Objective To study degradation of the antigen-extracted meniscus in PBS solution with no enzyme or with different enzymes. Methods Four types of enzymes (collagenase, hyaluronidase, trypsin, papain) were used to enzymolyze the antigen-extracted meniscus and the fresh meniscus for 3, 7, 15 and 30 days (37℃). The antigenextracted meniscus and the fresh meniscus were immersed in PBS solution (37℃) for 30 days. Weight loss measurement, UV spectrophotometry, and scanning electron microscopy (SEM) were used to characterize the degraded materials. Results The two types of the materials were remarkably digested under the enzymes, especially under trypsin. The degradation curves showed that the antigen-extracted meniscus was enzymolyzed less than the fresh meniscus. The degradation products were grouped as amino, peptide, and polyose by the analysis. Both of the materials could hardly behydrolyzed in PBS solution without the enzymes. The four different enzymes had different surface morphologies under the examination of SEM. Conclusion The antigen-extracted meniscus is enzymolyzed more slowly than the fresh meniscus in vitro, and the result can be used as a guideline to the further research.
Insufficient supply of organ for allotransplantation made the study on finding new organ resources from animal progress. Pig is regarded as one of the optimal donor animals for human. The major obstacle in this field is hyperacute reaction (HAR), which is triggered after the xenogenic natural antibodies preexisting in recipient blood combine to the antigens on the surface of the endothelium and activate the complement system. alpha-Galactose residues (alpha-Gal) on the endothelial cell have been identified as the major xenoantigens. NJZ Pig has been closely breed since 1938, whose family history is clear. Tissue samples from heart, liver, kidney, pancreas, lung, small intestine, skin, spleen, thymus and lymph node were obtained and embedded in paraffin. The sections were performed the immunohistochemical staining with the sera from health volunteers (including all the blood types) as the primary antibodies as well as the biotin labeled bandeirae simplicifolia I isolectin B4 (BS I-B4), which has specific affinity to alpha-galactose. All the staining sections were compared with the tissues digested with alpha-galactosidase. There was no difference between the antigens recognized by sera of different blood types. alpha-Gal was still the major xenoantigen on the endothelial cells. There might exist non-alpha-Gal antigens on the distal convoluted tubules and collecting tubules of the kidney. There was no alpha-Gal distributing on the secreting part of pancreas, either the islet cells or the matrix cells, but surely on pancreatic duct and vessels. All the antigenity was destroyed after the enzyme digestion except that the small intestine gland still positive with the BS I-B4. alpha-Gal is the major xenogenic antigen in NJZ Pigs. There exist some unknown antigens on the distal convoluted tubules and collecting ducts of the kidney. The blood type of recipient is not the first affair to be considered in pig-to-human xenotransplantation. The specificity of BS I-B4 for the alpha-galactose needs more detail research.
Limitation of donor source for allograft makes the research on xenograft progress. Pig is regarded as one of the ideal donor animals. The major obstacle in xenograft is hyperacute rejection, which is caused by complements after they are activated by xenogeneic antigens combined with natural antibodies. It has been confirmed that alpha-Gal is the major target antigen, whose expression is incharged by alpha-1,3 galactosyltransferase (alpha-GT). The approaches to overcome hyperacute rejection against alpha-Gal included: immunoadsorption of xenogeneic natural antibodies, lysis of antigen by enzyme and genetic manupilation to obtain animal lack of alpha-GT. Besides alpha-Gal, there were other antigens binding to human serum antibody, such as gp65 and gp100, which was expressed on PAEC after induced by TNF, the A-like antigen. But their function was still unknown. It was debatable on the role of MHC in xenograft. Both direct and indirect pathway were involved in cellular response in xenograft.
OBJECTIVE: Porcine stress syndrome (PSS) is one kind of molecular genetics defect diseases of pig which will cause malignant hyperthermia syndrome (MHS) and is the first index should be excluded in screening of a pig species for xenotransplantation. It was reported that mutation of pig rynodine receptor(RYR1) gene is the main reason for PSS. In this study, RYR1 genotypes of the Chinese Banna mini pig inbred line and inbreeding closed colony Wuzhishan pig were investigated with polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) technique. METHODS: Antevenocaval whole blood samples were collected from 50 Banna mini-pig inbred-line(BMI), 15 inbreeding Wuzhishan pig (WZSP) and 25 Neijiang pigs (NJP) as negative control, the primer were designed and synthesized, PCR reaction was conducted following the sequence of 94 degrees C (1 min), 58 degrees C (1 min) and 72 degrees C (1 min) for 30 cycles. The PCR products were digested with restriction endonuclease HhaI and then electrophoresis check. RESULTS: A 659 bp DNA fragment was amplified with these two primers, the HALNN sample fragment was cut into fragments as 493 bp and 166 bp individually after the digestion, indicates no point mutation at site 1,843 in RYR1 gene in all tested BMI pig and WZSP. Namely, the RYR1 genotype of 50 cases of BMI and 15 cases of WZSP were HALNN, therefore their phenotype is PSS negative. CONCLUSION: It indicates that the genotype of Banna mini pig inbred line and inbreeding Wuzhishan pig are HALNN therefore PSS absolutely negative, the group penetrance is 0. This is consistent with experimental observation. It suggests that Banna mini pig inbred line and inbreeding Wuzhishan pig may be the alternative donor for xenotransplantation.
Objective To explore the histological changes of bio-derived bone prepared by different methods after implantation, and to provide the scaffold material from xenogeneic animal for tissue engineering. Methods Theextremities of porcine femur were cut into 0.5 cm×0.5 cm×0.5 cm. Then they were divided into 5 groups according to different preparation methods: group A was fresh bone just repeatedly rinsed by saline; group B was degreased; group C was degreased and decalcificated; group D was degreased, acellular and decalcificated; group E wasdegreased and acellular. All the materials were implantated into femoral muscle pouch of rabbit after 25 kGy irradiation sterilization. The cell counting ofinflammatory cells and osteoclasts, HE and Masson staining, material degradation, collagen and new bone formation were observed at 2, 6, and 12 weeks postoperatively. Results The residue level of trace element in biomaterials prepared by different methods is in line with the standards. All the animals survived well. There were no tissue necrosis, fluid accumulation or inflammation at all implantation sites at each time point. The inflammatory cells counting was most in group A, and there was significant difference compared with other groups(P＜0.05). There was no significant difference in osteoclasts counting among all groups. For the index of HE and Masson staining, collagen and new bone formation, groups C and D were best, group E was better, and groups A and B were worse. Conclusion The degreased, acellular and decalcificated porcine bone is better in degradation,bone formation, and lower inflammatory reaction, it can be used better scaffold material for tissue engineered bone.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
OBJECTIVE: To observe the heart anatomic and histological structure of the Banna mini-pig inbred-lined and to provide the morphological data for heart xenotransplantation and breeding transgens pig. METHODS: Ten Banna mini-pigs (12-18 months old) were affused and fixed by common coratid artery. The heart were observed and measured by gross anatomy and histology. RESULTS: There were many similarities between the Banna pig heart and the human heart in anatomy and histology. However, the following differences were observed in the Banna pig heart: 1. Azygos vein directly drew into right atrium cordis. 2. The intercalated disk of cardiac muscle was less than that of human. 3. The Purkinje’s fibre was bigger than that of human. CONCLUSION: On the morphology and histology, the structure of Banna pig heart is similar to the heart of human being. It is possible that Banna minipig heart becomes organ donors for xenotransplantation.
Objective To investigate the mechanism of hyperacute rejection (HAR) in pig to rhesus monkey vein xenograft. Methods Porcine femoral vein was transplanted into rhesus monkey. Deposits of IgM, IgG, C3 and C4 on the grafts were observed by immunoflurescence. Results Great deal of IgM, C3 and C4 were seen along the endothelium of donor vein, but IgG was not seen. ConclusionIn pig to monkey xenograft model, HAR is intiated by the binding of xenoreactive IgM to donor xenoantigens and followed by the activation of complement via the classical pathway.