Effects Of Cyclic Tensile Stress On Expression Of Periostin And Collagen Type â…  In Human Periodontal Ligament Cells

Background and objection:Periodontal tissue remodeling under stress state is the biological basis of orthodontic clinical treatment, As stress-sensitive cells, hPDLCs are the main component of periodontal tissue, through the signal transduction system into a biological signal which can cause a series of biological reactions after mechanical force stimulation. hPDLCs not only participate the synthesis of hPDLCs in the extracellular matrix of most various types of collagen and proteoglycan, but also trigger a number of extracellular and intracellular signaling network through secretion and synthesis of a variety of cytokines and enzymes, together involved in the regulation of metabolism and periodontal tissue remodeling. Studies have shown that tumor necrosis factor, fibroblast growth factor, prostaglandins, etc, which are related to periodontal tissue remodeling.Periositn (PN), bunched protein (fasciclin) family, a molecular weight of 90kD, from the amino terminus (EMI domain),4 repeated bundling protein domain (fas1 domain) and carboxy-terminal (C-terminal) form, in which the tooth periodontal ligament and periosteum specific expression, is recognized as the extracellular matrix protein, known as periodontal ligament cell properties evaluation of the key factors is one cell marker gene ligament.Its supporting cell adhesion, regulating fiber formation and cross-linking of collagen plays an important role in the integrity of the periodontal tissues, tooth development and eruption, as well as tissue repair and regeneration caused by mechanical stress,It is considered to be a kind of necessary extracellular matrix proteins for tissue integrity and maturity of periodontal ligament homeostasis.Between PN and collagen type Ⅰ there are a common distribution area, which can directly combine collagen type Ⅰ, The study also found that the diameter of the collagen fibers in PN-knockout mouse decreaseed, reducing the level of cross-linking of collagen, suggesting that PN regulating collagen type Ⅰ synthesis, thus becoming an important mediator of the biomechanical properties of fibrous connective tissue. PN is currently only limited in vivo experimental studies in rodents, but in vitro studies on the gene and protein levels of orthodontic tooth movement of hPDLCs on PN expression has little reported.In this study, Flexcell-5000 Tension System stress loading system for cultured hPDLCs loaded mechanical force simulates the process of orthodontic tooth movement, qRT-PCR and Western blot were used to detect expression of PN mRNA and protein; qRT-PCR was used to detect expression of collagen type Ⅰ mRNA. To preliminarily explore the mechanism of PN and collagen type Ⅰ on the cell mechanics signal transduction, to study the signal transduction pathway between PN and collagen type Ⅰ in the study of molecular mechanisms of orthodontic force periodontal tissue remodeling of the foundation, to further clarify the conversion mechanism and the adaptability of periodontal ligament fibroblasts during orthodontic force and molecular mechanisms of mechanical signals transtruction in cell for theoretical basis.The paper includes the following two parts:The first part Construction of culture-tensile stimulate models of hPDLCsObjective:Modified tissue explant collagenase method can be successfully cultivated hPDLCs, by observing cell morphology, proliferation, mineralization ability and identified by immunohistochemistry. hPDLCs loaded mechanical force were observed cells growth, morphological changes and proliferation, to determine whether mechanical stimulation model was successfully constructed.Methods:1. Healthy permanent wisdom teeth and premolars extracted for orthodontic reasons were collected from young adults, scraping periodontal ligament tissue from the roots, hPDLCs were isolated and cultured by combination of explants method and enzymatic separation method.2. The cell morphology was observed under the phase contrast microscope; For MTT assays, the cells were seeded, the absorbance of each sample was analyzed at 490 nm using a microtiter plate reader at the same time in 1 to 7 days, then we have astatistical analysis and paint the growth curve; The cells of the third generation were mineralization induced for 21 days, fixed, stained with alizarin red, then mineralized nodules were observed; cells detected by immunochemical methods of expression of vimentin and keratin.3. In this experiment the cells were randomly divided into four experimental groups and one control group. Experimental group were applied with 6h,12h,24h,48h, with no loading group (0h) for the control group.4. This study used Flexcell FX-5000 Tension System stress loading system. The setting is:sine wave, the maximum deformation of 10%, the frequency of 0.5 Hz. hPDLCs morphology and growth was observed under the phase contrast microscope after cells loaded cyclic tensile stress5. MTT for hPDLCs loaded stress:four experimental groups after cyclic tensile stress,every hole by adding 5g/1 of MTT 200ml, cultured 4h, added 3ml of dimethyl sulfoxide (DMSO), thoroughly shaken 10min, the crystal was fully dissolved, then measured absorbance of each well 490nm wavelength (OD) by enzyme-linked immunosorbent assay.Results:1. hPDLCs had higher growth rate and survival rate cultured by modified tissue explant collagenase method. Morphological observation showed:The cells were spindle or start-shaped, bigger volume and fibroblastic-like. The represented cells attached after 24h, gradually extended into irregular circle, polygon or spindle, and showed radial proliferation.2. The cells entered logarithmic phase after cultured for 24h that meaned the cells activity was good; The mineralization nodules could be detected in the presence of mineralization medium after 3 weeks of culture; Immunohistochemistry showed vimentin intracellular staining cells Pulp visible yellow granules, keratin staining cytoplasm no yellow particles. All albove showed that the cells from mesenchymal.3. hPDLCs loaded cyclic tensile stress were in good condition, long shuttle; the arrangement of cells gradually palisade parallel growth of in response to the force direction of arrangement, and consistent with the direction of the force.4. MTT analysis for hPDLCs loaded stress:Compared with the control group, proliferation of hPDLCs of experimental group decreased at 6h, proliferated manifestly at 12h, then reached the peak at 24h, at 48h proliferation decreased.5. Culture-tensile stimulate models of hPDLCs were established successfully.The second partEffect of cyclic tensile stress on cells expression of PN and collagen type Ⅰ in hPDLCsObjective:To observe effect of cyclic tensile stress on cells expression of PN and collagen type Ⅰ in hPDLCs at different times, to preliminarily explore the mechanism of PN and collagen type Ⅰ on the cell mechanics signal transduction.Methods:1. In this experiment the cells were randomly divided into four experimental groups and one control group. Experimental group was divided into cyclic tensile stress is applied with 6h,12h,24h,48h, with no loading group (Oh) for the control group.The experimental groups were applied cyclic tensile stress(sine wave, the maximum deformation of 10%, the frequency of 0.5 Hz).2. qRT-PCR:The total RNA of cells was extracted using the Trizol Reagent, and the first-stand cDNA synthesis was performed according to the relative manufacture’s protocol. SYBR Green kits were used to detect real-time polymerase chain reaction. The relative quantitative method 22-ΔΔCt was used to accurate expression of PN and collagen type Ⅰ.3. Western blot:Cells were rinsed and scrapped in radioimmunoprecipitation assay (RIPA)lysis buffer supplemented with protease inhibitors. The total protein was quantitated using BCA assay. Equivalent protein amounts were separated in 10% SDS-polyacrylamide gels and transferred to PVDF membranes. The blots were then hybridized with specific primary PN antibodies, Actin and secondary antibodies labeled with an IRDye800-conjugated affinity-purified anti-rabbit/mouse immunoglobul in M antibody. The membrane was washed several times and scanned using an Odyssey infrared imaging system at a wavelength of 800μm. The data were analysed with ImageJ software.4. Repeat the results of three times, using SPSS 13.0 software for statistical analysis, and the results measured normality and homogeneity of variance test. Among groups were compared using ANOVA, if homogeneity of variance between groups are using LSD test, variance arrhythmia selection Dunnett’s T3 test. Test level for a=0.05, P<0.05 considered significant.Results:1. qRT-PCR analysis:Expression of PN mRNA:PN mRNA expression in normal human periodontal ligament cells. Compared with the control, after 10% cyclic stretch for 6 h PN mRNA expression was decreased, then upregulated after 12 hours; For 24h, PN mRNA expression was in the highest level (P<0.05), but after 48 h began to decline; Expression of collagen type Ⅰ mRNA:Compared with the control, after 10% cyclic stretch for 6 h,12h, collagen type Ⅰ had no different;but, For 24h, collagen type Ⅰ mRNA expression began to increase, then up to the highest level (P<0.05).2. Western blot analysis:Compared with the control group, after 10% cyclic stretch for 6 hours, expression of PN protein was decreased, then upregulated after 12 hours; For 24hours, PN protein expression was in the highest level (P<0.01). After 48 hours, PN protein expression began to decline, consistent with the expression level of the control group (P>0.05).Conclusions:1. Modified tissue explant collagenase method can be successfully cultivated hPDLCs, and culture-tensile stimulate models of hPDLCs were established successfully in vitro.2. Within a certain time, a certain periodicity tensile stress of 10%,and frequency of 0.5Hz can promote hPDLCs expression of PN and collagen type Ⅰ.3. Under cyclic tensile stress, PN and type Ⅰcollagen involved in hPDLCs mechanical stress signaling processes.4. PN and collagen type Ⅰ may be as the main factor involved in the regulation of orthodontic periodontal tissue remodeling.