Study On Biological Safety And Immunogenicity Of Human Amniotic Membrane Preparations

Objective: Evaluate the microbial safety of human amniotic membrane (HAM) in preparing and preservation, to establish effective methods of microbe control for HAM bank. Investigate whether HAM preparations lead to typeⅠhypersensitivity, to measure the allergenicity. Investigate the biocompatibility and immunologic reaction difference between fresh and preserved HAM grafting, and evaluate the immunologic safety for HAM transplantation. The study according to the relative methods and regulations of medical devices prescribed by the International Organization for Standardization (ISO) and GuoBiao/Technique (GB/T), evaluate the biological safety of HAM preparations, to provide theory and experimental evidence for HAM preparations entering clinical use as new typical biological materials. The study concludes three parts: ExperimentⅠ: study on microbial safety of HAM in preparation and preservation. ExperimentⅡ: study on allergenicity of fresh HAM for typeⅠhypersensitivity. ExperimentⅢ: study on biocompatibility and immunologic reaction of HAM grafting. Methods Experiment Ⅰ : Human amnions were collected from elective caesarean sections in 20 healthy women, divided into groups based on different sterilization process (soak in NS. or gentamicin – amphotercin B solution), and preservation methods (glycerol at -4oC , DMEM at -4oC , DMEM/glycerol at -80oC or vacuum drying at -4oC). Bacteria, fungi and mycoplasmas were detected at 24h, 1m, 3m and 12m after preservation. Other amnions were from eutocia in 5 healthy women, after soak in gentamicin – amphotercin B solution, detected bacteria, fungi, mycoplasmas. ExperimentⅡ: In allergic test model, 30 guinea pigs were divided into three groups of 10 each. Guinea pigs were immunized with fresh HAM homogenate, albumen solution (positive control) or PBS (negative control). After the animals were challenged with corresponding allergen respectively, observe their reaction till dying or 3h, then obtain blood samples, to determine histamine concentrations using fluorometry and four hemorheologic markers by hemorheology analysis system. ExperimentⅢ: Subcutaneous implant models were established in 150BALB/C mice, which were randomized into five groups of 30 mice each, based on different implants---FAM, DFAM, GPAM, chorion (positive control) or merely operation (negative control). Each group was divided into five sub-groups of 6 mice each, respectively according to the observing time---1w, 2w, 4w, 8w and 12w after surgery. The expression of CD3~+CD25~+ in peripheral blood was identified by FACS. The tissue samples from grafted area were observed with HE staining and the typing of immune cells were determined by immunohistochemistry. The inflammatory cells were calculated with light microscopy. Results ExperimentⅠ: Two of HAM samples only soaked in NS. were detected contamination with E. coli and/or S. epidermidis, respectively from two caesarean sections. There's no any microbe detected in all of the amnions from caesarean sections which were soaked in gentamicin – amphotercin B solution. One of HAM samples after soaked in the antibiotics solution was yet detected lactobacilli from eutocia. ExperimentⅡ: The guinea pigs responded to fresh HAM homogenate in almost the same manner as to PBS, and no obvious allergic reaction was observed in the animals except those in positive control group. The blood histamine concentration and four hemorheologic markers showed no significant differences between HAM and PBS (P>0.05), both much lower than positive control group (P<0.01).ExperimentⅢ: In all groups, the mice lived normally and their wounds healed at one stage. The expression of CD3+CD25+ in peripheral blood, the typing of immune cells and histological observation for grafted area tissues showed similar reaction tendency. In all of AM groups, there were only unspecific inflammation and mild immunological reaction as xenograft during early grafting stage. In addition compared with each other, there were no significant differences for the expression of CD3~+, CD4~+, CD8~+ and C...