The Preliminary Study Of Osteogenetic Effect Of Rat Osteoblast Cell Sheets On The Different Titanium Surfaces

Objective: To observe preliminarily the osteogenetic effect of rat osteoblast cell sheets on the different titanium surfaces for analyzing if the cell sheet of bone tissue engineering technology and titanium surface modification combined utilization could promote the interface form of implant-bone osseointegration.Methods:Separate the skull of 24 h newborn SD rats in vitro to extract the primary cell,Culture and passage to the third generation,then observe the cell morphology on a regular basis and identify osteoblast cell by HE staining,alkaline phosphatase(alkaline phosphatase staining,ALP)staining and alizarin red(alizarin red staining,ARS)staining,type I collagen immunohistochemical.Static water contact Angle test the wettability of different titanium surface.Observate different titanium surface morphology features and cell growth characteristics by scanning electron microscope(SEM).Plant the third generation osteoblast cells which has been identified on the different titanium surface to build osteoblast cell sheets with the density of 0.5x106 cells/ml.Routine observe the sheet morphology and detect the osteoblast cell sheets differentiation by Alkaline phosphatase activity.Detect the Gene and protein expression of OPN,OPG,RANKL,which is Osteogenesis iconic factor,by qRT-PCR and Western blot respectively.Plant the osteoblast cell sheets which culte 14 days in vitro into the subcutaneous tissue of Nude mice.Remove the specimen at the eighth and the sixteenth weeks respectively,evaluate the ectopic ossification ability of osteoblast cell sheets on different titanium surface by HE staining,Von kossa staining and type I collagen immunohistochemical.Results:After cultivation and identifying of primary cells,the morphology characteristics conform to be osteoblasts;The static water contact Angle test of the three titanium surfaces,between 5 °to 90 °,which are hydrophilic surfaces(Less than 5 ° is super hydrophilic,greater than 90 ° is hydrophobic,between 5 °to 90 ° is hydrophilic).the experimental groups are closer to hydrophilic than the control group,but between the tow experiment groups have no statistical significance(P>0.05).Observing by SEM,the group of SMO-Ti is smooth and the osteoblast cells growth were monolayer after cell attachment,cellular matrix incontinuity;the group of AD-Ti are full of array micropores,cell growth intensive orderly,pseudopodia hole edge crawling;the group of SLA-Ti become rough and multistage micropores,which were sizes and uneven.cell three dimensional laminated growth after attachment.Each time point,the experimental group activity were higher than that of the control group(P<0.05).But between the tow experiment groups have no statistical significance on 7 Days or 14 Days(P>0.05),Each groups of osteoblast cell sheets ALP activity on Day 14 is lower than that on Day 7(P<0.05).Regarding RT-qPCR and Western blotting detection:In terms of comparison among groups,mRNA and protein expression of OPN and OPG of osteoblast cell sheets of experiment groups are higher than those of control group on each time point(P<0.05);But between the two experiment groups have no statistical significance on each time point(P>0.05),In terms of Intro-group comparison,mRNA and protein expression of OPN and OPG of osteoblast cell sheets of both experimental groups and control group are raising trend from the 7th day to the 14 th day(mRNA and protein expression of OPN of osteoblast cell sheets of both experimental groups and control group:P<0.05,mRNA and protein expression of OPG of osteoblast cell sheets of control group: P>0.05,mRNA and protein expression of OPG of osteoblast cell sheets of experimental groups:P<0.05).The mRNA and protein expression of RANKL on the different titanium surfaces has no(P>0.05).Tissue staining results of ectopic osteogenesis experiments in vivo shows,control group failed to form bone matrix at the 8th weeks,however,the experimental groups were formed osteoid matrix,but the difference is not significant between experimental groups.At the 16 th weeks,the Three groups all can form class osteoid tissue and osteogenesiscan organization structure which the form and maturity relatively better compared with the control group.Contrasting between experimental groups,comprehensive analysis by the three tissue staining results,formation of bone tissue by AD-Ti and SLA-Ti were not significant difference.Conclusion:(1)AD-Ti and SLA-Ti have better osteogenetic effect than that of SMO-Ti;(2)The effect of the different titanium surface for the OPG/RANKL signaling pathways of osteoblast cell sheets mainly through raising OPG mRNA and protein to adjust the ratio increased,but seemingly has no effect on RANKL mRNA and protein.(3)At the eighth weeks,AD-Ti and SLA-Ti were formed osteoid matrix,but SMO-Ti failed to form bone matrix,all of the groups could formed bone tissue at the sixteenth weeks.