Objective To evaluate the effect of WO-1 on repair of the bone defect in the New Zealand rabbit radius by an oral or local administration. Methods Bone defects were surgically created in the bilateral radii of 36 Zealand rabbits (1.6-2.0 kg), which were randomly divided into3 groups. In Group A, the defective areas were given WO-1 0.1 ml (50 mg/ml) by the local injections; in Group B, the rabbits were given WO-1 5 mg each day by the oral administration. Group C was used as a control group. Among each of the 3 groups, 4 rabbits were randomly selected and were sacrificed at 20, 30 and 60 days after operation, respectively. Then, the serological, X-ray and histological examinations were performed. Results The serum alkaline phosphatase and bone glaprotein levels were significantly higher at 20 and 30 days after operation in Groups A and B than in Group C, but significantly lower at 60 days after operation in Groups A and B than in Group C（Plt;0.01）. The X-ray and histological examinations at 20, 30 and 60 days after operation revealed that the callus formation and remodeling were earlier in Groups A and B thanin Group C, and the remodeling was earlier and better in Group A than in Group B. Conclusion WO-1 can promote the repair of the radial defect in a rabbit; however, further studies on the doseeffect relationship, administration time, and administration route are still needed.
Objective To study the effect of various doses of estrogen on tissue injury, blood supply and survival area of skin flap and to investigate its mechanism. Methods Thirty New Zealand white rabbits aged 3-4 months old and weighing 1.5-2.2 kg (male or female) were used. Random pattern skin flap (12 cm × 3 cm in size) taking the central l ine of the rabbit dorsum as axis and with the pedicle attached at the proximal end was prepared, and the flap pedicle division was performed 7 days after operation. The rabbits were divided randomly into three groups (n=10 rabbits per group). At 2, 4, and 6 days after operation, the proximal edge of flap in group A and B received 100 ?g/kg and 50 ?g/kg subcutaneous injection ofestradiol benzoate, respectively, while group C received no further treatment serving as control group. General condition ofthe rabbits was observed after injection, gross observation was performed 3 and 7 days after injection, survival area of the skin flap was measured 7 days after injection, contents of malondialdehyde (MDA) and nitric oxide (NO) were tested 5 days after injection, and the flaps were harvested 4 and 7 days after injection to receive histology and no significant difference was noted between group A and group B (P gt; 0.05). The NEU counts 4 days after injection were (18.20 ±6.24) cells/HP in group A, (21.27 ± 5.34) cells/HP in group B, and (28.78 ± 7.92) cells/HP in group C, and at 7 days after injection, there were (15.16 ± 7.02) cells/HP in group A, (18.12 ± 6.44) cells/HP in group B, and (29.67 ± 9.12) cells/HP in group C. The VEGF score 4 days after injection was (4.02 ± 0.48) points in group A, (4.19 ± 0.66) points in group B and (3.67 ± 0.49) points in group C, and at 7 day after injection, it was (4.96 ± 0.69) points in group A, (5.12 ± 0.77) points in group B, and (3.81 ± 0.54) points in group C. Significant difference was evident between 4 days and 7 days after injection in group A or B in terms of NEU counts and VEGF score (P lt; 0.05), and difference between 4 days and 7 days after injection in group C was not significant (P gt; 0.05), and the differences among 3 groups were significant (P lt; 0.05). Conclusion Estrogen injection can increase VEGF expression and NO content of flap, decrease MDA content and NEU infiltration of flat, and improve survival area of flap.
【Abstract】 Objective To build nano-biomimetic tissue engineered blood vessel (NBTEBV) with nanotopology by using electrospinning (ELSP) technology. Methods Cony vascular endothel ial cell(VEC) on tubiform tooting in vitro was cultured. NBTEBV was built by use of multi-row nozzle with the suspension of cony vascular smooth muscle cell (VSMC) and mimic ECM (MECM) solution. NBTEBV was cultured with bioreactor in vitro . VEC and VSMC viabil ity and prol iferation were observed with MTT; and HE staining, scanning electron microscopy(SEM) observation and biomechanical test were carried out after 24 hours of static culture and 7 days of dynamic culture. Results After 7 days of culture, the length of NBTEBV was 57 mm, the external diameter was 4 mm and the thickness of wall was 0.4 mm. The NBTEBV’s color was white and the texture was even and flexible. MTT results indicated the viabil ity of cells cultured on NBTEBV for 7 days was normal(8.9 × 106 /mg, 3.5 ×105/mg for 24 hours). SEM and HE staining indicated that the topologic character of NBTEBV was similar to that of the naturalblood vessel. The NBTEBV showed a network scaffolds structure with 100 nm thick fiber and 600 nm aperture. The HE stainingresult showed that the NBTEBV was composed of VEC and VSMC by layer. Vascular mechanical results showed that the NBTEBVultimate hydrostatic pressure was 950 mmHg, the compl iance of the NBTEBV under physio-pressure (110/70 mmHg) was 3.0%; the ultimate tensile strength of 20 mm × 5 mm tissue sl ice was 18.5 MPa. Conclusion The technology of ELSP can use VSMC and MECM scaffold simultaneously to build tissue engineered blood vessel with nanotopology mimic native blood vessel.
ObjectiveTo study the long-term prevention effect of self-developed chitosan electrospun membrane on cerebrospinal fluid leakage.
MethodsTwenty-five healthy adult New Zealand rabbits were selected to prepare the bilateral dural defect (0.8 cm×0.8 cm in size) via midline incision of head.Defect of the right was repaired with chitosan electrospun membrane as the experimental group; defect of the left was not repaired as the control group.At 2-16 weeks after operation,one rabbit was sacrificed for the general observation of inflammatory response surrounding bone window and absorption of chitosan electrospun membrane; at 3 and 6 weeks after operation,5 rabbits were sacrificed for sampling to observe histological change and collagen expression by HE and Masson staining,and to measure the expressions of epidermal growth factor receptor (EGFR) and basic fibroblast growth factor (bFGF) by immunohistochemical staining.
ResultsNo inflammatory reaction of swelling,exudation,and sppuration appeared in the skin and subcutaneous tissue after operation in 2 groups.There was no adhesion around the chitosan electrospun membrane,and new fiber membrane formed under the chitosan electrospun membrane in the experimental group; no cerebrospinal fluid leakage happened; the chitosan electrospun membrane was gradually degraded with time,and was completely absorbed at 16 weeks.There was uneven scar around the dural detect in control group.Histological observation showed less inflammatory cell infiltration in the experimental group,showing significant difference in the number of inflammatory cells compared with control group at 3,6 weeks (P<0.05); capillary,granulation tissue and collagen fiber massively proliferated; collagen fiber arranged in line,and there was a clear borderline between chitosan electrospun membrane and adjacent collagen fiber.The immunohistochemical staining showed that there were high expressions of bFGF and EGFR in the experimental group,and low expressions of bFGF and EGFR in the control group.
ConclusionChitosan electrospun membrane for dural defect of rabbit can effectively reconstruct the dura,and it has exact long-term prevention effect on cerebrospinal fluid leakage.
Objective To find an ideal material for repairing bone defect by local implanting simvastatin compounded with poly-lactic acid (PLA) into the radial critical size defects of rabbits, and to observe the reparative effect and type of bone formation induced by simvastatin. Methods Twelve 4-months-old male New Zealand white rabbits (2.3-2.8 kg) with 22 mm radial critical size defects on both sides were randomized into 4 groups (all n=3). Right side and left side of every rabbit were set as controls with each other. The left defects (experimental groups) of groups A, B, and C were implanted with cyl inder-l ike compound scaffolds containing 50, 100, and 200 mg of simvastatin (fixed with 250 mg PLA), or auto-bonegraft as group D, respectively. The right defects of groups A, B, and C were implanted with scaffolds containing only 250 mg PLA. The right defects of group D were left without any treatment. Digital X-ray images of bone defects were taken 8 and 16 weeks after operation, X-ray was scored double bl ind and X-ray pixel value was measured. Animals were euthanized16 weeks postoperatively. CT was appl ied to analyze new bone formation volume in the defects. In addition, orphologicalcharacters of new bones were observed through micro-CT and histology. Results X-ray films showed that the bone defect of each experimental side had much cloud-l ike callus, and the bone stump were not clear 8 weeks after operation; and the cortex in the defect was continuous and the medullary was recanal ized 16 weeks after operation. In control sides, the cortexes were discontinuous and the ends of fractures were sclerified. At 8 and 16 weeks after operation, the X-ray scores, pixel values and the CT volume percentage of new bone in experiment sides were all significantly higher than those in control sides (P lt; 0.05). The X-ray scores of experimental sides in groups C and D were significantly higher than those in groups A and B 8 weeks after operation (P lt; 0.05), and the X-ray scores of experimental sides in groups B and D were significantly higher than those in groups A and C 16 weeks after operation (P lt; 0.05). The X-ray pixel values of experimental sides of group B were significantly higher than those of groups A, C, and D 8 weeks after operation (P lt; 0.05). The new bone formation volume of experimental side of groups B and D was higher than that of groups A and C (P lt; 0.05), and group D was significantly higher than that of group B (P lt; 0.05). Micro-CT showed bone defects of experimental sides of group B had totally healed, with connected medullary cavities and continuous bone cortex, on the contrary bone defects of control sides of group B did not healed completely. Histological observation showed better bone remodeling effects of the experimental sides than control sides, with connected medullary cavities and continuous bone cortex. And the osteogenetic type was endochondral ossification. Conclusion Local implantation of simvastatin can promote repairing rabbit radial critical bone defect, 100 mg is the best dose of repairing the bone defects.
Objective
To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro.
Methods
SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 ± 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining.
Results
SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells.
Conclusion
It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.
Objective To study the feasibility of core-binding factor α1 (Cbfa1) gene modified marrow mesenchymal stem cells (MSCs) composed with porcine acellular bone extracellular matrix in repairing the radial defects. Methods Radial defects of 1.2 cm in length were created in 40 Japanese white rabbits and they were divided into four groups. In group A, MSCs isolated from homogeneous rabbits were infected with Cbfa1 recombinant adenovirus and implanted into acellular bone exteracellular matrix, and then the complexes were implanted into defects. In group B, the complexes including the MSCs without Cbfa1 gene-modified and scaffoldmaterial were implanted into defects. In group C, only the scaffold material was implanted. In group D, defects were not treated as the control. The macroscopic, X-ray and histologic analysis were performed to evaluate the repair effect at 4, 8 and 12 weeks postoperatively. The repaired radius were examined by biomechanical test at 12 weeks postoperatively. Results By gross examination，mature hard new bone formed at grafted areas at 12 weeks postoperativelyin group A, osteotomized ends connected by much callus in group B and less callus in group C at grafted areas. In contrast, bone nonunion formed in group D. X-ray and histological examination showed that the repaired results of defects in the group A were better than those in others groups evidently in extracellular matrix degradation, new bone remodeling and marrow cavity rebuilding at 4 and 8 weeks postoperatively. At 12 weeks postoperatively, the cortical bone became mature lamellar bone, new bone remolding was complete and marrow cavity was smooth in group A. Only proximal end of defects showed that marrow cavity was remolded partially in group B. The continuous callus could be observed in bone defect, and no obvious marrow cavity remolding was observed in group C. Lots of fibrous connective tissue filled in defect and bone nonunion was shown in group D. There was no significant difference in the damage compress loading of repaired radius between groups A, B and D (Pgt;0.05), but there was significant difference between groups C and D(Plt;0.01).Conclusion These results demonstrate that Cbfa1 gene modified MSCs combined with acellular bone extracellular matrix can be used to repair rabbit radial defects.
Objective To assess the effect of topical appl ication of 5-fluorouracil (5-FU) on intimal hyperplasia in rabbit vein graft. Methods Sixty-four male New Zealand white rabbits, aged 5 months and weighing 2.8-3.0 kg, were randomly divided into group A, B, C, and D (n=16 rabbits per group). Artery defect model was establ ished by cutting about 1 cm artery from the middle part of the dissociated left common carotid artery. A section about 3 cm was cut from the right external jugular vein, and the harvested vein was inverted and end-to-end anastomosed to the artery defect with 9-0 non-traumatic suture. After anastomosis, the extima of the grafted veins in group A, B, and C was completely wrapped with cotton sheet (12 mm × 30 mm × 1 mm in size) immersed by 5-FU at a concentration of 50.0, 25.0, and 12.5 mg/mL, respectively, and eachvein was treated 5 times (1 minute at a time). In group D, the extima of the graft veins was treated with normal sal ine instead of 5-FU. The grafted veins were obtained 1, 2, 4, and 6 weeks after operation, HE staining and Masson staining were preformed for histological changes of grafted vein wall, prol iferating cell nuclear antigen (PCNA) immunohistochemistry staining and TUNEL label ing staining were conducted for prol iferation and apoptosis of smooth muscle cell of the grafted vein, and transmission electron microscope observation was performed for cellular ultrastructure. Results The HE staining, Masson staining, and PCNA immunohistochemistry staining showed that the thickness of intima in group A and B was obviously less than that in group C and D at 1, 2, 4, and 6 weeks after operation, and the prol iferation cells in group A and B were less than that in group C and D at 1, 2, and 4 weeks after operation. The thickness of the intima, the degree of intima hyperplasia, the degree of vessel lumen stenosis of four groups at different time points were as follows: at 1 week after operation, group A [(12.69 ± 1.68) μm, 0.73 ± 0.05, 0.025 ± 0.003], group B [(17.52 ± 2.01) μm, 0.86 ± 0.06, 0.027 ± 0.004], group C [(21.92 ± 1.85) μm, 1.06 ± 0.09, 0.036 ± 0.006] and group D [(26.45 ± 3.86) μm, 1.18 ± 0.08, 0.041 ± 0.005]; at 2 weeks after operation, group A [(24.61 ± 2.91) μm, 0.86 ± 0.06, 0.047 ± 0.003], group B [(37.28 ± 2.78) μm, 1.17 ± 0.09, 0.060 ± 0.004], group C [(46.52 ± 2.25) μm, 1.44 ± 0.08, 0.073 ± 0.003], and group D [(52.07 ± 3.29) μm, 1.45 ± 0.05, 0.081 ± 0.006]; at 4 weeks after operation, group A [(61.09 ± 6.84) μm, 1.38 ± 0.08, 0.106 ± 0.007], group B [(63.61 ± 8.25) μm, 1.40 ± 0.07, 0.107 ± 0.010], group C [(80.04 ± 7.65) μm, 1.64 ± 0.07, 0.129 ± 0.011], and group D [(84.45 ± 9.39) μm, 1.68 ± 0.10, 0.139 ± 0.014]; at 6 weeks after operation, group A [(65.27 ± 5.25) μm, 1.46 ± 0.07, 0.113 ± 0.005], group B [(65.82 ± 7.12) μm, 1.45 ± 0.05, 0.112 ± 0.011], group C [(84.45 ± 9.39) μm, 1.69 ± 0.09, 0.135 ± 0.007], and group D [(87.27 ± 8.96) μm, 1.76 ± 0.05, 0.140 ± 0.012]. Group A and B were inferior to group C and D in terms of the above three parameters and cell prol iferation index 1, 2 and 4 weeks after operation (P lt; 0.05). Group A and B were superior to group C and D in terms of cell apoptosis index of intima and media 1 and 2 weeks after operation (P lt; 0.05). Transmission electron microscope observation showed that the synthetic cell organelles such as rough endoplasmic reticulum, golgi apparatus, and ribosome in group A and B were obviously less than those in group C and D (P lt; 0.05). Conclusion Topicalappl ication of 5-FU can effectively inhibit intima hyperplasia of the vein grafts.
Objective To compare the difference of preparing the acellular larynx scaffold between perfusion method and immersion method, and find better way to make acellular larynx scaffold for tissue engineering. Methods Twenty 6-month-old male New Zealand rabbits, weighing 2.0-2.5 kg, were divided into perfusion group (n=10) and immersion group (n=10) at random. All the larynxes were excised in a sterile fashion. The acellular larynx scaffold was obtained by perfusionmethod and immersion method respectively, and then comparative examinations were performed by the macroscopicview, histological view, scanning electron microscope (SEM), cartilage vital ity assay and toluidine blue staining. ResultsMacroscopic view showed that the larynxes perfused by sodium dodecyl sulphate (SDS) became transparent after 2 hoursof perfusion, but the larynxes immersed by SDS over 16 hours still appeared pink-white. Histology and SEM indicated thatcompared with immersion group, perfusion group showed better acellular effect, more ventages and collagen fibers wereretained, no intact cell or nuclei remained in acellular matrix and chondrocytes were still survival. The porosity was 85.39% ± 3.16% in perfusion group and 34.72% ± 4.51% in immersion group, showing significant difference (P lt; 0.01). The chondrocyte vital ity rate of perfusion group (86.93% ± 1.52%) was higher than that of immersion group (77.73% ± 1.66%), showing significant difference (P lt; 0.01). Toluidine blue staining showed that the chondrocyte heterochromaty was ber in perfusion group than that in immersion group. Conclusion Compared with immersion method, perfusion method is a better way to construct acellular larynx scaffold because it can achieve better acellular effect and retain chondrocyte vital ity at the greatest extent in the acellular larynx scaffold.
Objective Platelet-rich plasma (PRP) secretes many growth factors, including transforming growth factor β1 (TGF-β1), platelet derived growth factor, vascular endothl ial growth factor, insul in-l ike growth factor 1, and so on, which can promote cell prol iferation, chemotaxis, and collagen synthesis in wound heal ing. To investigate the effects of PRPon the tendon heal ing, and to explore the mechanism of action so as to provide the experimental basis for the tissue engineered tendons. Methods Forty healthy New Zealand white rabbits, weighing 2.5-3.0 kg and male or female, were randomly divided into the experimental group (n=20) and the control group (n=20). PRP was prepared from arterial blood of rabbit’s ears through twice centrifugation method of Landesberg. The platelet concentrations of whole blood and PRP were determined. The right achilles tendons of the rabbits were transected to make rupture models. In experimental group, the tendon was sutured after PRP (0.5 mL) was immediately appl ied at repair site. In control group, the tendon was sutured directly after transection. At 1, 2, 4, and 6 weeks after operation, the tendons of 5 rabbits in each group were harvested for morphological, histological, and immunohistochemical observations; the fibroblast counting, the content of collagen fibers, and the expression of TGF-β1 were detected. Results The concentration of platelet of PRP was 4.03 times of whole blood. All the animals survived till the end of the experiment, and the incision healed well. No death, infection, and other compl ications occurred. With time, the tendons almost healed in 2 groups, and the fibrous tissue at anastomosis site was more remarkable in control group than in experimental group. The histological observation showed significant differences in fibroblast counting at 1, 2, and 4 weeks after operation between 2 groups (P lt; 0.05), while no significant difference at 6 weeks (P gt; 0.05). The contents of collagen fibers in the parenchyma at repair site in experimental group were significantly higher than those in control group at each time point (P lt; 0.05). Immunohistochemistry staining showed the expression of TGF-β1 in experimental group was upregulated at 1 week and 2 weeks and reached the peak at the 2nd week, and subsequently downregulated at 4 and 6 weeks in comparison with the control group, showing signficant differences between 2 groups at each time point (P lt; 0.05). Conclusion PRP can facil itate rabbit’ s tendons heal ing and significantly improve the heal ing qual ity, which may be associated with its advancing the peak time of the TGF-β1 expression in tendon.
