Effects Of Mechanical Strain On Biological Characteristics And Proteomics Analysis In Osteoblasts

The skeleton is a very complex tissue which can regulate its mass and architecture to adapt to functional environment. Orthodontic tooth movement results from alveolar bone remodeling process induced by mechanical strain. Osteoblasts, osteocytes and osteoclasts are specialized cells that orchestrate the process. As the osteoblast is highly sensitive to mechanical stimuli,it plays the leading role in the bone remodeling process. However,the mechanism of how these mechanical load is translated into intracellular signals is not very clear.In order to better understand the mechanotransdution process , we investigated proliferation and biological characteristics of osteoblasts under mechanical strain. Osteoblasts secrete alkaline phosphatase (ALP), osteopotin(OPN) and osteocalcin (OC) which involve in bone modeling. Recently, Wnt/β-catenin signaling has been shown to play a particularly crucial role in bone formation, bone growth and remodeling. Many studies have shown canonical Wnt/β-catenin signaling could promote osteoblast differentiation, proliferation, and mineralization. The central player of Wnt signaling isβ-catenin, which is a transcription cofactor with Tcf/Lef in the Wnt pathway and a structural adaptor protein linking cadherins to the actin cytoskeleton in cell-cell adhesion. The cytoplasmic accumulation ofβ-catenin leads toβ-catenin entering the nucleus and heterodimerizing with Lef/Tcf transcription factors to regulate Wnt target genes including COX-2, c-myc, cyclin D1 and so on. Previous studies have shown that Wnt/β-catenin signaling is a component of osteoblastic cell early responses to load-bearing.On the other hand, a proteomics approach,in which entire proteins in tissue or cells are identified and quantified directly,has been shown to be a valuable way to bring insight into the molecular basis of signal transduction. Compare the proteins of the osteoblasts in different conditions: mechanical strained and unstrained group,which will help us to find the key signal transduction protein and understand the responsive mechanism of the osteoblasts under the mechanical strain. Therefore in the study of this part, we quantitatively and qualitatively analyzed the differentially expressed proteins in Saos-2 osteoblastic cells of 24h after mechanical strain loading by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS), computer informatics and computer network communications technology. The aim of our study was to elucidate the molecular mechanisms of osteoblast signal transduction under mechanical strain.In the present study, Saos-2 osteoblastic cells were subjected to mechanical strain using Flexcell strain loading system and observed the effects of mechanical strain on cellular proliferation, differentiation. In addition, Wnt/β-catenin signal transduction pathway that mediated cellular responses to mechanical strain was investigated using different biological techniques. Furthermore, proteins from samples in the Saos-2 osteoblastic cells obtained under mechanical strain for 24h were subjected to two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The research works are as follows:1. Effects of mechanical strain on proliferation and differentiation of Saos-2 osteoblastic cells Objective: To investigate the effects of mechanical strain on proliferation and differentiation of Saos-2 osteoblastic cells. Methods: Saos-2 osteoblastic cells were subjected to 0%,6%,12%,24% elongation for 0h,4h,8h,12h,24h by using Flexcell strain loading system simultaneously. MTT colorimetric method was used to assess cellular proliferation;Biochemical analysis was used to study the effects of mechanical strain on alkaline phosphatase (ALP) activity;semi quatative RT-PCR was used to examine mRNA level of osteocalcin and osteopotin. Alizarin Red-S method was used to study the mineralization of Saos-2 osteoblastic cells. Results: The proliferation and ALP activity of Saos-2 osteoblastic cells were significantly increased after mechanical strain treatment,12% elongation rate showed the strongest stimulatory effects (P<0.01). The expression of OC,OPN mRNA and the formation of mineralization nodules in Saos-2 osteoblastic cells were promoted by 12% mechanical strain elongation rate. Conclusion: Our data indicated that mechanical strain stimulation increased the proliferation and differentiation markers such as ALP activity , mRNA expression level of OC and OPN,and the formation of mineralization nodules in Saos-2 osteoblastic cells. Mechanical strain may play a very important role in proliferation and differentiation of osteoblasts.2. The role of Wnt/β-catenin signaling in mechanotransduction of Saos-2 osteoblastic cellsObjective:To investigate the effects of mechanical strain onβ-catenin in Saos-2 osteoblastic cells and to examine whether mechanical stimuli modulate Wnt/β-catenin signaling and its mechanism. Method:Saos-2 osteoblastic cells were subjected to 12% elongation by using Flexcell strain loading system. Cells were immediately fixed and processed for immuno?uorescence and tested for translocation ofβ-catenin.β-catenin and target genes of Wnt/β-catenin signaling COX-2, cyclinD1, c-fos, c-Jun mRNA expression were examined by RT-PCR. Saos-2 osteoblastic cells were co-transfected with a wild type or mutant Tcf luciferase reporter gene and Renilla luciferase plasmid. Posttransfection cells were subjected to 1 hour of 12% elongation. Luciferase activities were then assayed.β-catenin and E-Cadherin activities were determined by Western Blot analysis. The interaction betweenβ-catenin and E-Cadherin was determined by Co-immunoprecipitation. Result: In cells subjected to mechanical strain, there appeared to be more nuclearβ-catenin than in cells held in static culture. Mechanical strain induced a significant increase in Tcf reporter gene activity (P<0.01). The expression of target genes COX-2, cyclinD1, c-fos, c-Jun mRNA in Saos-2 osteoblastic cells were promoted by 12% mechanical strain elongation (P<0.01). Co-immunoprecipitation analysis revealed less E-cadherin associated withβ-catenin in cells subjected to mechanical strain (P<0.01). Conclusion:Our data indicate that 12% elongation mechanical strain can activate Wnt/β-catenin signaling and decreases the interaction betweenβ-catenin and E-Cadherin in Saos-2 osteoblastic cells. Wnt/β-catenin signaling plays an important role in mechanotransduction of osteblasts.3. Two-dimensional electrophoresis analysis of Saos-2 osteoblastic cells stimulated by mechanical strainObjective: To establish two-dimensional eletrophoresis (2-DE) images for Saos-2 osteoblastic cells stimulated by mechanical strain. Methods: Saos-2 osteoblastic cells were subjected to 12% elongation for 24 hours by using Flexcell strain loading system. Cells were immediately lysated and the total protein of the Saos-2 osteoblastic cells was extracted. Total protein content was determined in cell cultures lysates using Bradford protein assay. 2-DE was carried out according to procedures. The silver stained 2-DE were scanned and analyzed with ImageMaster 2D software. Results: Saos-2 osteoblastic cells stimulated by mechanical strain showed significant difference in a 2-DE system compared with control group. A total of 1031±41or 928±25 protein spots were resolved by 2-DE of controls or experimental groups extractions,respectively. We found 30 statistically significant change amount of protein compared with controls samples. Conclusion: The Saos-2 osteoblastic cells stimulated by mechanical strain could express the differentially expressed proteins, and 2-DE technique is effective to investigate mechanics of osteoblasts in bone remodeling.4. MALDI-TOF-MS analysis of differential expression protein in Saos-2 osteoblastic cells stimulated by mechanical strainObjective: To identify the differentially expressed proteins of Saos-2 osteoblastic cells under mechanical strain loading and to clarify the major proteins involved in the molecular mechanism of osteoblasts under mechanical strain loading. Methods: Matrix-assisted laser-desorprtion ionization time of flight mass spectrotmetry (MALDI-TOF-MS) were used to separate and identify proteins in the whole cell lysate and peptide mass fingerprinting (PMF).The proteins involved in Saos-2 osteoblastic cells under mechanical strain loading were identified and searched with proteome NCBI database. Result: The PMFs of 30 proteins were obtained through MALDI-TOF-MS analysis and 26 proteins were further analyzed with the protein and peptide databases. These bone remodeling associated proteins fell into 6 groups, including Stress reaction (cyclophilin, Chain A, Dopamine Quinone Conjugation To Dj-1), energy metabolism(mitochondrial ATP synthase, Alpha-enolase), cell proliferation( NUMA1, peroxiredoxin 1), reconstruction of cytoskeleton(Cofilin 1), signaling(Protein phosphatase 1, Nuclear receptor interacting protein 1, Proteasome Activator) and osteogenesis(Elongation factor 2, eukaryotic initiation factor 2, Annexin I). Conclusion: Our study provided fundamental information on the differential expression of proteins of osteoblast under mechanical strain. These proteins described here may play important roles in mechanisms of biological bone remodeling process.