The damage effects of the pure tumor necrosis factor (TNF) on the normal animals were observed. Eighteeen rabbits were divided into two groups, eight in tested group and ten in control group. 0.5mg per kg of the pure rabbit TNF was given to each animal of the tested group. Results:The symptoms similar to that induced by endotoxin appeared after the TNF injection. The functions of the main organs were markedly damaged. The arterial blood pressure of most animal was low. The weight ratio of the orgen to the body was raised. The pathologic changes were similar to those of the multiple organ failure (MOF) model. Most of the animal died before the end of the experiment. The results suggest that pure TNF could indece multiple organ damages similar to those of MOF.
Objective To provide a reliable experimental model for gastroesophageal reflux (GER) study. Methods Twenty Japan 5-month-old male rabbits wererandomly divided into two groups: group cardiomyotomy(n=10), group partial cardiomyectomy(n=10). The operations of cardiomyotomy and parital cardiomyectomy were performed in 2 groups respectively. All the animals underwent intraesophagealpH detection 1 week before operation and 4 weeks after operation. The mean changes of reflux ratios were compared between before operation and after operation.Results In gastroesophageal reflux ratio between before operation and after operation, there was no significant difference in group cardiomyotomy (1.98%±1.52% and 4.32%±2.39%, Pgt;0.05) and there was significant difference in group partialcardiomyectomy（1.56%±1.57% and 13.56%±3.27%, Plt;0.05）. Conclusion The reliable experimental model of GER can be made with procedure of partial cardiomyectomy. It can be used in estimating the operative procedure of antireflux and is conducive to dynamic observation and study of esophagitis.
Objective To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 μmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties. Results Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05). Conclusion After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.
Schwann cells (SC) play an important role in nerve regeneration. The cultures of both human and rabbit SC (gt;99%) were obtained, and were separately derived from the sciatic nerve of the human fetus and the rabbit respectively by "the method of reexplantation". In addition, the cryostore and resuscitation of SC were carried out, and the resuscitated cells could retain their growth properties.
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.
Objective To give a prel iminary experimental evidence and to prove chitosan and allogeneic morsel ized bone as potential bone substitutions in repairing rabbit radius segmental defect. Methods Chitosan and allogeneic morsel ized bone were mixed with various ratios (1 ∶ 5, 1 ∶ 10, 1 ∶ 25, 1 ∶ 50, and 1 ∶ 100). After preparation, the physicaland chemical properties of the composites were prel iminary detected; the composites at the ratios of 1 ∶ 50 and 1 ∶ 25 had good physical and chemical properties and were used for the animal experiment. The radius segmental defects of 15 mm in length were made in 50 adult New Zealand white rabbits (weighing 2.5-3.0 kg), then the animals were divided into 2 groups. In groups A and B, chitosan/allogeneic morsel ized bone composites were implanted at the ratio of 1 ∶ 50 and 1 ∶ 25, respectively. After 1, 2, 4, 8, and 12 weeks of operation, the gross, histological, immunohistochemical observations were performed. Before the rabbits were sacrified, X-ray films were taken; the serum calcium and alkal ine phosphatase (ALP) concentration were measured; and the biomechanical measurement was carried out at 12 weeks. Results The results of gross observation were essentially consistent with those of the X-ray films. The histological observation showed that the bone formation was earl ier in group A than in group B; the amount of new bone formation in group A was more than that in group B; and the bone forming area in group A was bigger than that in group B (P lt; 0.05) at 4 and 8 weeks after operation. The immunohistochemical staining showed that vascular endothel ial growth factor and insul in-l ike growth factor receptor II proteins expressed in the cytoplasm of 2 groups after 4 and 8 weeks, and the expression in group A was higher than that in group B (P lt; 0.05). There was no significant difference in the serum calcium concentration between 2 groups at each time point (P gt; 0.05). After 4 and 8 weeks, the ALP concentration in group A was significantly higher than that in group B (P lt; 0.05). After 12 weeks, the radius maximum bending loads of groups A and B were (299.75 ± 27.69) N and (278.54 ± 17.09) N, respectively, showing significant difference (t=4.045,P=0.002). Conclusion The composite of chitosan and allogeneic morsel ized bone has good osteogeneic activity and can beused as a bone tissue engineering scaffold, and the optimum ratio of chitosan to allogeneic morsel ized bone was 1 ∶ 50.
Objective To compare the myogenic differentiation abil ity in vitro of rabbit adipose-derived stem cells (ADCSs) from different sites so as to provide ideal seed cells for repair and reconstruction of urinary tract. Methods Adipose tissues were obtained from the nape of the neck, post peritoneum, and vicinity of epididymis of a 4-month-old male New Zealand rabbit and ADSCs were harvested through collagenase digestion. ADSCs were purified by differential attachment method. The protein marker CD44 of rabbit ADSCs was used to identify the stem cells by immunocytochemistry, then the5th generation of ADSCs were induced to differentiate into adipogenic, osteogenic, and myogenic cells. Multi- differentiation was confirmed by Oil red O staining, von Kossa staining, and RT-PCR. Myogenic differentiation abil ities of ADSCs from 3 different sites were compared between the control group (L-DMEM medium containing 10%FBS) and the experimental group (myogenic medium) by RT-PCR method. Results ADSCs could be easily isolated from adipose tissues of the nape of the neck, post peritoneum, and vicinity of epididymis. ADSCs displayed a typical cobblestone morphology. Brown particles could be seen in ADSCs by CD44 immunocytochemistry staining. Oil red O staining showed red fat drops in ADSCs after 14 days of adipogenic culture. Black matrix could be seen in ADSCs by von Kossa staining after 28 days of osteogenic culture. RT-PCR detection showed moderate α-actin expression in the control group and b α-actin expression in the experimental group after 42 days of myogenic culture. The growth rate of α-actin from the adipose tissue of post peritoneum (28.622% ± 4.879%) was significantly lower (P lt; 0.05) than those from the adipose tissues of the nape of the neck (35.471% ± 3.434%) and vicinity of epididymis (38.446% ± 4.852%). Conclusion The ADSCs from different sites show different myogenic differentiation abil ities in vitro. ADSCs from the adipose tissues of the nape of the neck and vicinity of epididymis can be used as ideal seed cells for tissue engineering of lower urinary tract.
Objective To evaluate repair of critical-sized cranialdefect with tissue engineered bone fabricated by coral, bone mesenchymal stem cells(MSCs) and sustainedly released recombinant human bone morphogenetic -protein 2 (rhBMP-2) by collagen. Methods Three scaffolds of rhBMP-2+coral,collagen+rhBMP-2+coral and MSCs+collagen+rhBMP-2+coral were fabricated. Forty New Zealand rabbits were made the models of critical-sized defects and divided into5 groups according to different implants: group Ⅰ, auto-ilium; group Ⅱ,coral; group Ⅲ, rhBMP-2+coral; grop Ⅳ, collagen+rhBMP-2+coral; and group Ⅴ,MSCs+collagen+rhBMP-2+coral. Repair of bone defect was evaluated after 8 and 16 weeks of implantation by gross obeservation, X-ray,HE staining and Masson’s trichrome staining. Results Repair ofbone defect in group Ⅴ was similar to that in group Ⅰ, andwas better than that in group Ⅳ; and group Ⅲ was worse. The gross appearance showed that defect region filled with bony tissue which had similar strength to adjacent bone and formed bone union with surrounding bone. The X-ray result displayed high radiopacity(80.45%±2.52% in the 16thweek). Histological observation showed new lamellar bone tissue and with few pore blank area. However, only transpasent fibrous tissue filled the defect in group Ⅱ. Conclusion Collagen may be a suitable sustained release system for rhBMP-2. And MSCs may have important effect on enhancing repair of bone defect. Tissueengineered bone fabricated by MSCs+collagen+rhBMP-2+coral may be a useful material for bone defect repair.
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.