Enhanced collagen type I synthesis by human tenocytes subjected to periodic in vitro mechanical stimulation

BACKGROUND Mechanical stimulation (e.g. slow heavy loading) has proven beneficial in the rehabilitation of chronic tendinopathy, however the optimal parameters of stimulation have not been experimentally determined. In this study of mechanically stimulated human tenocytes, the influence of rest insertion and cycle number on (1) the protein and mRNA levels of type I and III collagen; (2) the mRNA levels of transforming growth factor beta (TGFB1) and scleraxis (SCXA); and (3) tenocyte morphology, were assessed. METHODS Human hamstring tenocytes were mechanically stimulated using a Flexcell® system. The stimulation regimens were 1) continuous and 2) rest-inserted cyclic equiaxial strain at a frequency of 0.1 Hz for 100 or 1000 cycles. Data were normalized to unstimulated (non-stretched) control groups for every experimental condition. qPCR was performed to determine relative mRNA levels and quantitative immunocytochemistry image analysis was used to assess protein levels and cell morphology. RESULTS Collagen type I mRNA level and pro-collagen protein levels were higher in tenocytes that were subjected to rest-inserted mechanical stimulation, compared to continuous stretching (p<0.05). Rest insertion and increased cycle number also had significant positive effects on the levels of mRNA for TGFB1 and SCXA (p<0.05). There was no direct relation between cell morphology and gene expression, however mechanical stimulation, overall, induced a metabolically active tenocyte phenotype as evidenced by cells that on average demonstrated a decreased major-minor axis ratio (p<0.05) with greater branching (p<0.01). CONCLUSIONS The incorporation of rest periods in a mechanical stretching regimen results in greater collagen type I synthesis. This knowledge may be beneficial in refining rehabilitation protocols for tendon injury.