Makoto Yoshitani

Experimental study on the regeneration of peripheral nerve gaps through a polyglycolic acid–collagen (PGA–collagen) tube

We have developed a bioabsorbable polyglycolic acid (PGA) tube filled with collagen sponge (PGA-collagen tube) as a nerve connective guide, and compared its effectiveness with that of autograft in terms of nerve regeneration across a gap. The PGA-collagen tube was implanted into 24 beagle dogs across a 15-mm gap in the left peroneal nerve. The right peroneal nerve was reconstructed with the autograft harvested from the left side, as a control. After the surgery, the connective tissue extended from both cut ends in the PGA-collagen tube and connected again at the center. Pathologically, the collagen sponge in the tube provided adequate scaffolding for nerve tissue extension, and the nerve tissue reconnected within 3 weeks. Electrophysiologically, muscle-evoked potentials (MEPs) and compound nerve action potentials (CNAPs) were detected 18 days after the surgery. For up to 6 months postsurgery, CNAPs and somatosensory-evoked potentials (SEPs) on the PGA-collagen side had a shorter latency and larger peak voltage than those on the autograft side. The myelinated axons on the PGA side were larger in diameter than those on the autograft side. It is suggested that the PGA-collagen tube has the potential to be an effective alternative to conventional autografting for the repair of some peripheral nerve defects.

Use of collagen sponge incorporating transforming growth factor-β1 to promote bone repair in skull defects in rabbits

The objective of this study was to evaluate the potential of collagen sponge incorporating transforming growth factor-beta1 (TGF-beta1) to enhance bone repair. The collagen sponge was prepared by freeze-drying aqueous foamed collagen solution. Thermal cross-linking was performed in a vacuum at 140 degrees C for periods ranging from 1 to 48 h to prepare a number of fine collagen sponges. When collagen sponges incorporating 125I-labeled TGF-beta1 were placed in phosphate-buffered saline (PBS) solution at 37 degrees C, a small amount of TGF-beta1 was released for the first hour, but no further release was observed thereafter, irrespective of the amount of cross-linking time the sponges had received. Collagen sponges incorporating 125I-labeled TGF-beta1 or simply labeled with 125I were implanted into the skin on the backs of mice. The radioactivity of the 125I-labeled TGF-beta1 in the collagen sponges decreased with time; the amount of TGF-beta1 remaining dependent on the cross-linking time. The in vivo retention of TGF-beta1 was longer in those sponges that had been subjected to longer cross-linking times. The in vivo release profile of the TGF-beta1 was matched with the degradation profile of the sponges. Scanning electron microscopic observation revealed no difference in structure among sponges subjected to different cross-linking times. The TGF-beta1 immobilized in the sponges was probably released in vivo as a result of sponge biodegradation because TGF-beta1 release did not occur in in vitro conditions in which sponges did not degrade. We applied collagen sponges incorporating 0.1 microg of TGF-beta1 to skull defects in rabbits in stress-unloaded bone situations. Six weeks later, the skull defects were covered by newly formed bone, in marked contrast to the results obtained with a TGF-beta1 free empty collagen sponge and 0.1 microg of free TGF-beta1. We concluded that the collagen sponges were able to release biologically active TGF-beta1 and were a promising material for bone repair.

Artificial trachea and long term follow-up in carinal reconstruction in dogs.

We have already reported “del” successful carinal reconstruction of the trachea with an observation period of 1 – 2 years. In this study, we evaluate the long-term safety and efficacy of the reconstruction after 5-years of follow-up. The Y-shaped Marlex® mesh tube was reinforced with a polypropylene spiral and coated with atelocollagen made from porcine skin. The prosthesis was 60 mm long with an outer diameter of 18 mm. Replacement of the tracheobronchial bifurcation was preformed through a right thoracotomy in a beagle dog. Bronchoscopical examination and sampling of the tracheal epithelium was performed periodically to check the function of cilia. The implanted prothesis was promptly infiltrated by the surrounding connective tissue and completely incorporated by the host trachea and bronchus. Bronchoscopically, sufficient epithelization was confirmed from the upper to the lower site of anastomosis. After 5 years neither stenosis nor dehiscence was observed. In spite of there being mesh-exposure at the luminal surface, the dog had no clinical symptoms until sacrifice for pathological examination. The bent frequency of the cilia was maintained within the normal range, indicating “del” functional recovery of the regenerating airway. Our tracheal prosthesis is promising for clinical “del” repair of the tracheobronchial bifurcation.

In situ tissue engineering for tracheal reconstruction using a luminar remodeling type of artificial trachea.

BACKGROUND After successful trials of tracheal reconstruction using mesh-type prostheses in canine models, the technique has been applied clinically to human patients since 2002. To enhance tissue regeneration, we have applied a new tissue engineering approach to this mesh-type prosthesis. METHODS The prosthesis consists of a polypropylene mesh tube reinforced with a polypropylene spiral and atelocollagen layer. The cervical tracheas of 18 beagle dogs were replaced with the prosthesis. The collagen layer was soaked with peripheral blood in 6 of the dogs, with bone marrow aspirate in another 6, and with autologous multipotential bone marrow-derived cells (mesenchymal stem cells) in another 6. The dogs were humanely killed at 1 to 12 months after the operation. RESULTS All 18 dogs survived the postoperative period. Bronchoscopically, 3 of 4 dogs in the peripheral blood group showed stenosis, whereas no stenosis was evident in all 8 of the dogs in the bone marrow and mesenchymal stem cell groups 6 months after the operation. Faster epithelialization and fewer complications, such as mesh exposure and luminal stenosis, were observed in these two groups than in the peripheral blood group. Histologically, the cells from autologous bone marrow were found to proliferate into the tracheal tissue during the first month. Cilial movement in these two groups was faster than that in the peripheral blood group and recovered to 80% to 90% of the normal level. CONCLUSIONS Bone marrow aspirate and mesenchymal stem cells enhance the regeneration of the tracheal mucosa on this prosthesis. This in situ tissue engineering approach may facilitate tracheal reconstruction in the clinical setting.

New type of tracheal bioartificial organ treated with detergent: maintaining cartilage viability is necessary for successful immunosuppressant free allotransplantation.

The present investigation examined the key factors in the preparation of low-antigenic tracheal allografts by detergent treatment in dogs. In group 1 (n = 5), the grafts were treated by detergent at room temperature for 48 h and rinsed with running water until use. In group 2 (n = 4), detergent treatment was performed at 4°C for 48 h, but the rinsing step was omitted. In group 3 (n = 6), the grafts were treated at 4°C for 48 h, rinsed thoroughly with physiologic saline, and stored at 4°C. The grafts were then used for tracheal replacement without immunosuppressant in dogs. The epithelium and mixed glands had been removed completely from the grafts in groups 1 and 2, and in five of the six grafts of group 3. In groups 1 and 2, cartilage viability appeared to have been lost and all animals died of airway stenosis within 38 days. In group 3, the chondrocytes were viable and all animals survived uneventfully. These results suggest that maintaining cartilage viability in a tracheal graft is necessary for successful immunosuppressant-free allotransplantation and, consequently, to maintain an open airway. The treatment temperature and sufficient rinsing are key factors for maintaining cartilage viability of grafts.

Influence of dehydrothermal crosslinking on the growth of PC-12 cells cultured on laminin coated collagen.

Recently, we have demonstrated canine peroneal nerve regeneration with functional recovery across an 80 mm gap using a polyglycolic acid (PGA) -collagen tube filled with laminin coated collagen fibers. In that study, the laminin coating was applied before a dehydrothermal (DHT) treatment designed to extend preservation of laminin in situ. To address concerns that the biological activity of laminin might consequently be reduced, the present investigation examined the influences of DHT crosslinking on the activity of laminin in terms of neural cell growth in vitro. DHT crosslinking was performed on collagen (type I or IV) spread on glass in three groups: (1) before coating, (2) after coating, and (3) both before and after coating. PC-12 cells were disseminated in each of the three groups. All three groups were cultured, and the number of cells were compared statistically. Cell growth achieved through application of laminin coating after DHT crosslinking was statistically greater than that achieved when laminin coating was performed before crosslinking. A reduction in laminin activity induced by DHT crosslinking was demonstrated. The optimal timing for the crosslinking of biomaterials treated with trophic factors such as laminin should be examined in terms of the effects of crosslinking on the activity of the trophic factors.

Tracheal allotransplantation in beagle dogs without immunosuppressants.

BACKGROUND The antigenicity of tracheal grafts is still unclear. We investigated the possibility of performing tracheal allotransplantation without immunosuppressants. METHODS Intrathoracic five-ring tracheal replacements were performed in beagle dogs without immunosuppressants (n = 18). The dogs were divided into 9 pairs, and grafts were exchanged within the pairs. In group 1 (n = 6), the paired dogs were blood relatives, whereas in group 2 (n = 12), the paired dogs were not related. Full-thickness skin transplantation was also performed in both groups. RESULTS In group 1, 5 animals survived uneventfully for more than 3 months. No stenosis was observed in any of the dogs. In group 2, the grafts were incorporated by the host trachea in 2 dogs. Four animals died of airway obstruction within 3 months. Moderate or slight airway stenosis was observed in 6 dogs. Rejection was confirmed by histologic examination. In both groups, all of the skin allografts were destroyed within 2 weeks. CONCLUSIONS After tracheal allotransplantation, long-term survival was achieved, especially in recipient dogs that were blood relatives of donors. We conclude that it is possible to perform tracheal allotransplantation using histocompatible matched grafts without immunosuppressants.

Regeneration of the junctional epithelium and connective tissue after transplantation of detergent-processed allo-teeth.

The authors have developed a new artificial dental implant and evaluated it in a dog model in terms of its potential to produce: I) regeneration of junctional epithelium; II) regeneration and attachment of connective tissue. The implants were constructed from allo-teeth. We removed the cell components from the periodontal ligaments of these teeth with a detergent (1% TritonX-100); the remaining acellular periodontal ligament acted as an extracellular matrix upon which regeneration and attachment could proceed. We placed 10 of these implants in the just-extracted sites of three beagle dogs. We observed regeneration of both junctional epithelium and connective tissue at all implant sites after 3 months. The connective tissue was attached in all cases. Use of the acellular periodontal ligament as an extracellular matrix may facilitate regeneration of host periodontal ligament tissue, thus contributing to recovery of host immunological defense and long-term oral function.

MECHANICAL PROPERTIES OF MESH-TYPE ARTIFICIAL TRACHEAS

1 緒 言 気管は気管軟骨によって形状を保持している.気管の 骨格は気管の円周に沿って存在する軟骨切片の連なりで 形成されており,吸気の際には気管の過度の狭窄を,呼 気の際には気管の過度の膨張を防ぐ役割を果たしている. 気管中の軟骨のない部分は主に平滑筋からなっており, 気管膜様部と呼ばれている. 疾患や外傷で気管が損傷を受けたとき,人工物を用い て気管を再建しようとする試みは以前から行われてきた. 1960年代後半にはシリコンチューブを用いた人工気管が 考案された.1)シリコンは化学的に安定で柔軟な素材であ るという点で臨床的に有用であった.2)~ 4)しかしながら, シリコンチューブの人工気管では内腔に線毛が再生しな いため,内腔に痰などの異物が溜まり狭窄を起こしてし まうという問題点があった.最近,新しいタイプのメッ シュ型人工気管が開発された.5)~ 7)このメッシュ型人工気 管は円筒状のメッシュにステントをらせん状に巻きつけ ることによって内腔を保持している.また,気密性を向 上させるとともに結合組織・粘膜組織の進入を促すため, メッシュの両面をコラーゲンで被覆してある.この人工 気管は,埋入後は生体組織に害を与えることなく体内に 留まり,内腔には線毛を持つ粘膜が再生することが確認 されている. これまで生体の気管の力学特性に関する研究は数多く なされてきた.8)~ 19)人工気管の力学特性は実用化の上で大 変重要な要素であったが,上述のメッシュ型人工気管の 物性についての実験・解析はほとんど行われていなかっ た.20), 21)メッシュ型人工気管は内外から受ける力に耐え うるかたさを必要とするため,人工気管の形状を保持す る役割を担うステントの変形特性を明らかにすることは 非常に重要となる.本研究ではステントの材質,断面形 状,巻き方などが異なるメッシュ型人工気管を作製し, 側面圧縮試験を行った.各試料の力と相対たわみ量の関 係を比較することによってステントがメッシュ型人工気 管の力学特性に及ぼす影響を調べた.イヌの気管につい ても圧縮特性を調べ,メッシュ型人工気管と比較検討を 行った. 2 実 験 2・1 試料 ポリプロピレン (PP) 製のMarlexメッシュの円筒に, ナイロン (NY)(直径;0.70mm)をらせん状に巻きつけ た人工気管試料 (NY-s) と NY糸のリングを連ねた人工 気管試料 (NY-r) を作製した.NY-rについては単位長さ 当たりのリング数が異なる 3種類の試料 (NY-ra, NY-rb, NY-rc) を作製した.NY-rcの写真を Fig. 1.に示す.PP 糸(直径;0.80mm)のリングを連ねた試料 (PP-r) も作 製した.円筒状のメッシュに PPの板から切り出した長 方形の切片(断面の厚さ 1.0mm,幅 3.0mm)をリング 状に巻きつけた試料 (PP-R) と,生体の気管の形に倣っ て,円周の約 4分の 3にわたって PPの切片を巻きつけた 試料 (PP-A) も作製した.ステントに PPを用いた試料は ステントを円筒状のメッシュの外側に溶着した後,6-0プ ロリン縫合糸(直径;0.070~ 0.099mm)を用いて補強 した.ステントに NYを用いた試料はステントをメッ メッシュ型人工気管の力学特性 野田澤 俊 介 中 村 達 雄 清 水 慶 彦 瀧 川 敏 算 Mechanical Properties of Mesh-Type Artificial Tracheas