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 compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
【Abstract】 Objective To investigate the in vivo osteogenic feasibil ity of tissue engineered periosteum constructedby porcine SIS and BMSCs in allogenic New Zealand rabbit. Methods The tissue engineered periosteum constructed by SIS scaffold and BMSCs was prepared in vitro .Twelve 2-month-old New Zealand rabbits were used in the experiments. The 1.5-2.0 cm critical bone defects were made in the both sides of radius of the animals. The tissue engineered periosteum was grafted into one side defect randomly, while the other side defect was only grafted SIS. Four weeks after operation, the forearms of all animals were checked by X-ray. Then, animals were sacrificed to harvest the specimen which were treated promptly for HE and Masson staining.The X-ray film and the morphological tissue staining outcome were evaluated qual itatively. Results After operation,all animals had a normal behavior and diet; the incision healed normally; the forearm could move normally for bearing weight.The tissue engineered periosteum constructed by allogenic BMSCs and heterogeneic SIS scaffold could form new bone tissue, andbridged the bone defect which could be confirmed either in X-ray film or histological staining. The newly formed bone tissue had similar bone density to normal bone. A lot of irregular newly formed vessels and medullary cavity inserted in the newly borned tissue. No lymphocytes infiltrated in histological examination. While the control side had no any osteogenesis neithter in X-ray, nor in HE and Masson staining inspecting; the defect space only occupied with some connective tissue. Conc lu sion Tissue engineered periosteum can form new bone in allogenic rabbit and has the feasibil ity to repair the segmental diaphysis defect.
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.
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 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 explore the biocompatibility of poly(lacticacid/glycolic acid/asparagic acid-co-polyethylene glycol) biomaterials (PLGA-ASP-PEG) and biological behaviors of cultured marrow stroml stem cells (MSCs) combined with this new type of scaffold in tissue engineering. Methods The PLGA-ASP-PEG tri-block copolymers were obtained through bulk ringopening copolymerization method.MSCs were isolated from the bone marrow of 4 week old New Zealand rabbits. The 3rdgeneration MSCs were cultured combining with PLGA-ASP-PEG in vitro, while cells cultured in PLGA as control group. The cell adhesion rate and the adhesivepower were examined by conventional precipitation method and micropipette aspiration technique respectively. The morphological features were studied by scanning electron microscope. The proliferation behavior of the cells was analyzed by MTT assay. The cell cycle, proliferation index, DNA index and apoptosis of the cells were detected by flow cytometry. The synthesis of protein and collagen were examined by Coomassie Brilliant Blue dyes and 3H-Proline incorporation test. Results The MSCs adhered and grew well on the surface of the biomaterial PLGA-ASP-PEG. The powers of cell adhesion, proliferation and protein and collagen synthesis of the cells were all significantly higher than those of PLGA group (P＜0.05), but the apoptosis rate was significantly lower than that of PLGA group (P＜0.05). The DNA indexes showed the cells of both PLGA-ASP-PEG group and PLGAgroup were normal diploid cells. Conclusion PLGA-ASP-PEG showedgood biocompatibilityand the biological properties improved greatly compared with the PLGA scaffold materials. These results demonstrated that the promise of PLGAASPPEG canbe used as an ideal scaffold material for construction of tissue engineered bone to restore bone defects in bone tissue engineering．
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 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.
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.
