| Background:The ability of the alveolar bone regeneration is fine,however,once the defect reaches the critical size,the process of bone healing always fails.Large alveolar defects can easily occur as a result of injury,tumors,or surgery.Bone grafting procedures are widely used for the management of alveolar bone deficiencies in oral and maxillofacial surgery.Currently,the application of particulated xenografts is the most widely used and well-documented method for the augmentation of alveolar defects.However,particulated substitutes are sub-optimal for the augmentation of bone defects due to the unfavorable mechanical properties with low resistance to pressure and thus a risk for collapse.In a recent vitro study,It was found that wound closure and flap suturing had induced a considerable displacement of the particulated grafting material resulting in a partial collapse.In contrast,the sites augmented with block bone substitutes exhibited less collapse.The investigators concluded that the ability to maintain the contour of the augmented region during wound closure and flap suturing was significantly enhanced when using a block bone substitute as compared to GBR with particulated material.The use of blocks of bone substitute materials may represent an effective alternative to particulated substitutes,because they provide adequate volume rebuilding and space maintaining.Currently,there are only limited data reporting on the application of xenogeneic and allogeneic blocks.In a clinical study,blocks of deproteinized bovine bone mineral(DBBM)were applied to 12 patients to treat horizontal bone defects before implant placement.After 9–10 months,in 11 of 12 patients,the resulting bone volume was sufficient to allow implant placement in a prosthetically optimal position.It was concluded that the procedure was effective for horizontal ridge augmentation.These results are in agreement with a preclinical trial comparing autogenous bone blocks with deproteinized bovine bone mineral(DBBM)blocks for horizontal ridge augmentation,in which a similar increase of ridge width was measured in both groups.In fact,3 months after GBR,all sites treated with DBBM blocks clinically appeared suitable for implant placement.However,the limitation of bone block substitutes is their reduced efficacy in bone formation within the block,especially in large-scale defects.The clinical application of DBBM block grafting in critical-size alveolar defects was limited.Bone healing is a complicated vivo process;it involves the presence of a clot containing large numbers of mesenchymal cells,then a proliferation and high metabolic level of osteoblasts and macrophages,followed by the regeneration of blood vessels and woven bone.The recruitment of progenitor cells to the injury site,neovascularization near the injury site.Recombinant human bone morphogenetic-2(rhBMP-2)is commonly used to promote spinefusion in humans.Its use was approved in 2002 by the US Food and Drug Administration(FDA)for single-level anterior lumbar interbody fusion.The use of rhBMP-2 has increased significantly since this time and indications for its use have expanded to include posterior lumbar spinal fusion as well cervical spine fusion.Despite the efficacy of rhBMP-2,recent reports have called into question its safety when employed during spine fusion surgery.Reported complications have included seroma formation,soft tissue swelling,vertebral osteolysis,ectopic bone formation,and carcinogenicity.Moreover,airway edema has been observed with its use in the cervical spine,prompting the FDA to issue a Public Health Notification warning for its use in cervical spine operations.Bone healing is a complicated vivo process;it involves the presence of a clot containing large numbers of mesenchymal cells,then a proliferation and high metabolic level of osteoblasts and macrophages,followed by the regeneration of blood vessels and woven bone.The recruitment of progenitor cells to the injury site,neovascularization near the injury site.Du Bing ect reported that the nHA/coral blocks local delivery of VEGF can significantly improve neovascularization and mineralization of newly formed bone at the early stages of bone healing in this dog implantation model,while new bone could seldom be found in the center of blocks.Kha HT and Richardson reported that specific naturally occurring oxysterols have robust osteogenic properties.Hydroxycholesterols,also known as oxysterols,consist of a large family of 27-carbon oxygenated products of cholesterol.These molecules are present in the circulation and in human tissues.Endogenous hydroxycholesterols are formed in various ways,including auto-oxidation of cholesterol,lipid peroxidation,and enzymatically.Circulating hydroxycholesterols are also obtained from exogenous sources through dietary intake.Hydroxycholesterols are biologically active in diverse metabolic processes,including regulation of cholesterol homeostasis,atherosclerosis,sphingolipid metabolism,platelet aggregation,and apoptosis.Most relevant to this study,however,is the fact that hydroxycholesterols have been shown to have pro-osteogenic activity.Activation of the hedgehog signaling pathway has been implicated in the mechanism of action of oxysterols in modulating osteogenesis in murine cells.Various naturally occurring hydroxycholesterol analogues,namely,22(R)-,20(S)-,and 22(S)-hydroxycholesterol,have been shown to induce osteogenic differentiation in the M2-10B4 murine multipotent bone mesenchymal stem cell line,and in primary murine mesenchymal stem cells.Akishige Hokugo et al reported that water-solubilized oxysterol incorporated biodegradable hydrogel was implanted into rat calvarial defects and induced successful bone regeneration.The innovative significance of this study lies in the development of a bone graft substitute that couples the osteogenic activity of oxysterol with a scaffold designed for optimized oxysterol release kinetics,all of which lead to better repair of bone defects.Thus,the scaffolds by coating them may be one of the most feasible approaches for promoting osteogenesis and bone reconstruction in critical-sized alveolar defects.We hypothesized that:(1)porous collagen-containing bovine block mineral(CBBM)blocks can be an optimal grafting material in critical-sized mandible defects,with good bone regeneration performance;and(2)local delivery of 20 S with the collagen-containing bovine block mineral(CBBM)used as a carrier can promote osteogenesis at stages of bone healing.Objective:1.To investigate the osteogenic efficiency of collagen-containing bovine bone block mineral(CBBM).2.To study the effect of 20(S)-hydroxycholesterol on bone regeneration in the restoration of critical-sized chronic mandibular defect with collagen-containing bovine blocks mineral(CBBM).Methods:1.Commercial available collagen-containing bovine bone block were observed by scanning electron microscopy(SEM),and pore size,the surface morphology and diameter were studied.2.At room temperature in a super-clean worktable,20 S was dissolved in anhydrous ethanol and distributed into 3mg/ml 20S/anhydrous ethanol.Then night 20 S blocks were placed on the centrifuge tube independently,with each one incubated into 1ml 20 S solution(including 3 mg 20 S / CBBM)for noncovalent physical adsorption of 20S;The other night blocks were also placed on the anhydrous ethanol independently,with each one incubated into 1 ml of anhydrous ethanol.All blocks were incubated in about an hour and stored in a low temperature and aseptic condition.3.Animal experiments of 20 S /CBBM in the critical-sized chronic mandibular defect.(1)In the animal experiment,there were two groups,20 S /CBBM served as experimental group and CBBM served as the control group.The time points(4 weeks)were designed in both groups.Each animal was labelled randomly and two standardized critical-sized chronic defects were designed in each side of mandibular.And in a split-mouth design,20 S /CBBM were implanted into one side of mandibular randomly and CBBM were implanted into the other side.(2)Establishment of critical-sized chronic mandibular defect model.The study was carried out in two surgical processes.In the first process,after general anesthesia,second,third,and fourth pre-molars and the first and second molars(P2-M2)in the bilateral mandibular were extracted from each dog by root separation method.Then,the mucoperiosteal flaps were reflected and four standardized box-type defects(5 mm in height from the crestal bone,5 mm in depth from the surface of the buccal bone,and 10 mm in width mesiodistally)were created in the buccal way of mandibules at the distance of 5 mm with carbide burs.The corresponding lingual bone plates were left intact.All osteotomy procedures were carried out under irrigating with sterile 0.9%physiological saline.The wounds were closed with interrupted mattress sutures.All sites need submerged healing of 4 weeks.In the second process,after general anesthesia,make bilateral vestibular incisions,and reflect all-thickness flaps and expose the implant sites for block grafting in the lower jaws.Before grafting,all chronic defects were reshaped(5×5×10mm),and all defects were distributed into20 S /CBBM and CBBM in a split-mouth design(eight 20 S /CBBM and eight CBBM in total).Periosteal-releasing incisions were made,the mucoperiosteal flaps were repositioned coronally and closed with interrupted mattress sutures.(3)4 weeks after implantation,animals were sacrificed by overdose anaesthesia,samples with blocks and adjacent host bone tissues were investigated by histological observation,immunohistochemical observation and histomorphometric analysis.After preparation of decalcified section,slices of each sample were made and HE staining was randomly treated.Cells,blood vessels and bone matrix were studied by histological observation.The percentage of newly formed bone area was histomorphometrically measured by IPP software in HE stained section.The sample size in control group and experimental group was both eight.In undecalcified section,the condition of biodegradation of blocks and fluorescent labelled by double fluorescent labeling method,newly formed bone area in both groups were observed through laser scanning confocal microscope,and the percentage of calcified newly formed bone area was measured by IPP software.Furthermore,the newly formed bone tissues were observed by toluidine blue staining in undecalcified section.4.The percentage of newly formed bone area in HE stained decalcified section and the percentage of fluorescence marked calcified new bone area in undecaicified section,were statistically analyzed by SPSS 18.0 software.All data was present as mean ± standard deviation.Kolmogorov-Smirnov and Levene test were used to inspect the normal distribution and homogeneity of variation.When they could meet the normal distribution and then the homogeneity of variance was neat,factorial design was used to analyse the main effect and interaction.For the single factor data,when data could meet the normal distribution and homogeneity of variance was neat,two independent sample t-test was used to compare the overall means in different groups;When the data could not meet the normal distribution or homogeneity of variance was not neat,two independent sample t' test would be used.Hypothesis test for two-sided test,the test level was 0.05.Probabilities(p)<0.05 were considered to be statistically significant,on the contrary,probabilities(P)>0.05 would be not statistically significant.Results:1.Ideal wettable property has been showed in the process of soak-loading of collagen-containing bovine blocks.3mg/ml 20S/Absolute ethanol solution with the volume of 1 ml could just infiltrate into each CBBM,and every block contained 3 mg 20 S.2.CBBM and 20 S /CBBM in dog criticalsized chronic mandibular defects.(1)General observation four beagle dogs were in good health and recovery after surgery.All animals in two groups had no death with normal soft diet and water.There were no obvious complications such as fester and wound inflammation in mandible implant area,and no loosing and falling off of block bone graft.the region of block bone graft and adjacent host bone healed very well after 4 weeks.The host bone and block graft boundary were easily identified.(2)Histological observation and histomorphometric analysis of HE stained decalcified section At 4th week in 20 S /CBBM group,obvious newly formed bone,especially at the interface adjacent to host bone.On the contrary,there was very small amount of newly formed tissue in the core of all block bone graft.Some trabecular bone along with blood vessels grew in along the paths between the porous structure without obvious inflammatory cells infiltration.In new blood vessels structure,a lot of circular arranged vascular endothelial cells formed new blood vessels and blood cells were clearly visible.Of the new trabecular bone structure,lines of active osteoblasts were present at the surface of block bone graft through the paths of interconnected porous structure.The quantity of new bone tissue in 20 S /CBBM group was slightly more than that in 20 S /CBBM group.Histomorphometric measurement results showed that the percentage of newly formed bone area at 4 week in CBBM and 20S/CBBM group was 8.73%±3.46%and 12.34%±3.86%?respectively.Statistical data were normally distributed and the variation was homogeneous.Independent sample t-test results showed as follows: t=2.213,P=0.042?0.05,The difference was statistically significant.(3)Histomorphometric analysis and histological observation of double fluorescence marked undecalcified section.Fluorescers can chelate calcified new bone tissue,tetracycline hydrochloride showed yellow fluorescent light and calcein green.At 4 week in collagen-containing bovine block group,mesh green calcein fluorescence was obviously present at the peripery of blocks,especially in the border area close to the host bone.Small amount of green band was observed in some inner part of blocks,but seldom in the core.While,very little yellow tetracycline fluorescence can only be found at the interface between host bone and block bone grafts.And in 20(S)-hydroxycholesterol collagen-containing bovine block group,both green calcein fluorescence and yellow tetracycline fluorescence became more obvious,especially in some inner part of block bone grafts.In addition,green fluorescent band presented not only on the surface of block bone grafts,but also at the paths through porous structure.Histomorphometric measurement results showed that the percentage of calcified new bone area at 4 week in CBBM and 20S/CBBM group was 0.79%±0.23% and 1.08%±0.27%,respectively.statistical data were all normally distributed and the variation was homogeneous.The two independent sample t-test results showed: t=2.369,P=0.045<0.05.The difference was statistically significant.Conclusions:1.In vivo,optimal three-dimentional porous structure of CBBM implanted in the critical-sized chronic mandibular defect can provide suitable space for cells migration to the inner part of block bone graft and consequently be beneficial to blood vessels formation and bone regeneration.Moreover,CBBM shows good biocompatibility,osteoconductivity and osteogenic efficiency.2.The local delivery of 20 S bio-coated CBBM shows improvement in the calcification of newly formed bone at the early stage of bone healing. |
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