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Uniaxial Static Strain Promotes Osteoblast Proliferation and Bone Matrix Formation in Distraction Osteogenesis In Vitro.

ABSTRACT: Objective:We aimed at investigating the effects of uniaxial static strain on osteoblasts in distraction osteogenesis (DO). Methods:To simulate the mechanical stimulation of osteoblasts during DO, 10% uniaxial static strain was applied to osteoblasts using a homemade multiunit cell stretching and compressing device. Before and after applying strain stimulation, the morphological changes of osteoblasts were observed by inverted phase-contrast microscopy, Coomassie blue staining, and immunofluorescence. Alkaline phosphatase (ALP) activity, mRNA levels (proliferating cell nuclear antigen [PCNA], ALP, Runx2, osteocalcin [OCN], collagen type I, hypoxia-inducible factor- [HIF-] 1?, and vascular endothelial growth factor [VEGF]), and protein levels (Runx2, OCN, collagen type I, HIF-1?, and VEGF) were evaluated by using ALP kit, real-time quantitative reverse transcription-polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Results:After the mechanical stimulation, the cytoskeleton microfilaments were rearranged, and the cell growth direction of the osteoblasts became ordered, with their direction being at an angle of about 45° from the direction of strain. The proliferation of osteoblasts and the expression levels of mRNA and protein of ALP, Runx2, OCN, collagen type I, HIF-1?, and VEGF were significantly higher than in the nonstretch control groups. Conclusion:Our homemade device can exert uniaxial static strain and promote the proliferation of osteoblasts and bone matrix formation. It can be used to simulate the mechanical stimulation of osteoblasts during DO.

SUBMITTER: Li Z

PROVIDER: S-EPMC7443025 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Uniaxial Static Strain Promotes Osteoblast Proliferation and Bone Matrix Formation in Distraction Osteogenesis In Vitro.

Li Zhengqiang Z Zheng Junfa J Wan Di D Yang Xiaoqin X

BioMed research international 20200812

<h4>Objective</h4>We aimed at investigating the effects of uniaxial static strain on osteoblasts in distraction osteogenesis (DO).<h4>Methods</h4>To simulate the mechanical stimulation of osteoblasts during DO, 10% uniaxial static strain was applied to osteoblasts using a homemade multiunit cell stretching and compressing device. Before and after applying strain stimulation, the morphological changes of osteoblasts were observed by inverted phase-contrast microscopy, Coomassie blue staining, and ...[more]

PMID: 32855965

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Project description:Background: Interfragmentary movements have benefits in the improvement of bone formation during distraction osteogenesis (DO). Although several clinical studies reported positive outcomes regarding the application of the cyclic distraction-compression (CDC) dynamization technique in cases with poor bone formation during DO, they are mostly anecdotal without a detailed description. The purpose of this study was to investigate the effectiveness and potential mechanism of different amplitudes and rates of the CDC technique on bone regeneration in a rat femur DO model. Methods: A total of 60 adult male Sprague-Dawley rats underwent right femoral mid-diaphysis transverse osteotomy and were randomly and evenly divided into Control (no manipulation), Group1 (CDC therapy), Group2 (CDC therapy with larger amplitude), and Group3 (CDC therapy with a slower rate) after distraction. The CDC technique was performed during the middle phase of the consolidation period according to different protocols. Animals were sacrificed after 4 and 6 weeks of consolidation. The process of bone formation was monitored by digital radiographs, and the regenerate bone was evaluated by micro-computed tomography (micro-CT), biomechanical test, and histological analysis. The serum contents of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Bone regeneration after the CDC technique was improved significantly during DO. The digital radiograph, micro-CT, histomorphological analysis, and biomechanical evaluation showed better effects regarding volume, continuity, and mechanical properties of the regenerate bone in Group2 and Group3 when compared to Group1. The angiogenic and osteogenic markers were more highly expressed in Group2 and Group3 than in Group1 according to the immunohistochemical analysis. As for ELISA, the serum contents of HIF-1α and VEGF were also increased after the CDC technique, especially in Group2 and Group3. Conclusion: The CDC dynamization technique has benefits on the improvement of bone formation during DO, and the mechanism may be due to tissue hypoxia activating the HIF pathway followed by the augmentation of osteogenic-angiogenic coupling. Better outcomes may be achieved by moderately increasing the amplitude and slowing down the rate of the CDC technique.

Project description:Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

Project description:Nearly all exogenous loading models of bone adaptation apply dynamic loading superimposed upon a time invariant static preload (SPL) in order to ensure stable, reproducible loading of bone. Given that SPL may alter aspects of bone mechanotransduction (eg, interstitial fluid flow), we hypothesized that SPL inhibits bone formation induced by dynamic loading. As a first test of this hypothesis, we utilized a newly developed device that enables stable dynamic loading of the murine tibia with SPLs ≥ -0.01 N. We subjected the right tibias of BALB/c mice (4-month-old females) to dynamic loading (-3.8 N, 1 Hz, 50 cycles/day, 10 s rest) superimposed upon one of three SPLs: -1.5 N, -0.5 N, or -0.03 N. Mice underwent exogenous loading 3 days/week for 3 weeks. Metaphyseal trabecular bone adaptation (μCT) and midshaft cortical bone formation (dynamic histomorphometry) were assessed following euthanasia (day 22). Ipsilateral tibias of mice loaded with a -1.5-N SPL demonstrated significantly less trabecular bone volume/total volume (BV/TV) than contralateral tibias (-12.9%). In contrast, the same dynamic loading superimposed on a -0.03-N SPL significantly elevated BV/TV versus contralateral tibias (12.3%) and versus the ipsilateral tibias of the other SPL groups (-0.5 N: 46.3%, -1.5 N: 37.2%). At the midshaft, the periosteal bone formation rate (p.BFR) induced when dynamic loading was superimposed on -1.5-N and -0.5-N SPLs was significantly amplified in the -0.03-N SPL group (>200%). These data demonstrate that bone anabolism induced by dynamic loading is markedly inhibited by SPL magnitudes commonly implemented in the literature (ie, -0.5 N, -1.5 N). The inhibitory impact of SPL has not been recognized in bone adaptation models and, as such, SPLs have been neither universally reported nor standardized. Our study therefore identifies a previously unrecognized, potent inhibitor of mechanoresponsiveness that has potentially confounded studies of bone adaptation and translation of insights from our field. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Dataset Information

Uniaxial Static Strain Promotes Osteoblast Proliferation and Bone Matrix Formation in Distraction Osteogenesis In Vitro.

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Uniaxial Static Strain Promotes Osteoblast Proliferation and Bone Matrix Formation in Distraction Osteogenesis In Vitro.

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