Immunogenicity Clearanceof Animal-based Collagen And Preclinical Applications

Animal-based collagen can be extracted and processed from animal bone,skin,tendons and amniotic membrane etc,as the main components of the extracellular matrix,which has multiplebiomedical functions and canbe applied in tissue regeneration and fibrosis etc.However,animal-based collagenwith certain immunogenicityis unsafety and greatly limitedin clinical applications.In order to extend its application in clinic,it is necessary toinvestigatethe immunogenicity of animal-based collagen,and the mechanism of the body's immune toxicologyafter implantation,and the removal methods and systematical evaluationof the immunogenicity.In this paper,we investigated methods of immunogenicity removal of type I bovine collagen.Firstly,C-peptide and N-peptide were removed by using acid and enzyme for removing the immunogenicity caused by telopeptide.Non-collagen proteins content was less than 0.1% by eliminating residual cells and surface antigen in the tissue with hypertonic saline solution,surfactant and H2O2 treatment.The protein purity was not less than 97%,and the residual DNA content was less than 1 ?g/m L after purification.Virus from animal tissue was inactivated by surfactant,alkali treatment and low p H hatches.The bacteria lipopolysaccharidein particularly from exogenous microbeswas removed by low p H hatches and gamma rays.With type Ibovinecollagen from sigma as control group,we got a preliminary understanding about hypersensitive reaction,humoral immune response,cell-mediated immunity,complement activation function and NK cell activation functions under the condition of different doses of type ? bovine collagen.The results showed that 1)the immunogenicity reaction of experimental group is similar to that of the control group;2)Skin allergic reactions and maximization test reactions were negative for guinea pigs when contacted with type ? bovine collagen;3)Both of the test and control group had no significant difference in the terms of lymphocyte proliferation,NK cell killing functions and the percentage of CD3+ cells,CD4+ cells,CD8+ cells,and the ratio of CD4+ and CD8+ cells after BALB/C mice subcutaneously dailyinjected with collagen for 12 days.And the spleen with histological analyseswas enlargedand the lymphatic sheath surrounding splenic artery appeared obvious thickening,and there was no obvious change about the splenic germinal center after after the injection of type I bovine collagen.This meant that animal collagen may have certain effect on the cellular immunity,and the spleen of BALB/C micewould be normal afterabsence of injection for 2 weeks;4)the mice serum total Ig E,Ig G,Ig M and collagen specific antibodylevel kept stableafter BALB/C mice subcutaneously injected with collagen 3 times,each time interval of 7 days.There were no obvious changes about the splenic germinal center(mainly for B lymphocytes),which meant BALB/C mice did not cause the humoral immune responseafter the injection.And also C3 a and C5 a levels in serum did not change after the injection,which meant that type I bovine collagen could not activate the complement system;the phagocytosis of mice reticuloendothelial system had no significant change after KM mice subcutaneously injected with collagen daily for 14 days.But both groups could increase the spleen index,there was no significant difference between experimental group and control;6)antinuclear antibodies,white blood cell classification,and the pathological histological analyses of multiple organs(except spleen)showednormal results after continuous injection,which meant that collagen has less possibility to cause the body to produce their own immune response and other toxicological effects.We prepared the collagen hydrogel dressing and collagen membrane respectively for diabetic rats wound repair and radial bone defect(3 mm in diameter)filling in New Zealand rabbits to verify the security of clinical application after immunogenicity removal of type I bovinecollagen.Wound healing rate,fibroblasts and angiogenesis count results showed that collagen hydrogel dressing could effectivelyimprove the microenvironment of diabetes chronic wound,promotingproliferationof fibroblasts and formation of new blood vessels.And the collagen hydrogel dressing did not cause wound stimulation,hypersensitivereaction,infection and other related adverse reactions.The role of collagen hydrogel dressing in wound repairingwas similar to recombinant growth factors.The lymphocytes in experimental group decreased significantly compared with the control group(P < 0.05)within 3-60 d during collagen membrane for radial bone defect filling.The serum antibody didn't increase significantly compared with the control during implantation.There was no significant difference between two groups in X-rays score when implanted in 30 days and 60 days.The fracture line has disappeared,and a lot of callus appeared in bone defect site for the experimentalgroup at the time of 60 days.However,the fracture line of the control group was fuzzy,and there was only a small amount of skeleton in defect parts.These proved that the role of collagen materials for wound healing was obviously better than the control group after the removal of immunogenicity.Histopathological results showed that tissue of the experimental group repaired faster than the control.A lot ofinflammatory cells and osteoclasts were found within the bone marrow cavity of part of the animal in control group.,which meant that type I bovine collagen with immunogenicity removal could reduce the local tissue inflammation to a certain extent and would not increase the serum antibody levels.Collagen could promote the fracture healing by inducing the osteoblast formation,inhibiting the osteoclast formation and reducing the inflammation reaction in medullary cavity withthe degradation of the collagen.The above results proved that hypersensitive reaction,humoral immunity and cell-mediated immunity,autoimmunity,histopathologic study of the immune toxicity to organ,effect of phagocytes,complement activation function,NK cell killing function,the influence of implant materials on the blood system and the histologic reaction of material in the specific medical application could be used as immunogenicity evaluation methods for animal-based collagen.We expected to further reduce the incidence of allergic reactions,embedded parts nodules,redness,foreign body reaction,embolism and other adverse reactions by removing the immunogenicitythrough strictly controlling extraction and processing.These studies has helpto improve the evaluation system of immunogenicity for animal-based collagen and medical equipments,and to further expand its medical applications inclinical safety.