| Periodontitis is a common oral diseases and mainly cause tooth-supporting apparatus damage. The periodontal regeneration technology has been widely used to promote the healing of teeth support groups. Bone graft, which is a key of regeneration techniques,is often used to treat bone defects. Now the main clinical bone graft include autografts allografts, xenografts and alloplasts. Autogenous bone is the ―gold standard’’, which is frequently harvested from intraoral sites. However, intraoral donor sites typically yield comparatively limited graft volume, create additional intraoral surgical sites easy absorption and make it easily absorption. Allograft bone has good osteoinduction and osteoconduction, but the main problem are the immune rejection and the risk of contamination disease. Bio-Oss and chitosan are both good bioactive scaffolds and have good biocompatibility.On account of its good biocompatibility, biodegradable and anti-inflammatory effect, more and more attention is paid to chitosan in recent years and widely used as graft, drug carrier and wound-healing dressing. Bio-Oss is composed of deproteinized, sterilized bovine bone and is categorized as a calcium deficient carbonate apatite, it don’t elicit anyallergic reaction. Since it is physically and chemically identical to natural human bone mineral, it is used as a highly effective osteoconductive grafting material, promoting the regeneration of the new bone, blood vessels formation and the stability of a blood clot.The natural polymer composite materials are playing an increasingly important role in bone tissue engineering. The next generation of biological scaffolds should possess biological activity and biological degradation characteristics, including simulating the natural bone physiology and activating the body program of tissue regeneration. Based on the above requirements, composite materials are the combination of biodegradable polymer materials and biological activity grafts, such as combining chitosan and Bio-Oss. The chitosan is lower than the natural bone in mechanical strength and can’t support the external force. Additionally, chitosan in itself does not have osteoinduction. It is need to add the bioactive scaffolds to improve the mechanical strength and osteoinduction. Many materials combined with chitosan to improve their mechanical and biological properties, such as hydroxyapatite, β-TCP, calcium sulfate, alginate, etc. This kind of composite materials have specific physical, bioactivity, mechanical properties and controlled degradability.Bone marrow mesenchymal stem cell is a mature seed cell in bone tissue engineering, which has a multipotential differentiation. In previous studies, the bone marrow mesenchymal stem cells comes from long bone or iliac. Due to its availability, simple proliferation and identification, it is combined with a variety of of biological materials to promote tissue regeneration. At present some new researches show that the jaw bone marrow mesenchymal stem cells had a stronger biological characteristics than iliac bone marrow mesenchymal stem cells.Will the biological composites and stem cells can achieve the advancement of theory? How about clinical practicality? There is still no conclusion. Especially the application in human body is limited by criterion and ethics, and it is unable to observe whether form a real histologic regeneration process. The present study explores the composites and cytology in vitro, the histologic observation and histometric analysis to evaluate chitosan/Bio-Oss composites combined with the jaw bone marrow mesenchymal stemcells on the feasibility and prospects of tissue regeneration.Objective:1. To prepare different proportions of chitosan/Bio-Oss composites and compare their physical and chemical properties. To compare the biological impact of chitosan/Bio-Oss composites on the adhesion and proliferation of jaw bone marrow mesenchymal stem cells and other physiological characteristics in vitro.2. To observe osteogenic properties of chitosan/Bio-Oss composites with hJBMMSCs in rat cranial defect.3. To choose a suitable periodontal defect model by comparing four type of periodontal defects.4. To evaluate periodontal regeneration after chitosan/Bio-Oss composites implanted into chosen periodontal defect.5. To exploit a novel scaffolds for promoting periodontal and oral tissue regeneration and provide more theories and evidence.Method:1. The physicochemical properties of chitosan/Bio-Oss compositesAccording to the proportion of chitosan and Bio-Oss(100:0, 75:25, 50:50, 25:75), the compressive strength, swelling rate and degradation rate of four kinds of grafts were detected after the Bio-Oss was added in chitosan/β-GP solution and freeze-dried.2. The evaluation of hJBMMSCs cultured with chitosan/Bio-Oss composites on biological characteristics in vitroThe hJBMMSCs were separated and cultured by tissue block method and were purified by limited dilution method cells; The self-renewal ability of the cell was detected by Colony-forming unit-fibroblast(CFU-F) methods and their surface markers were analyzed by flow cytometry; Their multipotent differentiation ability was identified using the adipogenic and osteogenic induction. The cell proliferation on the materials were detected by CCK-8 method; The cells on the composites were observed by scanning electron microscopy; The alkaline phosphatase activity was detected by kits.3. The effect of Bio-Oss/chitosan composites and hJBMMSCs on bone formation in critical-size rat cranial defectSprague-Dawley rats were used to provide Micro-CT and light microscopy observations of bone formation/maturation and immunohistochemical stain of osteoblasts differeneiation markers(OCN) and healing at 8 weeks following application of Bio-Oss/chitosan composites with h JBMMSCs into critical-size, ?8-mm, through-through, calvarial defects.4. Effects of acute furcation and intrabony defects on periodontal healingBeagles were used to provide Micro-CT and light microscopy observations of bone formation/maturation and healing at 8 weeks following application of Bio-Oss/Bio-Gide into different size of 1-, 2-, 3-wall intrabony defects and supraalveolar defects. The appropriate defect was selected for evaluating the defect of periodontal tissue regeneration.5. The effect of Bio-Oss/chitosan composites and h JBMMSCs on bone formation in one-wall periodontal defectformation/maturation and immunohistochemical stain of osteoblasts differentiation markers(OCN) and healing at 8 weeks following application of Bio-Oss/chitosan composites with h JBMMSCs into one-wall intrabony defects.Results1. Chitosan group was higher than chitosan/Bio-Oss groups [C/B(75:25), C/B(50:50), C/B(25:75)] in pore diameter and porosity, swelling rate and degradation rate, and C/B(75:25) group and C/B(50:50) group was higher than C/B(25:75) group in porosity, swelling rate and degradation rate. Three chitosan/Bio-Oss groups were higher than chitosan group in compressive strength, and C/B(25:75) group and C/B(50:50) group was higher than C/B(75:25) group in compressive strength;2. The hJBMMSCs were successfully obtained in the present study; The cells showed abilities of self-proliferation and clone formation and positively expressed mesenchymalstem cell(MSC) markers(CD90, CD105, CD29, CD146, STRO-1) while negative for hematopoietic stem cells markers(CD34 and CD45). Following osteogenic/adipogenic induction, the calcified nodule and fat drops were observed after Alizarin red staining and Oil red O staining; The h JBMMSCs were exhibited good adhesion and growth on three kinds of materials(chitosan, chitosan/Bio-Oss, Bio-Oss), and there was no difference significant among three kinds of materials in ALP activity.3. At 8 weeks, the experimental group(C/B+cell, C+cell, B, C/B, C) were higher than the control group and the groups(B and C/B+cell) were higher than the groups(C+cell, C/B, C) in bone volume/tissue volume by Micro-CT observation(p<0.05); All the experimental groups(B, C/B+cell, C/B, C+cell) were higher than the control group and the groups(B, C/B+cell, C/B, C+cell) were higher than chitosan group in light microscopy observations of bone area(p<0.05). The immunohistochemical analysis at 8 week showed OCN antigen reactivity within the newly formed trabecular bone and new bone surface in groups with JBMMSCs(C/B+cell, C+cell), which were far more than those in groups(C, C/B, B, Control).4. At 8 weeks, experimental group for 1-, 2-,3-wall intrabony defects and acute furcation were higher than corresponding of controls in Micro-CT and light microscopy observations of bone height/area(p<0.05); Only was the experimental group for one-wall intrabony defect higher than corresponding of controls in connective tissue attachment, junctional epithelium, cementum regeneration and periodontal ligament regeneration(p<0.05).5. At 8 weeks,the experimental group(C, C+cell, C/B, C/B+cell, Bio-Oss) were higher than the control group and the Bio-Oss group was higher than the chitosan group, chitosant/h JBMMSCs group and chitosan/Bio-Oss group in bone volume/tissue volume by Micro-CT observation(p<0.05); The chitosan/Bio-Oss/h JBMMSCs group, chitosan/h JBMMSCs group, Bio-Oss group and chitosan/Bio-Oss group were higher than the control group, the chitosan/Bio-Oss/h JBMMSCs group, Bio-Oss group and chitosan/Bio-Oss group than the chitosan group in light microscopy observations of height/area(p<0.05); The chitosan/Bio-Oss/h JBMMSCs group and Bio-Oss group werehigher than the chitosan/Bio-Oss group, chitosan/h JBMMSCs group, chitosan group and control group in light microscopy observations of new cementum/new periodontal ligament(p<0.05). The immunohistochemical analysis at 8 week showed OCN antigen reactivity within the newly formed trabecular bone and new bone surface in groups with h JBMMSCs(C/B+cell, C+cell), which were far more than those in groups(C, C/B, B, control).Conclusion:1. Chitosan combined with Bio-Oss could obviously increase the compressive strength and reduce the degradation of chitosan. The ratio of Chitosan and Bio-Oss was 50:50, it showed good properties in the pore size, compressive strength, swelling rate and degradation rate.2. The hJBMMSCs could grow and proliferate well on Bio-Oss/chitosan composites. The chitosan/Bio-Oss composites had no effect on h JBMMSCs in ALP activity and had a good biocompatibility.3. In four types of periodontal defect, one-wall intrabony defect had larger size and less source of tissue regeneration compared with two-, three-wall intrabony and supraalveolar defect, but it may act as a more discriminative and a clinically more relevant defect model for further evaluation of new procedures that promote periodontal regeneration.4. As Bio-Oss/chitosan composites with JBMMSCs implanted, some JBMMSCs were differentiating into osteoblasts and better promoted bone healing in rat calvarial defect and periodontal regeneration in one-wall intrabony defect compared with chitosan and chitosan/Bio-Oss. Although Bio-Oss/chitosan composites with JBMMSCs promoted no more the new bone volume than that Bio-Oss promoted, it provides a perspective with potential applications to bone and periodontal regeneration from the comprehensive considerations such as the amount of residue materials and basis of biological repair. |
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