| With the development of the modern implantation theory, it is more and more popular with the oral doctors and patients in the process of the immediate implantation which is characterized by short course of treatment and small wound. However, due to the shapes of implants and teeth socket morphology can not exactly match, in order to obtain a good primary stability of implants and effective final bone osseointegration, there are stringent requirements for immediate implant indications and surgical technology. In addition, it is also needed to implant bone materials, autologous bone tissues, bone regenerative active stimulating factors(PRF), guided bone regeneration membranes. Besides that, corresponding surgical operations are also neccesary to fill the alveolar bone defect, protect the implant and promote bone regeneration. All of these further increase the complexity of immediate implantation, affecting the widespread use this technology.Self curing calcium phosphate cement(CPC) also known as bone cement. It has good biocompatibility and it is a mature guided bone regeneration material. This material can be mixed into paste and randomly shaped, filling the complex shape of bone defects area. It is also capable of curing at body temperature; and after curing the material is with a certain level of strength under certain pressures. We believe that these characteristics of CPC make it has good application prospects in immediate implantation area. CPC can fill the gap between immediate implant and alveolar socket in addition with enriching the primary stability of implants. It could also be used to make individual prefabricated immediate implants and simplify the immediate implant surgery. However, the CPC lacks the activity of bone regeneration inducing capability, as a result, its degradation rate in vivo is slow. In order to solve these problems, there are some studies to add BMP and other bone growth factors into CPC and achieved good results. However, there are many limitations in the cost of preparation, activity preservation, and safety control for large molecular protein with biological active.P substance(SP) is a neural polypeptide consisting of eleven amino acid sequences. Recently, it is found that SP is also the signal molecules of bone regeneration and remodeling. It can induce bone marrow mesenchymal stem cells to differentiate into osteoblasts and induce angiogenesis. As small molecular peptide, substance P can be synthesized conveniently by peptide synthesizer with a low cost. It has no sp ecial three-dimensional structure and its activity is relatively stable. In addition, its degradation speed is quick in vivo making it safe to be applied. These characteristics of Substance P make it have good application prospects in tissue engineering re search. It could be taken as the active inducing factor of bone regeneration and angiogenesis. Through in vivo releasing of artificial composite scaffolds, SP could promote stem cells, realizing in situ tissue regeneration.In this study, authors explored the ideal method of self curing calcium phosphate loaded with P material, and the feasibility of using it as an auxiliary material for immediate implantation. Modular designing method was used in this experiment. According to the requirements of scaffolds in bone regeneration tissue engineering of porosity and pore diameter, mechanical properties, biological compatibility and bone inducing activity, the natural bone tissue composition, CPC was used as inorganic bone guide scaffold while substance P as the active bone inducing factor, type I collagen as organic bone guide component. Different models were performed to construct combinated material scaffolds and to detect the ultrastructural morphology and mechanical properties. Authors observed the osteogenesis of the composite in vivo by using a rabbit model of mandibular bone defect. According to the above results, the composite material was selected, and then coated on the surface of the pure titanium implant to make prefabricated immediate implants. The rabbit distal femoral metaphyseal bone defect model was constructed. The bone defect diameter and depth were the same with prefabricated immediate implants to stimulate the immediate implantation. To observe the degradation, bone guided or induced regeneration situation of biological material on the surface of pure titanium implants, this study aimed to determine whether the healing results can be achieved in the early stage.Experiment 1: In vivo and in vitro studies on the construction mode of self curing calcium phosphate compositeAims: The natural bone tissue composition was imitated. CPC was taken as bone guided inorganic scaffold while type I collagen as bone guided assisted organic component. Substance P was loaded as a bone inducing active factor. Effective methods of constructing the composite scaffolds with bone regenerative activity were explored.Methods: 1) Preparation: P material was prepared with polypeptide synthesis instrument. Rat tail tendon was extracted to prepare type I collagen solution with a quality volume ratio of 3%. The P material was mixed with a covalent cross-linking agent EDC, NHS with the molar ratio of 1:1.2:0.6. The mixture was added into type I collagen solution in MES buffer; thus P material-type I collagen covalent cross-linking solution was made. The collagen solution was made into collagen sponge, and the collagen sponge was cut into 1x1x1 mm collagen sponge grain. Stainless steel mould was made with 8mm diameter and 2mm thickness. 2) Grouping: The CPC was treated as control group. Mixing the filling composite into the stainless steel mold in a powder to liquid ratio of 3.0 g:1 ml; the cured specimens were labeled as group A. The CPC powder was mixed with the SP-collagen type I collagen covalent cross-linking solution, and the samples were recorded as group B. The P substance was added directly into the 3% collagen solution and then recorded as the group C. Substance P was added into the ordinary curing solution. After mixing the CPC powder, paste was prepared. Then 50% of the total volume proportion of type I collagen sponge particles was added into the mixture and recorded as group D. The P substance was added to the ordinary curing liquid, and then mixed with the CPC recorded as group E. The CPC powder was mixed with 3% collagen solution and the specimens were recorded as group F. The prepared specimens were observed under 300- 1000 times of scanning electron microscope. 3) Animal experiment: 30 adult New Zealand rabbits were divided into 6 groups. After anesthesia, a single cortical bone defect with a diameter of 8mm and depth of 2mm was made in the mandibular. Then the prepared 6 specimens were implanted. Animals were sacrificed after 8 weeks. Micro-CT test was performed to mersure the Tb Th, TbSp, BV/TV and Tb N values; in addition, the degradation rate of the materials was calculated.Results: Scanning electron microscope observing results indicated that after self curing of CPC, there exhibited nano crystalline structure and micron micro pore structure. CPC and SP mi xture did not change the CPC ultrastructure. After CPC and collagen solution mixed, the crystal structure changed but still kept the micron micro pore structure. After mixing CPC and collagen sponge, there showed 300 um level of large pore structure. In vivo experiments showed that loading substance P and mixed with collagen could improve the induced bone regeneration ability of scaffold materials and promote the biodegradation of CPC composite materials. Especially those mixed with collagen sponge with large pore structures. Further improve the in vivo bone transformation process. However, using the covalent cross-linking methods to fix substance P in collagen molecules and CPC composite scaffolds would lead to decreased material degradation and bone regeneration rate.Conclusions: 1) Loading of SP could promote the induced bone regeneration activity and the degradation rate of CPC composite scaffold. 2) Collagen could enhance the induced bone regeneration ability of CPC composite scaffolds, especially in the for m of collagen sponge. 3) Using covalent cross-linking methods to fix substance P in scaffold would affect its in vivo release, thereby further affecting the induced bone regeneration ability of scaffold materials.Experiment 2: In vitro study of se lf curing calcium phosphate composite for imme diate implantationAims: The CPC material was filled into the alveolar socket. The effect of CPC composite material on the early stability of immediate implants was detected in vitro.Methods: 1) Preparation of mold: Maxillary permanent canines were got. After coating their roots with separating agent, the teeth were then inserted into the self curing denture powder / liquid with a 10x10x20 mm cube shape. After the curing of the mold, the teeth were extracted to make in vitro simulated natural socket. 2) Grouping: Pastes from CPC powder mixing with the original curing liquid was attached to the 3.5x10 mmCamlog implant and they were filled into the mold. This group was considered as the control group, group A. According to the same method, the CPC powder was mixed with the 3% collagen solution, which was recorded as group B. The CPC powder was mixed with the SP- type-I collagen covalent cross-linking solution and labeled as group C. The CPC powder was mixed with the 50% total volume type I collagen sponge particles and the group was recorded as the group D. 3) Mechanical experiment of implant extraction in vitro: Making the simulated crown on the implant abutment with light cured resin(maxillary incisor morphology); and a hole was made in the upper 1/3 in the crown. The specimens were fixed on the multifunctional testing machine. Steel wire was made through the hole in the crown to fix it on the lifting device of the testing machine. Set the loading speed of 1mm/min. When the implant has a 2mm position change, it was considered as fully extracted. Four groups of specimens were tested with each group were recorded 10 times, the maximum bearing capacity of each implant was recorded. 4) In vitro implant stability test: T he sensor of the implant stability measuring instrument was installed on the implant body. The measuring probe of the implant stability measuring instrument was taken to close to the sensor; and then the ISQ value was obtained. The measuring was performed in mesio, distal, buccal and palate directions. The average of the ISQ value was recorded as the final value.Results: The maximum bearing capacity of group B and C group were increased compared to the control group; while the mechanical properties of group D was significantly decreased compared with the control group. The results showed that the ISQ value in the group D(68.5±1.9) was lower than that in the control group(85.6±3.7); but it was still higher than the lowest standard value(65) for the initial stability of the implant.Conclusions: 1) The mechanical properties of CPC-P composites with large pore structure decreased; but in vitro experiments showed that it could still provide basic initial stability support for immediate implants. 2) Covalent cross-linking fixed substance P with collagen CPC composite material has high mechanical strength; it could guarantee the immediate implant has a better stability.Experiment 3: In vivo study of self curing calcium phosphate composite for imme diate implantationAims: In the simulation of immediate implant in vivo, CPC composite was used to coat the titanium implants. The feasibility of gaining osseointegration of pure titanium implant through induced bone regeneration was studied.Methods: 1) Preparation and processing 10 mm long pure titanium implant whose roots(thread) with a diameter of 4mm, length of 6mm. Its crown(non threaded portion) was with a 4 mm length and 2.5 mm hexagon in cross-sectional face. According to the results of experiment 1 a nd experiment 2, CPC from group A and CPC composite in group D in experiment 1 were used to coat the threaded part of the implant body with a diameter of 8mm and length of 6mm cylinder. The crown part of the implant was exposed. The two groups were assigned as the control group and the experimental group respectively. 2) 12 adult New Zealand rabbits were divided into 2 groups with 6 rats in each group. After anesthesia, incision was made along the rabbit femoral metaphyseal line to expose the inside face of the rabbit femoral metaphysic. Experimental specimens were implanted with implants from the 2 groups in the bone defect area which had a diameter of 8 mm and a depth of 6 mm. At the end of the 4th weeks, 3 rabbits were sacrificed in each group and the rest 3 rats in each group were sacrificed after 12 weeks. Take out the rabbit femoral metaphysic to Micro-CT scanning of BV/TV, TbTh, TbN and TbSp value respectively in the 4th and 12 th week. Besides, the degradation rate of the material was also calculated.Results: Micro-CT showed that the edge of material in experimental group at the 4th week has been degraded with growing trabecular bone. While the material in the control group had a clear interface with the bone surface without inducing osteogenesis. At the 12 th week, the composite material in the experimental group was absorbed obviously, replacing by regenerated bone tissues. However, there still exhibited implants coated by material in the control group.Conclusions: CPC composites with large pore structure loading of substance P showed good results in maintaining implant primary stability, inducing bone regeneration and osseoingegration. This could provide new idea for immediate implanting technology and materials development. |
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