| Objective:By using of Konjac glucomannan (KGM) and hyaluronic acid (HA) as raw material, through the reasonable control KGM and HA in composite films in the material content. a new biodegradable preparation, non-toxic (KGM-HA) blend membrane; Through the in vitro and in vivo tests, and discuss (KGM-HA) blend membrane of cells in vitro degradation characteristics, biocompatibility and body organization, biological compatibility degradation characteristics and prevent adhesion properties. So as to find out a more effective clinical new the adhesion materials.Methods:1.To culture and amplify the rabbit's BMSCs by using the method of red blood cells cracking and adherence. The BMSCs is identified by morphology, biological functions and flow cytometry.2.Blending HA and KGM, Lactic acid as modified additives and the mixed solution of Ammonia and absolute ethyl alcohol as crosslinking agent to prepare blended membrane. Using the orthogonal experiment method to analysis its influencing factor, and using the Physical and chemical methods to detect the viscosity, thickness,absorption rate, mechanical property, Degradation Property and Micro Morphology of the membrane. Planning to find out the best anti-adhesion agent parameters of HA and KGM blended.3.Taking the third generation of mesenchymal stem cells and KGM-HA blended membrane to composite culture, using the inverted phase contrast microscope and scanning electron microscope to observe both composite degree, MTT method to detect cell proliferation and toxic effects, inducing Directional BMSCs and KGM-HA blended membrane differentiate to adipocyte in vitro, staining by oil red O to detect its differentiation effect.4.36New Zealand big white rabbit were randomly divided into three groups:the KGM-HA blend membrane group (group A), the hyaluronic acid group (group B).the blank control group (group C), each group only12. Laminectomy was performed on L2segment in each rabbit. After that, A group of epidural implanted KGM-HA blend membrane, group B epidural injection2ml medical hyaluronic acid; Group C for negotiations, then layered suture incisions. After six weeks materials, general observation epidural adhesion, Rydell classification and slice line HE dyed, observation epidural scar hyperplasia, parallel into fiber cell count. From the histologic and ultrastructural Angle into the biological materials prevent epidural adhesion of function and mechanism.Results1. we can detect a great quantity of adherent cells with great activity and generation capacity by inverted phase contrast microscope. Flow cytometric analysis results show that the cultured cells witn high level expression of CD2,CD44,CD90and low level expression of CD34,CD45. To confirm the cultured cells is rabbit BMSCs.2. KGM-HA has good water absorption rate, Composite can slow down the degradation rate of HA(24days of film the decomposition rate distribution in13～31%, and the only use of hyaluronic acid degradation time for4weeks), Excellent mechanical properties (the maximum tensile strength is20.73MPa, elongation22%); KGM-HA composite membrane has no fixed form, with lower crystallinity, and after the effect of crosslinking the acetyl of composite membrane is removed, the intermolecular hydrogen bond formation, and the crystallinity is improved:Through the lactic acid assisted microwave treatment can reduce the viscosity of the fluid film, the film liquid has a certain mobility, convenient film, and lowers the viscosity in favor of vacuum deaeration treatment, prevent excessive viscosity form appear deaeration incomplete.3. BMSCs and KGM-HA blend membrane combined cultured cells grow well, inverted microscope and scanning electron microscope visible cell growth; MTT detects the cell proliferation ability that shows:The experimental group and control group cell proliferation differences between not statistically significant (P>0.05):, BMSCs and KGM-HA composite culture and blank control group have no significant difference in growth and proliferation. BMSCs and KGM-HA blend membrane in in vitro culture conditions have adipogenic potential, which can differentiate into adipocytes.4. Six weeks after general observation:A, B group of epidural visible sparse and scattered the membranous adhesion, scar tissue and dural easy blunt separation, dural smooth and complete. A and B group of macroscopic observation were no material residues, art weeks without inflammatory exudate; One group A dural smooth complete degree is A group B. Reference Rydell classification:A, B group Rydell classification for0-the main class I; Group C epidural scar tissue density, adhesion widely, scar tissue and dural not easy blunt separation, after separation dural not more complete, score concentrated in II-III level. A and B group and group C are statistically significant difference (P<0.5). and the difference between A and B group was not statistically significant (P>0.5) Six weeks after the microscope:A, B group of hard with the scar tissue present between have obvious gap, A local materials left, epidural collagenous tissue hyperplasia less, scar tissue structure loose, the collagen fiber with hard film parallel arrangement, capillary A few hyperplasia, and fibroblasts and fiber cell confounding, quantity, group C epidural collagen organizations of proliferation, A compact scar, present A hard with the scar tissue closely adhesion, capillary and expansion of hyperplasia significantly, and fibroblasts and fiber cell number not much. But three composition fiber cell count difference was statistically significant (P>0.5).Conclusions:1.Using red blood cells lysis and adherent culture can culture and expand of rabbit BMSCs in vitro, Confirm that the cultured cells are rabbit BMSCs.2.KGM-HA blend film has a certain mechanical properties; KGM-HA blend film has a certain amount of water absorption, the degradation rate was significantly lower than the simple use of HA. excellent mechanical properties.3. BMSCs composite KGM-HA blend membrane in vitro culture, BMSCs can in KGM-HA film good adhesion and hyperplasia, and in particular conditions to fat cells differentiation. KGM-HA film no significant cytotoxicity, cell biocompatibility.the author concluded that, KGM-HA blend membrane is expected to be a new type of tissue engineering materials.4.KGM-HA blend membrane in prevention rabbit lamina resection of the membrane surrounding fibrosis and hard has good effect in the adhesion. A new "adhesion of materials used in the film is expected to be clinically. |
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