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）
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 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 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.
Objective To verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor β1 (hTGF-β1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring. Methods Rabbit models of disc degeneration were established by injecting the 25 μL fibronectin fragment (Fn-f, 1 mmol/ L), 4 weeks later,saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n=8). Group A received the 25 μL rAAV2-hTGF-β1 (1 × 1012 vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-β1 was performed on the sl ices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection. Results Immunohistochemical staining showed that extensive and intense positive immunohisochemical staining for hTGF-β1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P lt; 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks. Conclusion The discs injected with rAAV2-hTGF-β1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
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 explore the feasibility of high-pressure injection to transfer human thrombomodulin (hTM) gene into arterial wall of rabbits.Methods Eighty-four healthy New Zealand rabbits were randomly divided into three groups: pcDNA3.1/hTM plasmid group (n=28), pcDNA3.1(+)/neo plasmid group (n=28) and untransfected group (n=28). After gene transfection, the model of arterial injury-blocking was established. Then, the expressions of hTM mRNA and protein in arterial wall were examined by RT-PCR and immunohistochemistry at 3 d, 7 d, 14 d and 28 d after operation. Results Seventeen rabbits died accidentally from the day of operation to 3 d after operation. The expressions of hTM mRNA of different time points in pcDNA3.1/hTM plasmid group were significantly higher than that in pcDNA3.1(+)/neo plasmid group and untransfected group (Plt;0.01). For the expressions of hTM mRNA at different time points in pcDNA3.1(+)/neo plasmid group and untransfected group, the difference of inter-group and intra-group was not significant (Pgt;0.05). hTM protein was expressed in every group and mainly localized in the inner lining of arterial wall. The expressions of hTM protein at different time points in pcDNA3.1/hTM plasmid group were significantly higher than that in pcDNA3.1(+)/neo plasmid group and untransfected group (Plt;0.05). The expression of hTM protein at different time points in pcDNA3.1(+)/neo plasmid group and untransfected group kept relative constancy, the difference of inter-group and intra-group was also not significant (Pgt;0.05). Conclusion High-pressure injection is feasible to transfer pcDNA3.1/hTM plasmid into arterial wall of live animals.
