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 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.
ObjectiveTo compare the biomechanical differences between the kidney-shaped nano-hydroxyapatite/polyamide 66 (n-HA/PA66) Cage and the bullet-shaped n-HA/PA66 Cage. MethodsL2-L5 spinal specimens were selected from 10 adult male pigs. L2, L3 and L4, L5 served as a motor unit respectively, 20 motor units altogether. They were divided into 4 groups (n=5):no treatment was given as control group (group A); nucleus pulposus resection was performed (group B); bullet-shaped Cage (group C), and kidney-shaped Cage (group D) were used in transforaminal lumbar interbody fusion (TLIF) through left intervertebral foramen and supplemented by posterior pedicle screw fixation. The intervertebral height (IH) and the position of Cages were observed on the X-ray films. The range of motion (ROM) was measured. ResultsThere was no significant difference in the preoperative IH among 4 groups (F=0.166, P=0.917). No significant change was found in IH between at pre- and post-operation in group B (P>0.05); it increased after operation in groups C and D, but difference was not statistically significant (P>0.05). There was no significant difference in the postoperative IH among groups B, C, and D (P>0.05). The distance from Cage to the left margin was (3.06±0.51) mm in group C (close to the left) and (5.68±0.69) mm in group D (close to the middle), showing significant difference (t=6.787, P=0.000). The ROM in all directions were significantly lower in groups C and D than in groups A and B (P<0.05), and in group A than in group B (P<0.05). The right bending and compression ROM of group C were significantly higher than those of group D (P<0.05), but no statistically significant difference was found in the other direction ROM (P>0.05). ConclusionThe bullet-shaped and kidney-shaped Cages have similar results in restoring IH and maintaining the stability of the spine assisted by internal fixation. Kidney-shaped Cage is more stable than bullet-shaped Cage in the axial compression and the bending load opposite implant, it can be placed in the middle and back of the vertebral body more ideally.
After escaping from the hyperacute rejection (HAR), the xenograft has to be faced the challenge of acute vascular, acute cellular and even chronic rejection. Endothelial cells have been confirmed as a kind of antigen processing cell (APC) in allo-rejection. The porcine aortic endothelial cell (PAEC) expressed SLA-II and B7 which are the characteristics of professional APC. PAEC also has plenty of alpha-Gal residues, whether the antigen play any role in the post-HAR is still unknown. Human and porcine peripheral blood lymphocyte (PBLC) were isolated and divided into two parts, one for the effectors and the another were incubated with mitomycin C (MMC) as stimulators. The two kinds of PBLC were mixed-cultured within five days. Cultured PAEC from NJZ Pig was incubated with MMC and divided into two: One digested with alpha-galactosidase. The two kinds of PAEC were taken as stimulators to mixed-culture with human PBLC for five days. All the proliferation was detected with 3H-TdR intermingled in the system. The results showed that allo-MLR was ber than xeno-MLR in the cases. The proliferation was much ber when PAEC was used as the stimulator than that of porcine PBLC. However, the response was remarkably decreased after the digestion of alpha-Gal with alpha-galactosidase. The conclusion was that the low response of porcine-to-human MLR in vitro might be related to the predominant indirect pathway of antigen recognition in this system. While PAEC was used as the stimulator the proliferation in MLR was ber which might be concerned that PAEC itself was an APC as well as xeno-antigen sources, thus the direct pathway was predominant and worked more efficiently. The alpha-Gal might induce T cell proliferation through the linkage with the biological big molecules working as a complete antigen. The other post-HAR antigen might also exist in PAEC such as SLA-II, etc.
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.
To overcome the disadvantages of the artificial materials, to design pedicled demucosal small intestinal sheet to repair full-thickness abdominal wall defect. Methods The porcine model of full-thickness abdominal wall defect by resecting 10 cm × 7 cm abdominal wall tissue (from skin to peritoneum) in 20 female animals, which were randomizedto jejunum and ileum sheet groups(n=10). Defect of abdominal wall were repaired with pedicled demucosal jejunum/ileum sheet respectively and immediate spl it-thickness free skin grafting. The general condition was observed and the tension strength of the repaired abdominal wall was measured 30 days postoperatively. In another 5 models, defect was repaired with pedicled demucosal small intestinal sheets and immediate spl it-thickness free skin grafting. The histological change and tissue thickness of the pedicled demucosal small intestinal sheet, spl it-thickness free skin graft and the repaired abdominal wall were observed and measured respectively after 30 days of operation. Results The operations were successful and no operative death occurred in all animals. All pedicled demucosal small intestinal sheets primarily healed to the edge of defected abdominal walls. Neither infection nor wound dehiscence occurred. All the spl it-thickness free skin grafting were successful. Regeneration of the intestinal mucosa occurred 4 days to 5 days postoperatively in 3 animals (2 of jejunum sheet group and 1 of ileum sheet group) at the initial stage andwere successfully treated. No postoperative herniation occurred in all animals. The cel iac pressure of herniation of the repaired abdominal wall jejunum/ileum sheet was (24.8 ± 3.4) kPa in jejunum sheet group and (21.3 ± 2.8) kPa in ileum sheet group, and the difference was significant (P lt; 0.01). No rupture of the repaired abdominal wall occurred in jejunum and ileum sheet groups when the cel iac pressure was 40 kPa. Before repairing the abdominal wall defects, there was a l ittle residual mucosal tissue on the surface of all pedicled demucosal small intestinal sheets. At the 30th day after operation, conspicuous hyperplasia and thickening occurred in all parts of tissue of the repaired abdominal walls and the residual mucosal tissue disappeared completely. Conclusion Because of simple operation, satisfactory achievement ratio, good effect, no important compl ication, and no use of expensive prosthetic materials, it is a feasible method to repair the full-thickness abdominal wall defect with pedicled demucosal small intestinal sheet.
【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 investigate the feasibil ity of preparing the porous extracellular matrix (ECM) by use of some chemicals and enzymes to decellularize the porcine carotid artery. Methods The porcine carotid artery was procured, and warm ischemia time was less than 30 minunts. The porcine carotid artery was decellularized with 1% sodium dodecyl sulfate (SDS) for 60 hours to prepare common ECM; then common ECM was treated with 0.25% trypsin (for 6 hours) and 0.3 U/ mL collagenase (for 24 hours) to prepare porous ECM. The common ECM and porous ECM were stained with HE,Masson’s trichrome, and Orcein to evaluate the histological features. Then the mechanical property, cytotoxicity, and pore size of ECMs were determined. After 4 weeks of subcutaneous implantation in dogs, the histological examination was used for the study. Results Histological observation confirmed that 2 kinds of ECMs were decellularized completely and more porous structure was observed in porous ECM. Scanning electron microscope showed the pores in porous ECM were greater and the length of shorter axis in porous ECM ranged from 5 to 30 μm, the length of longer axis from 40 to 100 μm. The porosity of porous ECM (99.25%) was greater than that of common ECM (91.50%). The burst pressure of porous ECM decreased when compared with common ECM, showing significant difference [(0.154 3 ± 0.012 7) MPa vs [0.305 2 ± 0.015 7) MPa, P lt; 0.05]. There was no significant difference in suture retention strength between 2 kinds of ECMs (P gt; 0.05). The cytotoxicity test showed no obvious cytotoxicity in 2 kinds of ECMs. In vivo implantation test showed that the deeper host cells infiltration and more neo-microvessels in porous ECM were observed than in common ECM. Conclusion SDS and some enzymes can be used to prepare porous ECM as the scaffold for tissue engineered blood vessels.
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.
【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.