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.
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.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
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.
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.
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 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 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 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.
【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.
