| BackgroundsBone defect, especially the complex anatomical structure of bone defect, resulted from trauma, infection, tumors, or dysplasia, has been a difficult problem in clinical for a long time. Currently, the mainly treatments for bone defect in clinical are autogenous bone graft and allograft bone transplantation, however, the sources of autologous bone are limited, and autologous bone graft can also cause residual pain and deformation of the supply parts; allogeneic bone has antigenicity, there is a risk of infection, too. And it is also difficult to create a matching contour of the bone defect from autologous bone or allogenic bone. The emergence of bone tissue engineering in the 1980s of the 20th century, has opened up new channels for the treatment of bone defect, especially the complex anatomical structure of bone defect.The scaffold is one of the key elements of bone tissue engineering. To prepare a perfect scaffold for bone tissue engineering, in addition to select the appropriate biological materials, the scaffold forming process is also very important.The scaffold should not only have good mechanical strength and biological activity, but also have to provide rational three-dimensional structure the ingrowth of new tissue and cells, and match with the complex defect shape. While the application of the traditional manufacturing methods of the scaffold (fiber bonding, phase separation, solvent casting particulate filter, film lamination method, melt molding, gas foaming particulate filter method and the combined use of these methods) has been very extensive, but they have fatal flaws, they mainly appear in the manually manufacturing process, the use of toxic organic solvents, lack of the control of the pore structure (such as hole size, spatial direction, connectivity, etc.), it is difficult to create a specific contour of the scaffold, which is match with the bone defect. In the 80s of the 20th century, the emergence of a new manufacturing technology, with computer aided design (CAD) based-Rapid Prototyping (RP) technology, can create any complex three-dimensional solid shape for bone tissue engineering scaffolds, and it makes the profiling and bionic manufacturing of the scaffolds for bone tissue engineering to be possible.Since 1995, our study group has synthesized the Porites coral into coralline hydroxyapatite by "hydrothermal exchange reaction", and has proved its excellent biocompatibility. Its natural pore-like structure is extremely similar to the human cancellous bone structure, so it is beneficial for the ingrowth of fibers and vascular tissue. But the brittleness of the CHA artificial bone is so large, that is difficult to prepare personalized scaffold according to the shape of bone defects, so there is a certain shortage.If you can take advantage of rapid prototyping technology, while retaining the natural coralline hydroxyapatite porous-like structure, and prepare the personalized scaffold according to the shape of bone defects. It will avoid cumbersome process of the second preparation of the holes, which will greatly promote the development of bone tissue engineering scaffold. Biocompatible and biodegradable polymer biomaterials provide key "adhesives" for the preparation of the DCHA. L-Polylactic acid is a synthetic polymer material, which has been widely used in medicine, and it has been certified by the U.S. Food and Drug Administration as the body implants. It has been widely used in devices such as bone plate fixation, bone screws, surgical sutures, spinning and so on. In this study, we used L-PLA as "adhesives", and made the CHA into the DCHA artificial bone scafflold. Through a series of experiments, the physicochemical properties, biocompatibility of the DCHA artificial bone scafflold were studied. At the same time, by repairing the bone defect of the animal model, its clinical feasibility was investigated.Objectives1. To study the preparation methods of the DCHA artificial bone scafflold, and analyze heir structural characteristics, physical and chemical properties, and explore the ideal material ratio for preparing the DCHA artificial bone scafflold.2. To study the degradation performance of the DCHA artificial bone scafflold in the simulated body fluid in vitro, and explore its feasibility as a tissue engineering material.3. Perform the cell toxicity test, hemolysis test, pyrogen test, acute toxicity test, and delayed type hypersensitivity test, in order to evaluate bio-security of the DCHA artificial bone scafflold, and provide experimental basis for further research.4. Isolated and cultured the bone marrow stromal cells (BMSCs) to the osteogenic induction in vitro, and cocultured the cells with the DCHA artificial bone scafflold in vitro, in order to detect its cell biocompatibility.5. To explore the osteogenesis and the feasibility of as bone substitute materials for the clinical treatment of bone defects of the DCHA artificial bone scafflold, in order to provide a new artificial bone material.Methods1. The coralline hydroxyapatite was smashed into CHA particles with 250μm～ 500μm, and then the CHA particles were mixed with L-PLA in different mass ratios (2:1,3:1,4:1,5:1). With Simpleware 3.1 software, a digital cylindrical model was built by computer-aided design (CAD). Through the digital conversion, the model was transformed into STL file format, and then was imported into rapid prototyping machine. The DCHA artificial bone scafflold was prepared by sintered the mixture of the CHA particles and L-PLA, using selective laser sintering rapid prototyping (SLS RP) process. Using vernier caliper to measure the height and diameter of the DCHA artificial bone scafflold and then compared them with the height and diameter of CAD model, and analyzed its accuracy. Detection the hydrophilic rate of the DCHA artificial bone scafflold by soaking them in the simulated body fluid. Physicochemical, structural and mechanical properties of the DCHA artificial bone scafflold were analyzed by mercury porosimeter, mechanical testing machine and scanning electron microscopy (SEM).2. We used the mass ratio of 3:1 and 4:1 of the CHA particles and L-PLA as raw materials to prepare the DCHA artificial bone scafflold, and then they were placed in the 50mL stimulated body fluid (SBF) with the initial pH value of 7.4 in incubator at 37℃for degradation, and observed the changes of the specimen shape, color and gloss. Detected the pH value, calcium and phosphate ions concentration of the SBF in 2W,4W,8W,12 W and 16W, respectively, and detected the material degradation rate, compressive strength changes of the sample materials at different time points. And performed SEM of the DCHA artificial bone scafflold in 16W to observe the material changes in the microstructure.3. Referenced to GB/T 16886.1-2001 idt ISO 10993-1:1997 and GB/T 16175-2008, the DCHA artificial bone scafflolds, which were prepared at the mass ratio of 3:1 and 4:1 of the CHA particles and L-PLA, were performed the biological evaluations of the cell toxicity test, hemolytic reaction test, pyrogen test, acute toxicity, delayed type hypersensitivity test.3.1 Cell toxicity test:L929 cells were cultured with the extracts of the DCHA artificial bone scafflolds with different masses in vitro. The morphologic changes of L929 cells were observed by inverted microscope. MTS tests were performed to evaluate the cell proliferation, and the relative growth rate (RGR) was calculated to evaluate the cytotoicity of the digital coral hydroxyapatite artificial bone with different masses.3.2 Hemolytic reaction test:detected the hemolytic rate of the leaching liquor of the DCHA artificial bone scafflolds, and evaluated their hemolytic reaction.3.3 Pyrogen test:Intravenous injection the leaching liquor of the DCHA artificial bone scafflolds, and detected the temperature changes of the rabbits at different time points, in order to evaluat their pyrogen reaction.3.4 Acute toxicity test:Intravenous injection the leaching liquor of the DCHA artificial bone scafflolds, and observed the toxicity reaction in mouse, in order to evaluat the toxicity of the materials.3.5 Delayed hypersensitivity test:Intradermal injection the leaching liquor of the DCHA artificial bone scafflolds, and observed the skin reactions in guinea pigs, in order to evaluat the sensitization of the materials.4. Cell Compatibility Evaluation.4.1 Isolation and culture of rabbit BMSCs, osteoblastic induction and identification.Took suction 5mL bone marrow of the New Zealand white rabbit, by the combined methods of density gradient centrifugation and adherent cell separation, BMSCs were obtained. Performed adherent culture, amplification and passage in vitro, observed the morphology and growth of the primary and every generation cells under the inverted microscope, and depicted the growth curve. The 3rd generation cells were cultured with the conditioned medium, containing 1×10-8mol/L dexamethasone; 10mmol/Lβ-glycerophosphate and 0.2mmol/L L-ascorbic acid, in order to make them orientation osteogenic cultured and amplified. The cells morphological changes were observed by inverted microscope, and the cells proliferation was measured by MTS method. Alkaline phosphatase (ALP) activity test, Von Kossa staining and Alizarin red staining and other methods were employed to assess BMSCs’ osteoblastic differentiation and the generation of calcified extracellular matrix.4.2 The osteogenic cells and the DCHA artificial bone scafflolds were cocultured in vitro.The rabbits BMSCs were osteogenic cultured to 3 generations, and then the 3rd osteogenic cultured BMSCs were inoculated into the DCHA artificial bone scafflolds, which were prepared at the mass ratio of 3:1 and 4:1 of the CHA particles and L-PLA, using coverslip as a negative conteol group. By inverted microscope and scanning electron microscopy, the growth of the osteogenic induced cells in the materials was observed. Cells proliferation activity in the materials was detected by MTS, and alkaline phosphatase activity determination was used to to evaluate the osteogenic capacity. All of these were used to perform biocompatibility evaluation of the DCHA artificial bone scafflolds.5. Cavities bone defects were created at two tibial metaphyseals in each rabbit. And then used the DCHA artificial bone scafflolds, which were prepared at the mass ratio of 3:1 or 4:1 of the CHA particles and L-PLA, were implanted into the bone defects in rabbits through open operation. The curative effect was evaluated by radiographic examination, histology analysis and eyes observation in experimental groups, and blank control group at post-operative 2d,4 weeks,8 weeks,12 weeks, respectively.Results1. For the mixtures of CHA particles and L-PLA with different mass ratios, using CAD and SLS RP process, the DCHA artificial bone scafflolds could be prepared. The scafflolds were structured like small cylinders, uniform thickness, and their surface was roughness, its machining accuracy was +0.1 cm. And with the increase of the CHA content, the scaffold surface roughness increases, the particles in the surface is also easy to call off from the scafflolds, and the hydrophilic rate (35.00%～53.07%), porosity (31.93%～55.36%) and density (1.61g/mm3～2.29g/mm3) of the scafflolds gradually increased, while its compressive strength (1.04～3.70MPa) gradually decreased. The scafflolds were with microporous structure of interlocking between the hole and hole, the pore size was 150μm-350μm, and as the content of CHA increased, the proportion of large pore size gradually increased.2. With the in vitro degradation time, the surface roughness and porosity gradually increased, we could find fine granular material dissolution. In the course of 16 weeks degradation, pH values of the scafflolds groups decreased just slightly, maintaining of 7.34～7.36, and they were higher than the L-PLA’s (P<0.01), lower than the HA’s (P<0.01). Calcium, phosphorus concentration increased gradually with prolonged degradation and they increased rapidly within 2 weeks. The calcium concentration of the mass ratio of 3:1 of the scafflolds was higher than the calcium concentration of the mass ratio of 4:1 of the scafflolds, at 4W,8W,12W,16W (P<0.01), and phosphate concentration showed no significant difference (P>0.05). There was no significant difference in degradation rate between the DCHA artificial bone scafflolds with the mass ratio of 3:1 and 4:1 (P>0.05). As the degradation time, the compressive strength of the DCHA artificial bone scafflolds with the mass ratio of 3:1 and 4:1 decreased slowly, and the compressive strength were 2.89±0.22 MPa and 1.62±0.07 MPa at 2 weeks; 1.15±0.03 MPa and 0.69±0.08 MPa at 12 weeks. There was a significant difference between the two groups in the compressive strength at every degradation time (P<0.01).3. As to the extracts of the digital coral hydroxyapatite artificial bone scafflolds, with the mass ratio of 3:1 and 4:1, the cell toxicity grade were 0; hemolytic rate was 2.22%, which was less than 5%, and was meeting the requirements of the bio-medical. And their pyrogen was in the original compliance. Their acute toxicity grade was 0; allergic reaction score was 0 point.4. BMSCs cultured in vitro showed obvious osteogenic capacity in mineralization DMEM. Von Kossa staining and Alizarin red staining of the mineralized nodules and alkaline phosphatase detection of the passaged cells both yielded positive results. MTS assay showed the osteoblast proliferation was normal.The osteogenic cultured BMSCs could adhere to the digital coral hydroxyapatite artificial bone scafflolds, with the mass ratio of 3:1 and 4:1, and proliferate and grow on the surface of the scafflolds normally. The cellular activity and function were not affected by the materials, and no statistical difference was found between the groups (P>0.05).5. The bone defects that had been treated with DCHA artificial bone scafflolds, with the mass ratio of 3:1 and 4:1, exhibited new bone formation increased with time by radiography, histology and eye observation. The rate and quality of new bone formation were no statistical difference in the experimental groups (P>0.05), but better than the blank control groups (P<0.01). In blank control groups, there were no formation of new bone after operation and bone defects were finally repaired only by fibrous tissue. Conclusions1. By crushing CHA into 250μm～500μm CHA particles, and then mixing the CHA particles with L-PLA in different mass ratio (2:1,3:1,4:1,5:1) as raw materials. And with Simpleware 3.1 software, build a digital cylindrical model by computer aided design (CAD). And last through SLS RP process, the DCHA artificial bone scafflold can be prepared, its machining accuracy is +0.1 cm. All of the scafflolds maintain the natural porous coral-like structure, which can avoid of the cumbersome process of making holes.2. Through a series of methods to exosyndrome the DCHA artificial bone scafflolds, the ideal mass ratio is 3:1 or 4:1. By this mass ratios, the scafflolds are of pore size 150μm～350μm, irregular holes, porosity of 46.55%'～52.65%, hydrophilic rate of 42.66%～46.29%, compressive strength of 1.82MPa～3.02MPa.3. During the in vitro degradation in SBF, the pH value of the DCHA artificial bone scafflolds maintain around 7.35 in 16 weeks, the degradation rate of 16 weeks is 37%, which is between the CHA and L -PLA between. With the in vitro degradation time, the porosity gradually increase, and the compressive strength decrease, which is 0.32 MPa～0.55 MPa at 16 weeks, still maintaining a basic natural cancellous bone strength (0.4 MPa～11 MPa).4. The DCHA artificial bone scafflolds, with the mass ratio of 3:1 and 4:1, are with non-cell toxic and non-pyrogenic, do not cause hemolytic reaction and delayed hypersensitivity. They are full compliance to GB/T 16886.1-2001 idt ISO 10993-1:1997 standards. So the scafflolds has good biological properties, which meets the basic conditions for tissue engineering materials.5. The digital coral hydroxyapatite artificial bone scafflolds, with the mass ratio of 3:1 and 4:1, have a good cell biocompatibility and can be used as a tissue engineering scafflold. 6. The DCHA artificial bone scafflolds, with the mass ratio of 3:1 and 4:1, can repair the cavities bone defects of the tibial metaphyseal, showing good bone conduction and biocompatibility; the DCHA artificial bone scafflolds in bone defect sites can be degraded gradually, which is adapt with the bone repair, and the osteogenic effect was good. All of these show that the DCHA artificial bone scafflold is a good tissue engineering scafflold. It is supposed to be a good way to repair clinical bone defect. |
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