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 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.
【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.
Objective To study the protective effects of bone marrow mesenchymal stem cells (BMSCs) of rhesus monkeys on porcine islets from hypoxia/reoxygenation (H/R)-induced injury. Methods BMSCs were isolated and cultured from the marrow of 5 adult rhesus monkeys (weighing, 6-10 kg) by adherent monocytes. Islets were isolated and purified from the pancreas of 5 neonatal porcine (3-5 days old) by collagenase V digestion method, and were cultured with or without BMSCs, and exposed to hypoxia (1%O2) for 12 hours and reoxygenation for 24 or 48 hours, respectively. The experiment was divided into 4 groups: normal islet group (group A), normal islet + BMSCs group (Group B), H/R islet group (group C), and H/R islet + BMSCs group (group D). The survival rate of islets was calculated by fluorescein diacetate/propidium iodide (PI) staining. The viability of the islet cells was detected by cell counting kit 8. Apoptotic rate of islet cells was tested using Annexin V-FITC/PI labeling and flow cytometry. The stimulation index (SI) of islet function was analyzed by glucose-stimulated insulin secretion assay. Results The islet cell cluster of group C was more dispersed than that of groups A and B, and group C had more death cells; and the islet cell cluster of group D was more complete and the survival rate was higher than those of group C. The survival rate of islet was 90.2% ± 9.1%, 88.3% ± 5.9%, 52.3% ± 12.1%, and 71.4% ± 11.5% in groups A, B, C, and D respectively, it was significantly lower in groups C and D than in groups A and B (P lt; 0.05), but it was significantly higher in group D than in group C (P lt; 0.05). After coculture of BMSCs and islet at the ratio of 1 ∶ 10 and 1 ∶ 20 in group D, the viability of islet cells was significantly higher than that in group C (P lt; 0.05). The apoptotic rate was 27.1% ± 3.2%, 24.0% ± 1.0%, 64.3% ± 1.8%, and 46.2% ± 1.4% in groups A, B, C, and D respectively, it was significantly higher in groups C and D than that in groups A and B (P lt; 0.05), but it was significantly lower in group D than in group C (P lt; 0.05). There was no significant difference in SI between groups A and B at each time point (P gt; 0.05), but it was significantly lower in group C than in groups A and B (P lt; 0.05); and it was significantly higher in group D than in group C at 24 and 72 hours (P lt; 0.05). Conclusion BMSCs of rhesus monkeys can protect islet vitality and function from H/R-induced injury.
Objective To evaluate tissue regeneration, body reaction, and biological safety of xenogeneous bladder acellular matrix (BAM) that can be used to repair rabbit bladder. Methods Porcine BAM was prepared through physical, chemical, and enzymatic methods, and the effects of acellularization and the structure were observed with HE staining and scanning electron microscope (SEM). Eighteen New Zealand white rabbits (weighing, 2.5-3.0 kg) undergoing partial cystectomy were randomly divided into 2 groups. After partial (about 30%) cystectomy, the porcine BAM was used to replace partial rabbit bladder in the experimental group (n=12), and the incision was directly sutured as control group (n=6). The survival condition of animals was observed after operation. At 15 days, 1, 2, 3, and 6 months after operation, the blood routine, renal function, and electrolyte were tested by collecting the blood samples. At 1, 2, 3, and 6 months after operation, maximum bladder capacity, bladder leak point pressure, and bladder compliance were measured through urodynamic studies. Then gross observation was performed for regeneration of bladder, and the specimens of the bladder were harvested for HE staining and immunohistochemical staining. The surrounding organs and local lymphoid tissues were harvested for gross observation and HE staining. Results Cell components were completely removed in the porcine BAM, showing three-dimensional porous structure under SEM. All the animals survived during the experiment. At 15 days after operation, white blood cell count increased, and then returned to normal level in 2 groups, showing no significant difference between 2 groups (P gt; 0.05). The tests of renal function and electrolyte suggested no significant difference between 2 groups (P gt; 0.05). The level of serum creatinine showed a tendency of increase, but it remained within normal range at 6 months after operation. The maximum bladder capacity and compliance in experimental group were significantly higher than those in control group at 3 and 6 months after operation (P lt; 0.05), but no significant difference in bladder leak point pressure at each time point between 2 groups (P gt; 0.05). The urothelial regeneration, smooth muscle regeneration, and blood vessel regeneration were seen by histological observation in 2 groups. In the 2 groups, chronic inflammatory cells infiltration could be observed at 1 month postoperatively, and then chronic inflammatory cells decreased significantly (P lt; 0.05), until complete disappearance. There was no significant difference in score of chronic inflammatory cell infiltration between 2 groups at 3 and 6 months after operation (P gt; 0.05). The α-smooth muscle actin expression was significantly increased with time passing in 2 groups (P lt; 0.05), and it was significantly higher in control group than in experimental group at each time point (P lt; 0.05). In addition, gross and HE staining observations showed no abnormalities in surrounding organs and local lymphoid tissues. Conclusion No immune rejection response occurs when porcine BAM is used for xenotransplantation. It is indicated that porcine BAM is relative safety for xenotransplantation.
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 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.
Objective To establ ish a porcine model of articular full-thickness cartilage defect characterized byremaining cartilage calcified zone on femoral trochlea, so as to provide a considerable and comparative control group forinvestigating repair effects of tissue engineered scaffolds in articular cartilage defects with cartilage calcified zone remaining.Methods The full-thickness cartilage column defects (6 mm in diameter, 0.2-0.5 mm in depth) without damage on calcifiedcartilage zone were made on the femoral trochlea in 9 clean-grade 6-month-old Guizhou mini pigs by standard cartilage-defectmakingsuites. Microscopical observation was performed after modeling. Scanning were made by 3.0T MRI at 4 weeks. Thengeneral observation, stereomicroscope, and histological staining were used to observe cartilage repair. Results All animals wereal ive. No infection of incisions or patellar dislocations occurred; they were able to walk with partial weight-bearing immediatelyafter surgery and could move freely without limp at 1 week. Obvious signal discontinuity in trochlea and subchondral bone couldbe observed in MRI, without deep signal change in defects surrounding. Microscopical observation showed a few repair tissueand petechia at base of the defect with clear boundary. Nearly intact calcified zone of cartilage and zonal collapse of subchondralbone in defects could be observed with stereomicroscope. Under common microscope, no chondrocytes was found in defects,as well as negative staining of fast green-safranin O and alcian blue. Under polarized microscope, the bottom of defects werefilled with a l ittle of fibrous tissue presenting continuous and b l ight-refraction by sirius red staining. Conclusion Theanimal model of articular full-thickness cartilage defect on femoral trochlea by standard cartilage-defect-making suites can beapplied for the research of cartilage disease in early human osteoarthritis and function of calcified cartilage zone in pig.
Objective To prepare and study the biocompatibil ity of selectively decellular xenoskin which has the character of the lower antigen, continuous epidermis, and the dermal matrix without any cellular components. Methods The porcine skin was treated with glutaraldehyde solution, trypsin, and detergent solution TritonX-100 to prepare the selectivelydecellular xenoskin. The cytotoxicity was tested according to GB/T16886.5-2003 biological evaluation of medical devices for in vitro cytotoxicity, and the levels of cytotoxicity were evaluated with the United States Pharmacopeia. Subdermal implantation was tested according to GB/T16886.6-1997 biological evaluation of medical devices for local effects after implantation. Seventytwo mature Wistar rats were randomly assigned to groups A, B, and C (n=24). Three kinds of materials were implanted into subcutaneous of rats back. Selectively decellular xenoskin was transplanted into group A, fresh porcine skin was transplanted into group B, and allogeneic skin was transplanted into group C. The samples were collected to make the observation of gross and histology after 1, 2, 4, 8, 12, and 16 weeks. Results The cytotoxicity was proved to be first grade by biocompatibil ity test. The gross and histological observation of subdermal implantation: after implantation, the most severe inflammatory reactions were seen in group B which dispersion was very slow. Inflammatory reactions in groups A and C alleviated gradually. In groups A and C, there was an increased collagen fiber density and angiogenesis at late stage; the transplanted skin was gradually degraded and absorbed. In group B, no obvious degradation and absorption were observed. Conclusion Selectively decellular xenoskin, prepared with glutaraldehyde solution, trypsin, and detergent solution, possesses characteristics of integral skin structure andexcellent biocompatibil ity, so it can be used as a new type substitute to repair the burn wound.
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.