The Effects Of DNCPs On Bone Formation Of BMSCs In Vitro And Bone Regeneration After Sinus Floor Elevation And Simultaneous Implant Placement In Dogs

Maxillary sinus floor elevation and simultaneous implant placement with bone augmentation is a rapid and effective solution for patients with insufficient bone mass in the posterior maxillary region.At present,the commonly used bone substitute materials are only osteoconductive and weak in an osteoinductive activity.They are different in absorption and degradation rate and cannot promote bone formation and mineralization around the implant in a short term.The osteogenic ability and degradation performance of bone substitute materials can be improved by adding seed cells and growth factors with osteoinductive activity into bone substitute materials using tissue engineering technology.Dentin non-collagen proteins(DNCPs)are a group of complex proteins secreted by dentin,including dentin-specific proteins,mineralized tissue proteins and various growth factors,which participate in the composition of the osteogenic microenvironment and play a regulatory role in bone formation and mineralization.In this study,DNCPs were extracted from canine teeth,made into a composite with bone substitute ?-tricalcium phosphate(?-TCP)and combined with bone marrow mesenchymal stem cell(BMSCs)to construct a new tissue engineering bone.The effects of DNCPs on bone formation of BMSCs in vitro and bone regeneration after sinus floor elevation and simultaneous implant placement in dogs were explored.The specific contents of the experiment were divided into the following four parts:Experiment IThe effect of ?-TCP on bone formation of BMSCs in vitroObjective:At present,the bone substitute material deproteinized bovine bone mineral(DBBM),which was commonly used in the clinic,degrades slowly and forms new bone slowly in the maxillary sinus.In a short period of time,there is not enough osseointegration around the implant.The three-dimensional porous structure of synthetic bone substitute ?-TCP is similar to that of cancellous bone,and it is an ideal osteogenic spatial structure.In this part of the experiment,the effects of DBBM and?-TCP on BMSCs were compared in vitro,the suitable scaffold was selected for subsequent experiments,and the key regulatory factor for inducing BMSCs on osteogenic differentiation was explored.Methods:Rat bone marrow was isolated and cultured in vitro and inoculated on DBBM and ?-TCP respectively.The effect of cell inoculation on scaffolds was observed by a scanning electron microscope(SEM).The cell adhesion rate was detected by crystal purple staining,the proliferation of cells was detected by CCK8 assay,and the osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase(ALP)staining and activity.The mRNA expressions were detected in three factors:ALP,Core-binding factor alphal(Cbfal)and the osteocalcin(OCN)gene,by Real-time RT-PCR test The protein expressions of ALP,Cbfa1 and OCN were detected by Western blot test.BMSCs were treated with BMP1 siRNA to silence the BMP1 gene.The silenced group and the control group were inoculated on the surface of ?-TCP material respectively.The relative expression levels of ALP,Cbfal,OCN gene and protein were detected.Results:The adhesion rate of the P-TCP group was higher than that in DBBM group(p<0.05)24 hours after inoculating cells.SEM observation suggested that the two materials showed three-dimensional porous structure.The pore size of ?-TCP material was larger than that of DBBM.BMSCs can attach to the surface of both materials,and there was no significant difference in cell morphology and attachment mode between the two materials.The results of CCK8 analysis showed that there was no significant difference between the two groups on the first day of inoculation.From the 3rd day,the cell proliferation efficiency of ?-TCP group was higher than that of DBBM group,and the difference was statistically significant(p<0.05).The results of ALP staining and activity revealed that the expression and activity of ALP in BMSCs inoculated with the two groups increased with the time.The expression and activity of ALP in ?-TCP group were higher than those in DBBM group at each time point(p<0.05).The results of Real-time RT-PCR and Western blot test showed that the?-TCP group was more able to up-regulate the expressions of bone formation related factors ALP,Cbfal and OCN than those of the DBBM group(p<0.01).When the cell line silencing BMP1 gene was inoculated on the surface of ?-TCP material,the expressions of bone formation related factors ALP,Cbfal and OCN were down-regulated(p<0.01).Conclusion:Compared with DBBM,?-TCP is a suitable scaffold material for BMSCs.?-TCP promotes osteogenic differentiation of BMSCs through the regulation of BMP1.Experiment IIThe effects of DNCPs on bone formation of BMSCs in vitroObjectives DNCPs are a group of complex proteins secreted by dentin which play a crucial role in the formation of bone and dentin.At present there are few reports of DNCPs as a compound growth factor used in tissue engineering bone.The osteogenic promoting mechanism of DNCPs remains unclear.This part of the experiment discusses the effects of DNCPs on bone formation of BMSCs and provides the growth factor for subsequent experiments.Methods:DNCPs were extracted from canine dentin and were analyzed by Western blot test.Bone marrow was extracted from the canine ilium and BMSCs were isolated and cultured by density gradient centrifugation.DNCPs with the concentration of 10?g/mL were used to induce BMSCs,and the proliferation ability of cells was detected by CCK8 assay.The osteogenic differentiation ability of BMSCs was detected by ALP staining and activity,and the mineralization ability of BMSCs was analyzed by Alizarin red staining and cetylpyridine chloride quantitative detection.The expressions were detected in three osteogenic related factors:RUNX2,OPN and COL-1,by Real-time RT-PCR and Western blot test.The secretion of transforming growth factor 1(TGF-?1)in culture medium was detected by Elisa kit.The expression of signal transduction molecule pSmad3,which was the downstream signaling molecule of TGF-?1,was detected and the expressions of osteogenic related factors were detected by blocking Smad3 phosphorylation by inhibitor,to explore the signal pathway which promoted the osteogenic differentiation of BMSCs by DNCPs.Results:The protein expressions of OPN,DMP1,BSP and BMP1 could be found in DNCPs.The results of CCK8 showed that there was no significant difference between the experimental group and the control group when adding DNCPs at the concentration of 10 ?g/mL(p<0.05).The results of ALP staining and activity on the 7th and 14th day after induction showed that DNCPs could up-regulate the expression of ALP in BMSCs.Compared with the eontrol group,the differences between the two groups and the time points were statistically significant(p<0.01).The results of Alizarin red on the 14th and 21st day after induction showed that the number and size of calcium nodules in DNCPs group were better than those in control group.The concentration of calcium ion was also higher than that of the control group,the difference was statistically significant(p<0.01).The results of Real-time RT-PCR and Western blot test showed that DNCPs could up-regulate the expressions of osteogenic related factors RUNX2,OPN and COL-1(p<0.01).The results of mechanism analysis showed that the concentration of TGF-pl in experimental group added with DNCPs was higher than that in control group,and the difference was statistically significant(p<0.01).The results of Westerm blot test showed that DNCPs could up-regulate the expression of pSmad3,which was significantly different from that of the control group(p<0.01).After Smad3 phosphorylation was blocked by inhibitor SIS3,the expressions of osteogenic related factors in DNCPs-SIS3 group were significantly lower than that in DNCPs group(p<0.01),but there was no significant difference compared with the control group(p>0.05).Conclusion:DNCPs promote osteogenic differentiation of BMSCs through TGF-?1/Smad3 signal pathway.Experiment ?The effect of DNCPs-?-TCP composite on bone formation of BMSCs in vitroObjective:DNCPs with different mass ratios were combined with ?-TCP to screen the optimal mass ratio of protein to material.The cell compatibility and osteogenic ability of BMSCs inoculated on the composite were measured,which provided the composite for subsequent maxillary sinus elevation and simultaneous implant placement in dogs.Methods:DNCPs-?-TCP composites with DNCPs content of 2.5%,5%and 10%were prepared by mixed freeze-drying method.The release rate of DNCPs from the 1st to 28th day was detected in simulated body fluid,and the best mass ratio was screened.BMSCs was inoculated on the composite to detect its adhesion,proliferation and differentiation and the morphology of cytoskeleton was observed by SABC-Cy3 fluorescence staining.The expressions were detected in three related factors:RUNX2,OPN and COL-1,by Real time RT-PCR and Western blot test.Results:The simulated release results in vitro showed that the release rate of DNCPs-?-TCP composites was highest in the first 3 days.The release rate remained higher in the following days until the 10th day,then slowed down gradually,and there were unreleased DNCPs in the composites with three contents on the 28th day.The release effect of 5%DNCPs-?-TCP composites in vitro was the best,and there was still a high concentration of DNCPs in the composites on the 28th day.The results of adhesion and proliferation of BMSCs showed that there was no significant difference between DNCPs-?-TCP composite and ?-TCP alone(p>0.05).But DNCPs-?-TCP composite could up-regulate the ALP activity of BMSCs and promote the differentiation of BMSCs,which was significantly different from that of the control group(p<0.05).Fluorescence staining showed that BMSCs grew well in both experimental group and control group,and there was no significant difference between the two groups.The results of Real-time RT-PCR and Western blot test showed that compared with ?-TCP alone,DNCPs-?-TCP composite could up-regulate the expressions of osteogenic related factors RUNX2,OPN and COL-1.Conclusion:?-TCP can be used as the carrier material of DNCPs,and the release effect of 5%DNCPs-?-TCP composite in vitro is the best.DNCPs-?-TCP has good cell compatibility with BMSCs,which can promote osteogenic differentiation of BMSCs in vitro.Experiment IVThe osteogenic effect of DNCPs-?-TCP composite combined withBMSCs after maxillary sinus floor elevation and simultaneous implant placement in dogsObjective:To evaluate the osteogenic effect of DNCPs-?-TCP composite combined with BMSCs after maxillary sinus floor elevation and simultaneous implant placement in dogs.Methods:Bilateral M1 extractions were performed in 6 beagle dogs,and maxillary sinus floor elevation and simultaneous implant placement were performed 3 months later.Design a double-blind controlled experiment.DNCPs-?-TCP-BMSCs and?-TCP-BMSCs were implanted into the bilateral maxillary sinus of each beagle dog.The implant stability index(ISQ)was measured during operation,6 weeks after the operation and 3 months after the operation.The bone implant contact and bone density of the implant bone interface were detected by CT Scan.The ossification in maxillary sinus and mineralization around the implant were detected by histology.The bone implant contact rate was detected by bone histomorphometry.Results:The results of ISQ analysis suggested that 6 weeks after the operation,the experimental group of DNCPs-?-TCP-BMSCs showed a slow upward trend,while the control group of ?-TCP-BMSCs showed a downward trend.3 months after the operation,the experimental group increased significantly,the value of ISQ was as high as 78.3±4.3,the control group increased slowly,and the ISQ was 58.7±7.9,and the difference was statistically significant(p<0.01).CT observation showed that there was no obvious radiation transmission around the implant and no abnormal image in maxillary sinus.The CT value of implant bone interface in DNCPs-?-TCP-BMSCs group was significantly higher than that in ?-TCP-BMSCs group,and the difference was statistically significant(p<0.01).HE staining of decalcified sections was performed on the bone tissue at the central position of the peri-implant maxillary sinus.The results showed that the bone matrix in DNCPs-?-TCP-BMSCs group was dense and orderly,the bone tissue was mature and the bone trabeculae in the bone marrow cavity were thick and formed.In the control group of ?-TCP-BMSCs,the bone matrix was disordered,the bone tissue was immature and the bone trabeculae in the bone marrow cavity were scarce.The hard tissue sections with implants were stained with Masson.The results showed that the mature bone tissue around the implant thread was mostly red-stained in the experimental group of DNCPs-?-TCP-BMSCs and the structure of bone trabeculae is clear and orderly and multiple bone marrow cavities can be seen.In the control group of ?-TCP-BMSCs,there are obvious blue braided bone strips around the thread of the implant and the bone trabecula is irregular and the bone marrow cavity is rare.Bone histomorphometry results showed that the bone contact rate of DNCPs-?-TCP-BMSCs group was significantly higher than that of P-TCP-BMSCs control group(p<0.01).Conclusion:DNCPs-?-TCP-BMSCs tissue engineering bone can promote early osteogenesis in the maxillary sinus and early bone mineralization around the implant.