We compared the sensitivities of human embryo conjtmctival fihroblasts(HECF)and rabbit conjunctival fibroblasts(RCF)m five anlineoplasties in vitro.When the concentration of vincrismum and doxorubicin was 0.001~10mg/L.5-FU was 1~1000mg/L and cisplatin was 0.01~10mg/L,the sensitivities of HECF to tile drugs were lower than that of RCF (Plt;0.01).while the difference of the sensitivilics be;ween HECF and RCF to VP-16 was not significant (P＜0.05). The results suggested that the selection of therapeutic agents for intraocular proliferative disease wilh'hunmn conjunctiva fihroblasts may be more valuable than that with RCF. （Chin J Ocul Fundus Dis,1994,10:223-225）
ObjectiveTo explore the effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits.MethodsFifty mature New Zealand rabbits (weighing, 2.5-3.0 kg) were randomly divided into control group (n=10), model group (n=20), and experimental group (n=20). The rabbits of model and experimental groups were injected with lipopolysaccharide and methylprednisolone to establish the animal model of early steroid-induced osteonecrosis of the femoral head. The rabbits of experimental group were feeded with icariin solution once a day for 6 weeks since the first injection of methylprednisolone, whereas the rabbits of control and model groups were given normal saline at the same time points. The left femoral heads were removed after 6 weeks and gross morphological features were evaluated. Micro-CT scan was performed to analyze the trabecular microstructure with the following parameters: trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp). The Micro-CT scan was also converted to three-dimensional reconstruction images for observation. HE staining was applied to observe the trabecular structure and morphological changes of osteocytes and marrow adipocytes. It was also used to determine whether the samples of femoral heads occurred osteonecrosis based on the criteria for pathological diagnosis, and calculate the rate of empty lacunae.ResultsSeven rabbits died during the study, and 9, 16, and 18 rabbits in the control, model, and experimental groups, respectively, enrolled the final analysis. Compared with control group, the femoral head collapse and trabecular breaks were more obvious, and the trabeculae were sparse with irregular arrangement in the model group according to the results of gross observation, Micro-CT scan, and three-dimensional reconstruction images. But in the experimental group, the surface of femoral head was slight shrinking without obvious collapse, and the degeneration of trabecular structure was mild. According to bone microstructures analysis, the Tb.N, Tb.Tn, and BV/TV of femoral head in model and experimental groups were lower than those in control group, while the Tb.Sp in the model and experimental groups were significantly higher. The Tb.N, Tb.Tn, and BV/TV of femoral head in experimental group were higher than those in model group, while the Tb.Sp in the experimental group was significantly lower. The differences between groups were all significant (P<0.05). In the model group, HE staining showed that the number of osteocytes reduced, the number of empty lacunae increased, and the marrow adipocytes piled up in the space between femoral trabeculae, some even mashed together like a cyst. In the experimental group, the trabecular structure was still relatively complete compared with model group, no obvious apoptosis of osteocytes was observed, the size and number of adipocytes were basically normal. None of the animals in control group occurred osteonecrosis of the femoral head based on the criteria for pathological diagnosis, and the incidence of osteonecrosis were 81.3% (13/16) in the model group and 66.7% (12/18) in the experimental group, and the difference was not significant (P=0.448). The rate of empty lacunae of osteonecrotic femoral heads in the model group was 33.1%±1.4%, which was higher than that in experimental group (18.9%±0.8%) and in control group (12.7%±1.5%), and the differences between groups were significant (P<0.05).ConclusionThe icariin has a protective effect on the early steroid-induced osteonecrosis of the femoral head in rabbits, which can decrease osteocytes apoptosis, improve the bone microstructure, and delay such disease processes.
An experimental model of proliferative vitretinopathy(PVR) induced by macrophages was used for the evaluation of drug efficacy of daunomycin encapsulated in liposomes in the treatment of PVR.Five mu;g daunomycin(n=40),10mu;g daunomycin-liposome(DL,n=30)and 0.1 ml saline or empty liposomes(n=40,as controls)were injected into the rabbit vitreous after macrophage injection.Retinal detachment developed in 77.5% of the control eyes on day 28,compared to 33.3% of the eyes treated with DL(P＜0.01)and 50% of the daunomycin-treated eyes(P＜0.05).The results suggest that encapsulation in liposomes of cytotoxic agents can enhance drug efficacy.The phasic course of development of PVR is important in the selection of particular drugs. （Chin J Ocul Fundus Dis,1993,9:77-80）
ObjectiveTo investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO4).MethodsBone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A1, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B1, n=5, decellularized by improved NaClO4 immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A2, n=5) and experimental group (group B2, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation.ResultsMTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B1 was well, showing no significant difference when compared with group A1 and negative control group with pure culture medium (P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A1 and B1) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A1, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B1, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A1, and the growth trend was better. In vivo experimental results showed that the rejection of group B2 was lower than that of group A2. The contens of IgM and IgG in group A2 were significantly higher than those in group B2 at each time point after operation (P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B2.ConclusionThe decellularized matrix treated by NaClO4 has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.
Objective To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Methods Thirty 3-month-old New Zealand rabbits (2.5-3.0 kg in weight) were randomly divided into 5 groups (n=6) and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected (0.3 mL/kg) and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm2, and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively (in 1, 2, 3, 4, and 5 minutes groups respectively). The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. Results The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance (A) value showed significant differences when compared 1 minute and 2 minutes groups with other groups (P<0.05), but no significant difference was found between 3, 4, and 5 minutes groups (P>0.05). Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. Conclusion EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm2, and duty ratio of 20% for 2 minutes in bone defects of rabbits.
ObjectiveTo explore the osteogenesis effect of advanced-platelet-rich fibrin (A-PRF) and β-tricalcium phosphate (β-TCP) composite. MethodsThirty-two healthy female New Zealand rabbits were randomly selected. A-PRF was prepared by collecting blood from middle auricular artery. Rabbits were randomly divided into 6 groups: groups A, B, C, D, and E (6 rabbits in each group) and group F (2 rabbits). Bone defects (6 mm in diameter, 8 mm in depth) were drilled into femur condyle of each rabbit’s both back legs. Then A-PRF and β-TCP composites manufactured by different proportion were planted into bone defects of group A (1∶1), group B (2∶1), group C (4∶1), group D (1∶2) and group E (1∶4) (V/V). The bone defect was not repaired in group F. The specimens were collected at 8 and at 12 weeks after operation. Then gross observation, X-ray examination, Micro-CT examination, and biomechanical test were performed. The bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp), compressive strength, and modulus of elasticity were calculated. ResultsThe gross observation and X-ray examination showed that the osteogenesis effect at 12 weeks was better than that at 8 weeks. At the same time point, the repair of bone defect and the formation of new bone in group B were better than those in other groups. Micro-CT examination showed that the trabeculae of new bone in group B were the most and the trabeculae arranged closely at 8 and 12 weeks. Besides there were significant differences in BV/TV, Tb.N, and Tb.Sp between group B and the other groups (P<0.05). There were significant differences in Tb.N and Tb.Th in group B, BV/TV and Tb.Sp in group C, Tb.Sp in group D between 8 weeks and 12 weeks (P<0.05). Biomechanical tests showed that the compression strength and elastic modulus of group B were the highest, and the compression strength and elastic modulus of group C were the lowest at 8 and at 12 weeks, showing significant differences (P<0.05). There were significant differences in compression strength and elastic modulus of each group between 8 weeks and 12 weeks (P<0.05). ConclusionThe A-PRF and β-TCP composite can repair femoral condylar defects in rabbits, and the osteogenesis is better in proportion of 2∶1.
ObjectiveTo evaluate the bone repair efficacy of the new nano-hydroxyapatite (n-HA)/polyurethane (PU) composite scaffold in the treatment of chronic osteomyelitis in tibia.MethodsA novel levofloxacin@mesoporous silica microspheres (Lev@MSNs)/n-HA/PU was successfully synthesized. Its surface structure was observed by scanning electron microscopy (SEM). Fifty adult female New Zealand rabbits were randomly selected, and osteomyelitis was induced in the right tibia of the rabbit by injecting bacterial suspension (Staphylococcus aureus; 3×107 CFU/mL), which of the method was described by Norden. A total of 45 animals with the evidence of osteomyelitis were randomly divided into 4 groups, and the right medullary cavity of each animal was exposed. Animals in the blank control group (group A, n=9) were treated with exhaustive debridement only. The remaining animals were first treated by exhaustive debridement, and received implantations of 5 mg Lev@PMMA (group B, n=12), 1 mg Lev@MSNs/n-HA/PU (group C, n=12), and 5 mg Lev@MSNs/n-HA/PU (group D, n=12), respectively. At 12 weeks postoperatively, the right tibia of rabbits were observed by X-ray film, and then gross observation, methylene blue/acid fuchsin staining, and SEM observation of implant-bone interface, as well as biomechanical test (measuring the maximal compression force) were performed.ResultsX-ray films showed that the infection were severer than those of preoperation in group A, while the control of inflammation and bone healing of rabbits in group D were obviously better than those at preoperation. The gross observation showed extensive bone destruction in group A, a significant gap between bone tissue and the material in groups B and C, and close combination between bone tissue and the material in group D. The histology of the resected specimens showed that there was no obvious new bone formation around the materials in groups B and C, and there was abundant new bone formation around the periphery and along the voids of the materials and active bone remodeling in group D. The SEM observation of the bone-implant interface demonstrated that no new bone formation was observed at the bone-implant interface in groups B and C. However, bony connections and blurred boundaries were observed between the material and host bone tissue in group D. The biomechanical test showed the maximal compression force of groups B and D were significantly higher than that of groups A and C (P<0.05), but there was no significant difference between groups B and D (P>0.05).ConclusionThe novel synthetic composite Lev@MSNs/n-HA/PU exhibit good antibacterial activities, osteoconductivity, and biomechanical properties, and show great potential in the treatment of chronic osteomyelitis of rabbits.
ObjectiveTo investigate the bone repair and regeneration abilities of biomimetic mineralized collagen bone graft material and autologous bone marrow in rabbit posterolateral spinal fusion model.MethodsTwenty-seven 20-week-old male New Zealand white rabbits were used to establish the posterolateral spinal fusion model of L5 and L6 segments by stripping the transverse process and exposing cancellous bone with electric burr. The rabbits were randomly divided into 3 groups, 9 in each group. Groups A, B, and C were implanted 1.5 mL autologous iliac bone, 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material, and 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material and autologous bone marrow in each bone defect. At 4, 8, and 12 weeks after operation, the apparent hardness of the bone grafting area was observed by manipulation method, in order to evaluate bone graft fusion effects. Three animals were sacrificed in each group at each time point, the vertebral body specimens were excised and the bone defect repair and fusion were observed by X-ray films, and three-dimensional CT examination was performed to evaluate whether new bone was formed in the body. HE staining was performed at each time point to observe the formation of new bone and the repair and fusion of bone defects.ResultsThe manipulation test showed that bone graft fusion was not found in all groups at 4 weeks after operation; 3 (50.0%), 2 (33.3%), and 4 (66.7%) of groups A, B, and C reached bone graft fusion at 8 weeks after operation; 5 (83.3%), 4 (66.7%), and 5 (83.3%) of groups A, B, and C reached bone graft fusion at 12 weeks after operation; the fusion rate of group C was similar to that of group A, and all higher than that of group B. X-ray film observation showed that the fusion rate of group C at 8 and 12 weeks after operation was higher than that of group B, which was similar to group A. Three-dimensional CT observation showed that the degree of bone fusion in group C was better than that in group B, which was close to group A. HE staining observation showed that large area of mature lamellar bone coverage appeared in the bone graft area of groups A, B, and C at 12 weeks after operation, the material was completely degraded, and the marginal boundary of the host bone disappeared and tightly combined.ConclusionBiomimetic mineralized collagen bone graft material mixed with autologous bone marrow has good osteoinduction and osteogenesis guidance. Compared with biomimetic mineralized collagen bone graft material, it has better and faster osteogenesis effect, which is close to autologous bone transplantation.
ObjectiveTo observe and compare the effects of peptides on the repair of rabbit skull defects through two different binding modes of non-covalent and covalent, and the combination of carboxyl (-COOH) and amino (-NH2) groups with materials.MethodsTwenty-one 3-month-old male ordinary New Zealand white rabbits were numbered 1 to 42 on the left and right parietal bones. They were divided into 5 groups using a random number table, the control group (group A, 6 sides) and the material group 1, 2, 3, 4 (respectively group B, C, D, E, 9 sides in each group). All animals were prepared with 12-mm-diameter skull defect models, and bone morphogenetic protein 2 (BMP-2) non-covalently bound multiwalled carbon nanotubes (MWCNT)-COOH+poly (L-lactide) (PLLA), BMP-2 non-covalently bound MWCNT-NH2+PLLA, BMP-2 covalently bound MWCNT-COOH+PLLA, and BMP-2 covalently bound MWCNT-NH2+PLLA were implanted into the defects of groups B, C, D, and E, respectively. At 4, 8, and 12 weeks after operation, the samples were taken for CT scanning and three-dimensional reconstruction, the ratio of bone tissue regeneration volume to total volume and bone mineral density were measured, and the histological observation of HE staining and Masson trichrome staining were performed to quantitatively analyze the volume ratio of new bone tissue.ResultsCT scanning and three-dimensional reconstruction showed that with the extension of time, the defects in groups A-E were filled gradually, and the defect in group E was completely filled at 12 weeks after operation. HE staining and Masson trichrome staining showed that the volume of new bone tissue in each group gradually increased with time, and regenerated mature bone tissue appeared in groups D and E at 12 weeks after operation. Quantitative analysis showed that at 4, 8, and 12 weeks after operation, the ratio of bone tissue regeneration volume to total volume, bone mineral density, and the volume ratio of new bone tissue increased gradually over time; and at each time point, the above indexes increased gradually from group A to group E, and the differences between groups were significant (P<0.05).ConclusionThrough covalent binding and using -NH2 to bound peptides with materials, the best bone repair effect can be achieved.
Objective To investigate the effect of autologous osteochondral tissue and periosteum transplantation on tendon-bone healing of rotator cuff in rabbits. Methods Twenty-four male New Zealand white rabbits were randomly divided into autologous osteochondral tissue and periosteum transplantation group (experimental group, n=12) and simple suture group (control group, n=12). Both groups were subjected to acute supraspinatus tendon injury and repaired with corresponding techniques. At 4, 8, and 12 weeks after operation, 4 specimens from each group were taken from the right shoulder joint for histological examination (HE staining, Masson staining, and Safranin O-fast green staining), and the left shoulder was subjected to biomechanical tests (maximum tensile load and stiffness). Results Both groups of animals survived until the completion of the experiment after operation. At 4 weeks after operation, both groups showed less collagen fibers and disorder at the tendon-bone junction. At 8 weeks, both groups showed reduced inflammation at the tendon-bone junction, with more organized and denser collagen fibers and chondrocytes. The experimental group showed better results than the control group. At 12 weeks, the experimental group showed typical tendon-bone transition structure, with increased generation of collagen fibers and chondrocytes, and the larger cartilage staining area. Both groups showed an increase in maximum tensile load and stiffness over time (P<0.05). The stiffness at 4 weeks and the maximum tensile load at 4, 8, and 12 weeks in the experimental group were superior to control group, and the differences were significant (P<0.05). There was no significant difference in stiffness at 8, 12 weeks between the two groups (P>0.05). Conclusion Autologous osteochondral tissue and periosteum transplantation can effectively promote the fiber and cartilage regeneration at the tendon-bone junction of rotator cuff and improve the biomechanical effect of shoulder joint in rabbits.