Development And Study Of A Novel Porous PLA/BMG Composite Bioactive Material

Part 1 Preparation of the Porous PLA/BMG Composite Biomaterials and its Physical and Chemical Characteristics StudyObjective: To develop a novel porous poly (lactic-acid) (PLA)/bone matrix gelatin (BMG) bioactive composite biomaterial with supercritical carbon dioxide fluid technique (SC-CO₂) and to choose the optimal PLA/BMG proportion by evaluating its physical and chemical characteristics and cellular compatibility.Methods: The bone allografts procured from the qualified donor were processed into bone powder with the size of 50μm～200μm by deep-freezing, cleaning according to the processing standards of bone allograft in Shanxi Provincial Tissue Bank. Then the powder was processed into Bone Matrix Gelatin (BMG) by defatting and demineralization as described by Urist. The PLA and BMG, mixed with the PLA:BMG proportion of 9: 1,8: 2, 7: 3, 6: 4, and the 100μm～200μm NaCl was added in the SC-CO₂ equipment in order to make pores. Tightly closing the reactor kettle and CO₂ under 0℃was pumped into kettle. When the internal pressure reached 20MPa, the temperature was regulated at 36℃. The pressure was kept in (20±0.1) MPa and the temperature in (36±0.5)℃. After 30 minutes, the gas was released at the rate of 5 MPa/min.Opening the reacting kettle and the samples were taken out. After the samples were leached in deionised water to remove the NaCl in material, they were packaged and sterilized by radiation. The macroscopic observation, porosity, mechanics and scanning electron micrograph (SEM) were done to evaluate the physical and chemical properties. Then referring to the cellular compatibility, the optimal proportion was chosen.Results: In macroscopic observation, the PLA/BMG composite biomaterial, prepared with the SC-CO₂ technique, have porous structure and good interconnective pores. The structure is like that of human cancellous bone. The porosity was more than 60 % and increasing with the BMG proportion being improved. While the compressive strength and elastic modulus was in inverse proportion to the proportion of BMG in PLA/BMG. When the BMG proportion was more than 30 %, the mechanics properties dropped significantly. In cell compatibility, PLA biomaterial did harm to proliferation of rat L6 cells. With the increasement of BMG proportion, cell compatibility was getting better. When the proportion reached 40 %, there was no difference between the PLA/BMG composite biomaterial group and the culture medium group. The SEM of PLA/BMG with 30 % BMG showed PLA and BMG were mixed uniformly, the pores diametesr were between 200μm and 400μm and the pores were distributed uniformly and interconnected each other. On the wall of pores, there still a number of about 10μm pores.Conclusion: The percent of BMG in the BLA/BMG composite biomaterials prepared with SC-CO₂ technique was in direct proportion to its cellular compatibility and porosity, and in inverse proportion to its mechanics. The results show the PLA/BMG containing 30 % BMG was the optimal.Part 2 Evaluation of Biocompatibility on the Porous PLA/BMG Composite BiomaterialsObjective: To evaluate the tissue biocompatibility in vivo by animal experiments and cell biocompatibility in vitro by cell experiments and provide the biocompatibility datas for PLA/BMG composite biomaterial.Methods: Tests for cytotoxicity in vitro methods: Referring to the biological evaluation of medical devices-Tests for cytotoxicity: in vitro methods, the PLA/BMG and PLA porous biomaterials were immersed in DMEM culture medium with 0.1 g biomaterial each ml DMEM. After 72 hours in 37℃, the supernatants were collected by centrifuge. 200μl of the MC3T3-E1 cell suspension with a cell density of 5×10⁵ cells/disk was seeded evenly into each well in 96-Well Plate. When the cell adhered after 24 hours, the culture medium in wells was removed and 200μl supernatant were added according to groups respectively, with DMEM culture medium being the control group and no cells well being the blank control group. The cells were cultured in 37℃humidified atmosphere of 5% CO₂. The cell culture mediums were exchanged every two days. On the 1^st day, 2^nd day, 3^rd day, 4^th day, 5^th day, 6^th day and 7^th day respectively, 30μl 2mg/ml MTT solution was added into each well. After being incubated for 4 hours, 150μl DMSO was added and the plate was shaken for 10 minutes. The OD of the medium was read at 570 nm with a plate reader. The cell proliferation rates were calculated and transformed into the toxicity grades. The cellular affinity of PLA/BMG composite biomaterial: The 3 mm×3 mm×3 mm PLA/BMG blocks processed were placed in 96-Well Plate. 500μl of 1×10⁴/ml MC3T3-E1 cell suspension was seeded into each well and DMEM was added to immerse the PLA/BMG blocks. Cell morphology and adhering to materials was observed on the 1^st, 3^rd, 5^th day after seeding under phase contrast microscope and on the 5^th day under SEM. Biological evaluation in vivo: 20 Wistar mice, PLA biomaterials were implanted both in nape subcutaneous tissue and thigh muscle on the left side, and PLA/BMG biomaterials on the right side. The gross observation about animal situations was made. At the 2^nd, 4^th, 6^th, 8^th week after implantation, the 5 mice were chosen to be killed randomly. The implanted material and surrounding tissue were procured and stained with HE. The histological changes were made observation under light microscope.Results: Tests for cytotoxicity in vitro methods: In PLA/BMG group, the cell proliferation rates were over 100 % and the cell cytotoxic grades were Grade 0 from the 1^st day to the 7^th day. While in PLA group, the cell proliferation rates were less than 100 % and the cell cytotoxic grades were Grade I on the 2^nd, 4^th and 7^th day. The cellular affinity of PLA/BMG composite biomaterial: on the 1^st day after seeding, there were few cells on PLA/BMG. On the 3^rd day, a few cells adhered to the edge of PLA/BMG and the cells surrounding kept normal morphology. On the 5^th day, the number of cell adhered to PLA/BMG increased, even converged somewhere. SEM showed there were cells both on the surface and in the pores of PLA/BMG. The cellular morphology was triangle or polygon with abundant microvillus on the surface. Some cells connected and overlapped by protrusions. The cells adhered to the surface of PLA/BMG tightly. Some cells were keeping at mitosis phase. Biological evaluation in vivo showed PLA were enclosed with connective tissue containing a lot of lymphocytes and neutrophilic granulocytes. The cells and tissue grew into PLA slowly. The PLA/BMG materials were enclosed with little connective tissue including a few inflammatory cells. The surrounding tissue immersed into the center of PLA/BMG easily. Some mesenchymal cells migrated to the edge of BMG.Conclusion: the PLA/BMG composite biomaterials prepared with SC-CO₂ technique is of nice cellular affinity. The cytotoxicity grade is Grade 0, better than that of PLA. It shows good cell compatibility. Biological evaluation in vivo also suggests the tissue compatibility of PLA/BMG is better than that of PLA. Part 3 Experimental Study on Osteoinductive Activity of the Porous PLA/BMG Composite BiomaterialsObjective: To evaluate the osteoinductive activity of PLA/BMG by calcium depositions, ALP activity and Ca content by Mouse osteoblast-like MC3T3-E1 cells be cultured in DMEM with the different biomaterials.Methods: The PLA/BMG group: with 100μg of the scrushed PLA/BMG each well; The PLA group: with 100μg of the scrushed PLA each well. The DMEM group serves the control. There were 6 wells in each group. Cells suspensions were prepared and adjusted to 2×10⁶ cells/ml. 20μl cells suspension was delivered into each well of 24 well plate with the different group materials. After the cells were adhered, 1 ml of culture medium containing 10μmol/ml disodiumβ-glycerophosphate (β-GP) and 5μg/ml vitamin C was added. 2/3 of Culture medium was changed every 3 days. After 2 weeks, the cells were collected. Calcification depositions assay: After being washed three times with PBS buffer, the cell cultures were fixed with formalin and stained by alizarin red solution. The microscopic images were taken with the converse phase microscope and the macroscopic ones with the digital camera. The percentage of the stained area, standing for the calcification deposition, was measured with the image processing and analysis software. ALP activity and Calcium content measurement: After the harvested cells were washed twice with Hanks solution. The cells were resuspended in 1 ml of 0.2 % Nonidet P-40 (NP-40) and sonicated in an ice bath for 2 min using an ultrasonic processor. The measurement of the ALP activity and calcium content were taken according to the instructions of kits.Results: The ALP activity, Ca content and percent of calcification area in PLA/BMG group [ (325.59±70.40) U/g prot,(3.51±1.64) mmol/g prot),(42.98±4.44) % ] were more than those in PLA group [ (63.62±30.01) U/gprot,(1.04±0.21) mmol/g prot, (9.55±1.94)% ] and DMEM control group [ (2.40±1.47) U/g prot,(0.70±0.24) mmol/g prot,(0.86±0.41) % ] (P＜0.05). Meanwhile, there was statistic difference between PLA group and DMEM group in the ALP activity and calcification area.Conclusion: The PLA/BMG prepared by SC-CO₂ technique has good osteoinductive activity, better than that of PLA. And it is worth studying further as bone biomaterial and bone tissue engineering scaffold.Part 4 Experimental Study of the Porous PLA/BMG Composite Biomaterialsfor the Repair Radial Segmental Bone Defect of RabbitObjective: To evaluate the repair of rabbit radial defects by PLA/BMG biomaterials prepared by SC-CO₂ technique and provide the experimental date for the possible clinical applications.Methods: Twenty four New Zealand male rabbits with the Wt of about 2.5 kg were obtained for this study. After the rabbits were anesthetized and the forelegs were sterilized, the diaphysis of both radii were exposed through a longitudinal extensile incision, the periosteum was elevated circumferentially, and the 12 mm long middle shaft of the radius was cut with a power-driven oscillating saw. Then a 12 mm bone defect was created. The biomaterials were filled according to the different groups. Then the wound was closed in layers and the animals were put back into their cages. The animals were divided into 2 groups randomly. In one group, nothing was filled in defect on the left and the bone autograft cut was re-implanted on the right; in another group, PLA/BMG was filled on the left and PLA/BMG was done on the right. 4 animals of each group were put to death at week 4, 8, 12 after operation. Radiographic observation and evaluation: Radiographs were taken when the animals were put to death at week 4, 8, 12 respectively. The situation of bone reconstruction were observed each group and evaluated in a blind condition according to the Standard of Lane's X-ray Scores. Histological observation: the radial samples from rabbits were harvested and fixed in formalin solution. Then the dehydrated samples were embedded with plastic and sectioned longitudinally by 5 mm thickness, stained with hematoxylin and eosin, and finally observed under light microscope for the formation of the new bone and the degradation of the material.Results: Gross observation: except forl bone fracture in the blank control group and 2 fracture in the PLA group, all the animal were in good condition after operation and the operated limbs was uneventful with no detectable complications. Radiographic findings: In the blank control group, at 4 week postoperatively, little callus formed and the bone defect was clear; at 8 week postoperatively, a little callus with low-density was seen and the length of bone defect became shorter; at 12 week postoperatively, some slim callus formed at the both ends of broken bone and the defect was seen easily. In the PLA group, at 4 week postoperatively, a little callus with low-density formed at the ends and some fog-like image was seen in the defect; At 8 week postoperatively, the callus at the both ends met near to the ulna, but the defect was clear far to the ulna; at 12 week postoperatively, the both ends were connected with uneven shadow, little callus formed circumferentially and the cavitas medullaris did not recanalize. In the PLA/BMG group, at 4 week postoperatively, the both ends were enclosed with callus and the defect full of high-density shadow can not be identified easily; at 8 week postoperatively, the both ends met by high-density shadow; at 12 week postoperatively, with the new bone with uniform density bridging the both ends, the bone was reconstructed and the cavitas medullaris recanalized. In the autograft group, at 4 week postoperatively, the implanted bone could be identified and some callus formed at the broken ends; at 8 weeks postoperatively, the implanted bone were connected with the host bone by high-density callus; at 12 week postoperatively, the both ends were bridged with uneven high-density callus and the avitas medullaris recanalized. Radiographic evaluation: The PLA/BMG group and bone autograft group had no statistic difference at week 4, 8, 12 postoperatively. Which was better than both the PLA group and the blank control group (P＜0.05). There was significant difference at week 8 (P＜0.05) and no difference at week 4 and 8 between the PLA group and the blank control group. Histological Findings: In the blank group, the defect was filled with fiber connective tissue and no new bone formed. In the PLA group, the PLA were enveloped with connective tissue with little new bone formation. In the PLA/BMG group, the PLA/BMG incorporated with the host bone tightly. The new bone immersed into the PLA/BMG and some mesenchymal cells attached the BMG. In bone autograft group, large amounts of callus formed at the interface and the bone had reconstructed and remodeled.Conclusions: The ability to repair bone defect of the PLA/BMG is same to that of bone autograft and better than that of the PLA.