Biocompatibility Of Nano - Hydroxyapatite / Chitosan / Konjac Glucomannan Bone Substitute Implanted Scaffolds

Background and Object: Bone defects that caused by all different reasons are common in clinic,especially the bone defects that merge osteomyelitis. The problems of low cure rate and the high recrudescent rate have not been completely resolved. The classic treatment is:1. Completing remove the lesions;2. The application of antibiotics:we should maintain effective bactericidal concentration long enough in order to achieve the desired therapeutic effect, often adopting the method that topical application combined with systemic. Topical uses gentamicin PMMA beads often.3. Eliminating dead space and repairing soft tissue. Otherwise completing remove the lesions can easily lead to bone defects and recurrent infection,ILizarov technology can solve some of bone defect problem tha merge osteomyelitis. However, most patients are not satisfied with the treatment. In recent years, researchers hope to resolve the issue by biological tissue engineering methods, many scholars try to develop a kind of scaffold that has characteristics of anti-infection, osteoinductivity, suitable mechanical strength and degradation to treat bone defect.Hydroxyapatite (HAP) is the major inorganic constituents in bones of hurnan and animal. Nano-hydroxyapatite (nHAP) have better biological activity, and has osteoinductivity; chitosan (CS) has a good antibacterial property; konjac glucomannan (KGM) that has three-dimensional network structure has an excellent mechanical strength, water holding capacity. The composite material KHC that made by nHA, KGM, CS three materials with a certain percentage has good biocompatibility, mechanical strength, and we can achieve the desired degradation rate by adjusting the content of KGM. KHC has drug properties and its liposomal that complex with vancomycin has been proven in animal experiments.This study is to test the compatibility and safety of KHC materials by cytotoxicity, hemolysis experiments, heat test, mouse bone marrow micronucleus test, skin sensitization test and sperm abnormality test in vitro. Then KHC may play a role in the treatment of bone defects, especially that bone defect that merge osteomyelitis.Method:According to the standards of GB GB/T16886-1997 (Biological Evaluation of Medical Devices) to test and evaluate results.1. Cytotoxicity in vitro: GB recommends extracts experiment, direct contact experiment, indirect contact experiments, we adopt extracts experiments in this study. The concentration of extracts is 0.2g/ml, using 10% DMEM complete medium to extract at 37±2℃ environment 72h and then get supernatant. Then we can observe the cell morphology and measure the cell proliferation to evaluate the toxicity of the materials.2. hemolysis experiments: Carried out by hemolysis experiments in hematological section of implantation instrument experiment,adopting making the material directly immersed in the fresh diluted rabbit blood to measure the dissolution rate of RBC.3. The pyrogenic test: Injecting the extracts into the rabbits, measure the rabbits’temperature in three to evaluate the defect of Pyrogenic.4. sperm abnormality test: The experiments that using mammalian or non-mammalian cells, bacteria, fungi or yeasts to test whether the materials, equipments or extracts can cause gene mutations, chromosomal abnormalities and other DNA or genetic changes is called genetic toxicity Test. In this study, we inject the extracts into stomach to see whether the extracts can cause abnormal sperm to confirm whether extracts cause mutations in experimental animals.5. Skin sensitization test: According to the method of irritation and sensitization test in GB/T16886,10-1997"Biological Evaluation of Medical Devices" to evaluating the potentialof the materials that rise skin sensitization in guinea pigs under experimental conditions, an experimental basis, using the method of intradermal injection combined with smearing.6.The mouse bone marrow micronucleus test in the genotoxicity test is to determine the effect of experimental materials to structure of chromosome in experimental animals.Results:A series of experiments in this study, the cytotoxicity of the materials in vitro is class Ⅱ, the reasons for it is that high levels of calciurn induces cytotoxicity, and cells grow well, according to the GB the toxicity of materials is safe, but the toxicity of materials must be still study on. The results of hemolysis experiments, mice sperm abnormality test, skin sensitization test and micronucleus test in mice bone marrow cells are all within the scope of GB.Conclusion:This study confirms that nano-hydroxyapatite / chitosan / KGM have a good biocompatibility. And we can carry out the next step in clinical trials, hoping these materials will play a role in the treatment of osteomyelitis.