Experimental study of bone morphogenetic proteins-2 slow release from an artificial trachea made of biodegradable materials: evaluation of stenting time

We manufactured an artificial trachea that slowly releases bone morphogenetic protein 2 (BMP-2) and used it to replace a section of the canine trachea. We made a three-layered prosthesis composed of an outer layer of gelatin sponge, a middle layer of collagen sponge, and an inner silicone tube. BMP-2 solution was soaked into the gelatin sponge layer. An approximately 3 cm length of the canine trachea was resected, and the artificial trachea was inserted into the resulting gap and anastomosed. The implanted portion was covered by periosteum. At 2, 4, and 8 weeks after surgery, the inner silicone tube was removed. Soon after removal of the silicone tube at 2 and 4 weeks, the dogs died of choking because of collapse of the trachea. One dog whose silicone tube was removed at 8 weeks was able to survive without choking. At 6 months after removal of the silicone tube, the bronchoscopic findings revealed that the gap in the trachea had been closed by regenerated tissue and covered by mucosa. We have demonstrated that our artificial trachea slowly releasing BMP-2 requires at least 8 weeks to achieve regeneration of solid tissue to support the tracheal gap.     

大鼠坐骨神经缺损聚吡咯膜植入试验

了解大鼠神经组织对长期埋入其中的聚吡咯膜的生物学反应 ,同时观察聚吡咯涂层的硅胶管作为桥接物 ,修复周围神经缺损的可能性。在硅胶管内壁用电化学方法合成聚吡咯膜管涂层 ,并以桥接方法修复大鼠的坐骨神经缺损 10mm。术后 2 4周 ,对再生组织进行电生理学测试、组织形态学观察和计量学统计。在聚吡咯膜长期植入缺损神经期间 ,实验动物仅出现轻微炎症反应 ;聚吡咯膜管中可见到再生神经 ;聚吡咯膜管内再生的神经在电生理学、组织形态学及计量学方面的结果均略优于单纯的硅胶管桥接组。实验表明在体内周围神经组织对长期埋入的聚吡咯未产生不良反应。     

Collagen coated Y-shaped prosthesis for carinal replacement promotes regeneration of the tracheal epithelium

Carinal reconstruction has the highest operative mortality rate in pulmonary surgery, and high tension at the anastomoses is one of the reasons. We have reported previously that a new Y-shaped artificial trachea gives acceptable short-term results. Here, we report our long-term observations and histologic findings. A Y-shaped Marlex mesh tube reinforced by a polypropylene spiral was coated with collagen, which was extracted from porcine skin, to increase the biocompatibility and airtightness of the prosthesis. Replacement of the carina was performed in 20 beagle dogs. The prosthesis was covered by the omental pedicle in all dogs. Six of the 20 dogs survived past 6 months, and 4 were in good condition when they were killed. The other 13 dogs died within 11 days of the operation. The main cause of death was air leakage from the prosthesis. Bronchoscopic examination of the surviving dogs 12 months after surgery revealed that the prosthesis was covered with regenerated tissue, and neither stenosis nor dehiscence was recognized; 15 months after implantation microscopic examination showed the growth of ciliated epithelial cells over the lumen of the prosthesis. These results suggest that our Y-shaped artificial trachea might be useful for reconstruction of the carina with tissue regrowth, although some improvement of the prosthesis is necessary.     

人工组织神经移植物修复狗缺损坐骨神经后腓肠肌的形态观察

目的 了解复合型医用可降解材料制成的人工组织神经移植物辅加神经再生素修复狗坐骨神经缺损后，腓肠肌的形态变化。方法 将人工组织神经移植物连接在狗的坐骨神经缺损30mm处。以自体神经桥接和神经缺损的狗为对照组Ⅰ和Ⅱ。术后6个月时取腓肠肌进行称重、特殊染色和组织化学染色，显微镜下了解腓肠肌的形态变化并进行图像定量分析，同时了解肌纤维的超微结构变化。结果 术后6个月，实验组腓肠肌的萎缩形态指标变化均轻于对照组Ⅱ，而与对照组Ⅰ相似。结论 经人工组织神经移植物修复缺损的坐骨神经后，使腓肠肌又重新获得神经支配，肌肉萎缩明显减轻。     

The geometric design of micromachined silicon sieve electrodes influences functional nerve regeneration

A neural interface could be used to control a limb prosthesis. Such an interface can be created by facilitating axonal regeneration through a sieve electrode and then register nerve signals intended to control the prosthesis. A key question is how to design the electrodes to ensure the best possible regeneration. Our previous studies have indicated that regeneration can be achieved using electrodes with square-shaped, 100 x 100 microm, via holes (holes that axons will regenerate through). Other reports have indicated a suitable range of these holes between 40 and 65 microm. In the present study we used silicon sieve electrodes with via holes of either 30 or 90 microm. The transparency, i.e. the percentage of the total via hole area, of these electrodes was either 20 or 30%. The electrodes were inserted into a silicone chamber which was used to bridge a gap in a rat sciatic nerve. After 12 weeks of nerve regeneration electrodes with a hole size of 30 microm and a 30% transparency had the most favourable result as judged by the regained gastrocnemius muscle force and the formation of reactive tissue inside the chamber. The sieve electrode transparency is crucial for ensuring regeneration.     

Evaluation of biocompatibility of polypyrrole in vitro and in vivo

In this study, the biocompatibility of the electrically conductive polymer polypyrrole (PPy) with nerve tissue was evaluated in vitro and in vivo. The extraction solution of PPy powder, which was synthesized chemically, was tested for acute toxicity, subacute toxicity, pyretogen, quantitative measure of cell viability, hemolysis, allergen, and micronuclei. The PPy membrane was synthesized electrochemically on the indium tin oxide conductive borosilicate glass. The dorsal root ganglia from 1-3-day-old Sprague-Dawley rats were cultured above PPy membrane and observed by light or scanning electron microscopy. The PPy-silicone tube (PPy membrane on the inner surface of the silicone tube) also synthesized electrochemically was used to bridge across 10-mm sciatic nerve gap in rats. Twenty-four weeks after the operation to rats, the regenerated tissues were observed by electrophysiological and histological techniques. PPy extraction solution showed no evidence of acute and subacute toxicity, pyretogen, hemolysis, allergen, and mutagenesis, and the Schwann cells from the PPy extraction solution group showed better survival rate and proliferation rate as compared with the saline solution control group. The migration of the Schwann cells and the neurite extension from dorsal root ganglia on the surface of PPy membrane-coated glass was better than those of bare glass. There was only lightly inflammation during 6 months of the postoperation, when the PPy-silicone tube bridged across the gap of the transected sciatic nerve. The regeneration of nerve tissue in the PPy-silicone tube was slightly better than that in the plain silicone tube by means of electrophysiological and histological examination. The results of this study indicate that PPy has a good biocompatibility with rat peripheral nerve tissue and that PPy might be a candidate material for bridging the peripheral nerve gap.     

A morphological study on the effects of collagen gel matrix on regeneration of severed rat sciatic nerve in silicone tubes

The present study is a chronological morphological examination on the effects of collagen gel matrix on regeneration of severed sciatic nerves. The nerves (5 mm length) were resected, and both the distal and proximal stumps were inserted into a silicone tube with 5 mm gap in between. In the test side, the gap in the tube was then injected with liquid collagen which gels in the tissue when reconstructed with a certain buffer solution. The gap space in the tube of the control side was left empty. In a chronological examination of the tissue in the tube, considerably more rapid growth of sprouting axons toward the distal stump in the test side was revealed in comparison with the control side. The cells, including both fibroblasts and larger Schwann cells, were less in number. More orderly directions were observed in the collagen matrix than in the control tube. The result indicates that regeneration of the peripheral nerves in the silicone tube can be improved, by using appropriate exogenous fine materials, collagen matrix.     

A MORPHOLOGICAL STUDY ON THE EFFECTS OF COLLAGEN GEL MATRIX ON REGENERATION OF SEVERED RAT SCIATIC NERVE IN SILICONE TUBES

The present study is a chronological morphological examination on the effects of collagen gel matrix on regeneration of severed sciatic nerves. The nerves (5 mm length) were resected, and both the distal and proximal stumps were inserted into a silicone tube with 5 mm gap in between. In the test side, the gap in the tube was then injected with liquid collagen which gells in the tissue when reconstructed with a certain buffer solution. The gap space in the tube of the control side was left empty. In a chronological examination of the tissue in the tube, considerably more rapid growth of sprouting axons toward the distal stump in the test side was revealed in comparison with the control side. The cells, including both fibroblasts and larger Schwann cells, were less in number. More orderly directions were observed in the collagen matrix than in the control tube. The result indicates that regeneration of the peripheral nerves in the silicone tube can be improved, by using appropriate exogenous fine materials, collagen matrix.     

In situ tissue engineering of the bile duct using polypropylene mesh-collagen tubes

Multiple attempts have been made to replace biliary defects with a variety of materials. Recently, successful biliary reconstruction using the Gore-Tex vascular graft has been reported experimentally and clinically. We designed a new artificial bile duct consisting of collagen sponge and polypropylene mesh. We presently evaluated the feasibility of using this prosthesis as a scaffold for bile duct tissue regeneration in a canine model. Our prosthesis, a sponge made from porcine dermal collagen, is reinforced with a polypropylene mesh cylinder. We used the prosthesis to reconstruct the middle portion of the common bile duct in seven beagle dogs to evaluate its efficacy. While one dog died of biliary stricture 8 months after operation, six survived without problems to scheduled time points for tissue evaluation at 1 to 12 months. All prostheses had become completely incorporated into the host. A confluent epithelial lining was observed within 3 months. In cholangiograms the prosthesis displayed long-term patency in the six dogs and provided satisfactory bile drainage for up to 12 months. Our graft thus shows promise for repair of biliary defects and should lead to development of a new treatment for biliary reconstruction.     

Tubulation with dental pulp cells promotes facial nerve regeneration in rats

Dental pulp is an easily obtainable source of viable cells for potential use in peripheral nerve regeneration. We prepared artificial conditions for nerve regeneration using a silicone tube containing a collagen gel embedded with rat dental pulp cells, and we examined its effectiveness for repairing a gap in the rat facial nerve. Twelve days after transplantation, defective facial nerves connected with silicone tubes containing dental pulp cells were repaired more rapidly than control tubes containing the collagen gel alone. When a tube containing green fluorescent protein (GFP)-positive dental pulp cells was transplanted into a facial nerve gap in a GFP-negative rat, we observed regenerated nerves with GFP-positive cells at 2 weeks posttransplantation. The regenerated nerves included Tuj1-positive axons, RECA1 and GFP double-positive blood vessels, and S100 and GFP double-positive Schwann-like supportive cells. Osmium-toluidine blue staining revealed that the regenerated nerves contained myelinated fibers. Moreover, fluorescent retrograde tracing analysis by application of Fluoro-Gold into the regenerated nerves demonstrated the presence of Fluoro-Gold-positive motor neurons in the facial nucleus of the rat brain. These results suggest that the transplanted dental pulp cells formed blood vessels and myelinating tissue and contributed to the promotion of normal nerve regeneration.     

修复周围神经缺损的组织工程研究

取有限的自体可牺牲的神经组织剥去外膜及束膜制成粗品雪旺氏细胞，并保留其中剖分基质，与２％壳聚糖凝胶制成混材料，加入外源性神经生长因子，注江表管内用以套接修复大鼠坐同有神经１００ｍｍ之缺损，术后１２周神经电生理测试及再生神经横面图像分析形态计量提示。     

Peripheral nerve regeneration using silicone rubber chambers filled with collagen, laminin and fibronectin

A 10 mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with an extracellular gel containing collagen, laminin and fibronectin. Empty silicone rubber tubes were used as controls. Six weeks after implantation, all extracellular elements were completely degraded and absorbed, and 90% of the animals from the extracellular gel group exhibited regeneration across the nerve gaps, whereas only 60% in the control group. Both qualitative and quantitative histology of the regenerated nerves revealed a more mature ultrastructural organization with 28% larger cross-sectional area and 28% higher number of myelinated axons in the extracellular gel group than the controls. These results showed that the gel mixture of collagen, laminin and fibronectin could offer a suitable growth medium for the regeneration of axons.     

Experimental study on in situ tissue engineering of the stomach by an acellular collagen sponge scaffold graft

We carried out an experimental study to clarify the feasibility of stomach tissue engineering for reconstruction after partial gastrectomy. A collagen sponge scaffold was implanted to support the regeneration of stomach tissue. A 4 cm square area of the anterior wall of the stomach was surgically resected in three beagle dogs, and then reconstructed using the collagen sponge scaffold. The dogs received intravenous hyperalimentation for 14 days after the operation, and a silicone sheet was used as a patch on the luminal side to protect the scaffold from degradation by digestive juice. The silicone sheet was removed endoscopically 4 weeks after the operation, and the surgical defect was observed endoscopically at various time points. At 4 weeks after surgery, the stomach wall had regenerated, but was not yet covered by stomach mucosal tissue. However, at 16 weeks after surgery, mucosa totally covered the regenerated area. Microscopic findings confirmed regeneration of the stomach wall, mucosa, and thin muscular layer. These results show that tissue engineering of the stomach will be feasible in the near future for reconstruction after partial gastrectomy.     

Novel approach to regeneration of periodontal tissues based on in situ tissue engineering: effects of controlled release of basic fibroblast growth factor from a sandwich membrane

To regenerate periodontal tissues, a sandwich membrane composed of a collagen sponge scaffold and gelatin microspheres containing basic fibroblast growth factor (bFGF) in a controlled-release system was developed according to the new concept of "in situ tissue engineering." A three-walled alveolar bone defect (3 x 4 x 4 mm) was made bilaterally in edentulous regions created mesially to the canines in both the maxilla and mandible of nine beagle dogs. A sandwich membrane with or without bFGF (100 microg) was implanted in each defect (each group, n = 18). During weeks 1, 2, and 4, histologic evaluation and histometric analyses were performed on three dogs. Throughout the 4 weeks, vascularization and osteogenesis were active only in the bFGF-treated group (p < 0.01). New cementum was formed (2.4 +/- 0.9 mm) on the exposed root surface at 4 weeks, and functional recovery of the periodontal ligament was indicated in part by the perpendicular orientation of regenerated collagen fibers. In the control group, epithelial downgrowth and root resorption occurred and the defects were filled with connective tissue. Thus, our sandwich membrane induced successful regeneration of the periodontal tissues in a short period of time.     

Effects of chitosan nonwoven membrane on periodontal healing of surgically created one-wall intrabony defects in beagle dogs

The purpose of this study was to investigate the periodontal regenerative effects of a chitosan nonwoven membrane applied to surgically created preclinical one-wall intrabony defects in beagle dogs. One-wall intrabony defects (4 x 4 x 4 mm) were surgically created bilaterally in the mandibular second and fourth premolars of six beagle dogs. The surgical control group received a flap operation only. The resorbable-membrane (RM) group was treated with resorbable membrane. The chitosan-nonwoven-membrane (CNWM) group was treated with chitosan nonwoven membrane. The amount of junctional epithelium migration and the amount of connective tissue adhesion did not show any statistically significant differences among the groups. However, the amount of suprabony cementum regeneration, intrabony cementum regeneration, and alveolar bone regeneration showed significant differences (p < 0.05) between CNWM site group and control group. The results demonstrate the regenerative effects of the chitosan nonwoven membrane in one-wall intrabony defects of beagle dogs. The chitosan nonwoven membrane has the potential to support the cementum and bone regeneration, possibly by providing the conditions needed for guided tissue regeneration in the one-wall intrabony periodontal defects of beagle dogs.     

Regeneration of canine peroneal nerve with the use of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge: a comparative study of collagen sponge and collagen fibers as filling materials for nerve conduits.

A novel artificial nerve conduit was developed and its efficiency was evaluated on the basis of promotion of peripheral nerve regeneration across an 80-mm gap in dogs. The nerve conduit was made of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge. Conduits filled with either sponge- or fiber-form collagen were implanted into an 80-mm gap of the peroneal nerve (five dogs for each form). Twelve months postoperatively nerve regeneration was superior in the sponge group both morphometrically (percentage of neural tissue: fiber: 39.7 +/- 5.2, sponge: 43.0 +/- 4.5, n=3) and electrophysiologically (fiber: CMAP 1.06 +/- 0.077, SEP 1.32 +/- 0.127 sponge: CMAP 1.04 +/- 0.106, SEP 1.24 +/- 0.197, n=5), although these differences were not statistically significant. The observed regeneration was complementary to successful results reported previously in the same model, in which collagen fibers exclusively were used. The results indicate a possible superiority of collagen sponge over collagen fibers as filling materials. In addition, the mass-producibility, superior scaffolding potential, and capacity for gradual release of soluble factors of the sponge provide make it an attractive alternative to fine fibers, which are both technologically difficult and costly to produce. This newly developed nerve conduit has the potential to enhance peripheral nerve regeneration across longer gaps commonly encountered in clinical settings.     

Replacement of a tracheal defect with autogenous mucosa lined tracheal prosthesis made from polypropylene mesh

Reliable prosthetic or tissue grafts for the trachea have not, as yet, been developed for reconstruction of large, circumferential tracheal defects. Major limitations are anastomotic dehiscence and stenosis, attributed to the poor epithelialization and vascularization of the prosthetic graft. We have developed a new tracheal prosthesis that has a well vascularized and viable mucosa. The prosthesis consists of a Prolene mesh reinforced with polypropylene rings, and coated with gelatin. We lined the luminal surface of the prosthesis with transplanted autogenous oral mucosa, wrapped the prosthesis with greater omentum, and placed it in the peritoneal cavity for 2 weeks. Complete surgical resection and replacement of a segment (5 cm in length, 8 to 10 tracheal rings) of the thoracic trachea was then performed in nine adult mongrel dogs. The transplanted mucosa was well vascularized and maintained its normal histology in prereplacement analysis. Dogs with tracheal replacement regained their full activity and did not show any respiratory problems until sacrifice at 1, 2, and 6 months. After 6 months, the prostheses were completely incorporated by the host trachea in all dogs and confluent epithelialization was confirmed histologically from the upper to the lower anastomotic site of the prosthesis; furthermore, the transplanted mucosal cells had changed to ciliated columnar epithelium.     

A novel use of genipin-fixed gelatin as extracellular matrix for peripheral nerve regeneration

Application of combining herbal medicine and biomedical material science to nerve regeneration is a new approach. In this study, we describe a novel use of purified genipin, which can be extracted from Gardenia jasminoides Ellis, fixing the gelatin to be an extracellular matrix for peripheral nerve regeneration. A 10-mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with either the genipin-fixed gelatin or collagen gel. Silicone rubber tubes filled with saline were used as controls. Six weeks after implantation, regeneration across the nerve gaps occurred in 80 and 90% of the animals from the groups of genipin-fixed gelatin and collagen, respectively, whereas only 30% in the control group. Large numbers of myelinated axons were also seen in the genipin-fixed gelatin (5104 +/- 3278) and the collagen groups (8063 +/- 1807). These findings indicated that the genipin-fixed gelatin could be an acceptable extracellular matrix for nerve regeneration.     

Use of small intestinal submucosal implants for regeneration of large fascial defects: an experimental study in dogs.

A single layer of porcine small intestinal submucosa (SIS) was sutured into a 5 x 5 cm window created in the fascia lata of ten adult mongrel dogs in order to determine the efficacy of this material in promoting tissue regeneration of large fascial defects. A similar defect in the contralateral limb was left empty and served as a negative control. Tissue regeneration was examined grossly and histologically at 6 and 12 weeks. By 6 weeks, marked fibroplasia and angiogenesis had occurred throughout the SIS scaffold. The regenerated tissue was well organized and showed good integration with the adjacent fascia while the control specimens were filled with loose areolar connective tissue. At 12 weeks the experimental defects were filled with a regenerated tissue that grossly and histologically resembled normal fascia. There was no evidence of adhesions to the underlying musculature. Conversely, the tissue that filled the control defects remained disorganized and was markedly thinner than the adjacent fascia. The results of this study suggest that SIS is capable of supporting tissue regeneration in large fascial defects. The ability of this material to induce regeneration of a substantial area of tissue grossly and histologically similar to normal fascia and without adhesions to the underlying musculature makes its application in reconstructive surgery appear promising.     

Regeneration of canine tracheal cartilage by slow release of basic fibroblast growth factor from gelatin sponge

We investigated the efficiency of basic fibroblast growth factor (b-FGF) released from a gelatin sponge in the regeneration of tracheal cartilage. A 1-cm gap was made in the midventral portion of each of 10 consecutive cervical tracheal cartilages (rings 4 to 13) in 15 experimental dogs. In the control group (n = 5), the resulting gap was left blank. In the gelatin group (n = 5), a gelatin sponge alone was implanted in the gap. In the b-FGF group (n = 5), a gelatin sponge containing 100 mug b-FGF solution was implanted in the gap. We euthanatized one of the five dogs in each group at 1 month after implantation and one at 3 months and examined the implant sites macroscopically and microscopically. In the control and gelatin groups, no regenerated cartilage was observed in the tracheal cartilage gap at 1 or 3 months. The distances between the cartilage stumps had shrunk. In the b-FGF group, fibrous cartilage had started to regenerate from both host cartilage stumps at 1 month. At 3 months, regenerated fibrous cartilage filled the gap and had connected each of the stumps. The regenerated cartilage was covered with regenerated perichondrium originating from the host perichondrium. Shrinkage of the distance between the host cartilage stumps was not observed in the b-FGF group. We succeeded in inducing cartilage regeneration in the gaps in canine tracheal cartilage rings by using the slow release of b-FGF from a gelatin sponge. The regenerated cartilage induced by b-FGF was fibrous cartilage.