Mechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation. Mouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated. The mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts’ miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation. In osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.

Mechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-β (β1, β5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-β in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear. After transfection with integrin-β1 siRNA or integrin-β5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-β1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-β5 siRNA had little effect on the osteoblastic differentiation induced by the strain. At the same time, the result of ECM formation promoted by the strain, was similar to the osteoblastic differentiation. Integrin-β1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-β5 is not involved in the osteoblasts response to the tensile strain.

Background: In heart, the extracellular matrix (ECM), produced by cardiac fibroblasts, is a potent regulator of heart^,s function and growth, and provides a supportive scaffold for heart cells in vitro and in vivo. Cardiac fibroblasts are subjected to mechanical loading all the time in vivo. Therefore, the influences of mechanical loading on formation and bioactivity of cardiac fibroblasts^, ECM should be investigated.

Different cell/tissue derived extracellular matrix (ECM) display subtle differences that might provide important cues for proliferation and differentiation of cells in vitro or in vivo. However, the bioactivities of different ECMs in vitro were not fully understood. In this study, osteoblasts-derived and fibroblast-derived ECM-coated cell culture dishes were prepared respectively by culturing osteoblastic MC3T3-E1 cells and rat fibroblast then decellularizing the cultures. We investigated the bioactivities of the two different ECMs coated on cell culture plates using cellular, biochemical and molecular method. The proliferative activity of the bone marrow-derived mesenchymal stem cells (BMSCs) cultured on osteoblast-ECM was lower than for BMSCs grown on fibroblast-ECM. Compared with the BMSCs cultured on fibroblast-derived ECM, the cells grown on osteoblastic ECM showed enhanced alkaline phosphatase (ALP) activity, higher BMP-2 and osteopontin protein levels, increased secreted calcium content, and higher levels of runt-related transcriptional factor 2 (Runx 2) and osteocalcin (OCN) mRNA. Knockdown of BMP-2 or FGF-2 with shRNA transfection hardly effected osteoblastic differentiation or proliferation of MC3T3-E1 seeded on osteoblast-ECM or fibroblast-ECM. Therefore, the osteoblastic ECM had better osteoinductive potential and lower proliferative effect than fibroblastic ECM, and the two ECM presented enough bioactivity, knockdown of growth factors had no significant effect on differentiation and proliferation of re-seeded cells.