| ObjectiveTo develop a new artificial bone repair material with excellent biocompatibility, osteoconductivity and biodegradability and provide experimental data for its ultimate clinical use, the poly(amino acids)/calcium sulfate (PAA/CS) composite was synthesized and its sterilization method, cytotoxicity, biocompatibility, bioactivity, osteoconductivity and in vivo biodegradability were investigated.Methods1. The PAA/CS composite was synthesized from six kinds of amino acids including 6-aminocaproic acid, L-glycine, L-alanine, L-phenylalanine, L-proline and L-lysine with CS in a certain proportion by melt polycondensation method. After sterilized by five different methods of gamma-irradiation, autoclave, hypothermal plasma, ethylene oxide and ultraviolet, the change of the crystalization pattern, compressive strength and cytotoxicity of the extracts of the PAA/CS composite was analyzed, using the X-ray diffraction analysis, mechanical test and L929 cells co-culture test accordingly.2. The bioactivity of the PAA/CS composite was preliminarily evaluated using the MG63 cells adhesion test and in vivo bone implantation test. The human osteoblast-like MG63 cells were seeded on the surface of the PAA/CS, PAA and CS plate (10mm in diameter and 2mm in thickness), and the polyester plate was used as blank control, with 6 samples per group. The adhesion and proliferation of the MG63 cells on the material surfaces were observed by scanning electron microscopy, and the osteogenesis of MG63 cells was determined by assaying the expression of alkaline phosphatase (ALP) and osteocalcin. The PAA/CS rod (2mm in diameter and 6mm in length) was implanted into one side of the rabbits'tibia, and the polyethylene rod in same size into the contralateral side as control. After 2, 8, 16 weeks, the specimen were harvested. The biological reactions between the PAA/CS and the host were observed macroscopically and histologically.3. The bone defects of 6.5mm in diameter and 6mm in depth were created on the both sides of the rabbits'femoral condyles, totally 72 bone defects in 36 rabbits. All the defects were divided into 3 groups, as one implanted with PAA/CS grannules and the contralateral one on the same rabbit implanted with CS or nothing as control. The specimen were harvested at postoperative 2, 4, 12 weeks. The biological reaction, bone defect repair and degradation of the PAA/CS composite were investigated macrosopically, radiologically by micro-CT scanning and histologically by decalcified bone slices staning with masson tricolor and toludine blue.Results1. The X-ray diffraction test demonstrated that the number, morphology and localization of the crystallization peak had no visible change after sterilization. Therefore, the five sterilization methods had no apparent influence on the crystallization pattern of the PAA/CS composite. The compressive strength of the composite apparently decreased after sterilized by autoclave and hypothermal plasma, but not after sterilized by othe three methods. The PAA/CS extracts apparently influenced the growth of the L929 cells after sterilized byγ-irradiation, autoclave and hypothermal plasma, and the cytotoxicity was grade 3～4; but not after sterilized by ethylene oxide and ultraviolet, and the cytotoxicity was grade 0～1.2. The MG63 cells around the PAA/CS and PAA plates were in well growth and proliferated with time, indicating the good cytocompatibility of the two materials. The MG63 cells exhibited good adhesion and proliferation behaviors on the surfaces of PAA and PAA/CS but not of CS. After 14 days, the ALP activity in PAA/CS and PAA group were higher than the CS group and control group, and the difference between the two groups was not statistically. The osteocalcin content in PAA/CS group was statistically higher than PAA group, CS group and control group. The PAA/CS was intimately contacted with the host bone after implantation for 2 weeks, and new bone were formed at 8 weeks. At 16 weeks, the maturely differentiated bone grew into the inferior part of the material. During the entire period of observation, no abnormal pathological reaction or immunological rejection were identified.3. All incisions healed after postoperative 3 days. No infection, obsess or ulcer presented during the entire period of observation. Most of the CS were absorbed at 2 weeks with some mucus formed, and were completely degraded at 4 weeks. Only at 4 weeks were the PAA/CS visibly degraded, and were almost completely degraded at 12 weeks, without any abnormal tissue reactions. The decalcified bone slices exhibited that osteoblasts adhered on the surface of the PAA/CS at early stage, and a few of new bone were formed. As the degradation of PAA/CS, the newly formed bone grew into the inferior part and differentiated into mature bone. Although the defects got repaired in all groups at 12 weeks, the micro-CT scanning disclosed that only the cortical defect got repaired in the blank control group with less amount of bone trabecula. The bone density in the control group was also statistically lower than that in PAA/CS and PAA group.Conclusion1. The ethylene oxide and ultraviolet can be used for sterilization of the PAA/CS composite as they have no apparent influence on the crystallization pattern, compressive strength and cytotoxicity of the material. But the gamma-irradiation, autoclave, hypothermal plasma are not suitable as they affect the compressive strength or cytotoxicity of the PAA/CS composite.2. The MG63 cells can adhere, proliferate and differentiate well on the surface of the PAA/CS composite. The PAA/CS composite causes no abnormal tissue reactions and can firmly bond with the host bone. Therefore, the PAA/CS composite is of good cytocompatibility, tissue compatibility and bioactivity.3. The degradation rate of the CS is evidently decreased after the incorporation of PAA, which is more matched with the osteogenesis rate. The PAA/CS composite displayed comparable ability for bone defect repair with CS and is expected to be an excellent bone repair material. |
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