PRIMARY STUDY OF REPAIRING PERIPHERAL NERVE GAP WITH PORCINE SMALL INTESTINAL SUBMUCOSA

Objective To investigate the role of porcine small intestinal submucosa （SIS） conduit in axonal regeneration of rat sciatic nerve with a 10 mm gap. Methods Forty-eight rats were randomly divided into three groups （n=16）. Following a 10 mm gap was made in one side of the sciatic nerve of each rat; previously prepared SIS and auto-nerve graft were interposed into the gap to reconnect the proximal and distal ends of the nerve, respectively. In the control group, the nerve gap remained unconnected. The samples of the SIS, graft, and distal nerve in group 1 and group 2 were harvested at 6 weeks and 10 weeks after operation, respectively. Axonal regeneration was evaluated by histology, electrophysiology, and quantitated by using computer-analyzed image.Results Regenerative nerve fibers were evident which contained much myelinated axons and grew over the gap in the SIS conduits at 10 weeks. Electrophysiological examination and computer-analyzed image showed that axonal regeneration in the SIS group was similar to that in the auto-nerve grafting group at 10 weeks. Conclusion SIS as a conduit possesses the ability for axonal regeneration of the peripheral nerve, thereby having a potential to be an alternative bio-material instead of the autograft to repair the peripheral nerve gap.     

Scanning electron microscopic observation: three-dimensional architecture of the collagen in hepatic fibrosis rats

Background In the process of hepatic fibrosis, the accumulation of collagen fibers is strongly related to the hepatic function. The aim of this study was to investigate the three-dimensional architecture of the collagen network in the liver of rats with hepatic fibrosis. Methods Healthy adult male Wistar rats （n=32） were randomly divided into a control group （n=16） and a hepatic fibrosis group （n=16）. In the control group, the rats were treated with peanut oil while the rats in hepatic fibrosis group were treated for 10 weeks with 60% CCI4 diluted in peanut oil. The quantity of collagen fibers was detected by Western blotting; distribution of the collagen was detected by sirius red staining and polarized microscope; the three-dimensional architecture of collagen in the liver was observed under the scanning electron microscope after fixed tissues were treated with cell-maceration using NaOH. Statistical analysis was performed using the u test. Results The quantity of collagen fibers increased significantly in the hepatic fibrosis group. With the aggravation of hepatic fibrosis, collagen fibers gradually accumulated. They interlaced the reticulation compartment and formed a round or ellipse liver tissue conglomeration like a grape framework that was disparate and wrapped up the normal liver Iobule. The deposition of collagen fibers was obvious in adjacent hepatic parenchyma, especially around the portal tracts. Conclusion Our experiment showed the collagen proliferation and displays clearly the three-dimensional architecture of collagen fibers in rat liver with hepatic fibrosis by scanning electron microscope. It can provide a morphological foundation for the mechanisms of changed haemodynamics and portal hypertension in hepatic fibrosis.     

An experimental study on nerve regeneration and spinal neurons after CO2 laser anastomosis of rat sciatic nerve

Objective: Many methods have been used in an attempt to seal the epineurium and to prevent axonal outgrowth.In this study, the rat sciatic nerves were repaired with CO2 laser, the nerve regeneration and the morphology of spinal anterior horn neurons were investigated. Methods: Seventy-two male Sprague-Dawley rats were randomly divided into 6 groups of 12 rats. The animals were designed to observe the electrophysiology, the histopathology and the morphology of spinal anterior horn neurons. One of the rat sciatic nerve anastomosed with CO2 laser, the contralateral nerve was reconstructed by microsuture technique. At 2, 4, 6, 8 weeks postoperatively, neuromuscular functions, the regeneration of axons and neurons were evaluated by the electro-physiological and histopathological studies. The rats were killed at 4, 6 weeks postoperatively. Results: The recovery of toe spread and myodynamia in laser groups was better than that in suture groups (P＜0.05). The latency of foot withdraw caused by radiate heat and neuromuscular conduction velocity in laser groups were faster than that in suture groups (P＜0.05). The density of nerve fibers, percentage of axons passing through anastomotic area and numbers of neurons were better in laser groups than in suture groups. At 8 weeks postoperatively, the first grade dendrites of anterior horn neurons grew well. Their diameter, length, volume and total volume were much higher than that in control group. (P＜0.05, P＜0.01). Conclusion: CO2 laser repairing was effective in promoting the regeneration and the recovery of sciatic nerves in its earlypost-trauma stage. In addition, laser repairing was found to reduce regenerating axons misdirection and forming neuroma.     

Effect of low-energy shock waves in microfracture holes in the repair of articular cartilage defects in a rabbit model

Background  Microfracture is a type of bone marrow stimulation in arthroscopic cartilage repair. However, the overall concentration of the mesenchymal stem cells is quite low and declines with age, and in the end the lesion is filled by fibrocartilage. The aim of this research was to investigate a novel method of enhancing microfracture by determining whether low-energy shock waves in microfracture holes would facilitate cartilage repair in a rabbit model.

Methods  Full-thickness cartilage defects were created at the medial femoral condyle of 36 mature New Zealand white rabbits without penetrating subchondral bone. The rabbits were randomly divided into three groups. In experimental group A, low-energy shock-wave therapy was performed in microfracture holes (diameter, 1 mm) at an energy flux density (EFD) of 0.095 mJ/mm² and 200 impulses by DolorClast Master (Electro Medical Systems SA, Switzerland) microprobe (diameter, 0.8 mm). In experimental group B, microfracture was performed alone. The untreated rabbits served as a control group. At 4, 8, and 12 weeks after the operations, repair tissues at the defects were analyzed stereologically, histologically, and immunohistochemically.

Results  The defects were filled gradually with repair tissues in experimental groups A and B, and no repair tissues had formed in the control group at 12 weeks. Repair tissues in experimental group A contained more chondrocytes, proteoglycans, and collagen type II than those in experimental group B. In experimental group B, fibrous tissues had formed at the defects at 8 and 12 weeks. Histological analysis of experimental group A showed a better Wakitani score (P <0.05) than in experimental group B at 8 and 12 weeks after the operation.

Conclusions  In the repair of full-thickness articular cartilage defects in rabbits, low-energy shock waves in microfracture holes facilitated the production of hyaline-like cartilage repair tissues more than microfracture alone. This model demonstrates a new method of improving microfracture and applying shock waves in vivo. However, longer-term outcomes require further study.

     

The Experimental Study of Repairing Cartilage Defects Using a Bioreactor with Chondrocyte and Osteoblast Composites

This work aimed to evaluate the repair of cartilage defects using a bioreactor with chondrocyte and osteoblast composites. Our study involved co-culturing chondrocyte osteoblast composites using a bioreactor, treating cartilage defects with cell-β-TCP composites implanted into osteochondral defects in canine models in vivo. Composites were implanted using techniques of mosaicplasty, in which chondrocyte-β-TCP scaffold composites are placed in the top of the defect and osteoblast-β-TCP scaffold composites are implanted in the bottom of the defect. Our results as following, observations were made using electron microscopy. The induced chondrocytes and osteoblasts demonstrated good adhesion progression and proliferation in the β-TCP scaffold. At 16 weeks post-operation, the repaired tissues in the experimental group had established their thickness to the full depth of the original defects. In the control group, chondrocytes were arranged irregularly, and only part of the repaired defect consisted of fibrous tissue. In contrast, the defects in the control group were repaired only with fibrous tissue. In conclusion, perfusion culture permitted continuous nutrient supply and gas exchange to the center of a large scaffold. The perfusion bioreactor facilitates chondrocyte and osteoblast survival and proliferation through utilization of a three-dimensional scaffold.     

Degradation of human hair keratin scaffold material used to repair in-jured skeletal muscles of rabbits

Objective: To explore the mechanism of the degradation of human hair keratin (HHK) scaffold material implanted in damaged skeletal muscle tissues. Methods : Six New Zealand rabbits with HHK scaffold material implants (composed of 3 different types of HHK material with varied degradation speed) after mus-clectomy were divided into 3 groups (2 in each group) to observe the degradation of the material at 1, 3, 6 weeks after operation. Another rabbit without operation was used as the control group. The degradation of HHK was observed with light microscopy, histochemistry of ubiquitin and electron microscopy. Results: Light microscopy showed that human hair cuticles fell off from the HHK material and emerged, and the macrophagocytes and multinucleate giant cells were attached onto the surface of the material, which became homogeneous at the first postoperative week. The HHK scaffold material was degraded into particles that was phagocytosed by macrophagocytes and multinucleate giant cells at the third week. Ubiquitin enzymatic histochemistry showed that the macrophagocytes and the multinucleate giant cells were positive at the first week. Under electron microscope, HHK scaffold material was degraded into particles, and at the sixth week, part of HHK scaffold material was further degraded. Conclusion : Large mass of the HHK scaffold material is degraded via ubiquitin system, and the resultant particles are phagocytosed and degraded with the cooperation of lysosome and ubiquitin.     

Repair of regional osteoporosis after removal of rigid fixing plate. An experimental investigation.

Twenty-four New Zealand white rabbits were plated on their intact left tibiae with stainless steel plates and 4 animals served as controls. The plates were removed 2 months after implantation in 20 plated animals. Of them, 4 were sacrificed immediately after plate removal and the other 16 were killed in successive groups with 4 in each group 1,2,3 and 4 months after plate removal respectively. The remaining 4 plated animals were killed at 6 months after implantation. Bone samples under the plates were harvested and prepared for polarized light microscopy to investigate the repair of the regional osteoporosis induced by rigid implant. The results indicated that the regional osteoporosis could recover gradually after plate removal. The repair process manifested itself mainly as the repair of resorption cavities and the remodeling of collagen fibers. The orientation of the collagen fibers remained disorganized when the resorption cavities had been repaired. It is suggested that delayed restoration of bone s     

An Experimental Study on the Optimal Timing for the Repair of Incomplete Facial Paralysis by Hypoglossal-facial ‘Side'-to-side Neurorrhaphy in Rats

Objective To investigate the optimal timing for the repair of persistent incomplete facial paralysis by hypoglossal-facial ‘side'-to-side neurorrhaphy in rats. Methods A total of 30 adult rats with crushed and bulldog-clamped facial nerve injury were randomly divided into 5 groups(n = 6 each) that were subjected to injury without nerve repair or with immediate repair, 2-week-delayed repair, 4-week-delayed repair, or 8-week-delayed repair. Three months later, the effects of repair in each rat were evaluated by facial symmetry assessment, electrophysiological examination, retrograde labeling, and axon regeneration measurement. Results At 3 months after injury, the alpha angle significantly increased in the group of rats with 4-week-delayed repair compared with the other four groups. Upon stimulation of the facial nerve or Pre degenerated nerve, the muscle action potentials MAPs were recorded in the whisker pad muscle, and the MAP amplitude and area under the curve in the 4-week-delayed repair group were significantly augmented at 3 months post-injury. Similarly, the number of retrograde-labeled motor neurons in the facial and hypoglossal nuclei was quantified to be significantly greater in the 4-week-delayed repair group than in the other groups, and a large number of regenerated axons was also observed. Conclusion The results of this study demonstrated that hemi HN-FN neurorrhaphy performed 4 weeks after facial nerve injury was most effective in terms of the functional recovery of axonal regeneration and activation of facial muscles.     

Urethral stricture of battle wound repair with biodegradable stents

Objective To develop an experimental model of warinjured urethral stricture and evaluate its reconstruction using a biodegradable stents in the rabbits.Methods Twenty-eight New Zealand male rabbits were randomly divided into study group （20） and control group （8）.In study group,bulbar urethra of the rabbits was bombed with a special instrument to develop the model of war-injured urethral stricture.     

Repair of Rabbit Femoral Defects with a Novel BMP2-derived Oligopeptide P24

In this study, the bioactivity of a novel BMP2-derived oligopeptide P24 was investigated by using the model of rabbit femoral defect after loaded in the biodegradable poly （lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol） （PLGA-[ASP-PEG]）. A 1.5-cm unilateral segmental bone defect was created in the left femoral diaphysis in each of the 30 new zealand white rabbits. The defects of 18 legs filled with BMP2-derived peptide P24 combined with PLGA-[ASP-PEG] scaffold serves as the experimental group, and the defects in the rest 12 rabbits filled with （PLGA-[ASP-PEG]） without P24 as control group. The bone-repairing capability in the target region of the two group was grossly, radiologically, histopathologically and biomechanically evaluated 4, 8 and 12 weeks after the operation. Our results showed that in each group, primary healing of incision was achieved in the two groups. Radiographically, in experimental group, defects were filled with induced callus within 8 weeks, and a cortical bone-like structure was observed in some animals at the 12th week. According to the standardized stage of bone defect repair, 9 （64.28%） achieved grade-4 healing. In contrast, little bone formation was seen in the defects even 12 weeks after the operation, and 5 （62.50%） had grade 0 healing in this group. Histologically, tissue engineering material was mostly absorbed and cartilage was found around implants in the experimental group at the 4th week; 8 weeks after operation, the engineering material was completely absorbed, and formation of woven bone was observed and typical trabecular bone structure could be seen. In control group, 8 weeks after operation, the defect was filled with fibrous tissues, and no bone-like structure was observed. Statistical analysis showed very significant difference in biomechanical indicators between the two groups （P〈0.05）. It is concluded that new oligopeptide P24 can induce excellent bone regeneration and promote bone repair.     

人发角蛋白导管修复周围神经缺损的实验研究

目的 探索用人发角蛋白（HHK）制成的导管对大段缺损的周围神经的修复效果。方法 将25只新西兰兔分成3组，对照组不接受手术；另2组切除胫神10mm，分别用缝合线（无HHK组）和HHK导管（HHK组）连接神经两断功能恢复快，解剖观察发现，神经两断端之间以及HHK导管的腔隙被白色新生组织充满，人发部分消失，残余的人发易脆易断。在光镜下可见人发周围大量再生的雪旺细胞和较幼稚的神经纤维，神经纤维无序 排列，人发被初步降解，术后1年，人发被完全降解。神经缺损部位修复完好。结论 HHK导管可诱导神经纤维再生，跨过10mm的缺损间隙，从而修复神经缺损，HHK是制作神经导管的较为理想的材料。     

人发角蛋白导管修复周围神经缺损的实验研究

目的探索用人发角蛋白（HHK）制成的导管对大段缺损的周围神经的修复效果。方法将25只新西兰兔分成3组,对照组不接受手术;另2组切除胫神经10mm,分别用缝合线（无HHK组）和HHK导管（HHK组）连接神经两断端,术后不同时间分别进行神经电生理检查、解剖和组织学观察。结果术后92d经电生理检查,HHK组较无HHK组功能恢复快。解剖观察发现,神经两断端之间以及HHK导管的腔隙被白色新生组织充满,人发部分消失,残余的人发易脆易断。在光镜下可见人发周围大量再生的雪旺细胞和较幼稚的神经纤维,神经纤维无序排列,人发被初步降解。术后1年,人发被完全降解,神经缺损部位修复完好。结论 HHK导管可诱导神经纤维再生,跨过10mm的缺损间隙,从而修复神经缺损。HHK是制作神经导管的较为理想的材料。     

人发角蛋白神经导管诱导兔胫神经修复时NGF和p75的表达及分布

目的 研究人发角蛋白(HHK)神经导管诱导兔胫神经缺损再生修复时神经生长因子(NGF)及其低亲和受体p75的表达及分布。方法 取正常胫神经、缝线连接的胫神经和：HHK导管桥接的胫神经术后不同时期的切口部位及相邻组织制作石蜡切片，用免疫组化法对切片进行染色。结果 正常胫神经组织中无NGF阳性染色。术后76d，HHK周围的新生组织中NGF免疫组化染色表现为强阳性，而缝线周围的组织中无阳性着色。术后100d，与正常胫神经组织无显著差异。正常胫神经组织中无p75阳性染色。术后76d，导管组HHK周围较成熟的神经组织中p75免疫组化染色为弱阳性，而新生组织中为强阳性。术后100d，导管组的p75免疫组化染色与正常神经组织无显著差别。结论 HHK及其降解产物能诱导NGF和p75的产生，为神经再生创造良好的微环境；而缝线无此诱导作用。在新生神经组织中NGF和p75含量多，较成熟的神经组织中含量少。     

人发角蛋白神经导管诱导兔胫神经修复时NGF和p75的表达及分布

目的研究人发角蛋白(HHK)神经导管诱导兔胫神经缺损再生修复时神经生长因子(NGF)及其低亲和受体p75的表达及分布。方法取正常胫神经、缝线连接的胫神经和HHK导管桥接的胫神经术后不同时期的切口部位及相邻组织制作石蜡切片,用免疫组化法对切片进行染色。结果正常胫神经组织中无NGF阳性染色。术后76 d,HHK周围的新生组织中NGF免疫组化染色表现为强阳性,而缝线周围的组织中无阳性着色。术后100 d,与正常胫神经组织无显著差异。正常胫神经组织中无p75阳性染色。术后76 d,导管组HHK周围较成熟的神经组织中p75免疫组化染色为弱阳性,而新生组织中为强阳性。术后100 d,导管组的p75免疫组化染色与正常神经组织无显著差别。结论HHK及其降解产物能诱导NGF和p75的产生,为神经再生创造良好的微环境;而缝线无此诱导作用。在新生神经组织中NGF和p75含量多,较成熟的神经组织中含量少。     

大鼠坐骨神经切除后人发角蛋白诱导的神经的再生机制

目的 观察人发角蛋白(HHK)诱导坐骨神经再生时的形态学变化,以揭示神经再生机制,方法制备坐骨神经损伤动物模型,植入HHK丝束桥接体,手术后2天、1、2、3、6、9、12周进行组织学观察.结果 术后第2天到2周,大量新生微血管长入HHK植入部位,切除后近远端的施万细胞发生去分化.去分化后的施万细胞沿着HHK丝束表面纵向分裂增殖.术后第3周人发开始降解,HHK周围可见很多巨噬细胞和多核巨细胞,大量增生的施万细胞有规则地排列于HHK丝间,有轴突和大量的微血管出现.术后第6周,在HHK丝周围可见大量新生的神经纤维,其间有微血管分布.术后第9周,人发角蛋白降解显著,再生神经纤维增多,有明显的神经外膜和束膜.术后第12周,实验组人发角蛋白基本完全降解,其部位被新的神经纤维所取代,并已贯通缺损部位,且外观形态接近正常神经.结论 1,HHK对缺损的坐骨神经修复具有良好的桥接作用.2、受损后高度分化的施万细胞通过去分化形成幼稚的施万细胞,其中胞质脱落起着关键作用.3、受损轴突立即发生保护性封闭、脱落.健康轴突形成膨大的带突起的生长锥,生长锥突起上伸出许多丝状生长芽.并与1到多个施万细胞嵌合,它们通过竞争性选择,最后只有一条生长芽可发育成完整的轴突.4、神经纤维屏障膜(神经外膜、束膜及内膜)是由最外侧的血管屏障膜中和束内的微血管间充质细胞演变而成的.总之,施万细胞、神经轴突、神经膜三者的再生模式是自身器官化过程所要求的,它们是同步进行的协调行为.     

壳聚糖导管复合雪旺细胞修复兔面神经缺损的实验研究

目的：应用壳聚糖导管作为神经再生室, 复合体外培养的雪旺细胞修复兔面神经缺损,观察神经再生的效果。 方法： 用壳聚糖和羧甲基壳聚按3：1的比例采用冷冻干燥法制成壳聚糖复合导管; 取胎兔坐骨神经,经体外增殖培养获得雪旺细胞,复合于壳聚糖导管,修复兔面神经0.8?cm的缺损,并与单用壳聚糖导管作对照,观察修复后4、8、16周的神经电生理及组织学检查情况。结果:术后8周新生面神经纤维已沿管壁通过缺损到达远端,术后16周导管大部分降解,新生神经变粗变直,神经纤维排列整齐规则,神经电生理检查记录到复合动作电位,复合雪旺氏细胞的导管再生神经较粗,动作电位幅值较高。结论：壳聚糖导管可引导神经再生,复合雪旺细胞的壳聚糖导管神经再生效果较好。     

应用普通橡皮管作为周围神经保护鞘的动物实验

作者采用普通橡皮管作为周围神经保护鞘做了动物实验,观察周围神经生长情况及光镜下组织学变化。结果表明,橡皮管可起到保护吻合口神经轴索对接,刺激再生的作用,今后可临床试用。     

含神经生长因子壳聚糖神经导管修复大鼠坐骨神经10mm缺损

目的:研究京尼平交联的含神经生长因子的壳聚糖神经导管修复大鼠周围神经缺损的可行性。方法:利用含神经生长因子的壳聚糖神经导管桥接修复大鼠坐骨神经10mm缺损为实验组共5只,缺损组对照共5只。术后6个月行电生理学检测,并对再生神经组织和靶肌行形态学观察。结果:术后6个月,实验组术侧均能记录到复合肌动作电位,而缺损组术侧均未能记录到复合肌动作电位。抗NF-200免疫组化染色显示,实验组再生神经中段再生轴突分布密集。肌肉三色染色显示,实验组腓肠肌无明显萎缩,亦未见明显增生的胶原纤维;缺损组腓肠肌明显萎缩,同时可见大量胶原纤维增生。结论:术后6个月,京尼平交联含神经生长因子壳聚糖神经导管,能较好修复大鼠坐骨神经10mm缺损,实现靶肌神经功能的重支配。     

Degradation of human hair keratin scaffold implanted for repairing injured skeletal muscles.

OBJECTIVE: To observe the in vivo degradation process of human hair keratin (HHK) scaffold after implantation in rabbits. METHODS: Seven New Zealand rabbits were divided into 4 groups including a control group and 3 operation groups. HHK scaffold was implanted, after partial resection of the skeletal muscles, in rabbits of the 3 operation groups, followed by observation 1, 3, and 6 weeks later respectively. Routine morphological observation, histochemistry with ubiquitin and electron microscopy were performed. HHK scaffold incorporated 3 types of HHK with different degradation speeds, respectively designated types F, B, and Z. RESULTS: Light microscopic observation revealed that human hair cuticles began to strip off at the first postoperative week, and the material was homogeneous on the surface of which macrophagocytes and multinuclear giant cells adhered. At the third week HHK scaffold was degraded into particles as seen under electron microscope and was phagocytosed by macrophagocytes and multinuclear giant cells. Ubiquitin enzymatic histochemistry demonstrated that macrophagocytes, multinuclear giant cells were positive at the first week. At sixth week, further degradation of HHK scaffold occurred when newly generated muscles were seen beside the HHK. CONCLUSIONS: HHK scaffold is initially degraded extracellularly by ubiquitin system into particles, which are phagocytosed by the cells and degraded by the cooperation of lysosome and ubiquitin; meanwhile the satellite cells are activated, beginning to proliferate and eventually fused into newly generated muscle fibers.     

生物型人工硬脑膜应用的实验研究

OBJECTIVE: To evaluate the safety and efficacy of a dural graft prepared using porcine membrane in duraplasty. METHODS: Eighteen New Zealand rabbits were randomly divided into groups A (n=4), B (n=4), C (n=5), and D (n=5) sacrificed 3, 14, 30 and 90 d after duraplasty, respectively. Each animal underwent bilateral parietal craniectomy behind the coronal suture and beside the midline to expose the dura, which was cut on the right side and substituted with the dural graft. The exposed dura on the left was kept intact as control. The rabbits were observed for WBC counts before the operation and before sacrifice by transcardiac formalin perfusion, respectively. The meninges and brain tissues were histologically examined after sacrifice. RESULTS: The WBC count varied little after the operation (P>0.05). Microscopic examination demonstrated tissue repair on both the implantation side and control side, without graft adhesion to the cortical surface. In group A, a large number of leukocytes were seen gathering on the lateral dura, suggesting acute tissue repair. In group B, endothelial cells covering the inner surface of the graft could be seen. Fibroblasts and fibrocytes were seen in the grafts between collagen fibers in group C, and in group D, fibroblasts and fibrocytes increased between the collagen fibers and the suture healed. CONCLUSION: The dura graft is safe and applicable for dural defect repair.