One eye each in 3 groups of 12 pigmented rabbits after bilateral vitrectomy received 0.5mg, 1mg or 2mg triamcinolone acetonide (TA), respectively. The fellow eye received only balance saline solution as control. Ophthalmoscopy and electroretinography were performed during 1 day to 38 days after vitrectomy and drug injection. Light and electronmicroscopic studies were done on the 28th day. The particles of drug were visible on day 28 in all TA-treated eyes. Administration
of 0. 5rug and 1mg TA did not result in different changes in ERG b-wave amplitudes compared with those in control eyes(P＞0. 05). There were significant elevations of ERG b-wave in 2mg TA eyes compared to the control eyes(Plt;0.05), Both ligbt and electronmicroscopy of the retina in these
groups were almost normal. The results showed no Toxielties in TA treated eye up to 2mg after vitrectomy. This offers the experimental evidence as a baseline for combining TA with vitrectomy to
reduce recurrence of proliferative vitreoretinopathy.
（Chin J Ocul Fundus Dis,1996,12: 105- 107）
ObjectiveTo observe the effect of preserving tibial residual fibers on the expressions of ligament remodeling related genes in rabbit anterior cruciate ligament (ACL) reconstruction model.
MethodsSixty healthy adult New Zealand white rabbits were randomly divided into 4 groups:normal control group (group A, n=6) , sham-operation group (group B, n=18) , non tibial remnant preserved group (group C, n=18) , and tibial remnant preserved group (group D, n=18) . At 2, 6, and 12 weeks after operation, the ligament tissue was harvested to detect the mRNA expressions of collagen type 1A1(COL1A1) , collagen type 3A1(COL3A1) , transforming growth factor β1(TGF-β1), vascular endothelial growth factor (VEGF), growth-associated protein 43(GAP-43) , and neurotrophin 3(NT-3) by real-time fluorescent quantitative PCR.
ResultsAt each time point, there was no significant difference in the mRNA expressions of COL1A1, COL3A1, VEGF, and NT-3 between group A and group B (P>0.05) . In group D, the mRNA expressions of COL1A1, COL3A1, TGF-β1, and GAP-43 significantly increased when compared with those of group C at 6 weeks after operation (P＜0.05) ; an increased level of VEGF mRNA was also detected in the group D at 12 weeks after operation (P＜0.05) ; and an increased level of NT-3 mRNA was also observed in group D at 2 and 12 weeks after operation (P＜0.05) .
ConclusionThere is a time-dependent manner of angiogenesis-promoting, repair-related, and nerve-related gene expressions after ACL reconstruction with preserving tibial residual fibers during the process of ligamentization. Furthermore, the remnant preservation in ACL reconstruction can promote the expressions of related genes in some time points.
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 repair of critical-sized cranialdefect with tissue engineered bone fabricated by coral, bone mesenchymal stem cells(MSCs) and sustainedly released recombinant human bone morphogenetic -protein 2 (rhBMP-2) by collagen. Methods Three scaffolds of rhBMP-2+coral,collagen+rhBMP-2+coral and MSCs+collagen+rhBMP-2+coral were fabricated. Forty New Zealand rabbits were made the models of critical-sized defects and divided into5 groups according to different implants: group Ⅰ, auto-ilium; group Ⅱ,coral; group Ⅲ, rhBMP-2+coral; grop Ⅳ, collagen+rhBMP-2+coral; and group Ⅴ,MSCs+collagen+rhBMP-2+coral. Repair of bone defect was evaluated after 8 and 16 weeks of implantation by gross obeservation, X-ray,HE staining and Masson’s trichrome staining. Results Repair ofbone defect in group Ⅴ was similar to that in group Ⅰ, andwas better than that in group Ⅳ; and group Ⅲ was worse. The gross appearance showed that defect region filled with bony tissue which had similar strength to adjacent bone and formed bone union with surrounding bone. The X-ray result displayed high radiopacity(80.45%±2.52% in the 16thweek). Histological observation showed new lamellar bone tissue and with few pore blank area. However, only transpasent fibrous tissue filled the defect in group Ⅱ. Conclusion Collagen may be a suitable sustained release system for rhBMP-2. And MSCs may have important effect on enhancing repair of bone defect. Tissueengineered bone fabricated by MSCs+collagen+rhBMP-2+coral may be a useful material for bone defect repair.
Objective To investigate the effect of allogeneic chondrocytes-calcium alginate gel composite under the intervention of low intensive pulsed ultrasound (LIPUS) for repairing rabbit articular cartilage defects. Methods Bilateral knee articular cartilage were harvested from 8 2-week-old New Zealand white rabbits to separate the chondrocytes by mechanical-collagen type II enzyme digestion. The 3rd passage chondrocytes were diluted by 1.2% sodium alginate to 5 × 106 cells/mL, then mixed with CaCl2 solution to prepare chondrocytes-calcium alginate gel composite, which was treated with LIPUS for 3 days (F0: 1 MHz; PRF: 1 kHz; Amp: 60 mW/cm2; Cycle: 50; Time: 20 minutes). An articular cartilage defect of 3 mm in diameter and 3 mm in thickness was established in both knees of 18 New Zealand white rabbits (aged 28-35 weeks; weighing, 2.1-2.8 kg), and divided into 3 groups randomly, 6 rabbits in each group: LIPUS group, common group, and model group. Defect was repaired with LIPUS-intervention gel composite, non LIPUS-intervention gel composite in LIPUS group and common group, respectively; defect was not treated in the model group. The general condition of rabbits was observed after operation. The repair effect was evaluated by gross and histological observations, immunohistochemical staining, and Wakitani score at 8 and 12 weeks after operation. Results Defect was filled with hyaline chondroid tissue and white chondroid tissue in LIPUS and common groups, respectively. LIPUS group was better than common group in the surface smooth degree and the degree of integration with surrounding tissue. Defect was repaired slowly, and the new tissue had poor elasticity in model group. Histological observation and Wakitani score showed that LIPUS group had better repair than common group at 8 and 12 weeks after operation; the repair effect of the 2 groups was significantly better than that of model group (P lt; 0.05); and significant differences in repair effect were found between at 8 and 12 weeks in LIPUS and common groups (P lt; 0.05). The collagen type II positive expression area and absorbance (A) value of LIPUS and common groups were significantly higher than those of model group (P lt; 0.05) at 8 and 12 weeks after operation, and the expression of LIPUS group was superior to that of common group at 12 weeks (P lt; 0.05); and significant differences were found between at 8 and 12 weeks in LIPUS group (P lt; 0.05), but no significant difference between 2 time points in common and model groups (P gt; 0.05). Conclusion Allogeneic chondrocytes-calcium alginate gel composite can effectively repair articular cartilage defect. The effect of LIPUS optimized allogeneic chondrocytes-calcium alginate gel composite is better.
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 observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.
Objective To investigate whether anterior cruciate l igament (ACL) reconstruction with the remnants and the remaining bundle preservation is beneficial for the revascularization of the graft or not. Methods Animal models of the ACL reconstruction in 18 healthy New Zealand White rabbits (2-3 months old) were made using about 2.5 cm long extensordigitorum longus tendon and randomly divided into three groups (n=6): remnants debridement group (group A), remnantspreservation group (group B) and remaining bundle preservation group (group C). The histological examination was made after 2, 4 and 8 weeks of operation. The intravascular injection of ink was used to observe the revascularization of the graft. The specimens were stained with HE method to observe the histological changes of the graft. Results All the animals were in good condition and had good knee functions in the observation period. There was no new vessel in groups A and B at 2 and 4 weeks postoperatively. And new vessels were observed in the synovium of the graft in group C. The areas of the vessels in group C were (505 ± 27) pixels at 2 weeks and (624 ± 23) pixels at 4 weeks. At 8 weeks postoperatively new vessels were observed in all the three groups. The new vessel areas of groups A, B and C were (674 ± 65), (836 ± 76) and (1 219 ± 146) pixels, respectively. The vessel areas of group C were significantly bigger than those of groups A and B (P lt; 0.05). The vessel area of group B was significantly bigger than that of group A (P lt; 0.05). The fibroblast amount in group B was significantly more than that in group A at 2 and 4 weeks postoperatively (P lt; 0.05). But there was no significant difference between them at 8 weeks postoperatively (P gt; 0.05). The fibroblast amount of group C was significantly more than that of group A (P lt; 0.05) at each observation time postoperatively. At 2 and 4 weeks postoperatively, the fibroblast amount of group C was significantly more than that of group B (P lt; 0.05), but there was no significant difference between them at 8 weeks postoperatively (P gt; 0.05). Conclusion ACL reconstruction with remnants and remaining bundle preservation is beneficial for the early revascularization and fibroblasts growing of the graft. So the course of necrosis, regeneration and remodel ing of the graft is shortened.
Objective
To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits.
Methods
Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule.
Results
The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm.
Conclusion
Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.
Objective To study the effect of decorin in the suppression of postoperative flexor tendon adhesion. Methods Eighteen Japanese large ear white rabbits underwent complete transection of the Ⅱ digit flexor digitorum profundus tendon in zone Ⅱ and defects immediately were repaired using the modified Kessler technique with -0 nonabsorbable monofilament suture. The site of the right repaired tendon was then injected with 100 μl of decorin(0.25mg/ml) as test toe, the site of the left repaired tendon with 100 μl of PBS as control toe. Inevery group, rabbits were killed and the feet were prepared for biomechanical testing, macroscopic examination and histological inspection. Results In every group, biomechanical testing demonstrates that the sliding distances and the rangs of motion significantly increased in the test toe compared with the control toe(Plt;0.05); macroscopic examination demonstrated that the tendon adhesions of the test toe were significantly reduced when compared with the control toe. In the tese toe, hematoxylin and eosin staining revealed that the hyperplasia of fibroblast was significantly delayed and the collagen fibrils arranged regularly and hadthe normal diameters. Conclusion Decorin can significantly reduce the flexor tendon adhesion formation, adjust collagen fibrillogenesis and promote the tendon healing.
