| BackgroundSkull defects is a common problem in neurosurgery, its causes are mainly including burns, trauma, cancer, and so on. To repair bone defects and rebuild its biological characteristics is one of the important issues to be need in neurosurgery. The repair of the skull defect, including the choice of repair materials and optimization the methods. In order to avoid the problems of the traditional materials ,many kinds of artificial bone materials for repair have been developed, Among them,"inducing factor + Carrier-delivery system "model already become a hot point in bone injure and bone tissue engineering research.A ideal bone-induced artificial material should have the following characteristics:①Good biocompatibility and biodegradability;②Osteoacusis and other active molecules induced osteogenesis together;③Be able to load a large number of cells , to support bone cell growth and differentiation;④Appropriate mechanical strength and plastic;⑤R aw materials have a wide source , low price and are easy to disinfect and save.Based on the research progress of bone tissue engineering materials at home and abroad ,The following types of natural or artificial materials have been gradually approved by many researchers,For example: bone morphogenetic protein (BMP) is a multifunctional growth factor, characterized by a stronger-induced osteoblast activity, and no species-specific; collagen (COL) is the main organic components in the bone matrix, the compound of COL and BMPs can delay the release of BMP and reduce the induction dose of BMPs, maintain a longer effect; Hydroxyapatite (HA) is a ideal bone repair materials with biocompatibility and immunogenicity, nano-hydroxyapatite (n-HA) have a better repair effect for crystal size is equal with the hydroxyapatite in bone in human body, calcium phosphate cement (CPC) has good properties in Plasticity , absorbency and isothermal auto-solidification.however, A separate application of the above-mentioned material shows different problem including plasticity, porosity and mechanical strength etc, in addition, no an ideal composite materials have been used in the practice at present.In order to find more effective tissue-engineered bone defect repair materials for clinical application, we carried out improvements on the CPC through a series of experiment. synthesized the CPC based onα-tricalcium phosphate (α-TCP) system as an Accessional Delivery (AD),Natural bone collagen and hydroxyapatite (HA) self-assembled into a nanometer Nucleus Delivery (ND) by the bio-mimetic mineralization technology. With the properties of ND and AD , the inherent relation between ND and AD , we constructed organically the Double Delivery System(DDS); Extracted the natural active protein as a core for induction and prefabricate the space of Active Growth Channel(AGC) in the DDS with precision and stability under the premise of enough material strength .Prepared the bBMP / ND / AD delivery system with AD, ND and bBMP complex, Applying the vacuum-negative pressure vacuum technology fill the space of AGC with the release system effectively for the construction of AGC. Finally obtain the main part of the artificial bone which its composition, space configuration and biological activity are extremely similar to normal bone. Based on above thinking, to solve the defects of the existing material vector, we have carried out the following work.1. Evaluation of ectopic induced osteogenesis activity of active proteinObjective: To determinate the ectopic induced osteogenic activity of self-maded bovine bone morphogenetic proteinMethods: 40 Kunming mice were randomly divided into 2 groups, bBMPs materials implanted into lateral inner muscle bag of the right thigh, carry out radiology examination respectively in 2 weeks, 3 weeks, 4 weeks and 8 weeks after operation, Evaluate the state of bone tissue growth by histology methodsResults: x-ray show the density increased with small bar in material implanted area in 4 weeks after operation, the density show a small blocky increase in 8 weeks,. Histology show that a large number of cells gather in 2 weeks; more bone cells and bone matrix generation in 3 weeks, clear formation of bone-like tissue in 4 weeks; significantly formation of bone tissue in 8 weeks but the control group did not show above resultsConclusion: The extracted and purificated bBMPs in this research show a good biocompatibility and ectopic osteoinduction ability in the mouse muscle bag.2. Preparation and physical property evaluation of Delivery MaterialObjective: Detection for micro-structure, mechanical strength and plasticity of the ND, AD and DDSMethod:①prepared the ND by wet method, measured its size and morphology by X-ray diffraction (XRD), scanning electron microscopy (SEM);②prepared CPC (AD) based onα-TCP (α-TCP) system by muffle calcination process, detected its mechanical strength with domestic multi-function pressure testing machine, measured the purity of TCP by XRD, detected the calcium/phosphorus ratio by energy spectrum, observed the microstructure by SEM;③DDS detection was same as above. Results:①ND: Particle size of ND is 98 nanometers close to the human bone; SEM show micro-structure of samples was like petals, have a same feature with collagen and HA②AD: The average hold pressure has reached 5,000 Newton / cm3 when material hydrated for 72h, the 8800 Newton / cm3 when material hydrated for 7d; Receiver operating curve (ROC) showed that the results were normal distribution; the content of TCP is 92.7% (standard samples are 94%,from Fluka company,NO:50553) by XRD detection; after 24h hydration, the materials transformed to HA, XRD showed that materials had no the harmful group, the porosity of the material was 45% , energy spectrum showed that calcium/phosphorus ratio was 1.52; With the prolong of hydration time (24h, 72h, 7d, 14d), SEM revealed that the first crystallization of the material were needle-like, eventually the crystallization formed plate,in low-crystalline state,③DDS: three groups respectively added 0.25%, 0.5%, 1% ND, pressure detection showed that the pressure of the composite material added 0.25% ND had went down to 4200 Newton / cm3; XRD of dynamic hydration process showed that hydration products had no obvious abnormalities when added 0.25% ND; the porosity of the material was 55%, calcium/phosphorus ratio was 1.6, SEM shows that micro-structure similar to ND.Conclusion: AD materials prepared in this study, the pressure-carrying capacity of the composite materials reduced when ND was added, the pressure drop in value had a positive correlation with the addition of ND. The pressure drop in value when 0.25% ND was added meet the requirements of pressure and strength as delivery material in this study. Under the conditions of maintaining the stability of the hydration product, calcium/phosphorus ratio of the composite materials is more close to the human bone, mechanical strength decrease and porosity increase, in favor of material degradation, is an ideal bone defect repair materials.3. Rabbit skull defects repair experiment with the induced artificial bone materialObjective: To observe the effect of the induced artificial bone material in the rabbit skull defect repairing procedureMethods: 51 healthy adult New Zealand white rabbits were divided into experimental materials group (composite material, M group), the experimental control group (freeze-dried bone, B group) and the blank control group ( N group); the materials of M group were "buttons" shaped samples by solidification to make, prefabricate Active Growth Channel(AGC) with release system (ND, bBMP); prepared the periosteum and bone defects models with 8mm size in bilateral parietal bone respectively, implanted corresponding materials and fixed, drawed materials respectively according to the time points of 2 weeks,4 weeks, 8 weeks,12 weeks and 16 weeks after the operation,carried out the general observation, blood tests, X-ray inspection, the fluorescent double marker detection, histology and immunohisto- chemical methods for compare evaluation of every group material in the rabbit skull defect repairing procedure.Results:①General observation:after the operation, there were 3 or 4 animals every group with mild swelling in overhead suture line, disappeared in 3 days; After removed the material, implanted material was clearly visible on defect surface in 2 weeks in M Group and B Group, with the tissue growth between host bone and material, there were the thin tissue cover defect surface in N group.There were the thin tissue coverage from the above observation in M group in 4 weeks, but material was still visible; there were transparent yellow tissue in AGC site on one side of material surface when observed from inside surface of parietal bone; in 8 weeks the callus can be seen on the surface of materials in M group, a small amount of connective tissue coverage can be seen in other groups. In 12 weeks and 16 weeks the bone defects surface were packed by tissue and were not easily distinguishable in every group.②Blood tests:In preoperative and postoperative 1 week, 2 weeks, blood LYM% value of M group, B group and N group animals statistical analysis showed that: the differences between LYM% of M group respectively with the B group, N group have statistics significance. The statistical analysis of total protein, alanine aminotransferase, urea nitrogen, alkaline phosphatase and calcium iron of the three groups animals in preoperative and in the time of blood drawn showed the differences between every group or five time point of the same group have no statistics significance .③X-ray observation:The density of the material in M group is higher than trabecular bone around material in postoperative 2 week, the implanted material was clearly visible ; Callus formation seems to seen on the left side of the material in 4 weeks, Vague outline of material had been indistinct in 8 weeks, the density of central district of material has declined than 4 weeks, and the decline is more obvious in 12-week, close to the surrounding tissue, there were continuous callus in the edges of the material, degradation had been observed in material in 12 weeks. the degradation of material is more obvious in 16 weeks, and material had blended into bone tissues around implants .The density of the material in B group is similar to trabecular bone around the material in postoperative 2 week, Callus formation can be seen in the edges of the material and vague outline of material had been indistinct in 8 weeks, the absorption had been seen in the central part of freeze-dried bone, bone sclerosis had been observed in the edges in16 weeks. The defect were obvious in N group in 2-8 weeks, The edge of defect became fuzzy and may be the growth of new bone in 12 weeks and 16 weels,, but the area of bone defect were bigger than B group.④Observation on morphometry of undecalcified bone tissue:The edge of defects had been irregular under the light microscope(4X) in postoperative 8weeks and 12 weeks in M group, The many irregular blue trabecule and osteoid can be observed in internal part of material,and the dark red calcium deposit show the growth of new bone; But B group material is incomplete, scattered to scrape, only in the edge of freeze-dried bone a small amount of new trabecule and osteoid can be observed. In N group ,most defects still existed, no new bone structure found in central district of defects.⑤Observation with fluorescence dual-labeling technique of undecalcified bone tissue:The many green and yellow fluorescent band can be seen in the implanted site in M group in postoperative 8weeks,12 weeks and 16 weeks, with the features of scattered spots, irregular ring shape and "two-track" sign. showed there were active growth of new bone. The spacing between green and yellow fluorescent band was close to 20 meters(the time interval between injection of two fluorescence tracer was 7 days),showed the growth speed of new bone was very quick. The fluorescence mainly located in marginal areas of defects in B group,in center of defects 1-2 fluorescent bands can be seen,the amount of bands was less than the M group. In N Group, the fluorescence was very few and only can be seen in marginal areas of defects.⑥observation on histology and morphology:A large number of cells gathered in implanted material area in M group in 2 weeks, there were new bone growth in marginal areas in 4 weeks, The bone-like tissue found in AGC, small blood vessels and a large number of cells gathered, the material had degraded and absorbed partially. the remaining material split into particles. The growth of bone cells were very rapid in AGC and new bone connected mutually into the new sheet in 8 weeks; The new bone had a further reconstruction in defect in 12 weeks, the formation of new bone showed " frame " in longitudinal section, and new bone extended into inside of"frame";The formation of marrow -Like structure could be seen in 16 weeks, surrounded by a large number of new bone tissue and blood vessels.The amount of new bone was less in every time point in B group than M group after operation, the material in defects areas gradually separated into sheet; the new bone could be seen in defects after 4 weeks. A small amount of new bone could be found in marginal areas of defects afer 4 weeks, no growth of new bone had been found but a large number of connective tissue fibers filled in the central areas of defects. No inflammatory cells had been found gathered around the defects in each group at different time points .⑦Observation on immunohistochemistrySet three check time point (4 weeks, 8 weeks, 12 weeks) in M group and drawed samples, analyzed the changes of BMP2 contents at the junction of material and defect in M group by immunohistochemical technology. The results showed that there were endogenous BMP2 distribution at the junction at three time point, meanwhile, there were exogenous BMP2 in holes located central areas of materials. the semi-quantitative analysis of gray levels of immunohistochemical results had been carried out by Motic Images Advanced3.2 image analysis software. Statistical analysis shows that the gray levels of BMP2 was no significant difference in the three time pointsConclusion:①Animal experiments show the complex materials have good biocompatibility,can be organically combined with bone growth factor , beneficial to slowly release of growth factor.②Because of the construction of AGC in complex material, there are multi-point growth in the process of bone induction, favorable to shorten the growth distance of new bone, accelerate bone defect repairing.③The material can degradate after implanted into body and promote the growth of new bone continuously, the capacity of bone induction is stronger than the allogeneic freeze-dried bone. |
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