| Background and purpose:The healthy periodontal condition is the guarantee for the tooth to bear the occlusal or orthodontic force.Both orthodontic force and occlusal force of physiological range can cause periodontal tissue remodeling,including periodontal ligament and bone tissue.It is a non-infectious,minimally invasive and traumatic inflammatory reaction process.Studies have shown that mechanical or inflammatory stimulation can cause changes of periodontal local microenvironment,such as changes in blood flow and expression of various inflammatory factors,causing periodontal tissue remodeling.The orthodontic or occlusal force beyond the physiological range may cause an irreversible damage of periodontal tissue.However,the physiological range for periodontitis patients would be greatly reduced.For periodontitis susceptible patients,the steady-state of periodontal microenvironment likes more easily to be damaged.The absorption and dispersion of mechanical force is necessary for the PDL to maintain periodontal stability.Studies have shown that periostin plays a very important role in this process.Periostin is named because of its specific distribution in periosteum and periodontal ligament.Recent studies found that periostin distributes in skeletal muscle,myocardium,lung,tendons and other organs rich for fibroblastic tissue and plays a very important role in organizations that continue to accept mechanical stress stimulation.As an important extracellular matrix protein,the expression of periostin is obviously superior in developing individuals to adults,but it increases in vascular injury and fracture.Periostin also plays a very important role in maintaining the integrity of the PDL.It was pointed out that the expression of periostin in the PDL increased significantly at the initial stage of orthodontic tooth movement.The cells that express the periostin in periodontal tissue are mainly fibroblasts,osteoblasts and osteocytes.Periostin is involved in the synthesis of extracellular collagen fibers and participates in the sense of mechanical stress in periodontal and bone tissues.It can also promote the migration and adhesion of many cells by binding to the receptor of integrin βvβ3 Studies showed that the rate of orthodontic tooth movement decreased after periostin knockdown,and the expression of Sclerostin and metabolism of collagen disappeared in the pressure and tension side of the teeth.At the same time,periodontal tissue defects in POSTN(-/-)mice,and the occlusal and orthodontic force in physiological range are all likely to cause alveolar bone and other periodontal tissue defects.Periodontal ligament stem cell(PDLSCs)has potential of multi-differentiation and periodontal regeneration,which play an important role in periodontal remodeling and periodontal tissue regeneration.It has been found that periostin can promote the migration,homing and osteogenic differentiation of BMMSCs,but the effect of periostin on PDLSCs has not been reported.Based on the above findings,wehypothesized that periostin can participate in periodontal tissue remodeling by regulating the biological behavior of the PDLSCs.Therefore,we intend to explore the effect of POSTN on proliferation,migration,adhesion and osteogenic differentiation of PDLSCs,so as to further understand the mechanism of periostin involved in periodontal remodeling.Methods:Standard isolated teeth(teeth extracted for orthodontic or healthy impacted wisdom teeth under 25 years of old)were clinically collected.Remove the excess of epithelial tissue and inflammatory granulation tissue,flushing periodontal tissue alternately with culture medium and PBS containing 20%penicillin and streptomycin.Trim the periodontal tissue in tUIe middle 1/3 root of the tooth into size of 1mm3,then selected and evenly placed in the bottom of the culture bottle by tweezers.Human periodontal ligament cells were isolated and cultured in vitro by tissue explant method,and the success rate and viability of cells were increased by using high concentration of serum medium.After 1-2 weeks of routine culture,cells were gradually separated from the edge of the tissue under inverted microscope,and the cells can be subcultured after the bottom was filled.hPDLSCs were isolated by the limiting dilution method.Surface markers were quantified by flow cytometry.P4 or P5 of hPDLSCs were selected for the experiment.Cell-counting kit-8(CCK8),Transwell,cell migration assay and cell adhesion test were used to detect the proliferation,migration and adhesion of hPDLSCs cultured with different concentrations of recombinant human periostin.ALP activity assay,alizarin red staining were used to determine the effect of periostin on osteogenic differentiation of PDLSCs.Meanwhile,the mRNA expression changes of Runx2,ALP,OPN,OCN,osterix and BSP in hPDLSCs were detected by qRT-PCR to preliminarily explore the mechanism of the effects of periostin on biological behavior of hPDLSCs.Results:(1)Isolation and identification of hPDLSCs:The primary cells of the periodontal membrane tissue swam out of the tissue block around 7 days and fulfilled the culture bottle after 2-4w.The cells were radiated and vortexed at the center of the tissue.After the passage,the cells grew exuberantly and the character was stable.Cell surface molecules was identified by flow cytometry.After induction of osteogenesis and adipogenesis,cells showed obvious mineralized nodules and lipid droplets.(2)CCK8 experimental results showed that:Compared with the control group,20ng/mL,50ng/mL,100ng/mL rhPOSTN can promote cell proliferation of periodontal ligament stem at 24h,48h,72h,and it increases with the increase of concentration.But the 250ng/mL rhPOSTN can inhibit the proliferation of hPDLSCs.(3)Transwell experiment,scratch test,adhesion experiment:Transwell and cell scratch test showed that compared with control group,50ng/ml and 100ng/ml rhPOSTN could significantly improve the migration ability of hPDLSCs.Cell adhesion assay showed that the periostin could obviously promote the initial adhesion and extension of the cells.(4)ALP activity assay,alizarin red staining and qRT-PCR experimental results:Periostin can promote the ALP activity in the cells and supernatant and improve the mineralization ability of the hPDLSCs after vitro osteogenic induction.PCR results showed that the mRNA expression of ALP,osterix,Runx2,OPN,OCN and BSP of hPDLSCs was improved by periostin at different degree.Conclusion:(1)Periostin can promote the proliferation of the periodontal ligament stem cells within a certain concentration,but the high concentration of periostin inhibits the proliferation of the periodontal ligament stem cells.(2)Periostin can stimulate periodontal ligament stem cell migration and adhesionin the appropriate concentration range,and the promoting effect presented a concentration gradient.(3)In the suitable concentration range,periostin can promote the osteogenic differentiation of the periodontal ligament stem cells by promoting the activity of ALP,the mineralization ability and the expression of various bone related factors. |
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