A Comparative Study Of Three Methods For Reparing Porcine Small Intestinal Submucosa Extracellular Matrix

ObjectiveBiological materials are a natural extracellular matrix (ECM), which use mechanical、chemical or enzymatic method ways to make cells to prepared from crosslinking treatment,with a certain mechanical strength, and low immune response. P-SIS is the vast majoritycollagen fibers of type I and type III which of is over90%. With conditions to retain acertain mechanical strength, P-SIS also contains a variety of cytokines, such as basicfibroblast growth factor (bFGF), transforming growth factor (TGF), epidermal growthfactor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1(IGF-1) and glycosaminoglycans, fibronectin class, chondroitin sulfate, hyaluronic acid,and tissue remodeling of these components play an important role in wound healing[1-2].P-SIS has been widely used in modern biomedical fields, such as tendon reconstruction,urinary tract reconstruction, vascular reconstruction, repairing partial or full thickness skininjury[3-5].Biological materials have the same synthetic material with a low recurrence ratehernia, biocompatibility, anti-blocking property, and are also more desirable thansynthetic materials. Significantly lower incidence of hernia recurrence after infection,obstruction, fistula formation and other adverse reactions[6].Despite of all the advantages of the extracellular matrix as described above, there isno uniform standard of a certain kind of ECM preparation method.Due to variousdifferences of ECM structure, applying the same method acellular is difficulty tocontrol.The method which currently used acellular divided into three categories: physicalmethods, chemical methods and enzymatic digestion method. If the application of themethod or reagent dosage is too low, residual cellular components can more easily causeimmune rejection, severe local tissue adhesions, and tissue wrapped calcification. If highdoses of reagents, cells can be removed and clean, and will also damage the structure ofECM[7]. Therefore, how to use a method to remove cells and DNA more exhaustivly, andminimize the organizational structure of the ECM, damage and mechanical properties ofbiological activity, retaining a more complete structure and function of many proteins, isthe current treatment methods acellular hotspot.Comparison of mechanical systems, machinery-acid method, mechanical-SDS method to prepare porcine small intestinal submucosa (P-SIS) for its tensile strength, theamount of residual cells, growth factor levels and the body’s immune response impact,providing experimental evidence to seek good preparationMethodsThree methods of preparation of porcine small intestinal submucosa: Take freshporcine jejunum, respectively using mechanical methods acellular, machinery-acid-baseacellular method, the mechanical-SDS method acellular prepare P-SIS biological patch.After each step, keeping specimens from sample (n=10), as A, B, C group; fresh porcinejejunum act as control.Animals in vitro test:①the three methods of material carry on uniaxial mechanicaltesting (by Shanghai Tongji Medical College, biomechanical testing laboratory to helpconsummation).②HE staining, Masson staining, Sirius red staining, PAS staining (by theShanghai Changzheng Hospital pathology assist), DNA gel electrophoresis experimentprove to removal cells or DNA content of different methods of preparating P-SIS;③usingElisa method for detecting growth factors VEGF and TGFβ1Animal testing and evaluation: preparation methods were used in three differentmethods of the P-SIS to repair abdominal partial wall defects in rats by intraperitonealinjection experiments, the repair area immunohistochemical determination of inflammatorycells, Ellisa method for the determination of inflammatory mediators, to evaluate its tensilestrength and the immune and inflammatory response of each group after surgery.Results1.An animal in vitro test: by uniaxial tensile test results of the mechanical analysis,P-SIS of acid-base group in tensile strength,and maximum elongation are better thanSDS group and mechanical group (P <0.05).Through various staining and DNAconcentration determination can be concluded that DNA in acid-base group is significantlylower than the other two groups (P <0.05), and the elastic fiber structure neat rows.Elasticfibers is loosen in SDS group, and part of the fiber has a fracture; through Ellisa methodfor determining the growth factorS TGF-β, and VEGF levels derived, acid groupcompared with growth factors levels is higher SDS group (P <0.05).2, animal testing and evaluation: After36SD rats were alive, no obvious infection, hematoma formation,and abdominal hernia, abdominal wall defects partially complete.Through the three groups of rats at8th weeks after intraperitoneal injection test, repairingarea showed no swelling, abdominal hernia formation in three group(P>0.05). Accordingto IL-6and TNF-α levels in serum by Ellisa methods, the levels in acid-base grou is lowerthan SDS group and mechanical group (P <0.05); through immunohistochemical detectionof inflammatory cells and endothelial proliferation cell in the partial restoration area,concluded that the content of acid-base group of inflammatory cells in general is lowerthan the SDS group and machine group (P<0.05).ConclusionThree different ways of preparing the P-SIS, growth factors levels and residual DNAconcentrations in acid-base grou are better than mechanical-group and SDS-group. Afterthe implantation of the animal, from the body’s immune response and hyperplasia ofendothelial cells, it is concluded that acid-base group is better than the other two groups.So P-SIS acid-base method to prepare P-SIS in three ways is the best suited as a method ofacellular extracellular matrix, it is worth further improved.