, we applied both isolated and combined HP-T stimuli to cartilage explants obtained from a non-inflammatory adolescent knee joint. Tissue and cellular responses were evaluated through histochemical staining and transcriptomic analyses, employing bulk RNA-sequencing complemented with signaling enrichment analyses. Our findings reveal that the thermal component of the coupled HP-T stimulation predominantly regulates the chondrocytes' transcriptional profile during the stimulation period. When coupled with HP stimulation, a peak in chondroinduction was observed. This coupling process notably boosted chondroprotection in a synergistic manner, as demonstrated by the corresponding enhanced negative regulation of apoptotic processes and increased levels of Heat Shock Protein 70 (HSPA). Our study suggests that the upregulation in protein translation and processing, triggered by thermal stimulation, may serve as an adaptive mechanism in chondrocytes to mechanical simulations, thereby contributing to the observed synergy during the coupling of these two biophysical stimuli. The results highlight the potential of integrating thermal stimulation, a natural accompanying process during cartilage deformation, in tissue engineering, cell therapy or physiotherapy. Using a previously developed customized bioreactor, various biophysical stimulation protocols were administered for 1.5 hours daily over five consecutive days. Four experimental groups were considered and the specific stimulation details for each group are: (1) Control group (“C”): Maintained at a steady 32.5 ℃ within the bioreactor, replicating the resting knee temperature, with no applied hydrostatic pressure (HP) or temperature increase. (2) Isolated pressure group (“P”): While kept consistently at 32.5 ℃, samples underwent sinusoidal cyclic HP loading with a 5 MPa amplitude at 1 Hz. (3) Isolated temperature increase group (“T”): Samples were exposed to a gradual temperature rise from 32.5 ℃ to 38.7 ℃ without any HP loading. This temperature profile mirrors the intra-articular temperature evolution observed during a 1.5-hour jogging. (4) Coupled HP-T stimulation group (“PT”): Samples were concurrently subjected to a 5 MPa cyclic HP loading (as in the “P” group) and a temperature increase from 32.5 ℃ to 38.7 ℃ (as in the “T” group) for the entire 1.5-hour stimulation duration. In each group, the stimulation medium was replaced every other day. Samples of all conditions were harvested immediately after the last stimulation and processed accordingly to the analysis described below.