| BackgroudFor the principle of fracture fixation changed from the strong fixation to the biological fixation, there is more requirements for the intraosseous devices. Traditional metal screws and plate system is complained of infection, stress shielding, plate shift, and subjective pain or sensory abnormalities and other issues. About 12% of patients after maxillofacial fractures require surgical removal of the secondary fixation, with the development of absorbable polymer materials research, such as polyvinyl acetate, polyglycolic acid, polylactic acid and glycolic acid. The absorbable devices can reduce the risk of secondary surgery, and the stress can gradually transferred to the bone to promote bone regeneration. These potential benefits prove there is a bright future for absorbable material in the treatment of fractures. However, these materials are also complained of a foreign body sensation, local self-limiting inflammation, infection and internal fixation removal.Silk is FDA approved, and shows a strong mechanical properties, good biocompatibility as well as biodegradable property, it can maintain a stable structure in high temperature more than 170 ℃, which made it can be autoclaved. Besides, it is also stable at extreme pH values. According to previous literature, silk has the great potential to be a new kind of medical biodegradable material. But when turn silk into materials of high strength, the making process include adding various chemical reagent to change the arrangement of silk fibers, such as from random curled structure into a β-sheet structure, to improve strength of materials in different conditions including temperature, concentration, and other experimental conditions. The harmful chemical agents such as methanol,1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) are used in the process, it is crucial to test it poisonous effects of cells, what’s more, changes of relevant physical and chemical properties,biocompatibility and mechanical properties still need further study.ObiectiveIn this study, we aim to make silk into the orthopedic fixation devices with high strength, and can be absorbed in vivo with good biocompatibility on the bases of the reported literatures, and to improve the materials by changing the preparation conditions and processes. We discussed in detail the preparation of the basic properties of new materials, and from in vitro cell morphology, cytotoxicity, cell proliferation, apoptosis, to blood compatibility to make evaluation of the in vitro biocompatibility of the orthopedic silk fixation materials and to provide guidance for the later animal experiments and clinical application.Methods1. Preparation of materials:The procedure included silk degumming, washing, drying, dissolving and then the silk were centrifuged, vacuum-dried in frozen condition and re-dissolved in HFIP. The mixture were remolded in methanol and replaced by water, silk blanks were made into screws or plates in factory.2. The basic properties of materials:The biomechanical properties were evaluated by three-point bending, shear force; the contact angle by water was measured; microstructure and cross section of the material surface were observed by scanning electron microscopy(SEM); The pH value of the extract was measured by pHS-2C digital pH meter; water absorption rate and change of the slice’s diameter were used to evaluate expansion properties; degradation in protease XIV in vitro was measured and the silk screws were implanted into rabbit femora.3. The biocompatibility of the materials:Material extract was prepared in accordance with ISO 10993-12 standard,10% FBS/a-MEM culture medium was used as control group. Cytotoxicity were determined by Calcein staining, lactate dehydrogenase (LDH) measuring, cck-8 assay was used to evaluate effects on cell proliferation, cell apoptosis was also measured, hemolysis test was used to determine whether the material’s effects on red blood cells.Results1. Characterization of Materials1.1 The maximum shear stress of silk cylindrical rod was 42.6 ± 13.5MPa (n=5). Flexural strength was 67.3 ± 8.4MPa (n=5),The elastic modulus was 45.7 ± 14.8 MPa (n= 5); which was closer to the bone’s elastic modulus compared with the metal material; 24 screws were successfully implanted into rabbit femoral trial, the specimen visible screws remain firmly fixed after removal in the femoral shaft in first three months with no prolapse or shift.1.2 The contact angle of the surface was 95.8±8.5°, which indicated the surface was of hydrophobicity.1.3 The flat surface and dense structure was observed by scanning electron microscopy. The silk slice was composed of carbon(62.81%), nitrogen(14.69%), oxygen(22.40%) and calcium(0.1%).1.4 The weight of the material has begun to increase in PBS in 10 min, and the absorption rate reached its peak rate at 2h, and eventually reached saturation in PBS in 48h, the maximum water absorption rate was 30.7±0.3%. The diameter of the material increased and reached to maximum (17.9 ± 1.5%) at 48h.1.5 Degradation test in protease XIV in vitro indicated the degradation was in line with the law of second order function, y=0.00087x2+0.2003x-0.495, R2=0.98, It can be derived that material in protease XIV in vitro for three months would lose 24.57% by weight, and the entire degradation in vitro takes 8 months according to the formula.1.6 The PH value of extracts increased slightly, but eventually stabilized between 7.0 and 7.2.2. The biocompatibility of the materials2.1 Cytotoxicity test:LDH activity reached 963.0 ± 90.0 U/L (silk slices extract) and 882.4 ± 106.0 U/L (the control group), no significant difference (P> 0.05) was found,. Calcein staining test (viable cells were stained green, dead cells were stained red):MC3T3-E1 cells spread well and no dead cells were found.2.2 Morphology:MC3T3-E1 cells cultured with the material or its extract for 48h showed normal shape and spread well on the plate.2.3 Cell proliferation assay:MC3T3-E1 cells were cultured with the material and each groups’OD values gradually increase, their OD values were lower than the control group at different time points, and the difference was statistically significant (P<0.05), but there was no significant difference (P> 005) among cells cultured the extract groups and control groups;2.4 Apoptosis Assay:The necrosis and apoptosis rate of silk group were 3.0%,2.3% respectively; the control group were 2.0%(necrosis) and 2.9%(apoptosis); while the positive control group were 3.5%(necrosis) and 6.4%(apoptosis) (Figure 4-5). The apoptosis rate and necrosis rate showed no significant difference among the silk groups and the control groups.2.5 The hemolytic test was carried out to assess the blood compatibility of the silk material and the Hemolytic ratio (HR) value was 1.9%. According to ISO 10993-4 standard that the HR of the materials to be used in blood environment has to be less than 5%, the results indicated that the samples had good hemcompatibility.Conclusion1. The silk material in this study showed high strength and closer elasticity modulus to the bone than the value of mental material, which could be a ideal implant material in the future.2. The reasonable water absorption ratio and degradation ratio of the silk material provided good potential for fracture fixation.3. The silk material’s surface was hydrophobic which is not conducive to cell adhesion; but cells surrounding the material showed non-cytotoxicity and good biocompatibility, the material did not affect cell proliferation and its apoptosis.4. The silk material in this study showed good blood compatibility. Therefore, the silk material in this study is a new choice for orthopedic fixation with high mechanical strength and reasonable biodegradable abilities. With the future improvement of the preparation process, the silk material is expected to replace the existing absorbable orthopedic fixation materials. |
PDF Full Text Request