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 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 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.
Objective To evaluate the immunological reaction and the outcome of allogeneic chondrocyte transplantation in repairing articular cartilage defects in porcins. Methods Full articular cartilage from the knee of two Shanghai white porcins about one-month-old was removed and cut mechanically, digested by 0.25% trypsin and 0.2% type Ⅱ collagenase and cultured in 10％ DMEM medium. Defects of 0.5 cm×0.5 cm involving the subchodral bone were created in both the left and right femur condyloid in 8 two-month-old Yunnai bama porcins. Allogeneic chondrocyte transplantation were implanted in defects at a density of (1.0-2.0)×106,0.2 ml. The lymphocytes from the receivers’ blood were collected before transplantation and after 3, 5, 7 and 12 weeks of transplantation, then mixed with allogeneic chondrocytes to determin the lymphocyte stimulation index(SI) in vitro. The histological observation in vivo was made after 5, 7 and 24 weeks of transplantation. Results Lymphocyte SI at 3, 5, 7 and 12 weeks(1.457±0.062,1.739±0.142,1.548±0.047，1.216±0.028) after transplantation was higher than that before transplantation(1.102±0.034，Plt;0.05). SI began to increase in the 3rd week and reached the peak value in the 5th week, then gradually declined at the 7th and 12th weeks, showing significant differences when compared with in the 5th week (Plt;0.05). Inflammation and lymphocytes infiltration could be seen in subchondral bone and the intergration area between repair tissue and normal cartilage in the 5th week, and then decreased and limited in subchondral bone in the 7th week. Defects were filled with cartilage tissue, which had good intergration with subchondral bone at 24 weeks after transplantation. Conclusion Immunological reactions can be found at early stage of allogeneic chondrocyte transplantation and then decreased with the time, the fullthickness articular cartilage defects could be repaired mainlywith hyaline cartilage by the allogeneic chondrocyte transplantation. This may provide a new method to repair articular cartilage defects clinically.
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 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.
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.
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 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.