Experimental Studies On Tissue Engineered Bone Repairing Rabbit Mandibular Defect And Implants Embedding Synchronously

IntroductionAdvanced and effective Functional reconstruction of jaw defect accompanied by absence of tooth caused by various kinds of reasons in clinical, i.e., reparation of jaw defect with simultaneous placement of dental implants, so that can shorten the time of tooth loss, lessen suffering of many times operation and recover functions such as articulation, chew, deglutation and pronunciation early, is a tough problem which maxillofacial surgeons, dental implants dentists and prosthodontists are facing with. Recently tissue engineering had a rapid development so did bone tissue engineering a branch of tissue engineering. The technology of bone tissue engineering, which used seed cells, scaffold and growth factors as its core content, construct cell-scaffold compound, i.e., tissue engineered bone in vitro by micro-trauma or even no trauma methods. Tissue engineered bone can substitute autogenous bone, allograft bone and xenograft bone for repairing of bone defect and be considered of the most potential and perspective bone defect repairing material. In order to improve the success ratio of repair jaw defect and implant embedding synchronously, ideal bone defect repairing material is needed so that dental implants obtain satisfactory initial stability, but the surface character of dental implants also play a very important role. In this study, we used BMSCs as the seed cells, nHAC as the scaffold material and PRP as the source of growth factors and constructed tissue engineered bone in vitro. We used this kind of tissue engineered bone to repair box shape defect of rabbit mandibular and embedded TA2 pure titanium implants at the same time, which had been treated with SLA and then formed HA coating by IBAD technology. There are five parts in our study:Part one: The effects of PRP on biologic characteristics of BMSCs of rabbit cultured in vitroObjective To explore the effects of PRP on biologic characteristics of rabbit BMSCs cultured in vitro. Methods Rabbit BMSCs were cultured in vitro and divided into experimental group and control group. In experimental group, the culture medium was contained 1% PRP. The morphological characteristic were observed, cell-DNA distribute and the proliferation index of cells were tested, ALP stain and activity, OCN content and the mRNA level of opn were measured. Results The experimental group cells had the osteoblasts-like morphological characteristic; the proportion of S phase were increased significantly and the proliferation index of cells were 33．15(?)1．02 compared with that of control group 22．89(?)1．24 (P <0．01) after treated with PRP for 7 days; ALP stain of experimental group cells were positive, ALP activity and OCN content of experimental group cells increased greatly compared with that of control group; the relative expression level of opn mRNA was 3．48 times greater than that in control group. Conclusions PRP could improve the proliferation of rabbit BMSCs and increase the potential of rabbit BMSCs induced into osteoblasts in vitro.Part two: Effects of nHAC on differentiation of BMSCs intoosteoblasts induced by PRP in vitroObjective To explore the effect of nHAC worked as scaffold material on PRP inducing BMSCs into osteoblasts. Methods Rabbit BMSCs co-cultured with nHAC and cultured conventionally were induced by PRP. The growth state of rabbit BMSCs co-cultured with nHAC was observed by scanning electron microscope; to compare with the ability of PRP inducing BMSCs co-cultured with nHAC and cultured conventionally into osteoblasts, ALP activity, OCN content and the mRNA level of opn were measured. Results The osteoblasts differentiated from BMSCs grew well on nHAC; compared with the cells cultured conventionally, ALP activity and OCN content of cells co-cultured with nHAC increased greatly; the relative expression level of opn mRNA under co-culture condition was 4．78 times greater than that under conventional culture condition. Conclusions nHAC as scaffold material could improve the ability of PRP to induce the BMSCs into osteoblasts.Part three: Influence of HA coating on the SLA surface of titanium dental implants on biologic characteristics of MOOBsObjective To explore the effects of HA coating deposited on the SLA surface of titanium dental implants on biologic characteristics of MOOBs. Methods Coating of HA on the SLA surface of titanium dental implants were formed by IBAD method. MOOBs cultured in vitro were seeded onto the surface of HA/SLA-Ti and SLA-Ti, the growth state of MOOBs on the two samples were observed by scanning electron microscope; the proliferation index, ALP activity, OCN content of MOOBs and mRNA relative expression level of opn were compared between two groups. Results MOOBs grew well on the surface of HA/SLA-Ti; the proliferation index, ALP activity and OCN content of MOOBs were obviously higher in HA/SLA-Ti group than in SLA-Ti group; the relative expression level of opn mRNA in HA/SLA-Ti group was 3．25 times greater than that in SLA-Ti group. Conclusions The HA coating formed by IBAD method on the SLA surface of titanium dental implants can improve the ability of proliferation and the expression of osteoblastic phenotype of MOOBs significantly; it will be a potential method to treat the surface of titanium dental implants.Part four: Experimental studies on tissue engineered bonerepairing rabbit mandibular defect Objective To explore the ability of tissue engineered bone constructed with rabbit BMSCs, nHAC and PRP repairing rabbit mandibular box shape defect. Methods The size of 15mm(?)15mm full-thickness defect in rabbit mandibular was repaired by following methods: Group A, repaired with tissue engineered bone; group B, repaired with autogenous iliac bone; group C, repaired with nHAC; group D, control group, do no repair to defect. Rabbit mandibular bone with defect repaired by different methods were harvested after 1(?)3(?)6 months of operation and assessed by means of gross observation, bone mineral density assessment, radionuclide bone imaging and histologic observation. Results Visible new bone tissue was formed in bone defect region of both group A and group B after 6 months of operation, but only chondroid tissue was formed in bone defect region of group C and no bone, only fibrous connective tissue was formed in bone defect region of group D. The results of radionuclide bone imaging showed that after 1 and 3 months of operation, bone metabolism of bone defect region was more active in group A and B than in groupC and D, but there was no significant deviation between group A and B, after 6 months of opration, there was no significant difference of bone metabolism of bone defect region between all groups. The results of bone mineral density assessment showed that bone density of bone defect region in group A, B and C increased gradually after operation and was higher than group D obviously. Bone density of bone defect region in group A and B was higher than group C after 3 months of operation, but there was no significant deviation between group A and B. The results of histological observation showed that little lamelliform preosseous tissue was formed in bone defect region and nHAC began to degrade after 1 month of operation, big lamelliform new bone was formed after 3 months of operation in group A. After 6 months of operationin, nHAC nearly degraded completely in group A and bone defect region was repaired by bone tissue. The ability of bone formation in group A was higher than group C, but there was no obvious different between group A and group B. In group D, bone defect region was repaired by fibrous tissue. Conclusions The ability of repairing bone defect by nHAC compounded with rabbit BMSCs and PRP was similar with autogenous iliac bone but better than only used nHAC, and nHAC could degrade completely in vivo. So tissue engineered bone constructed with BMSCs, nHAC and PRP can be a kind of favorable repairing material of bone defect.Part five: Experimental studies on tissue engineered bonerepairing rabbit mandibular defect and implants withdifferent surface treatment embedding synchronously Objective To investigate osteogenesis and intergration of tissue engineered bone repairing rabbit mandibular defect with HA/SLA-Ti and SLA-Ti implants embedded them synchronously. Methods Tissue engineered bone constructed with rabbit BMSCs, nHAC and PRP was used to repair the size of 15mm(?)15mm full-thickness defect in buccal wall of rabbit mandibular, and HA/SLA-Ti (group A) and SLA-Ti (group B) implants were embedded in them synchronously. After 1(?)3(?)6 months of operation, specimens were harvested and assessed by means of gross observation, X-ray examination, scanning electron microscope observation, histological observation, push-out test and uniaxial pull-out test of implants. Results Visible new bone tissue was formed in bone defect region and fine synostotic interface was formed around implants of group A, but in group B there were fibrous tissue between implants and newly formed bone. The results of X-ray examination showed that in group A and group B, after operation 1 month, bone density of bone defect region was lower than normal and there was almost low density image around implants; after 3 months of operation, bone density of bone defect region in group A and group B was increased, but compared with group B, there was more high density image around implants in group A; after 6 months of operation, bone density of bone defect region was similar with surrounding normal bone tissue in group A and there were all high density image around implants, but in group B, bone density around implants was not uniformity .The results of scanning electron microscope observation showed thatnew bone connected with HA/SLA-Ti directedly in group A and more tightly than in group B. The results of histological observation showed that bone formation on the surface of HA/SLA-Ti was earlier than on the surface of SLA-Ti. The results of push-out test and uniaxial pull-out test of implants showed that there was no obvious difference of push in stress force and extract out loading between group A and group B after 1 month of operation, but after 3 months of operation compared with group B, push in stress force and extract out loading of implants in group A increased obviously and the difference had statistical significance. Conclusions Dental implants embedded synchronously in tissue engineered bone which were used to repair rabbit mandibular defect could formed better synosteosis with newborn bone tissue. The ability of synosteosis of HA/SLA-Ti was better than SLA-Ti, and time of bone healing was earlier than the latter.