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 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.
To investigate the preventive effect of TGF-β1 neutral izing antibody on collagen production and adhesion formation of flexor tendon. Methods Tendon fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from 6 New Zealand rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-β along with increasing dose of TGF-β1 neutral izing antibody. Col I production was measured by enzyme-l inked immunoabsorbent assay after 3 days. Eighty-four adult New Zealand White rabbits forepaws underwent sharp transection of middle digit flexor digitorumprofundus and immediate repair. Then the rabbits were divided into three groups: the normal saline (NS group, n=36), 1.0 μg/ mL TGF-β1neutral izing antibody (1.0 μg/mL TGF-β1group, n=36) and 2.0 μg/mL TGF-β1 neutral izing antibody (2.0 μg/mL TGF-β1 group, n=12) were injected in tendon sheath respectively. Tendons were harvested at 4 and 8 weeks for biomechanics testing, histological evaluation and scanning electron microscope observation. Tendons were harvested at 1, 2, 4 and 8 weeks to determine the mRNA expression of TGF-β1 and Col I by in situ hybridization. Results ELISA exhibed that TGF-β1 enhanced Col I production and the neutral izing antibody significantly inhibited TGF-β1-induced Col I production in all 3 cell culture with a dose-dependent. At 4 and 8 weeks after operation the gl iding excursion of the tendon and the simulated active flexion in NS group were less than that of 1.0 μg/mL TGF-β1 group and 2.0 μ g/mL TGF-β1 group. There was significant difference between NS group and 1.0 μ g/mL TGF-β1 group, 2.0 μ g/mL TGF-β1 group (P lt; 0.05). The tendon anastomosis breaking strength showed no significant differences among three groups (P gt; 0.05). Scanning electron microscope and histological observation showed that collagen fibers arranged irregularly in NS group, but arranged regularly in 1.0 μ g/mL TGF-β1 group and 2.0 μ g/mL TGF-β1group at 4 and 8 weeks after operation. The in situ hybridization results revealed that TGF-β1 and Col I mRNA expression in 1.0 μ g/mL TGF-β1 group was lower than that in NS group at each time. There was significant difference between two groups (P lt; 0.05). Conclusion TGF-β1neutral izing antibody can inhibit the function of the TGF-β1 effectively and prevent adhesion formation after the flexor tendon injured and repaired.
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 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.
PURPOSE:To investigate the approaches for transplanting retinal pigment epithelium. METHODS,Retinal pigment epithelial eells(RPR)of pigmented rabbits' eyes prepared by rotalne preparation of our institute,were transphmted in 18 unpigmemed rabbits'eyes.Eight eyes were undergone outer approach, i.e., transplanting the RPR cells to the subretinal space of recipient eyes by way of perforating sclera and choroid;while 10 eyes were undergone internal approach by way of the routine procedure of vitrectomy with making artificial localized retinal delachment. Light and transmisskm electrone microscopy examination were done at 10th, goth, 40th and 90th day after the operation. RESULTS: In internal approach group,tbe operated eyes,revealed no difference in thickness of the neural retinal layer in transplanted and non-transplanted area 40 days after operation tinder light microscope. Transmission electrone microscopy revealed postoperatively the transplanted RPE cells attached to the Brucb's membrane and the outer segments of photoreeeplive ceils located at a normal position at the 40th dayland the secondary lysozymes with engulfed outer segment were found in the Iransplamed cells at the 90th day. Tbe outer approached operations in eight eyes were failed owing to ehoroid hemorrhage or perforation of retina. CONCLUSION:The internal appraach procedure is much effebtive and practical for transplantation of RPE cells. （Chin J Ocul Fundus Dis,1997,13:160-162）
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 study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro. Methods The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensitywere observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. Thecharacteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkal ine phosphatase (ALP) staining, Al izarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell prol iferation. Results The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before lebel ing and after lebel ing. After osteogenic induction of labeled BMSCs, ALP staining and Al izarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, l ipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS. Conclusion Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal abil ity and multipotent differentiation abil ity; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
The change of Ig-forming cells in the gallbladder mucoderm were studied in the rabbit models.One hundred rabbits were randomly divided into the control group(Con,n=10),simple biliary obstruction group(BO,n=45)and biliary obstruction and infection group(BOI,n=45).The results showed that only a few Ig-forming cells presented in the gallbladder mucoderm of normal rabbit.At the 3rd,7th and 14th day,the quantities of IgG and IgA-forming cells in the mucoderm in BO group remained unchange,but increased much higher in BOI group(Plt;0.001),especially in IgG formation.This study suggests that the gallbladder of rabbit may be the important place of Ig-formation.The quantities of Ig-forming cells in bilitary tract may have a close relationship with the gallstone formation.
Objective Platelet-rich plasma (PRP) contains high concentrations of platelets and leucocytes, which play a key role in antimicrobial host defense system. To evaluate the antimicrobial efficacy of autologous PRP in vitro and in vivo and to explore the mechanism of action so as to provide the experimental basis for the prevention and treatment of bone infection. Methods PRP was prepared with the method of two centrifugation from 15 health volunteers. Platelet-leukocytegel (PLG) was obtained after activation of PRP with bovine thrombin. Next, PLG was incubated with Staphylococcus aureus (1 × 106 cfu/mL) in vitro compared with PRP, platelet-poor plasma (PPP) and PBS. Samples were taken out after 2, 4, 6, 8, 12, and 24 hours for bacterial culture and colony count. Thirty-six New Zealand adult rabbits, weighing (2.85 ± 0.11) kg, were divided into 4 groups: PLG (n=10), antibiotic (n=10), infection (n=10), and PBS (n=6) groups. The osteomyel itis models were made by injecting 0.1 mL Staphylococcus aureus suspension (1 × 106 cfu/mL) into the tibial canal in PLG group, antibiotic group, and infection group; equal volumes of PBS was injected in PBS group as a control. Autologous PLG was injected immediately after operation in PLG group. Cefazol in (30 mg/kg) was injected through the auricular vein from 1 hour before operation to 72 hours after operation in antibiotic group, once per 8 hours. No treatment was given in infection and PBS groups. The efficacy of PLG for osteomyel itis prophylaxis was evaluated by microbiological, X-ray and histological observation within 28 days. Results The contents of leucocyte and platelet of PRP were 6.2 times and 5.5 times of whole blood, showing signficant differences ((P lt; 0.05); the contents of leucocyte and platelet of PPP were significantly lower than those of whole blood and PRP ((P lt; 0.05). In vitro test showed that PLG had the most obvious bacteriostasis effect. The bacterial count reached a minimum value at 4 hours after incubation in PLG and at 6 hours after incubation in PRP. PPP had slow and no obvious bacteriostasis effect and PBS had no bacteriostasis effect. At 2, 4, 6, 8, 12, and 24 hours of incubation, the bacterial count reduced significantly when compared PLG with PRP and PPP (P lt; 0.05), when compared PRP with PPP (P lt; 0.05). In PLG group and antibiotic group, 1 rabbit died, respectively; 34 rabbits survived to the end of the experiment. There was no significant difference (P gt; 0.05) in temperature, body weight, erythrocyte sedimentation rate and content of leucocyte between 28 days after operation andbefore operation in 4 groups. After 28 days, the X-ray scores were 2.78 ± 1.39, 1.55 ± 1.48, 4.17 ± 1.25, and 0 in PLG, antibiotic,infection, and PBS groups, respectively, which was significantly higher in infection group than in other 3 groups ((P lt; 0.05). Also, the histological scores were 5.89 ± 3.92, 3.00 ± 2.31, 10.33 ± 4.03, and 0, respectively, which was significantly higher in infection group than in other 3 groups (P lt; 0.05), and was significantly lower in antibiotic group than in PLG group ((P lt; 0.05). The results of bacterial culture showed that the infection rates of PLG group (44.4%) and antibiotic group (20.0%) were significantly lower ((P lt; 0.05) than that of infection group (88.9%). The quantitative analysis of bacteria showed that the number of bacteria was signifcantly lower ((P lt; 0.05) in PLG and antibiotic groups than in infection group. Conclusion PRP forms into PLG after activating, it can inhibit Staphylococcus aureus reproduction in vitro and can effectively prevent bone infection in vivo.