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 review the common methods of isolation and purification of porcine islets and research progress. Methods Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. Results The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. Conclusion The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.
Objective To research the effect of porcine acellular dermal matrix in the reconstruction of abdominal wall defects in rabbits, and to investigate the appl ication feasibil ity of xeno-transplantation of acellular dermal matrix. Methods The porcine acellular dermal matrix was prepared from a health white pig. Twenty-six Japanese white rabbits (weighing 2.2-2.3 kg, female or male) were randomly assigned to 2 groups: the control group (n=6) and the experimental group (n=20). In the control group, the full-thickness abdominal wall defect of 5.0 cm × 0.5 cm was made, and the defect wassutured directly; in the experimental group, the full-thickness abdominal wall defect of 5.0 cm × 2.5 cm was made, and the defect was repaired with porcine acellular dermal matrix patch at the same size as the defect. At 5 weeks after surgery, the incidence of hernia and the intra-abdominal adhesions were observed and the wound breaking strength was compared between the patchfascia interface and the fascia-fascia interface. The graft vascularization was evaluated through histological analysis at 6 months after surgery in the experimental group. Results No hernia occurred in all rabbits of 2 groups. At 5 weeks after surgery, heal ing was observed between patch and the muscularfascia; the vascularization was seen in the porcine acellular dermal matrix patch. There was no significant difference in the adhesion grade (Z= —0.798, P=0.425) between the experimental group (grade 2 in 1 rabbit, grade 1 in 5, and grade 0 in 12) and the control group (grade 1 in 1 and grade 0 in 5). No significant difference was found (t= —0.410, P=0.683) in the breaking strength between the patch-fascia interface in the experimental group [(13.0 ± 5.5) N] and the fascia-fascia interface in control group [(13.6 ± 4.0) N]. In the experimental group, the small vessels and the infiltration of inflammatory cells were observed in the porcine acellular dermal matrix patch after 5 weeks through histological observations. The junctions of the patch-fascia interface healed with fibrous connective tissue. At 6 months after surgery, the inflammation was subsided and the collagen fiber of the patch was reconstructed. Conclusion The porcine acellular dermal matrix patchhas good results in repairing full-thickness abdominal wall defect. The patch-fascia interface has siml iar breaking strength to the fascia-fascia interface. The collagen fibers of the patch are reconstructed.
Objective To study the development of a physiologic fixation method and investigate the effect of physiologic fixation method on porcine aortic root and aortic valve leaflets. Methods Physiological fixer of aortic root was manufactured in a factory. The fixers with different diameter were made of organic glass. Porcine aortic root with ascending aorta and anterior leaflet of mitral valve and partial ventricular septum were dissected out from the fresh heart. The roots were attached to appropriately sized inflow and outflow spigots. Physiologic fixation was utilized to maintain aortic root and leaflets natural anatomical shape, the aortic root was pressurized to the inflow and outflow portions simultaneously, and the leaflets floated freely at zero-pressure differential with in the pressurized root. Results The process of physiologic fixation retained the properties of a native valve. The leaflets were much softer and extensible than those from valves fixed under low pressure. The results of pulsatile flow testing indicated that the effective orifice areas of predilation at 80mmHg were significantly greater than those of predilation at 40 mmHg（P〈0.05）, while mean pressure differences were found to be lower comparatively（P〈0.05）. This difference translates into a mode of valve function that more closely approximates that of the native aortic valve. Conclusion Physiologic fixation process retains the valve＇s natural anatomical shape as well as the underlying structure of the leaflets, providing improved flow characteristics.
ObjectiveTo summarize the advances of precl inical research in xenogeneic (porcine) cell transplantation in recent years. MethodsThe literature about the precl inical research in xenogeneic (porcine) cell transplantation was analyzed and summarized. ResultsWith the application of new immunosuppressive agents and the generation of transgenic pigs, great progress has been achieved in xenogeneic transplantation of pig-derived nerve cells, islet cells, liver cells, and various types of stem cells. The survival time of xenogeneic cell (porcine) significantly prolonged, but there is still a long way to go before cl inical application. ConclusionThe source of xenogeneic (porcine) cells is abundant and the experiments are reproducible. However, how to effectively prevent rejection and prolong the survival time in the host, and avoid the spread of virus between species are still need to be solved in the future research.
ObjectiveTo study the biomechanical stability of neckwear-knot-loop-ligature fixation for tibial eminence avulsion fractures by comparing with cannulated screw fixation and suture anchor fixation. MethodsTwenty-four fresh porcine knee joints were selected. After the model of tibial eminence avulsion fracture (type Ⅲ) was made, 24 samples were randomly divided into 3 groups: neckwear-knot-loop-ligature group (group A), cannulated screw group (group B), and suture anchor group (group C), 8 samples in each group. The Universal electromagnetic and mechanical testing machines were used for the biomechanical tests. After 200 cyclic tests, pull-out test was done until fixation failure. The maximum failure load, yield load, stiffness, and displacement were measured. ResultsFailure mode: the displacement was beyond limit in 8 samples of group A; screws extraction (5 samples) and bone fragment re-fracture (3 samples) were observed in group B; and suture anchor extraction (4 samples), suture rupture (3 samples), and suture thread cutting (1 sample) were found in group C. Biomechanical test: From groups A to C, the maximum failure load and yield load showed significant decreasing tendency (P<0.05), but the displacements showed significant increasing tendency (P<0.05). The stiffness also gradually decreased, but differences was not significant (P>0.05). ConclusionCompared with cannulated screw and suture anchor, neckwear-knot-loop-ligature fixation for tibial eminence avulsion fracture has good biomechanical performance and the advantages of firm fixation and simple operation.
Objective To investigate the feasibility of using the porcine small intestinal submucosa (SIS) as a kind of the new tissue engineered materials to repair the rat full skin defect. Methods Twenty-eight 6-week-old SD rats weighing 300-350 g were selected in this experimental study. Two 2-cm-diameter round full skin defects were made on the rat back. The upper round defect was used as the blank group, which had no coverings, and the lower round defect was used as the SIS group. SIS that had been produced earlier was transplanted in the defected area. At 3 days, 1, 2, 3, 4, 6 and 8 weeks after the transplantation, the observation was made on the repaired skin conditions, the HE stain, and the repaired skin proportion. Results There was no infection in the two groups. The repairing speed in the SIS group was faster than that in the blank group at 2, 3, 4 and 6 weeks after the transplantation. The skin repaired by SIS was soft and elastic in texture, which had the same high level as the normal skin. The scar tissues in the SIS group were thinner than those in the blank group. The repaired skin proportions at 1, 2, 3, 4, 6 and 8 weeks after the transplantation were 15.72%±3.64%, 43.81%±4.87%, 65.35%±5.63%, 87.95%±4.78%，96.90%±6.89% and 100%, respectively in the SIS group, and 13.42%±5.63%，58.74%±4.48%，76.50%±5.23%，92.30%±5.75% and 100%, respectively in the blank group. Therewas a statistically significant difference between the two groups at 1, 2, 3 and 4 weeks after the transplantation(P＜0.05). Under the microscope, the SIS-repaired skin was observed to have more keratinocytes and collagen tissues, whichwas familiar to the normal skin.Conclusion Porcine SIS can be used as a new kind of the tissue engineered materials to repair the full skin defect.
【Abstract】ObjectiveTo develop a method of adult porcine pancreatic islet isolation.MethodsThe tails of adult porcine pancreas were perfused through the pancreatic duct with 0.1% cold collagenase(type Ⅺ) and incubated at 38.5 ℃.The digested tissue was dispersed in 4 ℃ Hanks balanced salt solution(HBSS).The tissue suspensions were filtered through a 600 μm mesh.The residual tissue was resuspended in cold HBSS,and put in the Ricordi’s chamber and shaken for 5 minutes,then filtered again.The isolated islets were divided into three groups: control group(n=14),Pefabloc(trypsin inhibitor,n=8) group and FOY(trypsin inhibitor,n=5) group.The collagenase solution of the Pefabloc and FOY group was supplemented with 1.0 mmol/L Pefabloc and FOY respectively. ResultsThe islet yields of the Pefabloc group and FOY group 〔(11 848±3 530) islet/g pancreas and (14 496±3 693) islet/g pancreas〕 were significantly higher than that of the control group 〔(8 505±3 349) islet/g pancreas〕,P＜0.05.The activity of pancreatic protein enzyme in digestive fluid after digestion in control group was higher than the activity of pancreatic duct before injection and Pefabloc group(P＜0.01),which the control group, pancreatic duct before injection and Pefabloc group were (114.7±50.0) BAEEU,(4.0±1.8) BAEEU and (5.5±2.7) BAEEU,respectively.The pancreatic duct before injection and Pefabloc group showed no significant difference in statistics. In control group,when the harvest of islet was more than 8 000/g,the activity of pancreatic protoin enzyme was less than that with the harvest of islet below 8 000/g 〔(78.3±26.7) BAEEU vs (137.5±48.4) BAEEU,P＜0.05〕.Islet after purification in control group,Pefabloc group and FOY group showed good insulin secretion ability for different concentration of glucose.ConclusionA higher porcine pancreatic islet yield can be obtained by this method of pancreatic islet isolation and prophylactic administration of trypsin inhibitors consistently produce excellent islet yields.
ObjectiveTo prepare the aortic extracellular matrix (ECM) scaffold by using different methods to decellularize porcine ascending aorta and to comprehensively compare the efficiency of decellularization and the damage of ECM, evaluation of biomechanical property and biocompatibil ity. MethodsThirty specimens of fresh porcine ascending aorta were randomly divided into 6 groups (n=5). The porcine ascending aorta was decellularized by 5 different protocols in groups A-E: 0.1% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA)/PBS was used in group A, 1%Triton X-100/0.02% EDTA/ distilled water in group B, 1% sodium deoxycholic acid/distilled water in group C, 0.5% sodium deoxycholic acid/0.5% sodium dodecyl sulfate/distilled water in group D, and 1% deoxycholic acid/distilled water in group E; and the porcine ascending aorta was not decellularized as control in group F. The ascending aorta scaffolds were investigated by gross examination, HE staining, DNA quantitative analysis, immunohistochemistry, and scanning electron microscopy were used to observe the efficiency of decellularization, microstructure of the ECM, the damage of collagen type Ⅰ and elastin, the structure of intimal surface, and biomechanical property. The 90 Sprague Dawley rats were randomly divided into 6 groups (n=15). Each scaffold was implanted in the abdominal muscles of rats respectively to evaluate the immunogenicity and biocompatibil ity. ResultsHE staining and quantitative analysis of DNA showed that the cells were completely removed only in groups A and D. The expression of collagen type Ⅰ in group A was significantly lower than that in the other 5 groups (P < 0.05), and serious damage of the basement membrane and decreased beomechanical property were observed. The maximum stress and tensile strength in group A was significantly lower than those in the other groups (P < 0.05), and elongation at break was significantly higher than that in the other groups (P < 0.05). The destruction of collagen type Ⅰ was significant (P < 0.05) in group D, but the basement membrane was integrity, the biomechanical properties were close to the natural blood vessels (group F) (P > 0.05). Implantation results showed that the scaffold of group D had superior immunogenicity and histocompatibility to the scaffold of the other groups. The inflammatory reaction was gentle and the number of the inflammatory cell infiltration was lower in group D than in other groups (P < 0.05). ConclusionIt is concludes that 0.5% sodium deoxycholic acid/0.5% sodium dodecyl sulfate/distilled water is more suitable for the decellularization of porcine aorta, by which the acquired ECM scaffold has the potential for constructing tissue engineered vessel.
ObjectiveTo investigate the histological changes and vascularization of the porcine acellular dermal matrix (P-ADM) processed with matrix metalloproteinase 7 (MMP-7) (P-ADM-pm) after implanted into rats. MethodsSixty-two pieces of porcine reticular layer dermis which were from the pig abdominal skin and obtained by using a mechanical method, were randomly divided into group A (n=31) and group B (n=31). The porcine reticular layer dermis in 2 groups were treated with decellularization (P-ADM), then the P-ADM in group B were treated with processing by MMP-7 (P-ADM-pm). Thirty adult male Wistar rats were selected. P-ADM (group A) and P-ADM-pm (group B) were subcutaneously transplanted into the left and right fascia lacuna, respectively. The implants were harvested from 6 rats at 3, 7, 14, 21, and 28 days after implantation, respectively. Gross, histochemical, and immunohistochemical observations, and scanning electron microscopy (SEM) examination were performed to observe host cells, microvessels infiltration and histological changes in the implants. ResultsNo rat died in the experiment, incision healed well and no obvious inflammatory reaction was seen in all rats. Gross observation suggested that the implants of 2 groups were encapsulated by a thin layer of connective tissue at 7 days after implantation. With the time of implantation, the microvessels increased and coarsened, and the changes of group B were more obvious than those of group A. At 21 days, the microvessels of 2 groups decreased, and the implants of group B showed complete vascularization. The histochemical and immunohistochemical observations showed that group A had more severe inflammatory response than group B. Fibroblasts and microvessels in group B appeared in the superficial zone of implant at 3 and 7 days after implantation and they could be observed in the center zone of implant at 14 and 21 days. However, fibroblasts and microvessels in group A appeared in the superficial zone of implant at 3 and 14 days and they could not be observed in the center zone of implant at 28 days. Fibroblasts and microvessels of group B were significantly more than those of group A (P < 0.05). SEM examination showed that more fibroblasts and new collagen fibrils were observed in group B at 14 days. ConclusionThe host response to P-ADM-pm is similar to normal wound healing, and P-ADM-pm as implantable scaffold material plays a good template conduction role.