The Research Of Injectable DBM/HA-TCP Composite Materials

The purpose of this research is to develop a kind of bone defect repair material with good osteogenic capacity, good capacity of injection and plasticity, and rich active osteogenic ingredient DBM. In addition, with good degradability and osteogenic capacity, it can provide calcium and phosphorous ions for the regeneration of new bones, and can be developed under X-rays, thus easing the shortage of bone resources to some extent.The research is divided into the following parts:Part I Preparation and evaluation of the spherical nanocrystallineHA and TCPPurpose:To synthesize spherical nanocrystalline HA/TCP materials with chemical approaches. The biological activity of the synthesized materials was evaluated in vitro with osteoblast.Methods: With Ca(NO3)2 and(NH4)2HPO4 as raw materials, adjust the p H value of Ca(NO3)2 solution to 10-11 and the p H value of(NH4)2HPO4 solution to 9-10; After mixing at the ratios of 9:5 and 3:5 of Ca/P, adjust the p H value to 10-11 and vibrate for 24 h at 25°C; and then age, clean and dry. Calcine the dry powder at 800°C for 2h. Represent the XRD and SEM of the powder before and after the calcination, and determine the phase, morphology as well as the partial size. Cultivate the MC3T3-E1 osteoblast and study the in vitro cytocompatibility of the two metarials after calcination with this cell. The observation targets are the protein expression levels of the cell proliferation and alkaline phosphatase, the gene expression level of functional osteogenesis index COL-1 as well as the growing status of the cells.Results: Test the XRD of synthetic powder, the result of which shows that the 9:5 and 3:5 Ca/P before the calcination are similar in the precursor crystal textures, and all the positons of diffration peaks correspond with the standard calories of HA. After calcination at 800°C, the Ca/P=9:5 is an ideal-crystallinity HA material, and the Ca/P=3:5 is a high-crystalline TCP material. The SEM shows that the precursor materials of Ca/P=9:5 and Ca/P=3:5 before calcination are all spherical nanocrystalline with a diameter of 44-248 nm, and are similar in morphology. After calcination at 800°C, the HA and TCP are still spherical nanometer structures. The MTT result shows that, compared to the control group, the cells’ growth rates of the two material groups have clearly risen, and the auxo-action of TCP to cell proliferation is better than that of HA(P<0.05). Besides, the activity of alkaline phosphatase of TCP group is clearly hgiher than that of HA group(P<0.05). The PCR result shows that the osteolasts on the surface of both material groups have COL-I expressions, the amount of which increases over time. Therfore, the result remains the same, namely, TCP group is better than HA.Conclusion: This experiment, through successful utilization of traditional synthetic process of chemical wetprocess, synthesizes high-purity nano-hydroxyapatite with specific ratios of raw materials and mixed methods of allocation and under certain reaction conditions. Hydroxyapatite and calcium phosphate of spherical nanocrystalline are obtained after calcination. The experimental verification of osteoblasts shows the cytocompatibility is pretty good, and the spherical nano crystalline TCP group is even better than similar nanocrystalline HA in cell proliferation, activity of ALP and gene expression of COL-I. Nano crystalline TCP should be given more priority.Part II Preparation and activity evalution of DBMPurpose: Prepare DBM of rabbits and verify the calcium content and bone induction activity to provide material for follow-up study.Methods: Choose 10 healthy long-eared six-month-old New Zealand rabbits. Execute the rabbits, obtain the tubular cortical bones of the four limbs, remove the metaphysis and soft tissues such as musules, freeze for four weeks at a profound hypothermia of-80℃ to reduce antigenicity, clean with ultrasound to remove fat and impurities, smash and sieve after freeze-drying and select 24-50 pieces for follow-up experiments. Put the bone meal in hydrochloric acid of 0.5mol/L and decalcify for 24 h at room temperature, wash with purified water repeatedly till the p H value reaches about 6.0. After freeze drying, sealing and irradiation sterilization at 25 KGy 60Co, the sample of decalcification bone matrix is acquired. Observe the appearance changes of the sample. Take 1g of sample, dilute 20 times with boiled water, concuss for 10 minutes with ultrasound and then test the p H value after another 10-minute standing. Then take 0.5g of bone meal and DBM, soak with dense HNO3, digest completely with the high temperature of electric stoves, and then put into 100 ml volumetric flasks seperately with constant volume; test the calcium content with plasma emission spectrometer(ICP). Conduct bilateral experiments with 10 nude mice, with the left experiment as control group using the rabbit powder and the right as DBM group. Anesthetize with 0.3% pentobarbital sodium, and implant the sample in the spatium intermusculare of the mice’s legs. Draw the materials 28 days after the operation, fasten, make specimen and cut sections after dehydration, embedding, section and other processes, dye with HE and observe the histology reactons. Observe if there is formation of cartilage or osteoid tissues and make active classification according to the OI standard of bone induction activity rating.Results: The DBM after decalcification is milk white, with a grain size of 150μm-700μm. The tested p H values of the bone meal and DBM are respectively 6.2±0.5 and 6.1±0.6, both conforming to the national requirements(5.8-7.5). By the measurement of the plasma emission spectrochemical analysis, the calcium content of powder of rabbits, namely the four limbs of normal rabbits, accounts for 21.34-25.34% of the total weight. The calcium content clearly decreases, with a minimum of 4.9%. The wounds of the nude mice heal well after surgery. Four-week observation discovers that there is no surgery trace on the skin, foreign matter can be seen in the muscle, and some thread residues on a few muscles are not completely assimilated; the X-ray iconography shows development at the surgical spots in both experimental groups, with similar strength and no distinct differences, but there is no development in blank control group. The histological observation shows no obvious inflammatory response around the implantation materials; in the control group, no new bone is generated four weeks after the implantation of bone meal, there is a large number of vacuole bodies formed by adipocyles, and fibrous connective tissues evole, while in DBM group, new bones and chondrogenesis are clearly formed, large numbers of osteoblasts and chondrocyles can be see, and medullary cavity structures are formed in the center.Conclusion: We get DBM by improved Urist methods and strict control over the preparation. The in vivo experiment also shows that this DBM is active in bone induction, which lays necessary material foundation for follow-up composite materials preparation and animal experiment.Part III Preparation and evaluation of glycerin gelatine excipientmaterialPurpose: In clinical surgeries, the mouldability of China’s current bone granules or powder is rather poor in operation. None of the known biological excipient materials can stand the irradiation sterilization of 60 Co. To solve the final sterilization of such materials, this research prepares a sol-gel excipient, which, with glycerol as solvent, can be sterilized with irradiation and is easy to preserve.Methods: Put different amounts of gelatin in the beakers, steep with appropriate amount of water till full swelling; place 25 m L of glycerol in a oven at 70℃, and after heating for 30 minutes, mix gelatin and glycerin while it is hot and stir evenly; continue heating for five minutes, take it out, cool it down naturally, and remove the bubble on the top; uviolize with 500 W/m2 for two hours, and place it in the refrigerator-20℃ for 72 h, then we can get glycerol gelatin matrix of different concentrations. Detect the p H values of every matrix; test the translative temperature of the materials with DSC-60 differential thermal analyzer; detect the rheological property of the matrix with MCR302 advanced rotational rheometer of Anton Paar; observe the four fluid viscosities of 4% chitosan, 1% sodium hyaluronate, 2% sodium alginate and 1% glycerin gelatin under irradiation sterilization and effects of the p H values; take PBS as the simulated body fluid, dilute the matrix of gelatin of different concentrations for 10 times, measure the in vitro dissolution rate of every matrix by observing every two hours; take the BALB/c- mice as test model, conduct implantation in vivo experiment, and observe the histological responses on the first, third and seventh day after operation as well as the biocompatibility of the dying observation matrix of HE.Results: With the increase of the concentration of gelatin in glycerin gelatin matrix, the p H value of the matrix remains at about 5.60. The matrix is flowable sol at room temperature when the content of gelatin is less than or equal to 2 %; when the content of gelatin is greater than or equal to 2.5 %, the matrix turns into gel and is no longer flowable, but it will become flowable sol again when heated to a certain temperature. Based on the DSC curve and temperature scan results, the transformation temperature is at 46~50 ℃. With the increase of gelatin concentrations, the modulus of matrix are also on the rise, with the elasticity modulus ranging from dozens to about 2500; the 3ITT test of excipient shows that the viscoelastic modulus of the colloid can be restored to 90% of the original within 50 s. After irradiation, the viscosity values of three excipients—1 % sodium hyaluronate(SH), 4% chitosan(CS), and 2% sodium alginate(SA) were lower than 10 Pa?s, with the 1 % sol as an exception. The viscosity of the 1% sol decreases to 583 Pa?s; the p H value varies from 5.66 to 7.43; the viscous modulus of the gel hardly changes before and after the irradiation, but the elasticity modulus slightly decline. The colloid can be fully dissolved within 12h(the activity of bone induction and infiltration of the cells are not affected if removed within 24h); the result of histology shows that the glycerin gelatine possesses very good histocompatibility and degradation property.Conclusion: With glycerol as solvent, an excipient that can be sterilized with irradiation is prepared in the research. Based on the change of gelatin content, this sol- gel shows a large modulus span, which endows this excipient with large potential. The sol-gel carrier material that can be sterilized with 60Co-γ X-ray irradiation will greatly facilitate the manufacturing technique of synthetic compounds, which, to a large extent, will reduce the production cost of aseptic operation facilities and technologies as well as the risks of the craft process. Given the stability and security of the matrix, the application can be further expanded to pharmaceutical and food industry.Part IV Preparation and evaluation of injectable DBM/HA-TCPcomposite materialsPurpose: Prepare injectable DBM/HA-TCP composite materials. Rabbit models with bone defect are chosen to conduct experiment, with ilium as the operation site. Determine the optimized ratio of DBM and HA-TCP by evaluating the effects of composite materials in repairing bone defects.Methods: The first is to mix the calcined HA and TCP materials by the proportion of 60:40, and BCP composite powder is acquired after intensive mixing; mix the DBM and BCP composite powder of the rabbitwere, and make five hybrid materials of 100% DBM, 25% BCP, 50% BCP, 75% BCP, and 100% BCP; mix the hybrid materials with glycerol gelatin evenly, with the ratio of glycerin and composite powder at 10:8, then five groups of samples are acquired and sterilized with irradiation after packaging. In the iliac bones of rabbits, drill hole-shaped defects with a diameter of 6mm, a length of 10 mm, and a depth of 5mm. Drill three defects on either side of the iliac bones, with a total of six ones. Place the five groups of materials in the bone defects of the rabbits seperately and keep one blank group for comparison. Inject penicilin for five consecutive days after operation, and observe at the fourth and eighth weeks. Evaluate the changes of local bone mass and bone changes with Micro-CT; after fluorescence labeling, observe the width difference between the two fluorescent labelings under the fluorescence microscope to calculate the osteogenic rate; in addtion, histological observation of VG dyeing is needed.Result: Expose the five materials respectively under X-rays for imaging, and the injected materials in 100% DBM hardly develop. With the addition of compound inorganic powder of HA and TCP, the development density of the injectable composite materials gradually increase. The surgical spots of some animals swell inordinately after surgery and will disappear in about two weeks. Based on the results of Micro-CT, the recovery modes of the surgical spots of all the other groups are in good condition at the eighth week except the blank group, and there is a defect of varying degrees in the center. Yellow green marks can be seen in all the samples under the fluorescence microscope. The new bone trabecula of each experiment group grows vigorously, and the rate of mineralization deposition in 75% BCP is the highest. The VG histological observations find that the new bone trabecula of each group grows vigorously, and no inorganic powder and DBM is found. The final result is that the new osteogenesis of 75% BCP group is the best.Conclusion: Based on the evaluation results, this research has prepared an injectable DBM/HA-TCP composite material with good capacity of injection and plasticity and high osteogenic capacity.