Optimal cyclic compressive loading promotes differentiation of 3D-cultured pre-osteoblasts

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Fracture healing is a special wound healing response. Wounded areas are not restored, but are regenerated and connected. There are four components to the injury site ; the cortex, the periosteum, the bone marrow, and the external soft tissues, all of which contribute to the healing process. The extent to which each component is involved depends on the conditions present at the injured tissue, such as the level of growth factors, hormones, nutrients, pH, oxygen tension, electrical environment, and mechanical stimuli of the fracture. Mechanical stimuli, applied to tissue, have various clinical effects on cells depending on the amount of stimulation. However, these have not been demonstrated by an in vitro experiment, and no standardized system has been established. In the present study, mouse preosteoblast-like cells (MC3T3-E1) were used to prepare 3D tissue cultures. The tissue cultures underwent repeated compressive loading using a cyclic compressive device, which can change the load, to examine bone differentiation markers and transcription factors. MC3T3-E1 cells were 3D cultured using a collagen scaffold to determine the conditions that induce osteoblast differentiation. The expressions of bone differentiation markers were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Cells to which no repeated compressive loading was applied were defined as the FS group. The FS group showed increased expressions of collagen Ia1 (Col I a1), alkaline phosphatase, osteopontin, and osteocalcin, over time. The day when the cells were 3D cultured was defined as day 0. Cells to which repeated compressive loading was applied on days 1-7 were defined as the EL group. This group showed decreased expressions of ALP, osteopontin, and osteocalcin in proportion to the load. The day when the 3D culture was started was defined as day 0. Cells to which repeated compressive loading was applied on days 8-14 were defined as the LL group. It showed significantly increased expressions of Col Ia1, ALP, RUNX2, osterix and Dlx5 at 5 kPa. The LL group also showed significantly increased expressions of Col Ia1, osterix, and Dlx5 at 20 kPa, and significantly decreased expressions of osteocalcin at 40 kPa. Early mechanical loading tended to decrease the expressions of differentiation markers to suppress osteoblast differentiation. Additional mechanical loading following 7 day static culture significantly increased the expressions of differentiation markers and transcription factors. Thus, the mechanical loading suppressed osteoblast differentiation at an early stage and promoted the differentiation of differentiated osteoblasts. Mechanical loading at 5 kPa promoted differentiation, suggesting that optimal cyclic compressive loading induced the bone differentiation of pre-osteoblasts.
2013-04-00

Optimal cyclic compressive loading promotes differentiation of 3D-cultured pre-osteoblasts

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