Objective To investigate the effect of repairing bone defect with tissue engineered bone seeded with the autologous red bone marrow (ARBM) and wrapped by the pedicled fascial flap and provide experimental foundation for cl inicalappl ication. Methods Thirty-two New Zealand white rabbits (male and/or female) aged 4-5 months old and weighing2.0-2.5 kg were used to make the experimental model of bilateral 2 cm defect of the long bone and the periosteum in the radius. The tissue engineered bone was prepared by seeding the ARBM obtained from the rabbits on the osteoinductive absorbing material containing BMP. The left side of the experimental model underwent the implantation of autologous tissue engineered bone serving as the control group (group A). While the right side was designed as the experimental group (group B), one 5 cm × 3 cm fascial flap pedicled on the nameless blood vessel along with its capillary network adjacent to the bone defect was prepared using microsurgical technology, and the autologous tissue engineered bone wrapped by the fascial flap was used to fill the bone defect. At 4, 8, 12, and 16 weeks after operation, X-ray exam, absorbance (A) value test, gross morphology and histology observation, morphology quantitative analysis of bone in the reparative area, vascular image analysis on the boundary area were conducted. Results X-ray films, gross morphology observation, and histology observation: group B was superior to group A in terms of the growth of blood vessel into the implant, the quantity and the speed of the bone trabecula and the cartilage tissue formation, the development of mature bone structure, the remolding of shaft structure, the reopen of marrow cavity, and the absorbance and degradation of the implant. A value: there was significant difference between two groups 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those three time points in groups A and B (P lt; 0.05). For the ratio of neonatal trabecula area to the total reparative area, there were significant differences between two groups 4, 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those four time points in group B (P lt; 0.05).For the vascular regenerative area in per unit area of the junctional zone, group B was superior to group A 4, 8, 12, and 16 weeks after operation (P lt; 0.05). Conclusion Tissue engineered bone, seeded with the ARBM and wrapped by the pedicled fascial flap, has a sound reparative effect on bone defect due to its dual role of constructing vascularization and inducing membrane guided tissue regeneration.
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.
In this experiment, two proximal ends of themedian and ulnar nerves of rabbit wereapproxirnated within the chitin tube for thepurpose to inhibit the neuroma formation. Byobservation under light and transmission electronmicrnscopo and immunohistochemistry, wefound that: (1) the axons of the two proximalstumpe could regenerate in the chitin tube for 2to 5mm, and then ceased to grow when anaxonal overlap happened resulting in inhibitingneuroma formation; (2) chitin tube could bedegradated a...
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 investigate the effect of simvastatin on inducing endothel ial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Methods Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 × 106 cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins(2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. Results All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% ± 4.07% in experimental group and 59.45% ± 5.43% in control group, showing significant difference (P lt; 0.05). Conclusion Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs homing and enhancing vasculogenesis.
【Abstract】 Objective Prostaglandin E2 (PGE2) production increases in human tendon fibroblasts after the tendon injuries and repetitive mechanical loading in vitro. To analyze the relations between PGE2 and tendinopathy by observing the changes of collagen content and proportion after the Achilles tendon of rabbits is repeatedly exposed to PGE2. Methods Twenty-four Japanese rabbits (aged 3-4 months, weighing 2.0-2.5 kg, and male or female) were equally randomized into 2 groups according to injection dose of PGE2: low dose group (50 ng) and high dose group (500 ng). Corresponding PGE2 (0.2 mL) was injected into the middle segment of the Achilles tendon of hindlimb, the same dose saline into the same site of the other side as controls once a week for 4 weeks or 8 weeks. The Achilles tendons were harvested at 4 and 8 weeks after injection. HE staining was used to observe the cell structure and matrix, and picric acid-sirius red staining to observe the distribution and types of collagen fibers, and transmission electron microscopy was used to measure the density of the unit area and diameter of collagen fibers. Results HE staining showed that collagen structural damage was observed in low dose and high dose groups. Picric acid-sirius red staining showed that the content of type I collagen significantly decreased while the content of type III collagen significantly increased in experimental side of 2 groups at 4 and 8 weeks after injection when compared with control sides (P lt; 0.05). The content of type I collagen was significantly lower and the content of type III collagen and ratio of type III to type I were significantly higher in high dose group than in low dose group (P lt; 0.05). Transmission electron microscopy showed that the collagen fibers density of unit area was significantly lower and the diameter was significantly smaller in high dose and low dose groups than in the controls (P lt; 0.05), and in high dose group than in low dose group (P lt; 0.05). Conclusion Repeat exposure of the Achilles tendon of rabbit to PGE2 can cause the decrease of type I collagen, the increase of type III collagen, the reverse ratio of type I to type III, reduced unit density of collagen fibers, and thinner collagen fibers diameter, which is related with tendinopathy.
Objective To observe the change of sino-atrial nodal tissue structure and ectopic pacing function after xenogenic sino-atrial nodal tissue transplanted into left ventricular wall, so as to provide new ideas for the treatment of sick sinus syndrome and severe atrioventricular block. Methods Seventy healthy rabbits were selected, male or female, and weighing 1.5-2.0 kg. Of them, 42 were used as reci pient animals and randomly divided into sham operation group, warm ischemia transplantation group, and cold ischemia transplantation group (n=14), the other 28 were used as donors of warm ischemia and cold ischemia transplantation groups, which were sibl ing of the recipients. In recipients, a 6-mm-long and about 2-mm-deep incision was made in the vascular sparse area of left ventricular free wall near the apex. In sham operation group, the incision was sutrued directly by 7-0 Prolene suture; in cold ischemia transplantation group, after the aortic roots cross-clamping, 4 ℃ cold crystalloid perfusion fluid infusion to cardiac arrest, then sinoatrial node were cut 5 mm × 3 mm for transplantation; in warm ischemia transplantation group, the same size of the sinus node tissue was captured for transplantation. After 1, 2, 3, and 4 weeks, 3 rabbits of each group were harvested to make bradycardia by stimulating bilateral vagus nerve and the cardiac electrical activity was observed; the transplanted sinus node histology and ultrastructural changes were observed. Results Thirty-six recipient rabbits survived (12 rabbits each group). At 1, 2, 3, and 4 weeks after bilateral vagus nerve stimulation, the cardiac electrical activity in each group was significantly slower, and showed sinus bradycardia. Four weeks after operation the heart rates of sham operation group, warm ischemia, and cold ischemia transplantation group were (81.17 ± 5.67), (82.42 ± 7.97), and (80.83 ± 6.95) beats/ minute, respectively; showing no significant difference among groups (P gt; 0.05). And no ectopic rhythm of ventricular pacing occurred. Sino-atrial nodal tissue survived in 6 of warm ischemic transplantation group and in 8 of cold ischemia transplantation group; showing no significant difference between two groups (P gt; 0.05). Two adjacent sinoatrial node cells, vacuole-l ike structure in the cytoplasm, a few scattered muscle microfilaments, and gap junctions between adjacent cells were found in transplanted sinus node. Conclusion The allograft sinus node can survive, but can not play a role in ectopic pacing.
Objective Platelet-rich plasma (PRP) secretes many growth factors, including transforming growth factor β1 (TGF-β1), platelet derived growth factor, vascular endothl ial growth factor, insul in-l ike growth factor 1, and so on, which can promote cell prol iferation, chemotaxis, and collagen synthesis in wound heal ing. To investigate the effects of PRPon the tendon heal ing, and to explore the mechanism of action so as to provide the experimental basis for the tissue engineered tendons. Methods Forty healthy New Zealand white rabbits, weighing 2.5-3.0 kg and male or female, were randomly divided into the experimental group (n=20) and the control group (n=20). PRP was prepared from arterial blood of rabbit’s ears through twice centrifugation method of Landesberg. The platelet concentrations of whole blood and PRP were determined. The right achilles tendons of the rabbits were transected to make rupture models. In experimental group, the tendon was sutured after PRP (0.5 mL) was immediately appl ied at repair site. In control group, the tendon was sutured directly after transection. At 1, 2, 4, and 6 weeks after operation, the tendons of 5 rabbits in each group were harvested for morphological, histological, and immunohistochemical observations; the fibroblast counting, the content of collagen fibers, and the expression of TGF-β1 were detected. Results The concentration of platelet of PRP was 4.03 times of whole blood. All the animals survived till the end of the experiment, and the incision healed well. No death, infection, and other compl ications occurred. With time, the tendons almost healed in 2 groups, and the fibrous tissue at anastomosis site was more remarkable in control group than in experimental group. The histological observation showed significant differences in fibroblast counting at 1, 2, and 4 weeks after operation between 2 groups (P lt; 0.05), while no significant difference at 6 weeks (P gt; 0.05). The contents of collagen fibers in the parenchyma at repair site in experimental group were significantly higher than those in control group at each time point (P lt; 0.05). Immunohistochemistry staining showed the expression of TGF-β1 in experimental group was upregulated at 1 week and 2 weeks and reached the peak at the 2nd week, and subsequently downregulated at 4 and 6 weeks in comparison with the control group, showing signficant differences between 2 groups at each time point (P lt; 0.05). Conclusion PRP can facil itate rabbit’ s tendons heal ing and significantly improve the heal ing qual ity, which may be associated with its advancing the peak time of the TGF-β1 expression in tendon.
ObjectiveTo detect the expression of human transforming growth factor β1 (hTGF-β1) gene mediated by adenovirus (Ad) in hamstring tendon after anterior cruciate ligament (ACL) reconstruction in rabbits. MethodsAd-hTGF-β1 and Ad-green fluorescent protein (GFP) were diluted to 5×108 PFU/mL with DMEM. Forty-eight New Zealand white rabbits were divided into 3 groups randomly (n=16) for ACL reconstruction with hamstring tendon autograft. Hamstring tendon was cultured and transfected with Ad-hTGF-β1 (group A) and Ad-GFP (group B) for 12 hours before ACL reconstruction, and was cultured with DMEM in group C. After 12 hours of transfection, green fluorescence was observed in groups A and B under fluorescence microscopy. At 2, 4, 6, and 8 weeks after operation, the hamstring tendon was harvested to detect the mRNA and protein expressions of hTGF-β1 by real time fluorescence quantitative PCR and Western blot. ResultsGreen fluorescence was observed after 12 hours of transfection in groups A and B. TGF-β1 protein level reached (221.0±12.2) ng/mL at 12 hours in group A. The hTGF-β1 mRNA expression could be detected in group A, but it could not be detected in group B and group C. The mRNA expression levels of hTGF-β1 were 1.004±0.072 at 2 weeks, 0.785±0.038 at 4 weeks, 0.469±0.053 at 6 weeks, and 0.172±0.021 at 8 weeks in group A, showing significant difference (P<0.05). Western blot results showed weakly positive band in groups B and C; the protein expression of TGF-β1 in group A was significantly higher than that in groups B and C (P<0.05), but no significant difference was found between groups B and C P>0.05). The protein expression of TGF-β1 gradually reduced with time, showing significant difference between different time points (P<0.05). ConclusionAd-hTGF-β1 can transfect the hamstring tendon successfully, and it can effectively express for a long time after ACL reconstruction.
Fluorescein angiography(FA)was performed in 31 pigmented rebbits.The angiograms were evaluated as prints and as negative film under a light microscope.The patterns of retinal pigment epithelial(RPE)cells were studied by scaning electron microscopy and fluorescein light one,compared with other rabbits belonging to the same species.In 58 eyes,we observed the hexagonal pattern of RPE cell.It showed central hypofluorescent area surrounded by hyperfluorescent rim,which was easily seen away from the medullary rays by three or more disc diameters and became larger in the periphery than that in the posterior pole.There were no finding in four lightly pigmented eyes. （Chin J Ocul Fundus Dis,1994,10:226-228）