| Background:In clinic, periodontal disease or tumor can lead to large sized alveolar defects, which the healing process frequently fails or is delayed and can not regenerate ideally by itself. Therefore, bone grafting is indispensible for these large defects. Currently, an autogenous graft is regarded as the gold standard for bone regeneration. However, it still does not have been widely applied clinically, because of donor site limitation, additional injury associated with the surgical procedure, and other complications, such as pain and infection. Among many xenogenous grafting materials, hydroxyapatite/coralline (HA/coral), also called coralline hydroxyapatite (CHA), is a biodegradable synthetic porous material, made by partially converting calcium carbonate of natural coral to hydroxyapatite via the hydrothermal exchange reaction. In this way, not only the three-dimensional porous structure of natural coralline but also good biocompatibility, mechanical strength, osteoconductivity of hydroxyapatite were retained. Previous studies showed that CHA particles have demonstrated good performance in promoting initial periodontal ligament cell (PDLC) attachment in periodontal regeneration. Moreover, numerous experiments about its mechanism and efficiency of osteogenesis have demonstrated that CHA particles can be enveloped by cells and collagen matrix and consequently newly formed bone can be well distributed in the space between particles. At present, CHA particles are often used for guided bone tissue regeneration in implant and periodontal surgery. However, they are susceptible to masticatory force and easily deformed. Hence, for the restoration of larger sized alveolar defect, predictable results have not been achieved with CHA particle grafts because of the weakness in shape remodeling and space maintaining. In comparison, CHA blocks may be advantageous in space maintaining and bone volume rebuilding. In addition, they may not be restricted by the size and can even be customized according to the shape of different defects.Although CHA block can overcome the above deficiencies of CHA particles, but it still can not be widely used clinically, because blocks graft offers a lower osteogenetic capacity than particles graft. Previous studies have found that the large sized bone defect was often followed with poor blood supply. Even if the ideal scaffold was used, uneven distribution of osteogensis and neovascularization between the peripery and center can also commonly exist due to the deficiency of nutrients and oxygen in the center of block grafting. Therefore, how to improve the angiogenesis of block graft is a key point to wide application of CHA block grafting in clinic.As we all know, bone defect repairing is a complicated process, which involves blood clot in the lung, migration of osteoblasts and macrophages to the wound, their proliferation and differentiation, the formation of vascular structures and woven bone. Among them, angiogenesis is one of the most important processes at the early stage of bone healing. Therefore, vascularization of scaffolds is regarded as a key point and vital means to the grafting of bone defect. In tissue engineering, there are several kinds of methods for scaffold vascularization, including stem cells transplant therapy, gene transfection cell transplants combination therapy, growth factors combined therapy, etc. Growth factors combined therapy means that angiogenic growth factors may be adsorpted onto the scaffolds using tissue engineering methods. By this means, angiogenic growth factors can be control-released and the vascularization of scaffold can be realized. Among so many growth factors, vascular endothelial growth factor (VEGF) is one of the most powerful growth factors which can have specific effects on vascular endothelial cells. It is well known to have high specificity to promote the mitosis of endothelial cells at the beginning of the angiogenesis, induce cell migration, proliferation and differentiation into vascular endothelial cells, control cells apoptosis, and improve new vessel formation. In addition, it also has capacity of up-regulating the expression of bone morphogenetic protein-2 (BMP-2), inducing the migration, proliferation, and differentiation of osteoblasts and enhancing new bone formation.Of course, local application of VEGF alone may always lead to fast degradation, which makes it difficult to play its biological role completely in the long term. Hence, a carrier is indispensable. Hydroxyapatite in the hydroxyapatite/coralline block has functional groups such as:-OH,-NH2,-COOH, which can non-covalently adsorb VEGF. In addition, the open and interconnected irregular porous structure can increase the superficial area of hydroxyapatite/coralline scaffold, which may be beneficial to the controlled release of growth factors. Therefore, as the scaffold material, CHA block can successfully combine VEGF by physical adsorption method and realize its vascularization.In bone tissue engineering, more and more studies pay constant attention to nanometer biomaterials. Among numerous nano-sized materials, nano-scaled hydroxyapatite can simulate the structure and chemical composition of natural bone, help for the specific recognition of human cells to biological macro molecules; bond collagen protein with the amide end and form a chemically bonded interface with a specific biological activity in the body; provide suitable micro environments for osteoblast adhesion, collagen and salt deposition. In addition, compared with micron-sized hydroxyapatite, the superficial area of nano-scaled hydroxyapatite can be obviously increased, which may be more fit for cells adhesion, migration and biological mineralization. Moreover, nano-scaled hydroxyapatite can also promote deposits of calcium phosphate and mineralization of osteoid. At present, there are only a few reports of CHA block grafting used in alveolar defects, and rare researches about nano hydroxyapatite/coralline block grafting and its vascularization studies. Whether nano hydroxyapatite/coralline block can be widely used in alveolar bone defect repairing in clinic still needs further study.In the present study, the critical-sized chronic mandibular defect model was established, and animal experiments were made through nano hydroxyapatite/ coralline block’s grafting into critical-sized chronic mandibular defect in order to investigate its osteogenetic mechanism and osteogenesis efficiency. In addition, the vascular endothelial growth factor was combined onto nano hydroxyapatite/coralline composite scaffolds and implanted into critical-sized chronic mandibular defect, and their performance in the improvement of blood vessels formation and bone regeneration was observed. The above fundamental research can provide theoretic support for the wide application of nano hydroxyapatite/coralline block in clinic.Objective:1. To investigate the osteogenic mechanism and efficiency of nano hydroxyapatite/coralline bone block grafting.2. To study the effect of vascular endothelial growth factor on neovascularization and bone regeneration in the restoration of critical-sized chronic mandibular defect with nano hydroxyapatite/coralline blocks.Methods:1. Commercial available coralline bone block, nHA/coral bone block, human cortical bone block, human cancellous bone block were observed by scanning electron microscopy (SEM), and the surface morphology, pore size, microscopic crystal shape and diameter were studied; The chemical element components on the surface of nHA/coral block and coralline block were studied by energy spectrum analysis; X-ray diffraction (XRD) detection and phase analysis were used to study crystal phase of nHA/coral block’s surface.2. At room temperature in a super-clean worktable, rhVEGF165 was dissolved in sterile PBS buffer, and distributed into 12 ug/mlVEGF/PBS solution. Then sixteen nano hydroxyapatite/coralline blocks were placed on the petri dishes independently, with each one incubated into 0.25 ml rhVEGF165 solution (including 3 ug rhVEGF165/bone block) for non covalent physical adsorption of VEGF; The other sixteen nano hydroxyapatite/coralline blocks were also placed on the petri dishes independently, with each one incubated into 0.25 ml of sterile PBS buffer. All nano hydroxyapatite/coralline blocks were incubated in about half an hour and stored in a low temperature and aseptic condition.3. Animal experiments of vascularized nano hydroxyapatite/coralline block grafting in the critical-sized chronic mandibular defect(1) In the animal experiment, there were two groups, VEGF/nHA/coral and nHA/coral. The VEGF/nHA/coral group served as experimental group, and the nHA/coral group served as the control group. Two time points(3 weeks and 8 weeks) were designed in both groups. Each animal was labelled randomly and four standardized critical-sized chronic defects were designed in each side of mandibule. And in a split-mouth design, VEGF/nHA/coral blocks were implanted into one side of mandibule randomly and nHA/coral blocks were implanted into the other side.(2) Establishment of critical-sized chronic mandibular defect model.The study was performed in two surgical processes. In the first process, after general anesthesia, the bilateral mandibular second, third, and fourth pre-molars and the first and second molars (P2-M2) were extracted from each dog by root separation method. Then, the mucoperiosteal flaps were reflected and four standardized box-type defects (9 mm in height from the crestal bone,6 mm in depth from the surface of the buccal bone, and 12 mm in width mesiodistally) were created in the buccal way of mandibules at the distance of 4 mm with carbide burs. The corresponding lingual bone plates were left intact. All osteotomy procedures were performed under irrigating with sterile 0.9% physiological saline. The wounds were closed with interrupted mattress sutures. All sites need submerged healing of 8 weeks. In the second process, after general anesthesia, bilateral vestibular incisions were made, and all-thickness flaps were reflected and the implant sites for block grafting in the lower jaws were exposed. Before grafting, the chronic defects were reshaped (6 x 9 × 12 mm), and all defects were distributed into nHA/coral block and VEGF/nHA/coral block group in a split-mouth design (16 nHA/coral blocks and 16 VEGF/nHA/coral blocks in totle). Following periosteal-releasing incisions, the mucoperiosteal flaps were repositioned coronally and closed with interrupted mattress sutures.(3) 3 weeks,8 weeks after implantation, animals were sacrificed by overdose anaesthesia, samples with blocks and adjacent host bone tissue were investigated by histological observation, immunohistochemical observation and histomorphometric analysis. After preparation of decalcified section, slices of each sample were made and HE, Masson and immunohistochemical vWF staining were randomly treated Cells, blood vessels and bone matrix were studied by histological observation, and the percentage of newly formed bone area was histomorphometrically measured by IPP software in HE stained section of both 3 and 8 weeks. The sample size in control group and experimental group were both eight. The secretion of collagen matrix was observed by masson staining; The condition of neovascularization was observed by vWF immunohistochemical staining method and the density of new blood vessels was histomorphometrically measured by IPP software. The sample size of control group and experimental group were both eight. In undecalcified section, by double fluorescent labeling method, the condition of biodegradation of nHA/coral blocks and fluorescent labelled newly formed bone at different time point in both groups were observed through laser scanning confocal microscope, and the percentage of calcified new bone area was measured by IPP software. The sample size in control group and experimental group were both eight. In addition, the newly formed bone tissue was observed by toluidine blue staining in undecalcified section.4. The percentage of fluorescence marked calcified new bone area in undecalcified section, the percentage of newly formed bone area in HE stained decalcified section, and neovascular density in vWF immunohistochemical stained decalcified section were statistically analyzed by SPSS 18.0 software. All data were present as mean±standard deviation. Kolmogorov-Smirnov and Levene test were used to checkup the normal distribution and homogeneity of variation. When they could meet the normal distribution and homogeneity of variance was neat, factorial design was used to analyze 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 and at different time points; When the data could not meet the normal distribution or homogeneity of variance was not neat, two independent sample t’ test was used. Hypothesis test for two-sided test, the test level was 0.05. Probabilities (P)<0.05 was considered to be statistically significant, and probabilities (P)>0.05 to be not statistically significant.Results:1. Scanning electron microscope observation results showed that coralline block, nHA/coral block, human cortical bone and cancellous bone all had similar microstructure in rough surface. Both coralline block and nHA/coral block had interconnected porous structure, and the pore size ranged from62 to 164μm. In addition, finger-shaped nano hydroxyapatite crystals were found well-distributed on the surface of nHA/coral block. The diameter of nHA crystals was in the range of 71-99nm. Energy spectrometry analysis results showed that the chemical elements on the surface of nHA/coral bone block include calcium, phosphorus, carbon and oxygen. The results of X-ray diffraction detection showed that crystals on the surface of nHA/coral block were hydroxyapatite and calcium carbonate.2. Ideal wettable property has been showed in the process of soak-loading of nHA/coral blocks.12 ug/mlVEGF/PBS solution with the volume of 0.25 ml could just infiltrate into each nHA/coral block, and every vascularized nHA/coral blocks contained 3 ug rhVEGF165.3. Vascularized nano hydroxyapatite/coralline block grafting in dog critical-sized chronic mandibular defect(1) General observationFour beagle dogs were in good health and recovery after each surgery. All animals in both groups had no death with normal soft diet and water. There were no obvious complications such as wound inflammation and fester in mandible implant area, and no loosing and falling off of nano hydroxyapatite/coralline block. After 3,8 weeks, the region of block bone graft and adjacent host bone healed very well. The host bone and block graft boundary was easily identified.(2) Histological observation and histomorphometric analysis of HE stained decalcified sectionAt 3 week in nHA/coral block group, obvious newly formed bone and neovascular tissue can be easily observed at the periphery of nHA/coral block, especially at the interface adjacent to host bone; On the contrary, there was very small amount of newly formed tissue in the core of nHA/coral block. Some trabecular bone along with blood vessels ingrew along the paths between the porous structure without obvious inflammatory cells infiltration. In new blood vessels structure, a large number of circular arranged vascular endothelial cells formed new blood vessels and blood cells inside vessels were clearly visible. Of the new trabecular bone structure, lines of active osteoblasts were present at the surface of scaffold through the paths of interconnected porous structure. And more new bone tissues were formed in VEGF/nHA/coral group, especially in the center of nHA/coral block, compared to nHA/coral group. However, the uneven distribution of new bone tissue was still obvious like that in nHA/coral group. At 8 week, the bone regeneration and angiogenesis in two groups were significantly increased; the trabecular bone became significantly wider, lamellar bone structure appeared. The quantity of new bone tissue in VEGF/nHA/coral group was slightly more than that in nHA/coral group.Histomorphometric measurement results showed that the percentage of newly formed bone area at 3 week in the nHA/coral group and VEGF/nHA/coral group was 21.7±4.6% and 27.3±5.8%, respectively; And the percentage of newly formed bone area at 8 week in nHA/coral group and VEGF/nHA/coral group was 32.6±7.2% and 39.3±7.8%, respectively. Factorial design analysis of variance showed no crossover effect between grouping and time (P=0.815). Statistical data at 3 week were normally distributed and the variation was homogeneous. Independent sample t-test results showed as follows:t=2.103, P=0.054>0.05. The difference was not statistically significant. In addition, statistical data at 8 week were normal distributed and the variation was homogeneous. Independent sample t-test results showed as follows: t=1.764, P=0.099>0.05. The difference was not statistically significant. Moreover, the percentage of newly formed bone area at 8 week in nHA/coral and VEGF/nHA/coral group was both significantly higher than that at 3 week. T-test results in nHA/coral group showed as follows:t=3.577, P=0.003<0.01; And t-test results in VEGF/nHA/coral group showed as follows:t=3.453, P=0.003< 0.01(3) Histological observation of masson stained decalcified sectionAt 3 week in nHA/coral group, newly formed trabecular bone was obvious with immature collagen fibers disorderly arranged and red stained. While, in VEGF/nHA/coral group, collagen fibers were relatively mature, red srained and regular arranged; Osteoblasts were surrounded by osteoid bone, which evoluted into bone cells and bone lacuna; Calcium salt deposited bone cells and bone lacuna structure were blue stained. At 8 weeks in both nHA/coral and VEGF/nHA/coral group, blue stained bone cells and mature bony with better aligned and wider collagen fibers were obviously visible; Mature collagen matrix secretion was significantly increased, and some woven bone formation could be easily found.(4) Immunohistochemical study of vWF stained decalcified sectionThe formation of new blood vessels was characterized by numerous vWF stained endothelial cells located in vessel basement membranes. The uneven distribution of newly formed blood vessels was easily observed (similar to newly formed bone). The neovascular structure at the periphery, adjacent to the host bone, was more evident than that at the center of the sample. At 3 weeks, the vWF positive expression areas were mainly distributed at the periphery adjacent to block-host bone interface, while seldom in the central part of blocks in both groups. The brown colored neovascular tissue presented in oval shape. The positive expression in VEGF/nHA/coral group was more than that in nHA/coral group. At 8 weeks, with the increasement of bone healing time, the quantity of new blood vessels in two groups significantly increased not only in the peripheral area of block, but also in the center. The positive expression of VEGF/nHA/coral group area was relatively more than that of nHA/coral group.Histomorphometric measurement results showed that at 3 week the neovascular density in the nHA/coral group and VEGF/nHA/coral group was 105±31.4 and 146±32.6 vessel/cm2, respectively; At 8 week the neovascular density in the nHA/coral group and VEGF/nHA/coral group was 269±66.6 and 341 ±70.8, respectively. Factorial design analysis of variance showed no crossover effect between grouping and time (P=0.419). Statistical data at 3 week were normally distributed and the variation was homogeneous. Independent sample t-test results showed as follows:t=2.56, P=0.023<0.05. The difference was statistically significant. Statistical data at 8 week were normally distributed and the variation was homogeneous. Independent sample t-test results showed as follows:t=2.098, P=0.055>0.05. The difference was not statistically significant. In addition, the neovascular density at 8 week in nHA/coral and VEGF/nHA/coral group was significantly higher than that at 3 week. T-test results in nHA/coral group showed as follows:t=6.303, P=0.001<0.01; And t- test results in VEGF/nHA/coral group showed as follows:t=7.081, P=0.001<0.01.(5) Histological observation and histomorphometric analysis of double fluorescence marked undecalcified sectionFluorescers can chelate calcified new bone tissue, calcein showed green fluorescent light and tetracycline hydrochloride yellow. At 3 week in nHA/coral 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, and seldom in the core. While, very little yellow tetracycline fluorescence can only be found at the interface between host bone and nHA/coral blocks. And in VEGF/nHA/coral group, both green calcein fluorescence and yellow tetracycline fluorescence became more and more obvious, especially in some inner part of scaffolds. In addition, green fluorescent band presented not only on the surface of scaffolds, but also at the paths through porous structure. At eight week in two groups, fluorescence labelled new bone at the periphery and internal of scaffolds significantly increased, yellow tetracycline fluorescence can be easily observed in the different parts of blocks. Compared with nHA/coral group, double fluorescence in VEGF/nHA/coral group significantly increased.Histomorphometric measurement results showed that the percentage of calcified new bone area at 3 week in nHA/coral group and VEGF/nHA/coral group was 0.79±0.22% and 1.08±0.29%, respectively; And the percentage of calcified new bone area at 8 week in nHA/coral group and VEGF/nHA/coral group was 4.25±1.14% and 5.21±1.07%, respectively. Factorial design analysis of variance showed no crossover effect between grouping and time (P=0.24S). Statistical data at 3 week were all normally distributed and the variation was homogeneous. The independent sample t-test results showed as follows:t=2.199, P=0.045<0.05. The difference was statistically significant. Statistical data at 8 week were normally distributed and the variation was homogeneous.The independent sample t test results showed as follows: t=1.725, P=0.107>0.05. The difference was not statistically significant. Additionally, the percentage of calcified new bone area at 8 week in nHA/coral and VEGF/nHA/coral group was significantly higher than that at 3 week. T’-test results in nHA/coral group showed as follows:t’=8.43, P=0.001< 0.01; And t’-test results in VEGF/nHA/coral group showed as follows:t’=10.497, P=0.001< 0.01.(6) Histological observation of toluidine blue stained undecalcified sectionAt 3,8 week time point, the configuration of scaffolds still kept regular in two groups, with no significant biodegradation. At 3 week in both groups, significant osteogenesis canbe observed at the periphery of blocks, especially in the area adjacent to host bone, and seldom in the core. At 8 week, new bone formation was significantly increased in two groups, and osteogenesis at the inner part of scaffolds became more significant than that at 3 week, but the phenomenon of uneven distribution betweenthe edge and center still existed.Conclusions1. Nano hydroxyapatite/coralline block has ideal performace in rough surface, chemical composition, nanoscale microstructure and three dimentional porous structure, which may be similar to those of natural bone, and can meet the demands of optimal scaffold materials in bone tissue engineering.2. In vivo, optimal three-dimentional porous structure of nano hydroxyapatite/coralline bone block implanted in the critical-sized chronic mandibular defect can provide suitable space for cells migration to the inner part of scaffolds and consequently be beneficial to blood vessels formation and bone regeneration. Moreover, nano hydroxyapatite/coralline block grafting showes good biocompatibility, biodegradation, osteoinductivity, osteoconductivity and osteogenic efficiency.3. The vascularization treatment by vascular endothelial growth factor’s combining to the nano hydroxyapatite/coralline block showes improvement in neovascularization and promotion in the calcification of newly formed bone at the early stage of bone healing, while without direct improvement in the bone formation in the whole process of bone healing. |
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