Objective To find out the recent progress in research of cl inical appl ication of fascia lata allograft. Methods The domestic and international articles were reviewed to summarize the princi pal properties, processing techniques, and various uses of fascia lata allograft. Results Histologically fascia lata is composed of parallel and compact bundles of collagen fibers with few cells and immunologically it is low-antigenic. After varied tissue processing and storage techniques, fascia lata, as the scaffold only with the extracellular matrix, has been used in cl inical practice and achieved good results, such as ophthalmology, urology, and orthopaedics. Conclusion Because of these unique properites in repairing defects and reconstructing functions, fascia lata allograft, as a natural biomaterial, is promising to be used in more aspects withthe development of the biomedical techniques.
Objective The immunogenicity of tissue engineered skins is still vague, though it has been appl ied cl inically for several years. To observe the evidence of immunologic rejection of tissue engineered skins transplanted to severe combined immunodeficiency (SCID) mice, which are implanted human splenic lymphocytes to construct human immunesystem. Methods Tissue engineered skins and acellular dermic matrix were constructed in vitro. Twenty SCID mice, aging4-6 weeks and weighing 16-17 g, were randomly divided into four groups equally (n=5). The tissue engineered skins, human foreskins from circumcision and acellular dermic matrix were transplanted to groups A, B, and C, respectively; group D was used as a control. After 2 weeks of transplanting, 3 × 107 human splenic lymphocytes were injected into every SCID mouse intraperitoneally. After 4 weeks, the morphology, histology, immunohistochemistry and human IgG immunofluorescence were used to observe immunologic rejection. Results Group A showed that transplanted tissue engineered skins had the bilayer structure of dermis and epidermis, which was similar to the normal human skin structure. Group B showed that the transplanted human foreskins still retained normal structure of human skin. Group C showed that acellular dermic matrix were located in situ and had no sign of degradation. After injecting human splenic lymphocytes into the SCID mice, no inflammatory cells infil itration were observed basically in groups A, C, and D; the inflammatory cells infil itration of group B were significantly higher than that of other 3 groups (P lt; 0.05). The results of anti human keratin 14 monoclonal antibody (mAb) staining and anti human type IV collagen mAb staining were positive in group A; no positive cells for CD3, CD4, and CD8 were observed in groups A, C, and D; and many positive cells for CD3, CD4, and CD8 were observed in group B. The results of IgG immunofluorescence staining was negative in group A, C, and D, and positive in the great vessel wells of group B. Conclusion The immunogenicity of tissue engineered skins is very weak, and tissue engineered skins would not be rejected by host immune system after transplantation.
【Abstract】 Objective To investigate the feasibil ity of applying enzymatic method to prepare decellularizedporcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibil ity. Methods 0.1% trypsin- 0.01% EDTA was appl ied to extract cells from porcine aorta under 37 continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothel ial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibil ity. Results Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson’ strichrome showed that the structure of matrix (fibrins) was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization (P gt; 0.05). However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductil ity after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observation, category and the numbers of the infiltrated cells between decellularized and non-decellularized tissues（P lt; 0.05). A cell monolayer was identified by HE staining and scanning electron microscopywhen the endothel ial cells were seeded onto the inner luminal surface of the scaffold, al igned at the same direction on the whole. Conclusion The decellularized porcine aortic scaffold, prepared by trypsin-EDTA extraction under continuously vibrating condition, could meet the requirements of tissue-engineering graft in biomechanical properties, immunogenicity and cell compatibil ity.
ObjectiveTo investigate the immunogenicity of freezing periosteum and bone marrow during allogeneic joint transplantation, and to explore proper pretreatment of allogeneic joint. MethodsThe allogeneic periosteum and bone marrow were harvested from knee joints of 5 New Zealand white rabbits (aged, 6 months; weighing, 2.6-3.0 kg). After gradient cooling, the tissue was cryopreserved for 1 month. The freezing periosteum and bone marrow were grinded to pieces after rewarming to prepare the suspension of periosteum and bone marrow. Eighteen Chinchilla rabbits (aged, 6 months; weighing, 2.1-2.8 kg) were divided into 3 groups randomly:normal saline injection group (group A, n=6), periosteum injection group (group B, n=6), and bone marrow injection group (group C, n=6). The normal saline, periosteum suspension, and bone marrow suspension were injected into the peritoneal cavity in groups A, B, and C, respectively. The concentrations of interleukin 2 (IL-2), IL-6, and tumor necrosis factor α (TNF-α) in serum and the ratio of CD4+ T cell/CD8+ T cell in venous blood were measured before injection, at 1 week and 2 weeks after injection. ResultsThere was no significant difference in the concentration of IL-2 between before and after injection in the same group (P=0.241), and between groups (P=0.055). The concentration of IL-6 after injection was significantly lower than that before injection in the same group (P=0.040), but no significant difference was found between groups (P=0.357). The concentration of TNF-α showed no significant difference between before and after injection in the same group (P=0.925), but the concentration of TNF-α in group B was significantly higher than that in groups A and C (P<0.05). The ratio of CD4+T cell/CD8+T cell of venous blood had no significant difference between before and after operation in the same group (P=0.248), and between groups (P=0.646). ConclusionThe freezing periosteum and bone marrow are lowly immunogenic. In order to decrease the immunogenicity of the joint, preserving the periosteum and removing the marrow cavity are recommended.
ObjectiveExtracting the endothelial cells or all endothelial cells and interstitial cells from the cryopreserved homograft valves (HV), to evaluate the immunogenicity of this two kinds of decellular HV. MethodsFor extracting the endothelial cells, the leaflet and wall of the HV were decellularized by a 4-step detergent-enzymatic extraction method involving the 1% triton in combination with RNase (1μg/ml) and DNase (10μg/ml). For extracting the endothelial cells and interstitial cells, the leaflet and wall of the HV were decellularized by a 3-step detergent-enzymatic extraction method involving the 1% deoxycholic acid (DOA) in combination with RNase (20μg/ml) and DNase (200μg/ml). HLA-DR antigen expression was detected by using immunohistochemical techniques. The valve and wall of the HV were transplanted subcutaneously in the mice for 8 weeks, and the histology, calcium assay and calcium content were examined. ResultsFor the staining of the HLA-DR antigens, the immunogenic potential of the HV with extracting all endothelial cells and interstitial cells or only the endothelial cells was lower than cryopreserved HV, but it more obviously decreased for the HV with extracting all endothelial cells and interstitial cells. After 8 weeks embedded in the mice, the histological signs of the inflammatory reactions and the calcification extent to the cryopreserved HV and the HV with only extracting endothelial cells were stronger than the HV with extracting all endothelial cells and interstitial cells predominantly. And calcification extent or the inflammatory reactions to the wall of the HV were more severe than those of the leaflet. ConclusionsThe immunogenicity of the HV with extracting all endothelial cells and interstitial cells is much less than HV with only extracting endothelial cells. The histological signs of the inflammatory reactions and the calcification extent in vivo experiments is obviously decreased. For the HV with only extracting endothelial cells, though the histological signs of the inflammatory reactions slightly decrease, the calcification extent in vivo experiments is more severe, especially for the wall. The interstitial cells may be the important factor for the donor-reactive immune responses that is related to the graft calcification or destruction after implantation.
ObjectiveTo construct a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A (FnBPA) r10-11 truncated fusion protein, and explore the immunogenicity of FnBPAr10-11. MethodsPloymerase chain reaction (PCR) amplification was carried out from the whole genome sequence of Staphylococcus aureus Newman strain by recombinant PCR technique. The amplified product was purified and transformed into Escherichia coli DH5α for cloning. The recombinant plasmid was extracted and identified by double enzyme digestion. The recovered fragment was ligated into the pET-32a plasmid and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The FnBPAr10-11 was purified by HIS protein purification column, identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and used to immunize mice, and the mice were divided into phosphate buffered saline (PBS) group, FnBPA group, and FnBPAr10-11 group. The serum levels of immunoglobulin G (IgG) and cytokines, and the immune protection rate of the mice were detected. ResultsSDS-PAGE result showed that the relative molecular mass of the protein was about 33.1×103. The titers of IgG antibody in FnBPAr10-11 group and FnBPA group reached 1∶128 000, and were significantly different compared with PBS group (P<0.05). The cytokine level in FnBPAr10-11 group was not significantly different compared with that in FnBPA group, and they were extremely significant (P<0.01) compared with that in PBS group. The immuno-protective effect of the FnBPAr10-11 group was over 50%. ConclusionsThe prokaryotic expression strain of Staphylococcus aureu FnBPAr10-11 truncated fusion protein was successfully constructed. The truncated protein has good immunogenicity.