| BackgroundsThe main internal fixation materials using to treating fracture is metal which has some limitations including repeat surgery, stress shielding effects and inflammatory cascades causing by metallics ions and/or particles through corrosion or wear. The alternative of metallic material used as internal fixation is biodegradable polymer which deragrades too quickly or slowly, lacks of mechanical strength, is poorly compatible in vivo and leads to tissue imflammation. The further research for the internal fixation materials aims to the materials that have good biomechanics, adequate degradation and safety.Magnesium and its alloy wih biocompatibility, bone conductibility, degradability, biomechanical similarity with bone are potential to be a kind of new biodegradable metal used as internal fixation in orthopaedic, but their defects is quick corrosion and generate massive hydrogen. Therefore, the major research is to improve corrosion ressistance.Magnesium alloying and coating in it are the important measures to improve its mechanical property and corrosion resistance. Open porous scaffold made of magnesium alloy (AZ91D) has even fast-degrading rate and good biocompatibility. Magnesium alloy LAE442 provides low corrosion rates and low reacts in vivo with an acceptable host response and its corrosion rate can be further reduced by MgF2 coating. Magnesium with Ca-P coating improves cytocompatibility and the phosphate layer can prevent magnesium from quick corrosion. Ca-P that can transforms into hydroxyapatite (HA) indicates good biocompatibility.In the current study, we used magnalium (AZ1B,3%Al,1%Zn) with or without HA or MgF2 coatings to evaluate their initial biology and biomechanics of interference scew.Objectives1. Evaluate cytotoxicity of magnalium with or without coatings;2. Evaluate haemolyticus of magnalium with or without coatings;3. Analysis element and pH of extract of magnalium with or without coatings;4. Evaluate sensitization of magnalium with or without coatings;5. Evaluate systematic toxicity of magnalium with or without coatings;6. Evaluate magnesium degradation and concentration of serum magnesium after rabbits were implanted pin made of magnalium with or without coatings;7. Evaluate biomechanics of magnalium interference scew with or without coatings.Methods1. There were 8 groups in the cytotoxicity test:blank group (N), extract of magnalium with 100% concentration group (100%M), extract of magnalium with 50% concentration group (50%M), extract of magnalium with 25% concentration group (25%M), extract of magnalium with 10% concentration group (10%M), extract of magnalium with HA group (H), extract of magnalium with MgF2 group (F) and positive control group(P). L929 cells were cultured in DMEM extract for 1,3,5 and 7 day. There were 6 parallel holes in every observing stage in every group with tatol 192 holes. Cell growth was observed using optical microscope and was measured optical density (OD) using WST-1. Cytotoxic reaction was calculated. The data was staticstically analysis with Factorial analysis, and S-N-K test was compared in pairwise comparison. Level of significanceα=0.05.2. There were 2 tests in haemolysis test. There were 5 groups in the first test: blank group (N1), magnalium group (M), magnalium with HA group (H), magnalium with MgF2 group (F) and positive control group (P1). There were 9 groups in the second test:blank group (N2), extract of magnalium with 100% concentration group (100%M), extract of magnalium with 50% concentration group (50%M), extract of magnalium with 25% concentration group (25%M), extract of magnalium with 10% concentration group (10%M), extract of magnalium with 5% concentration group (5%M), extract of magnalium with 2.5% concentration group (2.5%M), extract of magnalium with 1% concentration group (1%M) and positive control group(P2). There were 3 parallel operations in every group. The method is according to the 13th section hemolysis test in GBT 16175-2008 Biological evaluation test methods for medical organic silicon materials. OD was measured and haemolysis rate was caculated. The data was analysied with one way ANOVA. S-N-K test was compared in pairwise comparison. Level of significanceα=0.05.3. Extract of magnalium was diluted into 3 concentrations, extract of magnalium with concentration ranging from 100%,50% and 5% which were respectively measured the concentration of Na+ and Mg2+. Extract of magnalium was diluted into 7 concentration with ranging from 100%,50%,25%,10%,5%,2.5% and 1.0% which were respectively measured pH. The data was statistically analysis with one way ANOVA, and S-N-K test was compared in pairwise comparison. Level of significanceα=0.05.4. There were 5 groups in the The Guinea-Pig Maximization Test (GPMT) Blank group (N), magnalium group (M), magnalium with HA group (H), magnalium with MgF2 group (F) and positive control group (P). There were 8 guinea pigs in N and group P and 14 in group H, group F and group M. The method is according to Biological evaluation of medical devices-Part 10:Tests for irritation and delayed-type hyper sensitivity. The skin was observed and sensitization rate and mean response value were caculated.6h,24h,48h,72h after patch removal Skin was biopsied 72h after patch removal and was analysised with optical microscope.5. There were 4 groups in systematic toxicity test:blank group (N), magnalium group (M), magnalium with HA group (H) and magnalium with MgF2 group (F). There were 5 Mus musculus albus in every group. The method is according to the 8th section systematic toxicity test in GBT 16175-2008 Biological evaluation test methods for medical organic silicon materials. The Mus musculus albus weight were measured before and 3 days after injected. The data was staticstically analysis with one-way AN OVA. Level of significance a=0.05.6. There were 5 groups in implant test:magnalium group (M), magnalium with HA group (H), magnalium with MgF2 group (F), titanium (T) and polylactic acid (PLA).24 rabbits were divided into 4 groups in which there were 6. The pins were implanted into the condyles of femur.3 condyles of femur in every group contained implant were biopsied 4,12w after implant.3 condyles of femur opposite side inmediatly implanted pins in every group presented samples of the operative stage. Activity, eating, sleep, wound of entry and cutaneous emphysema were observed. Photographs and micro-CT scans were carried out that day,4w and 12w after implant. Serum Mg2+concentrations were examinated 1d before, 1d,3d, 1w,2w,4w,8w and 12w after implant. The data was staticstically analysis with one way ANOVA, and S-N-K test was compared in pairwise comparison. Level of significance a=0.05.7. For biomechanical test of magnalium interference scew with or without coatings, porcine tibias were used to test titanium interference screws, magnalium interference screws with or without coating fixation of bone-patellar tendon grafts. Displacement, stiffness, maximum load at failure and the mode of failure were determined under cyclical load from 25 N to 150 N then load to failure using a materials testing system. HAe coating was observed after test.Result1. Cytotoxicity test1.1 Morphous of cell:Cell Morphous was graded as 0 in N,10%M,25%M, 50%M, H and group F and as 3 in group P at every observing stage.Cell Morphous was graded as 1 at the 1st day and as 4 at the 3rd,5th and 7th day in group 100%M.1.2 There were statistical difference in OD among stages and groups. (F=230.883, P=0.000).At the 1st day:OD in group 100%M (0.286±0.008) was lower than group N (0.406±0.022) (P<0.05), and higher than group P (0.240±0.002) (P<0.05). OD in group 50%M (0.454±0.027), group 25%M (0.430±0.017), group 10%M (0.451±0.029) and group H (0.430±0.024) was higher than group N (0.406±0.022) (P<0.05). There was not statistical difference between group F (0.413±0.016) and group N (P>0.05).At the 3st day:OD in group 25%M (1.307±0.035), group 10%M (1.324±0.037) and group N (1.317±0.042) was higher than group 50%M (1.201±0.057) (P<0.05), group F (1.429±0.096) and group H (1.097±0.089) and OD in group P (0.292±0.010) was lower than the other groups (P<0.05).At the 5th day:OD in group 50%M (1.466±0.124), group 25%M(1.518±0.125), group 10%M (1.581±0.039), group H (1.379±0.067), group F (1.429±0.096) and group N (1.622±0.156) and OD in was higher than group P (0.270±0.015) (P<0.05).At the 7th day:OD in group 50%M (0.856±0.092), group 25%M (1.026±0.088), group 10%M (0.998±0.215), group H (0.845±0.068), group F (0.928±0.040) and group N (0.995±0.070) and OD in was higher than group P (0.281±0.006) (P<0.05).Growth curve of cell showed OD was increasing with time until the 5th day then decreased except group 100%M and kept lower value in group P at the 4 stages.1.3 Growth rate of cell culture.At the 1st day:Group 100%M (70.6%) and group P (59.2%) were graded as 2 and the other groups were as 0.At the 3rd day:group 50%M, group 25%M, group 10%M and group H were graded as 1. Although group F was degraded as 2, but its data was very close to 1. Group P were graded as 4.At the 5th day:group 50%M, group 25%M, group 10%M, group F and group H were graded as 1. group P were graded as 4.At the 7th day:group 25%M and group 10%M and group H were graded as 0. Group 50%M, group F and group H was degraded as 1. Group P were graded as 4.2. Haemolyticus test:Haemolysis rate was 68.3% in group M,-0.6% in group H and 0.8% in group F. Haemolysis was occurred in group 100%M (65.3%), group 50%M (66.7%), group 25%M (33.8%), group 10%M (11.8%) and group 5%M (6.2%), but not in group 2.5%M (0.3%) and 1.0%M group (-0.3%). 3. Extract analysis test:Mg2+concentration decreaded, but Na+concentration was not changed with dilution increased. pH in group 100%M (10.10±0.29), group 50%M (10.32±0.21), group 25%M(10.40±0.17), group 10%M (10.38±0.11), group 5%M (10.29±0.12) and group 2.5M (10.05±0.12) was higher than group 1%M (9.65±0.28) (P<0.05).4. Sensitization test:Erythema was observed 6h after patch removed and not found 24h,48h and 72h after patch removed in group N, group M, group F and group H. Erythema was observed in group P for all the time.Cutaneous edema, spongiosis, monocyte infiltration around vessel, dermis and epidermis were not found in group N, group M, group F and group H and observed in group P. Basophilic cell was found slightly in group N, group M, group F and group H and massively observed in group P.5. Systematic toxicity:Percent of body weight change was lower than 10% in group M (3.27%±9.19%), group H (0.15%±2.86%), group F (-0.71%±3.50%) and group N (1.57%±10.21%). There was not statistical diference among groups. (F=0.289, P=0.832)6. Magnesium degradation and concentration of serum magnesium after pin implanted.6.1 Condition of rabbit after operation:Abnormal activity, eating and sleep were not found. Wound of entry healed well and no cutaneous emphysema or fibrous capsule occurred in all the rabbits after operation.6.2 PhotographsThat day after implanted:Implant was combined with bone and there was not found blank gap between implant and bone in all groups.4 w after implanted:Implant was tightly combined with bone and its shape was not changed in group PLA, group H and group F. There was found blank gap between implant and bone in group M.12 w after implanted:Implant was tightly combined with bone and its shape was not changed in group PLA, group H and group F. Blank gaps between implant and bone were found wider in group M and slight in group F. 6.3 Micro-CT scansThat day after implanted:Implant was combined with bone and there was not found blank gap between implant and bone in all groups.4 w after implanted:A thin gap of high density was observed around implant in all the groups. Implant was tightly combined with bone and its shape was not changed in group PL A, group H and group F. There was blank gap between implant and bone in group M.12 w after implanted:A thin gap of high density was observed around implant in all the groups. Implant was tightly combined with bone and its shape was not changed in group PLA, group H and group F. Blank gap between implant and bone was found wider in group M and slight in group F.6.4 Concentration of serum magnesium was not altered among 1d before, 1d,3d, 1w,2w,4w,8w and 12w after implanted in group M, group H and group F. (F=1.081, P=0.410)7. Biomechanics of interference scewAll the samples were survived cycles. Significant differences were not observed between group M, group F, group H, T group and group PLA for displacement (1.70 mm±0.33 mm,1.89 mm±0.53 mm,1.73 mm±0.26 mm,1.97 mm±0.72 mm,1.49 mm±0.52 mm, F=1.389, P=0.253), stiffness (119.58 N/mm±25.71 N/mm,116.91 N/mm±19.34 N/mm,118.74 N/mm±24.10 N/mm,107.64 N/mm±23.24 N/mm, 125.18 N/mm±21.61 N/mm, F=0.774, P=0.548). Maximum load at failure was 522.75±80.33 in group M,573.60 N±109.73 N in group F,545.59 N±96.71 N in group H,484.72 N±97.06 N in group T,639.39 N±153.96 N in group PLA and there was statistical difference between them (F=2.772, P=0.038). Maximum load at failure in group PLA was higher than group T (P< 0.05). The mode of failure was not observed significant differences between group M, group F, group H, T group and group PLA (χ2=4.935, P=0.294). HA and MgF2 coating just sloughed at the sharp edge of screw threads and screw nut and were intact in the other parts.Conclusions1. Magnalium had cytotoxicity and haemolysis response in vitro, but it could not reprented the results in vivo.2. Magnalium coating with HA or MgF2 was not observed cytotoxicity and haemolysis response in vitro.3. Magnalium with HA or MgF2 was not observed sensitization and systematic toxicity.4.3 months after implanted there were just little degraded in magnalium without coating, MgF2 coating completely dispeared and HA coating still protected magnalium from degradation. Concentration of serum magnesium was not altered after implanted in vivo. Cutaneous emphysema and fibrous capsule were not observed in the 3 materials.5. Magnalium interference screw with or without hydroxyapatite or MgF2 coating can achieve enough fixation strength in the initial and most of HA and MgF2 coating on magnalium interference screw remains after anterior cruciate ligament reconstruction and test.6. HA and MgF2 coating on magnalium could reduce its corrosion rate magnalium and had good biocompatibility.7. The method of biomedical evaluation of the degradable magnalium may be revised.8. The result provides the basis of biological security in orthopedic and of experiments of the further researches and.clincal application.9. Magnalium with or without hydroxyapatite or MgF2 coating may be new medical metal implant material in orthopedics.
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