Preparation Of Acellar Peripheral Nerve Graft And Its Biocompatibility

Facial nerve is the mixed nerves, mainly in motor function. From central to peripheral parts of any damage, can lead to partial or complete facial paralysis, caused by muscle atrophy in patients with facial deformities, affecting the physical and mental health and quality of life of patients. The regeneration and functional recovery of peripheral facial nerve defect is always one of the hotspots in the plastic surgery, maxillofacial surgery, neurosurgery, pathology, traumatology, rehabilitation medicine and molecular biology.The autoallergic nerve graft could not satisfy the need to repair the long-defect nerve injury because of the limited resource and scarification of sensory function at donor sites.The acellular nerve allografts were prepared with fresh pig intercostal nerve by hypotension,freezing-thawing at -80℃repeatedly and NaOH splitting, evaluated its biocompatibility, further confirmed its weak antigenicity, and lay the foundation for clinical application .In this study, the xenogeneic nerve from pigs was used to get accellular nerve scafford,the biocompatibility of ANS was tested according to GB/T16886 biologiacal evaluation of medical devices , tested the host immune response to ANS in vivo and in vitro. The experiment could be divided to 5 parts:1. Xenogeneic and syngeneic acellular nerve scaffold were prepared via two sterilization methods : To obtain a good biocompatibility of ANS, Pig intercostal nerves and sciatic nerves of SD rats were cut and treated via two motheds,one is the hypotension,freezing-thawing at -80℃repeatedly and NaOH splitting(United methods),another one is the traditional chemical methods(sondell methods ), then observe characters of its structure under light and electron microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM).The frist method is a ideal paradigm to develop high quality acellular nerve allograft with the immunologic substances basically removed and the laminin and nerve structured well preserved. And this protocol offers a solution to the autologous nerve transplantation. Evaluation of biocompatibility on acellular nerve scaffold via two preparation methods: the biocompatibility of ANS was tested according to GB/T16886.4-2002:Selection of tests for interactions with blood, GB/T16886.5-1999: Test for cytotoxicity in vitro and GB/T16886.6-1997:Tests for local effects after implantation. The result of MTT test and Haemolysis test show that the ANS via different sterilization methods has statistical differences. Sondell group implanted rats showed an acute inflammatory response followed by chronic inflammation . United methods group implanted rats, however, showed an acute inflammatory response that diminished such that the graft ultimately became indistinguishable from native tissue, observations that are consistent with graft acceptance.2. Evaluation of biocompatibility on acellular nerve scaffold via three sterilization methods:Pig sciatic nerves were cut and treated using the NaOH maceration method. ANS are sterilized by ethylene oxide, Co60-irradiation, peracetic acid. Evaluate the biocompatibility of it by the cytotoxical MTT test, cellular compatibility test, collagenase susceptibility test in vitro and tests for local effects after implantation. peracetic acid sterilization offers a convenient alternative protocol for processing of ANS.ANS sterilized with PAA has good compatibility (cell, tissue) and biologic safety, so it is an ideal sterilization method for ANS.3. Immune response between the xenoantigenicity of ANS and xenogeneic sera in vitro: To detect the binding reaction btween antigen and antibody, the distribution of a-Gal in the ANS. Affinity immunohistochemistry assays were conducted following routine procedures on paraffin sections with normal human blood and BSI-B4 (a-Gal specific binding lectin) as the primary anti-bodies or affinity reagents. Sections digested by a-galactosidase were also examined as control. There was no significant difference both in the antigens recognized by sera of perineurium and epineurium or BSI-B4 and in the distribuion of a-Gal.The strongest a-Gal positive staining was appeared in perineurium and epineurium at all levels. After digested by a-Galactosidase, all samples were negative against BSI-B4 and human sera except few positions that showed different staining.4. Quantification of DNA in ANS:The methods used to remove the cellular component of each tissue vary widely. Decellularization is considered important because of the potential adverse immune response elicited by cell membrane epitopes, xenogeneic DNA, and damage associated molecular pattern (DAMP) molecules.The total DNA contents was assessed by a dot-blot hybridization assay. the amount of DNA within ANS was only 21.1+0.7 pg DNA/mg dry weight.5. Detect local and systemic immune response was caused by transplantation of ANS in Subcutaneous: ANS and fresh nerves was placed in a ventral midline subcutaneous pocket. the graft site, including implanted tissue, underlying abdominal wall, and overlying skin was excised. Cytokine RNA analysis in the the graft site was detected by RT-PCR. ELISA for determination of ANS specific antibodies. when the mice implanted with ANS, do show the presence of IL-4 and the absence of IFN-r.Through Anti-SIS antibody analysis of SIS-implanted mice,there are high levels of IgG1 and the absence of IgG2a and IgG2b , ANS-specific antibodies.