ObjectiveTo investigate the feasibility of establishing intervertebral disc degeneration (IDD) model by using minimally invasive acupuncture and rotary-cutting. MethodsForty New Zealand white rabbits [male or female, (2.9±0.3) kg in weight] were randomly divided into control group (n=20) and experimental group (n=20). No treatment was done in the control group; percutaneous puncture was performed on L4, 5 and L5, 6 intervertebral disc by using 18G needle under C-arm X-ray monitoring for rotary-cutting of nucleus pulposus to promote degeneration of the disc in the experimental group. At 4, 8, 12, and 16 weeks after operation, general observation and MRI observation were done, and intervertebral disc degeneration was accessed based on Pfirrmann grade; the specimens were harvested for Masson staining and Safranine O staining. ResultsThe nucleus pulposus showed dark colors and reduced elasticity in the experimental group when compared with the control group. T2-weighted MRI images indicated that the disc signal intensity of control group had no obvious change at early stage, and weakened slightly at late stage; disc signal intensity of the experimental group decreased with time. According to Pfirrmann grade for disc degeneration, disc degeneration degree was significantly aggravated with time in 2 groups (P < 0.05); degeneration was significantly more severe in the experimental group than the control group at the other time points (P < 0.05) except 4 weeks (P > 0.05). Masson staining results showed that irregular arrangement of annulus with integrate structure was observed in the control group with time; the annulus of the experimental group arranged in disorder, or even disc fibrous circle rupture appeared with time. Safranin O staining showed that the nucleus pulposus cells reduced significantly in the experimental group, but did not in the control group. ConclusionMinimally invasive acupuncture and rotary-cutting could successfully establish the IDD model in rabbits.
ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.
In this paper,the changes of activities of enzymes relating toenergy metabolism in rabbit's retina during acute ocular hypertension were observed.The activities of succinate dehydrogenase and adenosine triphosphatase were foud to be reduced,while the activities of the lactatic dehydrognease and glucose-6-phosphatase increased.The results reveal the disturbance of metabolism of energy in retina undergone acute ocular hypertension,and suggest that this might be the underlying factors relating to the defects of the functions and structures of the retina. （Chin J Ocul Fundus Dis,1993,9:141-144）
ObjectiveTo study the effect of transforming growth factor β3 (TGF-β3), bone morphogenetic protein 2 (BMP-2), and dexamethasone (DEX) on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells (SMSCs). MethodsSMSCs were isolated from the knee joints of 5 rabbits (weighing, 1.8-2.5 kg), and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups: TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3+BMP-2 in group C, TGF-β3+DEX in group E, BMP-2+DEX in group F, and TGF-β3+BMP-2+DEX in group G; group H served as control group. The diameter, weight, collagen type II (immuohistochemistry staining), proteoglycan (toluidine blue staining), and expression of cartilage related genes [real time quantitative PCR (RT-qPCR) technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation. ResultsSMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G (P<0.05). RT-qPCR detection results showed that the relative expressions of cartilage related genes (SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II) in group G were significantly higher than those in the other groups (P<0.01). Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 21 days. ConclusionThe combination of TGF-β3, BMP-2, and DEX can make the capacity of chondrogenesis of SMSCs maximized. The increase of the pellet volume is caused by the extracellular matrix rather than by cell proliferation.
The contents of lipid peroxides(LPO)and vitamin E(V.E)and some functional index and histologic changes in the lungs from the the rabbit models of acute cholangitis of severe type(ACST)were measured dynamically.The results revealed that the V.E content decreased strikingly from 6 hours and the LPO level increased progressivelg from 12 hours in the lungs.Simultanuosly,the congestion and neutrophil infiltreation in the lung mesenchyme,and the endothelial cell damage and thrombosis in the lung blood capillaries had been observed.These suggest that acute lung injury induced by ACST is referable to the lipid peroxidation damage to the lung blood capillaries which is due to increased LPO and decreased antioxidants including V.E.
Objective To investigate the curative effects of homograft of the mesenchymal stem cells(MSCs) compbined with the medical collagen membrane of the guided tissue regeneration(MCMG) on the full thickness defects of the articular cartilage. Methods MSCs derived from New Zealand rabbits aged 3-4 months weighing 2.1-3.4 kg were cultured in vitro with a density of 5.5×108/ml and seeded onto MCMG. The MSC/MCMG complex was cultured for 48 h and transplanted into the fullthickness defects on the inboardcondyle and trochlea. Twenty-seven healthy New Zealand rabbits were randomly divided into 3 groups of 9rabbits in each. The cartilage defects in the inboard condyle and trochlea werefilled with the auto bone marrow MSCs and MCMG complex (MSCs/ MCMG) in Group A (Management A), with only MCMG in Group B (Management B)and with nothing in Group C (Management C). Three rabbits were killed at 4, 8 and 12 weeks after operation in each group, and the reparative tissue samples evaluated grossly,histologically and immunohistochemically were graded according tothe gross and histological scale. Results Four weeks after transplantation, the cartilage and subchondralbone were regenerated in Group A;for 12 weeks, the regenerated cartilage gradually thicked; 12 week after transplantation, the defect was repaired and the structures of the carticular surface and subchondral bone was in integrity.The defects in Group A were repaired by the hylinelike tissue and the defects in Groups B and C were repaired by the fibrous tissues. Glycosaminoglycan and type Ⅱcollagen in Groups A,B and C were reduced gradually.The statistical analysis on the gross at 12 weeks and the histologicalgradings at 4 weeks,8 weeks and 12 weeks showed that the inboardcondylar repairhad no significant difference compared with the rochlearepair(Pgt;0.05).Management A was significantly better than Managements B and C (Plt;0.05), and Management B was better than Management C(Plt;0.05). Conclusion Transplantation of the MSCs combined with MCMG on the full thickness defects of the articular cartilage is a promising approach to the the treatment of cartilage defects. MCMG can satisfy the demands of the scaffold for the tissue-engineered cartilage.
Objective To explore the possibilityof constructing tissue engineering muscles by combining allogeneic myoblasts with small instestinal submucosa(SIS) in rabbits.Methods A large number of purified myoblasts were obtained with multiprocedure digestion and repeated attachment method from skeletal muscles taken from extremities of immature rabbits which were born 7 days ago. The myoblasts were labeled with BrdU, and then combined with SIS to construct tissue engineering muscles. This kind of tissue engineering muscles were grafted into the gastrocnemius muscle defect (1.5 cm in length, 1.0 cmin width) of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group. The rabbits were sacrificed 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic、histological and immunohistochemical observations, and by quantitative analysis of local immunocyte in the grafting site. Results Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious 4 weeks after grafting.The SIS began to break down and inflammation response became slight 6 and 8 weeks after operation. Compared with that of 8th week, the quantitative analysis oflocal immunocyte in 4th and 6th week in both experimental and control group hassignificance(Plt;0.05). Newly formed muscle tissues were found around SIS in the experimental group in 4th, 6th, and 8th week. Expression of BrdU and myosin immunohistochemical staining were positive in the experimental group and negative inthe control group.Conclusion Tissue engineering muscles of rabbits which are constructed by combining allogeneic myoblasts with SIS can survive and proliferate.
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.
To investigate the protective effect of propofol on ischemia/reperfusion induced spinal cord injury in rabbits and its influence on excitatory amino acid (EAA). Methods Sixty New Zealand white rabbits weighing 2.0-2.5 kg, half males and half females, were selected. The infrarenal circumaortic clamping model was used. And 6 mL/kg different fluids were continuously infused through a catheter into the aorta distal to the clamping site at a speed of 12 mL/(kg•h) during the 30 minutes ischemia period. According to the different infusing l iquids, the rabbits were randomized into 6 groups(n=10 per group): group A, normal sal ine; group B, 10% intral ipid; group C, propofol 30 mg/kg; group D, propofol 40 mg/kg; group E, propofol 50 mg/kg; group F, propofol 60 mg/kg. At 0, 6, 24, and 48 hours after reperfusion, neurologic outcomes were scored on a Tarlov scale system. At 48 hours after reperfusion, the number of normal neurons in the anterior spinal cord was counted, and concentration of EAA in the lumbar spinal cord was measured by high performance l iquid chromatography. Results The neuroethological score was better in groups C, D, E and F than that of groups A and B (P lt; 0.05), the score of group E was the highest (P lt; 0.05), and there was no significant difference between group A and group B (P gt; 0.05). The number of normal neurons in the anterior spinal cord of groups C, D, E and F was greater than that of groups A and B (P lt; 0.05), and group E was greater than groups C, D and F (P lt; 0.05). The concentration of EAA in groups A, B, C, D, E and F was greater than that of normal tissue, the group E was the lowest (P lt; 0.05), the groups A and B were the highest (P lt; 0.05), and there was no significant difference between group A and group B (P gt; 0.05). Concentrations of glutamate and aspartic acid were negatively correlated to normal neuron numbers in the anterior spinal cord and neuroethological scores 48 hours after reperfusion, and the corresponding correlation coefficient was — 0.613, — 0.536, — 0.874 and — 0.813, respectively (P lt; 0.01). Conclusion Propofol can significantly inhibit the accumulation of EAA in spinal cord and provide a protective effect against the ischemia/reperfusion injury induced spinal cord in rabbits.
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）