Research On The Effect Of Mechanical Stretch On Osteoblast And The Underlying Mechanisms

Background and objective:Bone tissue is a specialized form of connective tissue playing a major load-bearing role in the human body. It is composed of cells, extracellular matrix and inorganic compound. Bone is a dynamic tissue, throughout life bone tissue is continually being modeled and remodeled. Bone remodeling is a complex biological process which is dependent on genetic, hormonal, metabolic, age-related factors and mechanical environment. According to Wolff's law, mechanical loading plays an essential role in bone remodeling, removal of mechanical stimuli results in declined bone mass while a suitable dynamic mechanical strain promotes bone formation. Osteoblast, originating from mesenchymal stem cells (MSCs), plays a crucial role in bone development, bone remodeling and repair. Osteoblast and its precursor cells are sensitive to mechanical stimulation. They can translate mechanical signals into biological response and regulate bone homeostasis. Osteoblast is located on the surface of bone matrix and the mechanical environment of bone tissue is complex, thus, osteoblast can be influenced by many kinds of biological factors. For this reason, it is difficult to determine the precise effect of mechanical signal on osteoblast in vivo. The in vitro mechanical loading experiments can be performed using the cultured cells to exclude the influence of the biological factors on osteoblast. Furthermore, mechanical stimulation parameters such as the manner of loading, magnitude, frequency and time course can be designed, and the precise effect of mechanical stimulation on osteoblast can be detected. Though osteoblast can respond to mechanical signal, the influence of mechanical stimulation on osteoblast and the underlying mechanisms are still obscure. In our research, MC3T3-E1 cells, a mouse monoclonal osteoblastic cell line, were subjected to mechanical stretch of different magnitude and time via a four-point bending system. We aimed to detect the effect of mechanical signal on osteoblast proliferation, apoptosis, differentiation and mineralization; the underlying mechanisms involved in mechanical response in osteoblast were also investigated. In addition, based on our research, we want to provide theoretical basis for in-depth study of osteogenesis and bone remodeling under mechanical conditions.Methods:1. MC3T3-E1 cells were subjected to mechanical stretch of different magnitude (500με, 1000με, 2000με, 3000με, 5000με) at 0.5HZ via a four-point bending system. After mechanical stretch, cell proliferation was detected by MTT method; cell apoptosis was observed by DNA ladder and flow cytometry; expression of osteoblast differentiation marker genes was analysed by RT-PCR and WesternBlot; cell mineralization was studied by Von Kossa staining. 2. MC3T3-E1 cells were subjected to mechanical stretch at 2000μεfor different time (2h, 4h, 8h, 12h, 24h) at 0.5HZ. After mechanical stretch, cell proliferation, apoptosis, differentiation and mineralization were detected.3. After mechanical stretch, RT-PCR and Western Blot were employed to study the expression of molecules involved in BMPs/Smad signaling pathway; ELISA analysis was used to detect the content of BMP-2 and BMP-4 in the culture medium; activation and nucleus translocation of Smad proteins were observed by Western Blot.4. MC3T3-E1 cells were pretreated with Noggin to block BMPs signaling 2 hours before mechanical stretch, and then subjected to mechanical stretch for 8 hours. Smad activation and osteoblast differentiation marker genes expression were detected. Smad4 expression was inhibited by transfection with Smad4 siRNA; then MC3T3-E1cells were subjected to mechanical stretch for 8 hours. Nucleus translocation of p-Smad and expression of osteoblast differentiation marker genes were studied.5. The activation of p38MAPK and NF-κB signaling pathway in mechanical stimulated MC3T3-E1 cells was studied by Western Blot. PDTC was used to inhibit NF-κB pathway, then MC3T3-E1cells were subjected to mechanical stretch and BMPs expression was detected. Before mechanical stretch, MC3T3-E1 cells were pretreated with Noggin to block BMPs pathway, the relationship between p38MAPK pathway and BMPs signaling in mechanical stimulated cells was studied. SB203580 was employed to block p38MAPK pathway, the role of p38MAPK pathway in NF-κB activation, BMPs expression and osteobast differentiation was further explored.6. RT-PCR and Western Blot were used to study the effect of mechanical stretch on Smurf1, Smurf2 and CKIP-1 expression.7. To investigate the role of Smurf1 under mechanical conditions, MC3T3-E1 cells were transfected with Smurf1 siRNA. Smad, p-Smad and osteoblast differentiation marker genes expression were detected after mechanical stretch; the role of proteasome pathway in mechanical stimulated MC3T3-E1 cells were explored by inhibiting the proteasome activity with MG132.Results:1. Mechanical stretch of 1000με, 2000μεand 3000μεcould induce expression of osteoblast differentiation marker genes and 2000μεhad the most significant effect. However, different magnitude of mechanical stretch showed no significant effect on cell proliferation, apoptosis and mineralization.2. Different time course of mechanical stretch at 2000μεshowed no significant effect on cell proliferation, apoptosis and mineralization. The expression of osteoblast differentiation marker genes was up-regulated by mechanical stretch in a time dependent manne: ALP expression increased significantly 4 hours of mechanical stretch and returned to basal level at 24 hours, the maximum expression was detected at 8 hours; OCN expression started to significantly increase at 8 hours and the high-level expression lasted to 24 hours; Col I expression increased significantly at 4 hours and the expression level of Col I at 24 hours was still higher than that in control group.3. Expression and secretion of BMP-2 and BMP-4 were enhanced by mechanical stretch. mRNA expression of BMPRI, Smad1 and Smad5 was not influenced by mechanical stretch while their protein levels were elevated. Meanwhile, a significant increase in activated Smad1 and Smad5 both in whole cell and in nucleus was observed.4. Mechanical stretch induced activation of Smad proteins was attenuated by Noggin; p-Smad accumulation in the nucleus was also decreased by Smad4 siRNA. These events were followed by the down-regulation of osteoblast differentiation marker genes expression.5. p38MAPK pathway was activated by mechanical stretch at 15min while NF-κB was activated at 30min, these two pathways were both inactivated at 90min. Mechanical stretch induced expression of BMP-2 and BMP-4 was attenuated by NF-κB pathway inhibitor PDTC. Blocking BMPs signaling by Noggin had no effect on p38MAPK activation, indicating p38MAPK was activated by mechanical stretch in a BMPs independent manner. p38MAPK pathway inhibitor SB203580 inhibited mechanical stretch induced activation of NF-κB, expression of BMPs and osteoblast differentiation marker genes, suggesting that NF-κB activation and BMPs expression in mechanical stimulated cells was depended on p38MAPK pathway.6. Smurf1, but not Smrf2 and CKIP-1, was down-regulated by mechanical stretch.7. Transfection with Smurf1 siRNA and pretreatment with MG132 both augmented the effect of mechanical stretch on the content of BMPRI and Smad as well as the activation of Smad proteins and the expression of osteoblast differentiation marker genes.Conclusions:1. Mechanical stretch can promote osteoblast differentiation.2. BMPs/Smad signal pathway is activated by mechanical stretch and this pathway is involved in mechanical stretch induced expression of osteoblast differentiation marker genes.3. p38MAPK pathway and NF-κB pathway has cross-talk in mechanical stimulated osteobalst. Mechanical signal up-regulates BMPs expression via p38MAPK/NF-κB pathway and further activates BMPs/Smad pathway to induce osteoblast differentiaton.4. Smurf1 expression is down-regulated by mechanical stretch, the diminished Smurf1 levels lead to inhibition of degradation of BMPRI and Smad proteins and to subsequently enhanced BMPs/Smad signaling, induced by mechanical stretch. In summary, mechanical stretch induces the activation of BMPs/Smad pathway by activating p38MAPK/NF-κB pathway and down-regulating Smurf1 expression, and further promotes osteoblast differentiation.