| Objective:A novel nano-hydroxyapatite/poly-amino acid (n-HA/PAA) composite was prepared by using in-situ polymerization. The features of composition and construction, performance of degradation and biomechanics, biosecurity and biocompatibility in vivo and in vitro of the composite were evaluated synthetically. The model of femoral intercondylar fracture of experimental animal was eatablished, the feasibility of n-HA/PAA composite internal fixation bar as a novel internal fixation material was assessed, and we hope it can provide scientific experimental basis for n-HA/PAA composite's clinical practice.Methods:The experiments included four parts.1. Study on the preparation and performance of n-HA/PAA composite: In-situ polymerization was used to prepare n-HA/PAA composites of different mass ratios; The distributing of composite's inorganic components was observed by combustion test and scanning electron microscope; FT-IR and XRD was used to observe the composite's composition and construction features; The composite's initial biomechanical strength was inspected; SBF immersing experiment was performed to study on the composite's degradation function, mechanical strength after degradation and hydrophinicity.2. Study on the cellular compatibility of n-HA/PAA composite in vitro (the experiments included two parts).①The L929 cytotoxicity test of n-HA/PAA composite: The n-HA/PAA composites and L929 cell were cultivated together. The n-HA/PAA composite decoloration and cytolysis were observed by direct contact assay. The morphologic changes of L929 cells were observed by extract solution assay, MTT assay was performed to evaluation the cell proliferation at 24h, 48h and 96h, and apoptosis was detected by flow cytometry Annexin V/PI double staining method, and the ratios of normal cells, early apoptosis, advanced apoptosis and necrosis cells were calculated.②Biocompatibility evaluation of n-HA/PAA composite with osteoblast-like cells in vitro:The MG63 cell line was used to be cultured with n-HA and n-HA/PAA composite for 7 days. The MG63 cells morphologic changes of surrounding biomaterials were observed by inverted phase-contrast microscope, and SEM and DAPI fluorescent staining was used to observe the cells on the surface of biomaterials; MTT assay was performed to observe the growth of MG63 cells; IFCC recommendation method was used to evaluate MG63 cells' ALP activity; ELISA assay was performed to test the OCN content of MG63 cell; Flow cytometry was used to inspect MG63 cell cycle, and Annexin V-FITC/PI dual-label flow cytometry was used for testing the apoptosis.3. Study on the biocompatibility of n-HA/PAA composite in vivo (the experiments included two parts).①Study on the muscle implantation of n-HA/PAA composite: Polyethylene was used as the control group to carry out the following muscle implantation experiment. The changes of the muscle tissue around materials was observed by an unaided eye, HE staining and TEM, and at the same time, the biodegradation of composite was observed in vivo. Additionally, the method of degradation was analyzed by using XRD.②Study on the bone implantation of n-HA/PAA composite:Polyethylene was used as the control group to carry out the following tibia implantation experiment. The changes of the bone tissue around materials were observed by an unaided eye, hard tissue section and SEM. At the same time, the security of whole body was evaluate by comparing the blood routine and biochemical indicator tests.4 Study on the treatment of femoral intercondylar fracture fixed with n-HA/PAA internal fixation bar:The model of femoral intercondylar fracture of New Zealand rabbit was prepared, and it was fixed with n-HA/PAA internal fixation bar and Kirschner pin as control. The healing of fracture was observed by imaging, the connection of bone interface was observed by hard tissue section, and bone interface cohesion was measured by push-off test.Results:1 Study on the preparation and performance of n-HA/PAA composite:The n-HA/PAA composite could be obtained by in-situ polymerization. The distribution of n-HA component in the composite was homogeneous, and the composite has a good homogeneity. There was a connection of chemical bond between n-HA and PAA, not a mechanical bond with analysis of FT-IR and XRD. The biomechanical test showed that compressive strength of composite were 140.65±10.26Mpa, 128.44±9.92Mpa, 117.74±9.21Mpa, 62.41±6.25Mpa; flexural strength were125.46±9.96Mpa, 105.34±8.75Mpa, 92.16±8.59Mpa, 52.23±5.31Mpa; shear strength were 70.15±6.21Mpa, 60.61±5.36Mpa, 52.89±5.63Mpa, 22.11±4.21Mpa; and elastic modulus were 15.69±2.68Gpa,12.18±2.03Gpa,9.16±1.88Gpa,5.01±0.82Gpa with 10wt%, 20wt%, 30wt%, 40wt%n-HA, respectively. SBF immersing experiment showed that the composite has a good biodegradation, the ratio of degradation of composite were 6.97±0.63%,8.16±0.81%,17.98±1.69%,32.16±2.11% with 10wt%～40wt% n-HA at 12th week, respectively; and the flexural strength was decreased with the degradation, they were 102.39±8.21Mpa,90.1±7.89Mpa,75.59±7.66Mpa,25.63±3.69Mpa with 10wt%～40wt% n-HA at 12th week, respectively; The composite of 30wt% and 40wt% n-HA had excellent and stable hydrophilicity, and the hydrophilic ratio were about 10% and 15%, respectively.2 Study on the of cellular compatibility n-HA/PAA composites in vitro.①The L929 cytotoxicity test of n-HA/PAA composite: The cell morphologic observation showed that there was no obvious morphologic influence, decoloration and cytolysis in the n-HA/PA group and negative control group. MTT assay revealed that the extracts didn't inhibit the cell proliferation in n-HA/PA group and negative control group, there was no significant difference between the two groups (P<0.05), and the toxic level of n-HA/PA composite was ranked as 1. The cell scatter diagrams showed that there was no significant difference (P<0.05) in living cells, early apoptosis, advanced apoptosis and necrosis cells between the n-HA/PA group and negative control group.②Biocompatibility evaluation of n-HA/PAA composite with osteoblast-like cells in vitro:There was no obvious cell morphologic changes in the n-HA and n-HA/PAA groups, and the adherent MG63 cells around the n-HA and n-HA/PAA and others on the surface of them grew well. MTT assay revealed that the MG63 cells had obvious proliferation, the OD value increased gradually with the increasing time of culture in the two groups, and there was on significant difference (P>0.05) in the two groups at all time points. The ALP activity and OCN level of MG63 cells increased gradually with the increasing time of culture in the two groups, and there was on significant difference (P>0.05) in the two groups at all time points. The n-HA and n-HA/PAA composite couldn't inhibit the cell cycle of MG63 cells and promote the apoptosis, and there was on obvious difference (P>0.05) of the numbers of cell cycle and apoptosis in the two groups at all time points.3 Study on the biocompatibility of n-HA/PAA composite in vivo.①Study on the muscle implantation of n-HA/PAA composite:There was a similar result in the experimental group and control group during the muscle implantation. There were no obvious inflammatory response, immunoreaction, and rejection to the muscle tissue around the materials. The proportion of weight losses of the n-HA/PAA composite was 10% at 24th week. The result of XRD showed the composite would be degradable with external corrosion.②Study on the bone implantation of n-HA/PAA composite:There was no obvious inflammatory response, immunoreaction, rejection, and bone destruction or osteomyelitis to the bone tissue around the materials. The results of SEM showed that it was a osseously bond on the bone interface in 24 weeks, and the n-HA/PAA composite couldn't influence the liver, kidney, blood routine and biochemical indicator.4 Study on the treatment of femoral intercondylar fracture by fixed with n-HA/PAA internal fixation bar: Compared with the control group, the n-HA/PAA internal fixation bar has a similar internal fixation effect. The results of hard tissue section showed that there was a better osseously interface in the n-HA/PAA group. The results of push-off test showed that there was no obvious difference in the two groups (P>0.05) in the experimental early stage, but there was a better osseously interface in the n-HA/PAA group (P<0.05) after 2 weeks.Conclusion:1 The n-HA/PAA composite could be obtained by in-situ polymerization. There was a chemical bond between the n-HA and PAA, and this composite had good hydrophilicity, biomechanical property, and biodegradation.2 The n-HA/PAA composite was no obvious cytotoxicity to L929 cells.3 The n-HA/PAA composite had satisfactory biocompatibility with MG63 osteoblast-like cells.4 The n-HA/PAA composite had satisfactory tissue compatibility in vivo, and it could be degradable with external corrosion.5 The n-HA/PAA internal fixation bar has satisfactory internal fixation effect for femoral intercondylar fracture of experimental animal, and could provide a novel material of internal fixation with satisfactory biosecutity, efficiency and avoiding second operation for clinical cancellous bone fracture.
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