目的:建立兔VX2软组织肿瘤模型，研究其高频超声表现。方法:2006年2～6月在12只大白兔后肢建立软组织肿瘤模型，行高频超声检查。结果:12只大白兔成功建立软组织肿瘤模型，超声表现为等回声为主，肿瘤边缘区域血供较中央区域丰富。结论:兔软组织肿瘤模型易于建立，超声有一定的特征性表现。
Objective To investigate the effect of the injectable osteoinductive material with fibrin sealant(FS) as a carrier compounded with bone morphogenetic protein (BMP) on the proliferation and differentiation of marrow stromal cells (MSCs) towards osteoblasts and to provide the experimental foundation for the clinical application. Methods MSCs were extracted and cultured from bone marrow of the 3-day-old rabbit, and the third generation culturedMSCs were studied. The experiment included the experimental group(FS,including 1 μg/ml rhBMP-2), FS control group（FS）and blank control group (no material).The proliferation rate, the adhesive rate, the expression of the collagen Ⅰ and alkaline phosphatase, cell growth condition in the material and the ultrastructure of MSCs were investigated by electron microscopy, histochemistry and cell culture. Results The proliferation rate and the adhesive rate of MSCs in experimental group was significantly higher than those in blank control group ,but lower than those in FS control group （P＜0.05）. The expression level of thecollagen Ⅰ and alkaline phosphatase in the experimental group was significantlyhigher than those in all control groups（Marrow stromal cells Fibrin sealant Bone morphogenetic protein Cell culture Rabbits0.05）. Scanning electron microscope showed that the surface of material was rough and had many pores and that celland material mixed. Transmission electron microscope showed that MSCs of the experimental group were mostly of the phenotype of osteoblasts with relatively lowproliferation activity and high differentiation degree toward osteoblasts and with plenty of extracellular matrix and collagen fibers. MSCs of FS control group had low differentiation degree toward osteoblasts with few extracellular matrix and collagen fibers and high proliferation activity. MSCs of blank control group had low differentiation degree toward osteoblasts with few extracellularmatrix and collagen fibers, and low proliferation activity. Conclusion The injectable osteoinductive material with fibrin sealant as a carrier compounded with BMP could significantly accelerate the differentiation of MSCs towards osteoblasts. But it could not significantly accelerate the proliferation activity of MSCs.
Objective To investigate the effect of tissue engineering bone compounded in vitro by nanohydroxyapatite/collagen/ polylactic acid (nHAC/PLA) and recombinant human bone morphogenetic protein 2 (rhBMP-2) in repairing rabbit critical calvarial defects. Methods Forty eight New Zealand rabbits, weighting 2.0-2.5 kg, were made the models of critical cranial defects(15 mm in diameter) and divided into 4 groups randomly. Defects were repaired with autoflank bone in the positive control group; with no implant in the blank control group; with nHAC/PLA in the negative control; and with active nHAC/PLA(AnHAC/PLA) in the experimental group(the average quality of each AnHAC/PLA absorbed rhBMP-2 was 1.431 mg). The reapir results were observed through X-ray,HE dyeing and Masson’s trichrism dyeing after 8 and 16 weeks. Results The difference of bone formation was observed by X-ray block degree of skull defect area at 8 and 16 weeks. In the 8 th week and 16 th week, the radiopacities on cranial defect were 67.21%±2.06% and 86.48%±1.73% in the positive control group； 5.84%±1.92% and 9.48%±2.72% in the blank control group; 19.13%±2.51% and 35.67%±3.28% in the negative control group; and 58.84%±2.55% and 8561%±3.36% in the experimental group. There were significant differences between the negative control and the positive control group, and between the experimental group and the positive control group at 8 weeks(Plt;0.05) . There were significant differences between the negative control and blank group, and between the experiment and the blank group at 8 and 16 weeks(P＜0.05). The histology observation showed that the width of bone trabecula at 16 weeks was more than that at 8 weeks and bone defectwas full of bone tissue in positive control group. The bone defect was full of fibrous tissue at 8 and 16 weeks, and there was no new bone in the blank group. The bone defect was full of remnant material and fibrous tissue in the negative control group. The implanted area was replaced by the new bone at 8 weeks and the new bone was lamellar at 16 weeks in the experimental group; the residual material was less in defect area and there were more osteoblasts surrounding. Conclusion The nHAC/PLA is a good scaffoldmaterial of rhBMP-2 and AnHAC/PLA has agood ability in repairing bone defect. So it is hopeful to be applied in the clnical repair of large bone defect.
Objective To study the biological activities ofthe nerve regeneration conditioned fluid (NRCF), especially to further separateand identify the protein bands of the relative molecular mass of (232-440)×103. Methods The silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve in 6 New Zealand white rabbits (weight, 1.8-2.5 kg). The proteins in NRCF were separated by the native-polycrylamide gel electrophoresis (Native-PAGE), the protein bands of the relative molecular mass of (232-440)×103 were analyzed by the Shotgun technique, liquid chromatography, and mass spectrometry. Results The Native-PAGE result showed that there was 1 protein band of the relative molecular mass over 669×103, (232-440)×103 and (140-232)×103,respectively, and 6 bands of the relative molecular mass of (67-140)×103.Besides, 54 proteins were identified with at least 2 distinct peptides in 1 protein band of the relative molecular mass of (232-440)×103, including 4 unnamed protein products, mainly at the isoelectric points of 5.5-8.0 and of the relative molecular mass of (10-40)×103. Based on their functions in the protein database, allthe identified proteins in this study were classified into the following 5 groups: conjugated protein (43%), transport protein (30%), enzyme (6%), signal transducer (4%), and molecular function-unknown protein (17%). At the subcellular localization of the identified proteins, there was mainly a secreted protein (63%), and the remaining proteins were localized in the membrane and cytoplasm. Conclusion Native-PAGE and the Shotgun technique can effectively separate and identify proteins from NRCF, and can identify the components of the protein band of the relative molecular mass of (232-440)×103 and provide basicinformation on the unnamed protein products in NRCF.
Objective To separate each protein band from the nerve regeneration conditioned fluid(NRCF）and to study whether there are somenew and unknown neurotrophic factors in the protein bands with a relative molecular mass of 220×103. Methods The silicone nerve regenerationchambers were formed in the sciatic nerve of the 25 New Zealand rabbits (weight,1.8-2.5 kg), and NRCF was taken from it at 1 week after operation. The Nativepolyacrylamide gel electrophoresis (Native-PAGE) was used for separating the proteins from NRCF and detecting the relative molecular mass. The Western blot and ELISA were used to observe whether the protein bands ［220×103 (Band a), (20-40)×103(Band c)］ of NRCF could combine with the antibody of the known antibody of neurotrophic factor (NTF):nerve growth factor(NGF), glial cell-derived neurotrophic factor(GDNF), brainderived neurotrophic factor(BDNF), neurotrophin 3(NT-3)， NT-4, ciliang neurotrophic factor(CNTF). Results Separated by Native-PAGE, NRCF mainly contained two protein bands:Band a had a relative molecular mass about 220×103, and Band c had a relative molecular mass about (20-40)×103. Band a could not combine with the antibodies of the NGF, BDNF, CNTF, and NT-3, but could combine with the antibody of NT-4.Band c could combine with the antibodies of NGF, BDNF, CNTF and NT-3, but could not combine with the antibodies of NT-4 and GDNF. Conclusion The protein bands with a relative molecular mass of 220×103 have ber neurotropic and neurotrophic effects than the protein bands with a relative molecular mass of (20-40)×103, which contains NGF,CNTF, etc. NT-4 just has a weak or no effect on the sympathetic neurone. This indicates that there is a new NTF in the protein bands with a relative molecular mass of 220×103, which only combines with the antibody of NT-4.
Objective To investigate the outcome of repairing the peripheral nerve defects with the tissue engineered nerve constructed by Schwann cells and fibrin glue. Methods Wallerian degenerated sciatic nerve were harvested from the 4-week-old New Zealand rabbits for culture of Schwann cells. The Schwann cells were then separated, amplified and purified, and then were identified by the S-100 protein immunochemical staining. The cultured Schwann cells (1×106/ml) were mixed with fibrin glue to form the Schwann cell-fibrin glue compound, which was observed by the inverted phase contrastmicroscope. The compound filled some silicone tubes (Group A) and biomembrane (Group B) to fabricate the tissue engineered nerves with a purpose of repairing the 10-mm defects in the New Zealand rabbit tibia nerves. The autologous nerve grafting was performed in Group C. The electrophysiological examination and the histomorphological analysis were performed at 10 weeks after the transplantation. Results All the rabbits survived through the experiment. In Group A, all the rabbits developed an ulcer in the soles of their left feet at 3-4weeks after the transplantation, while less ulceration developed in Groups B and C. At 10 weeks after the transplantation, the electrophysiological examination was performed, the elective stimulation failed to pass through the nerve grafts, and no composed muscular action potential was found in all the rabbits in Group A; the elective stimulation could pass through all the nerve grafts in Groups B and C, and could evoke the composed muscular action potential; the composed muscular action potential and the nerve conduct velocity in the two groups were 4.21±0.82 mV and 3.40±5.40 m/s vs. 4.80±1.15 mV and 36.55±6.43 m/s(Pgt;0.05). In Group A, no regrown axon was found in the nerve grafts, but neuromawas found to have formed in the both ends of the silicon tube. In Groups B and C, there was no obvious neuroma formation but regrown axons could be found to have regenerated. The histomorphological analysis on the regrown axons showed thatthere was no statistically significant difference between Groups B and C. Conclusion The tissue engineered nerve fabricated with Schwann cells, fibrin glue, and biomembrane can promote the nerve regeneration, and its reparative effect is similar to that of the autologous nerves; therefore, the future of its clinical practice is brilliant.
Objective To observe the tissue engineered bonefabricated with the cultured mesenchymal stem cells (MSCs) by the green fluorescent protein (GFP) gene transfer. Methods The recombinant Adeno-XTM-GFP expression vector was purified after being packed and proliferated by the HEK293 cells, and then it was used to infect the rabbit’s MSCs directly afer the virus titer was assayed. The cell morphological changes were observed under the inverted phase contrast microscope, and the expression of GFP was observed under the fluorescence microscope to confirm success of the labeling of MSCs.The GPFlabeled MSCs and the pure MSCs were cultured together in the conventional osteogenic supplements for 3 weeks, and then they were seeded onto the compound scaffold of the calcium phosphate cement (CPC) and the fibrin glue (FG) to form a new kind of the tissue engineered bone. It was implanted into the donator rabbit subcutaneously to be used as the experimental group; in contrast, the pure compound scaffold of the CPC-FG without any MSCs was implanted in the same rabbit as a control. The alkline phosphatase (ALP) activity assay was performed respectively at 1, 2 and 3 weeks after operation. GFP was observed under the laserconfocal microscope at 4 weeks after operation, and the formed new bone was harvested at 4 weeks and evaluated by the Masson staining, the immunohistochemistry staining of osteocalcin (OC) and collagen typeⅠ.Results The virus titer was 3×108pfu/ml after proliferation and purification. Expresstionof GFP was confirmed 96 h after MSCs were infected by the Adeno-XTM-GFP expression vector and the infection rate was proximally 50%-70%. In contrast to MSCs, division and proliferation of the GPF-labeled MSCs were not significantly different. The ALP activity in the experimental group (12.546±1.091, 16.567±0.659, 20.443±0.706) was significantly higher than that in the control group (0.453±0.113, 0.243±0.018, 0.308±0.056), respectively at 1, 2 and 3 weeks after operation (Plt;0.05). The tissue engineered bone formed at 4 weeks. There were newly-formed trabeculae around the pore of the compound scaffold, and theimmunohistochemistry staining of OC and collagen typeⅠ were positive. The laser confocal microscope revealed that the GFP-labeled cells existed in many newlyformed tissues，and the compound scaffold of CPC-FG was partly degraded. Conclusion The engineered bone is similar to the spongy bone and the composed cells originate from the cultured MSCs, all of which can be confirmed by the GFP gene transfer technique.
Objective To compare the effect of guiding boneregeneration between l-ethyl-3(3-diaminopropyol)-carbodiimide(EDAC)crosslinked acellular bovine pericardium (ABP) and medical collagen membrane (CM). Methods Defects of 7 mm×7 mm×5 mm were created in both mandibles of 24 rabbits, which weighted 2.6～3.5 kg. One side defect was covered with EDAC-crosslinked ABP(EDAC-crosslinked ABP group), the other side defect with medical CM as control(CM group). The ability of bone defect repair and change ofboth membrane materials were evaluated by gross observation, histological study and computer graphic analysis in the 4th, 8th, 16th and 24th weeks after operation. Results The surface of bone defects was even, consistent with adjacent normal bonein EDACcrosslinked ABP group, while that of bone defects was of no evenness in CM group in the 16th and the 24th weeks. The histological observation showed that bone trabecula formed in the EDAC-crosslinked ABP group and fibrous connective tissue was seen in CM group in the 16th and the 24th weeks. There were no significant differences in new bone percentage of bone defects between 2 groups inthe 4th and the 8th weeks(P＞0.05). In the 16th week new bone percentage of bone defects was 81.99%±3.92% in EDAC-crosslinked ABP group and 76.35%±4.29% in CM group, showing significant difference (Plt;0.05). The average percentage of absorption in EDAC-crosslinked ABP group was 16.57%, 27.94%, 65.61% and85.72% in the 4th, 8th, 16th and 24th weeks respectively, while that in CM group was more than 50% in the 4th week and completely degraded at the end of 8 weeks. Conclusion EDAC-crosslinked ABP has a better effect on guiding bone regeneration than CM in the repair of bone defects.
Objective To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-t ransforming growth factor-β1 (TGF-β1) gene and to evaluate the feasibility of selfinduction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further “gene enhanced tissue engineering” research. Methods The rabbit MSCs was transfected with the reconstructed PGL3-TGF-β1gene by the Liposo mesMethod, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evalua ted by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-β1 and collagen Ⅱ were examined at 2 and 7 days of the cell culture afte r transfe ction with PGL3-TGF-β1gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was perfor med with the software of the SPSS 11.0, compared with the control group and the blank group. Results Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-β1gene by the Liposomes Method achie ved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (Plt;0.01). Tested by MTT, the living acti vity of the transfected cells was proved to be significantly decreased (Plt;0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF-β1 positive particles were detected in the experimental group，but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the ttest (Plt;0.01). By the immunohistochemistry me thod (SABC) to study collagen Ⅱ, there were more positive particles in the transfected cells in t he experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (Plt;0.01). Conclusion Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-β1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfecte d BMS cells with PGL3-TGF-β1 gene can express and excrete TGF-β1when cultured in vitro. The transfected MSCs that secret TGF-β1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.
Objective To explore the possibility of small intestinal submucosa (SIS) for reconstruction of urethral defect. 〖WTHZ〗Methods Twenty-four male rabbits weredivided into 4 groups: group A (the tubulate SIS graft for urethral repair), group B (control group, urethral tubulate defect), group C (the SIS patch graft forurethral repairs), group D (control group, urethral part defect). Then the regenerative segment was studied with histological technique by hematoxylineosin straining and immunohistological straining for α-actin after 6 and 12 weeks postoperatively. The retrograde urethrography and urodynamics were used to evaluate the function of the regenerative urethra at 12 weeks after operation. Results In groups A and C, at 6 weeks after operation, the luminal surface of matrix was completely covered by urothelium, minimal SIS graft was observed in the extracellular matrix, new smooth-muscle cells was confirmed; however, more inflammatory cells were observed in the host-matrix anastomosis in group A than in group C. At 12 weeks postoperatively, the regenerative tissue was equivalent to the normal urethral tissue and SIS disappeared in group C, but some minimal SIS grafts were observed in group A. In groups B and D, urethral strictures and fibrous connective tissue were observed except ３ cases. The urethrography showed wide smooth urethral in group A and C, meawhile urodynamic evaluation didn’t demonstrat significant difference(P＞0.05) in the bladder volume and the maximum urethral pressure between preoperation and postoperation in group A or group C. Conclusion SIS can be a useful material for urethral repair in rabbits, the SIS patch graft is superior to the tubulate SIS graft in urethra reconstruction.