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 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 isolate and characterize mesenchymal stem cells (MSCs) derived from bone marrow of Banna minipig inbred line (BMI). METHODS: BMI-MSCs was isolated from bone marrow by density gradient centrifugation and cultured in DMEM (containing 15% bovine serum) at 37 degrees C with humidified 5% CO2. These cultured stem cells were characterized in clonal growth, expression of specific markers and capability of differentiation. RESULTS: Mesenchymal stem cells were proliferative and could be expanded rapidly in vitro. Clonal growth of these cells can be observed when small amount of cells was inoculated. These cells were SH2, SH3, SH4, SB10 and SB21 positive. And it was proved that these cells possess osteo-differentiation ability, up-regulated alkaline phosphatase expression and calcium secretion after osteosupplement was added into the media for several days. CONCLUSION: Mesenchymal stem cells derived from bone marrow of BMI possess the general characters of stem cell.
OBJECTIVE: To explore the kidney anatomic structure of banna minipig inbred-lines, and to provide data for kidney xenotransplantation. METHODS: The fresh and infused kidneys of banna minipig (including the vessel and the ureter) were checked by anatomic microscope and vernier caliper in original location and away body. The tissue structure was observed by HE stain. RESULTS: The structure of kidney of banna minipig inbred-lines (including the vessel and the ureter) are similar to that of human being. The fascia propria of kidney is divided into three layers including capsula fibrosa, capsula adipose and fascia renalis. The thickness of cortex renalis is (20.0 +/- 2.4) mm. The average diameter of renal artery is 5.1 mm and is similar to that of human being. All the kidneys of banna minipig inbred-lines have a single branch renal artery. The diameters of left and right ureters are 5.1 mm and 4.7 mm, respectively. CONCLUSION: The kidney of banna minipig inbred-lines is an ideal replacement of human kidney for xenotransplantation.
On January 7, 2022, the University of Maryland Medical Center reported that the world’s first gene-edited pig heart was successfully transplanted into a 57-year-old man with end-stage heart disease, causing a global attention. The first gene-edited pig heart transplanted into a human successfully survived for 59 d without showing early signs of rejection, creating a history of xenotransplantation and marking a key step forward the clinical development of xenotransplantation. This article focuses on the role of gene editing in alleviating immune rejection, summarizes the case of xenotransplantation at the Maryland Medical Center, and outlines the current status of xenotransplantation and the unresolved issues of xenotransplantation. It is expected that xenotransplantation can successfully enter the clinic in the near future.
Objective To observe the characteristics and related gene expression of osteoblastic differentiation in porcine bone marrow mesenchymal stem cells (MSCs) during. Methods Bone marrow from 6 landrace pigs, 3-month-old about 50 kg, was aspirated from the medullary cavity of the proximal tibia. The MSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The MSCs from passage 1 were cultivated in DMEM with 1×10-8mmol/L dexamethasone (Dex), 10 mmol/L β-glycerophosphate (β-GP), 82 μg/ml ascorbic acid (Asc) and 10% inactivated fetal bovine serum (FBS) up to 21 days. The MSCs were cultivated in basic DMEM as a control. Cell morphology was observed by microscope. Cell proliferation was tested by using the fluorescent dye SYBR green I measurement. Osteoblastic differentiation was evaluated by alkaline phosphatase (ALP) histochemical staining, quantitative calcium deposit, and real-time PCR technology. Results Characterization of primary MSCs: At day 1, most cells depicted round and floating hematopoietic cells. Colonies consisting of fibroblastlike cells were observed at day 3 after removal of nonadherent cells, colonies grew to various sizes at day 7. Thirteen population doublings took place in primary culture. Osteoblastic differentiation: During osteogenic stimulation, cellular morphology of MSCs changed from a fibroblastic shape to a cubical form. Cell proliferation had no impact in osteogenic medium compared to basic medium (Pgt;0.05). At day 14, ALP staining presented b positive. Calcium deposit pronouncedly increased at day 21 (Plt;001). Furthermore, the mRNA levels of core binding factor α1 (Cbfα1), osterix, ALP, collagen Ⅰ(ColⅠ), osteonectin (ON) and osteocalcin (OC) increased gradually. Cbfα1, ON and ALP genes increased at early stage of osteoblastic differentiation. Osterix and OC at day 21 were significantly increased when compared with that at day 7 (Plt;0.05). ColⅠ was increased at day 14 (Plt;0.05). Conclusion Porcine MSCs harvested from bone marrow by density gradient centrifugation are capable of osteoblastic differentiation in vitro. The potential of osteoblastic differentiation relies upon upregulation of genes specific to this lineage under the osteogenic conditions.