| bjective:To evaluate the effect of human recombinant parathyroid hormone[rhPTH（1-34）]on the adhesion of MC3T3-E1 osteoblasts cultured on titanium surfaces of SLA,establish osteoblast model in vitro and preliminary study on the effect of rhPTH（1-34）,which whether can enhance the expression of integrinα1,α5,β1 in osteoblasts and promote the adhesion of osteoblasts on the surface of implants or not,by which will help to shorten the time of bone integration.Methods:（1）The Titanium disks are treated by the sand blasted Large grits and acid etched（SLA）technology,then text the roughness of Titanium surface and observate of titanium surface morphology by SEM,and Surface elements were analyzed by X-ray spectroscopy（EDS）.（2）The effect of different concentrations of rhPTH（1-34）on the adhesion of MC3T3-E1 cells to the surface of titanium disks at different times were analyzed by CCK8.The experimental group was cultured with a-MEM（10%FBS）containing10-7mol/L,10-8mol/L,10-9mol/L,10-10mol/L rhPTH（1-34）,while the control group was a-MEM（10%FBS）.After pre-cultured for 24h with a-MEM（10%FBS）containing with different concentrate rhPTH（1-34）,osteoblasts were planted on SLA-treated titanium disks.At 0.5h,1h,2h,4h and 8h after planting,assay the amount of MC3T3-E1 cells adhering to the titanium disks were indirectly reflected by CCK8,and the optimal concentration of rhPTH（1-34）was determined for subsequent experiments.（3）Using immunofluorescence and Scanning Electric Microscopy（SEM）,the cell counts and morphological changes of MC3T3-E1 cells cultured on titanium disks were observed.After pre-cultured for 24h with a-MEM（10%FBS）containing with optimal concentration rhPTH（1-34）,osteoblasts were planted on SLA-treated titanium disks.At 4h and 8h after planting,MC3T3-E1 cells were collected to co-culture titanium.Cell counting of MC3T3-E1 following DAPI immunofluorescence staining.Immunofluorescence method was used to observe F-actin and morphological changes of osteoblasts.Scanning electron microscopy was used to observe the morphology and spreading of MC3T3-E1 cells on titanium disks.（4）The expression of integrinα1,α5,β1 m RNA in MC3T3-E1 cells was detected by q PCR（Real-Time reverse transcriptase Polymerase chain reaction）.MC3T3-E1 cells were pre-cultured for 24h with rhPTH（1-34）medium at the optimal concentration.MC3T3-E1 cells were planted on SLA-treated titanium disks.The cultured MC3T3-E1 cells were collected on day 1,3 and day 5,and the expression of integrinα1,α5 andβ1 m RNA in MC3T3-E1 osteoblasts were detected by q PCR.Results:（1）EDS elemental analysis showed that the surface elements of titanium were Ti and Al,the content of Ti was 96.29%,the content of Al was 3.71%;the surface roughness was measured by Ra=1.0708um.（2）The results of CCK8 showed no difference in OD values between 0.5h and1h groups and between control groups（P>0.05）.At 2h,the OD value of the other experimental groups were all increased except the concentration of 10-7mol/L（P>0.05）.Compared with the control group,the OD value in 10-9mol/L and 10-8mol/L group was significantly increased at 8h（P<0.05）,there was significant difference between the two groups（P<0.05）.The comparison of 2h,4h,8h showed that the number of cells with the concentration of 10-9mol/L was the highest compared with the other groups,the difference was statistically significant（P<0.05）.（3）The cell counting results after DAPI staining showed that the number of cell adhesion in the experimental group increased more than the control group at4h and 8h（P<0.05）.（4）Morphological changes:SEM and immunofluorescence staining showed that the experimental group and the control group had different degrees of adherence on the surface of titanium,and the cells grew in monolayer,most of them were triangular or star-shaped.The experimental group showed that the number of osteoblasts adhering to the titanium surface was larger,the filopodia extending out into the foramen of the titanium surface formed a typical polygonal shape of osteoblasts,and the cell body had a large spreading area.The number of osteoblasts in the control group was less,the cell body was spindle-like,the surface initially extended filopodia.The cells in the control group showed oval-shaped parts,the pseudopodia were blunt and dull to form plate-like pseudopods,and the cells were not fully spreading.The experimental group showed complete cell expansion with cross-linking of the typical polygons of osteoblasts and the surrounding cells.The results of immunofluorescence showed that the fibroblasts in the experimental group showed more filopodia and the pseudopods of the experimental group cross-linked with each other at 4h,and the F-action of fibroblast actin filaments could be seen in some cells Beamed,arranged along the direction of cell expansion.The control group cell body part can be seen as oval-shaped,but also extended pseudopodia,but pseudopodia relatively blunt,poor spreading cells;F-action into a network,no obvious bundle structure.At 8h,the cells in the experimental group were longer than those in the control group,the cells were obviously polygonal,and the cell junctions were more closely connected.（5）qPCR showed that on the 1st,3rd,and 5th day respectively,the gene expression levels ofɑ1,ɑ5,andβ1 in the experimental group were significantly higher than those in the control group,and the difference was statistically significant（P<0.05）.Conclusion:（1）The surface of the titanium plate after sandblasting and etching is rough,which is favorable for the agglutination and aggregation of osteoblasts and can be used for the next in vitro experiment.（2）At a certain time,rhPTH（1-34）at different concentrations could promote the increase of the number of MC3T3-E1 osteoblasts adhering to the titanium plate,and use rhPTH（1-34）concentration of 10-9mol/L as the optimal concentration for Follow-up experiment.（3）rhPTH（1-34）can promote the remodeling of F-actin of osteoblasts,accelerate the morphological changes of osteoblasts and accelerate the process of adhesion on titanium plates.（4）Exogenous rhPTH（1-34）can promote the expression of integrinα1,α5,β1 m RNA in osteoblasts and promote the adherence of osteoblasts on the surface of implants.It may play a role in accelerating the process of osseointegration. |
