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 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.
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 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.
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.
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.
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.
Objective To explore an effective method to culture and purify porcine keratinocytes, to observe the morphological characteristics of porcine keratinocytes growing on acellular amnion and to offer the experimental basis for that the amnion is used for tissue engineering. Methods The primary porcine keratinocytes were cultivated with DKSFM(Defined keratinocyteSFM) containing 10% fetal bovine serum (FBS). The second passage porcine keratinocytes were cultivated with the medium of DKSFM containing different concentrations of FBS. Because of the speciality that keratinocytes stick to flask fast, we purified the keratinocytes by 0.02% EDTA and 005% trypsin step by step. The second passage keratinocytes were seeded on amnion, the keratinocytes/amnion composites were observed by dye directly, histopathology and immunohistochemical staining. Results The proliferation of the primry porcine keratinocytes cultured with the medium ofDKSFM containing 10% FBS was fast and the morphological characteristics were good. The cultivated porcine keratinocytes expanded to 60%70% of the total area of the bottle of the flask after 5 days. The proliferation of the second passage porcine keratinocytes cultivated with the medium that DKSFM containing 5% FBS was faster than the second porcine keratinocytes cultured with the medium of DKSFMcontaining 10% FBS, or DKSFM without FBS. The proliferation of the second passage porcine keratinocytes cultivated with DKSFM without FBS was the slowest one among the 3 medium. The porcine keratinocytes that were purified by 0.02% EDTA and 005% trypsin step by step were got with high pure. After the keratinocytes were cultivated on the surface of amnion 12 days, the keratinocytes form a single layer on the surface of amnion and the cells were polygong and arranged like slabstone. After 14 and 16 days,the cells contacted more closely. But at 16 days after the cells were seeded, some of the cells got aging. Conclusion To culture primary porcine keratinocytes with the medium that DKSFMcontaining 10% FBS and to cultivate the second passage with the medium containing 5% FBS, the proliferation of porcine keratinocytes are faster. The method that purify the porcine keratinocytes is effective. Acellular amnion offers excellent bioscafold to support keratinocytes to adhere and grow. After the porcine keratinocytes are cultivated on the surface of the acellular amnion 12 days, the morphologic characteristics are better than that of other groups.
Objective To study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92(NK-92). Methods By use of liposomes pack, the pcDNA3.0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by51Cr-release assays. Results HLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein andRNA. It also could be found that the survival rate of transfected PECs was muchhigher than that of non-transfected PECs, when both of them faced the lysismediated by human PBMC and NK-92.After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (Plt;0.05). Conclusion The PECs- transfected by HLAG1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.
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.