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Background: Critical obstacles must be addressed before clinical application of artificial tracheas. The major complications of long tracheal replacement include anastomotic dehiscence and stenosis owing to poor vascularity and incomplete reepithelialization. The objective of this report was to clarify whether preincubation of the prosthesis in the omentum could be applicable for reconstruction of a long segment of the intrathoracic trachea in a canine model., Methods: The framework of an artificial trachea was fabricated from a polypropylene mesh tube and coated with 1% neutral atelocollagen inside and outside the lumen. The prosthesis was placed in the omentum of 9 healthy male beagle dogs for 3 weeks. Then, the pedicled prosthesis was used to replace a 50-mm-long section of intrathoracic trachea. Results were evaluated bronchoscopically, macroscopically, and histologically., Results: After 3 weeks of abdominal incubation, the prostheses were incorporated into the host tissue. None of the dogs showed dehiscence of the anastomosis or infection of the prostheses during the postoperative period. Seven of the 9 dogs survived for more than 1 year. One dog died of a bowel obstruction resulting from a diaphragmatic hernia 3 months after replacement, and another died due to reasons unrelated to the prosthesis at 6 months. Bronchoscopic examination revealed no stenosis or dehiscence, and microscopic examination of all dogs showed that the luminal surface was covered by newly regenerated connective tissue and respiratory epithelium., Conclusions: Pedicled omentum-prosthesis complexes may allow successful reconstruction of a long segment of the intrathoracic trachea., (Copyright © 2022 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: It is unclear whether the movement and function of the regenerated cilia on collagen-conjugated artificial trachea are the same as those of normal cilia. This study assessed the ciliary beat frequency (CBF) and ciliary transport functions (CTFs) of regenerated cilia in a canine model., Methods: A tracheal defect introduced into the anterior portion of the cervical trachea of an adult beagle dog was covered with a collagen-conjugated prosthesis. Two months later, the trachea was harvested along the long axis, both from normal and regenerated regions. The cilia were stained with isothiocyanate-conjugated wheat germ agglutinin, and their movement was monitored with a high-speed camera to analyse CBF and CTF. Four samples each were obtained from the regenerated and normal regions for CBF analysis and 7 samples each were obtained for CTF analysis., Results: The wheat germ agglutinin-stained cells showed well-regulated beats in both the regenerated and normal regions of the trachea. Mean CBF in the regenerated and normal regions did not differ significantly (7.11 ± 0.41 vs 7.14 ± 1.09 Hz; P = 981). By contrast, CTF was significantly lower in the regenerated region than in the normal region (30.0 ± 6.62 vs 7.43 ± 0.58 μm/s; P = 0.005)., Conclusions: Mean CBF in the regenerated and normal regions did not differ significantly at 2 months. The CTF in the regenerated region recovered partially but remained lower than those in the normal region. Methods are needed to improve the CTF of regenerated cilia., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Tracheal resection is often performed for malignant tumours, congenital anomalies, inflammatory lesions, and traumatic injuries. There is no consensus on the best approach for the restoration of tracheal functionality in patients with tracheal defects. Artificial grafts made of polypropylene and collagen sponge have been clinically used by our group. However, 2 months are required to achieve adequate epithelialization of the grafts in humans. This study aimed to investigate the feasibility of transplantation therapy using an artificial trachea with human-induced pluripotent stem cell (hiPSC)-derived multiciliated airway cells (hiPSC-MCACs). Collagen vitrigel membrane, a biocompatible and absorbable material, was used as a scaffold to cover the artificial trachea with hiPSC-MCACs. Analyses of hiPSC-MCACs on collagen vitrigel membrane were performed by immunocytochemistry and electron microscopy and by assessing ciliary beat frequency. Along with the artificial trachea, hiPSC-MCACs were transplanted into surgically created tracheal defects of immunodeficient rats. The survival of transplanted cells was histologically evaluated at 1 and 2 weeks after the transplantation. The hiPSC-MCACs exhibited motile cilia on collagen vitrigel membrane. The surviving hiPSC-MCACs were observed in the endotracheal epithelium of the tracheal defect at 1 and 2 weeks after transplantation. These results suggest that hiPSC-MCAC is a useful candidate for tracheal reconstruction., (© 2019 John Wiley & Sons, Ltd.)

Tracheal epithelia have barrier and mucociliary clearance functions that prevent invasion of extraneous particles and infectious materials. Hence, following tracheal reconstructions, functional and morphological regeneration of epithelia is required to prevent respiratory declines and infectious diseases. Although growth factors (GFs) promote the regeneration of tracheal epithelial morphologies, it remains unclear whether tracheal grafts containing GFs are beneficial for regeneration of tracheal epithelial functions. Thus, we fabricated collagen sponge scaffolds containing insulin-like GF-1 (IGF-1) and the basic fibroblast, hepatocyte, and epidermal GFs (bFGFs, HGFs, and EGFs, respectively), and we evaluated the effects of the grafts on the functional regeneration of tracheal epithelia. Partial tracheal defects were imposed surgically, and collagen sponges containing IGF-1, bFGF, HGF, or EGF were then transplanted to defect sites. Subsequent immunofluorescence studies suggested that EGF and bFGF contribute to regular distributions of tight junction molecules, and tracer permeability assays suggested that EGF and bFGF promote regeneration of barrier function. Increased ciliogenesis was also observed using scanning electron microscopy in reconstructed regions treated with EGF- and bFGF-supplemented collagen sponges. However, bFGF-supplemented collagen sponges led to greater microsphere transport than did EGF-supplemented sponges. The present data suggested that collagen sponge scaffold containing bFGF promotes functional regeneration of tracheal epithelial tissues., (© 2019 John Wiley & Sons, Ltd.)

Background: Tracheal reconstruction is complicated by the short length to which a trachea can be resected. We previously developed a biocompatible polypropylene frame artificial trachea, but it lacked the strength and flexibility of the native trachea. In contrast, nitinol may provide these physical characteristics. We developed a novel nitinol frame artificial trachea and examined its biocompatibility and safety in canine models., Methods: We constructed several nitinol frame prototypes and selected the frame that most closely reproduced the strength of the native canine trachea. This frame was used to create a collagen-coated artificial trachea that was implanted into 5 adult beagle dogs. The artificial trachea was first implanted into the pedicled omentum and placed in the abdomen. Three weeks later, the omentum-wrapped artificial trachea was moved into the thoracic cavity. The thoracic trachea was then partially resected and reconstructed using the artificial trachea. Follow-up bronchoscopic evaluation was performed, and the artificial trachea was histologically examined after the dogs were sacrificed., Results: Stenosis at the anastomosis sites was not observed in any dog. Survival for 18 months or longer was confirmed in all dogs but 1, which died after 9 months due to reasons unrelated to the artificial trachea. Histological examination confirmed respiratory epithelial regeneration on the artificial trachea's luminal surface. Severe foreign body reaction was not detected around the nitinol frame., Conclusions: The novel nitinol artificial trachea reproduced the physical characteristics of the native trachea. We have confirmed cell engraftment, good biocompatibility, and survival of 18 months or longer for this artificial trachea in canine models., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Planar cell polarity (PCP) coordinates the patterning and orientation of cells and their structures along tissue planes, and although its acquisition during the formation of airway epithelium has been described, the mechanisms for its maintenance and reconstruction are poorly understood. We aimed to clarify whether ambient environment change by orthotropic autologous transplantation affected PCP at the cellular level., Methods: We performed orthotropic autologous transplantation by inverting tracheal segments in rats, and then performed morphological evaluation by microscopy. The PCP of the tracheal epithelium was assessed over time by analyzing the directions of mucociliary transport and ciliary beat, the positional relationship between the basal body and basal foot, and the bias of Vang-like protein 1 (Vangl1) at 2, 4, and 6 months postoperatively., Results: After 2 months, the directions of mucociliary transport and ciliary beat were preserved toward the lung in the inverted tracheal segments. The positional relationship between the basal body and the basal foot, and the bias of Vangl1, also indicated preservation of PCP in the inverted tracheal segments. Similar results were obtained at 6 months., Conclusion: The PCP of ciliated epithelium was preserved in reversed trachea, even after long-term observation.

Tracheal epithelial cells maintain airway homeostasis by mediating mucociliary clearance. Following tracheal reconstruction, timely epithelial regeneration is required to prevent respiratory compromise and infectious diseases. To achieve rapid tracheal epithelial regeneration, a heparin cross-linked collagen sponge containing fibroblast growth factor-2 (FGF-2) was prepared as a graft for tracheal reconstruction. The heparin cross-linked sponge exhibited a high FGF-2 retaining capacity, and tracheal epithelial and mesenchymal cells cultured in this sponge containing FGF-2 showed high proliferative capacities. Subsequently, heparin-free collagen sponge scaffolds (C/F scaffold) and collagen sponge scaffolds cross-linked with 10 μg/ml heparin retained FGF-2 (C/H10/F scaffold), and were transplanted into rats with tracheal defects. Invasion of both epithelial and non-epithelial cells was greater in rats treated with the C/H10/F scaffold at 1 week post-transplantation than in rats treated with the C/F scaffold. Moreover, at 2 weeks after transplantation, improved cilia formation was observed in the C/H10/F scaffold group, with higher motility and more potent posterior-anterior flow generation than in the C/F scaffold group. These results suggest that heparin improves functional regeneration of tracheal epithelium. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Conclusion: Tight fixation of the artificial trachea is important for epithelialization and tracheal stenosis., Objective: The authors have developed an artificial trachea and have used it for tracheal reconstruction. Although various studies on tracheal reconstruction have been conducted, no studies have examined the effect of artificial tracheal fixation on tracheal stenosis and regeneration. Therefore, the purpose of the present study was to evaluate the effect of artificial tracheal fixation., Study Design: Preliminary animal experiment., Methods: Artificial tracheae were implanted into rabbits with partial tracheal defects. Tracheal stenosis and regeneration of the tracheal epithelium on the artificial tracheae were evaluated by endoscopic examination, scanning electron microscopic analysis, and histological examination. The artificial tracheae fixed to the tracheal defects were classified into three groups (0-point, 4-point, and 8-point) by the number of fixation points., Results: At 14 and 28 days post-implantation, the luminal surface of the implantation area was mostly covered with epithelium in all fixation groups. However, a small amount of granulation tissue was observed in the 0-point fixation group at 14 days post-implantation. Moreover, tracheal stenosis did not occur in the 8-point fixation group, but stenosis was detected in the other groups.

Objective: We developed an in situ regeneration-inducible artificial trachea composed of a porcine collagen sponge and polypropylene framework and used it for tracheal reconstruction. In the present study, collagen sponges with different structures were prepared from various concentrations of collagen solutions, and their effect on the regeneration of tracheal epithelium was examined., Methods: Collagen sponges were prepared from type I and III collagen solutions. The structures of the sponges were analyzed using scanning electron microscopy (SEM). Artificial tracheae, which were formed using the collagen sponges with different structures, were implanted into rabbits, and regeneration of the tracheal epithelium on the artificial tracheae was evaluated by SEM analysis and histological examination., Results: The SEM analysis showed that collagen sponges prepared from 0.5% and 1.0% collagen solutions had a porous structure. However, the sponges prepared from a 1.5% collagen solution had a nonporous structure. After implantation of artificial tracheae prepared from 0.5% and 1.0% collagen solutions, their luminal surfaces were mostly covered with epithelium within 14 days. However, epithelial reorganization occurred later on artificial tracheae prepared from the 1.5% collagen solution., Conclusion: Collagen sponges with a porous structure are suitable for regeneration of the tracheal epithelium in our artificial trachea., (© The Author(s) 2015.)

Background: Mesenchymal stromal cells (MSCs) have been intensively investigated in regenerative medicine. Among the different types of MSCs, adipose tissue-derived stromal cells (ASCs) can be obtained with relatively less invasive techniques. Since ASC administration is a candidate strategy for the treatment of refractory diseases including pulmonary fibrosis, we investigated whether intratracheal injection of ASCs had therapeutic potential against bleomycin (BLM)-induced lung injury in rats., Methods: BLM was intratracheally administered to rats, and 1 week later ASCs were harvested. Two weeks after BLM treatment, ASCs or phosphate-buffered saline (PBS) were injected autologously into the rats via the trachea A semi-quantitative histological evaluation was conducted to assess the injured lungs, followed by cell tracing at 3 or 6 weeks after BLM instillation., Results: ASC administration did not affect the severity of lung damage on the third week after BLM exposure, but prevented further aggravation of the lung injury, as apparent on the sixth week. A fluorescent cell tracer revealed that the majority of ASCs did not appear to have penetrated inside the lung region injured by BLM on the third week after BLM instillation, but some of these cells sprouted deep into the thick distorted architecture of the injured lung on the sixth week after the BLM instillation., Conclusions: The results of the present study suggest that ASCs may play a role in the prevention of ongoing aggravation of lung injury in the long term.

Objectives: We herein report on development of a composite (synthetic and biological) tracheal scaffold with vascularized autologous connective tissue in the omentum, followed by in situ tissue engineering of the composite scaffold with the pedicled omentum. In this preliminary report, we focus on development and evaluation of the vascularized autologous connective tissue in the omentum., Methods: In animal experiment 1, a polypropylene framework as a synthetic component was placed in the omental sac for 3 weeks and another was placed in the pouch of Douglas as a control in five beagle dogs. In animal experiment 2, a polypropylene framework placed in the omental sac for 3 weeks was compared with a polypropylene framework coated with porcine atelocollagen, which was also placed in the omental sac in another five dogs, to investigate whether the coating of porcine atelocollagen contributes to development of more vascularized connective tissue. Macroscopic, radiological and histological evaluations were performed for developed autologous connective tissue on the frameworks, with a focus on its thickness and capillary vessels., Results: In animal experiment 1, the polypropylene framework in the omentum developed a composite tracheal scaffold with homogeneous and significantly thicker (2.6 ± 0.5 vs 1.2 ± 0.4 mm, P <0.0001) connective tissue in which more capillary vessels per 10-power field of view (3.5 ± 2.2 vs 0 ± 0, P = 0.015) were identified, compared with the control in the pouch of Douglas. In animal experiment 2, the omentum developed significantly thicker connective tissue on the polypropylene framework coated with porcine atelocollagen (3.6 ± 0.7 vs 2.2 ± 0.4 mm, P <0.0001) in which not significantly more capillary vessels were identified (3.5 ± 2.2 vs 5.0 ± 2.7, P = 0.12), compared with the framework that was not coated., Conclusions: Placement of the polypropylene framework in the omental sac resulted in development of homogeneous and vascularized autologous connective tissue on the polypropylene framework for a composite tracheal scaffold. The framework coated with porcine atelocollagen did not show an additional benefit in inducing vascularization. This preliminary report will be followed by the long-term evaluations of in situ tissue engineering of the composite tracheal scaffold., (© The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Objectives/hypothesis: In this study, a bioengineered trachea composed of autologous chondrocytes was developed, and its effect on cartilaginous regeneration was evaluated by implantation into tracheal defects in rabbits., Study Design: Prospective controlled trial in an animal model., Methods: The tracheal prosthesis used in this study was composed of polypropylene (the frame) and collagen sponge (the scaffold). Chondrocytes were harvested from the costal cartilage of rabbits and seeded into the tracheal prosthesis. The bioengineered trachea, consisting of the tracheal prosthesis with chondrocytes, was implanted into surgically created tracheal defects of rabbits in the bioengineered group, and a tracheal prosthesis without chondrocytes was implanted in the control group., Results: After implantation, the presence of regenerated cartilage was observed in the bioengineered trachea but not in the tracheal prosthesis without chondrocytes., Conclusions: It was confirmed in this study that the implanted chondrocytes proliferated in an appropriate portion of the tracheal defect and that the partially resected tracheal cartilage was repaired with regenerated cartilaginous tissue into a ring-shaped form as a whole. These results demonstrate the feasibility of cartilaginous regeneration using a bioengineered trachea with autologous chondrocytes., (Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Objective: The surgical procedure for subglottic stenosis is technically challenging when the vocal cords are involved and concomitant management for glottic involvement is required. After total cricoidectomy and laryngotracheal anastomosis, T-tube placement for 3 to 6 months is recommended. Bone grafts might shorten this period. We report the histologic and endoscopic changes after total cricoidectomy with or without bone grafts in a canine model to suggest an appropriate period for T-tube placement and the necessity for bone grafts., Methods: Ten dogs underwent total cricoidectomy and laryngotracheal anastomosis with or without bone grafts harvested from the ribs. Endoscopic examination was performed monthly, and 1 dog from both groups was humanely killed at 1, 2, 3, 6, and 12 months. The T-tube was removed before death in the dogs killed at 1, 2, and 3 months and at 3 and 6 months in those killed at 6 and 12 months, respectively., Results: Endoscopically, the glottic opening was in good condition in all dogs, except for 1 that had glottic stenosis. Histologically, active lymphocyte infiltration was observed in dense collagen fibers at the anastomosis at 1 month. At 2 and 3 months, fibroblasts were evident, suggesting active collagen fiber production. At 6 and 12 months, the collagen fibers had become looser. The bone grafts were intact and did not influence the surrounding tissue., Conclusions: In the canine model, 6 months of T-tube placement is probably sufficient; however, 3 months of placement might not be. Additionally, no difference was found between the dogs with and without a bone graft., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

The treatment of laryngotracheal stenosis remains a challenge as treatment often requires multistaged procedures, and successful decannulation sometimes fails after a series of operations. Induced pluripotent stem cells (iPSCs) were generated in 2006. These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue-engineering applications. We have previously reported tracheal wall regeneration using a three-dimensional (3D) scaffold containing iPSCs. However, the efficiency of differentiation into cartilage was low. In addition, it could not be proven that the cartilage tissues were in fact derived from the implanted iPSCs. The purpose of this study was to evaluate and improve the use of iPSCs for the regeneration of tracheal cartilage. iPSCs were cultured in vitro in a 3D scaffold in chondrocyte differentiation medium. After cultivation, differentiation into chondrocytes was examined. The ratio of undifferentiated cells was analyzed by flow cytometry. The 3D scaffolds were implanted into tracheal defects, as an injury site, in 24 nude rats. Differentiation into chondrocytes in vitro was confirmed histologically, phenotypically, and genetically. Flow cytometric analysis demonstrated that the population of undifferentiated cells was decreased. Cartilage tissue was observed in the regenerated tracheal wall in 6 of 11 rats implanted with induced iPSCs, but in none of 13 rats implanted with the control and noninduced iPSCs. The expression of cartilage-specific protein was also demonstrated in vivo in 3D scaffolds containing iPSCs. The presence of the GFP gene derived from iPSCs was confirmed in samples of cartilage tissue by the combination of laser microdissection (LMD) and polymerase chain reaction (PCR) techniques. Our study demonstrated that iPSCs have the potential to differentiate into chondrogenic cells in vitro. Cartilage tissue was regenerated in vivo. Our results suggest that iPSCs could be a new cell source for the regeneration of tracheal cartilage.

Conclusion: Our technique for tracheal regeneration using a collagen vitrigel scaffold affords a feasible approach for accelerating epithelial regeneration on the intraluminal surface of the host tracheal defect., Objectives: The purpose of the study was to quantitatively evaluate the effectiveness of a collagen vitrigel scaffold for the regeneration of the tracheal epithelium., Methods: The collagen vitrigel scaffold was developed by conjugating a collagen vitrigel membrane to a collagen sponge to promote both epithelial cell growth and mesenchymal cell infiltration. The collagen vitrigel scaffold was implanted into tracheal defects in rats as the vitrigel model, and a conventional collagen sponge was implanted as a control model. The structure of the regenerated tissue was observed and thickness of the epithelium was quantitatively evaluated by histological examination., Results: Histological findings showed the surface of the collagen vitrigel scaffold to be flat in comparison with that of the conventional collagen sponge. At 7, 14, and 28 days post-implantation, the average thickness of the regenerated epithelial layer in the vitrigel model group was greater than that in the control group.

Objectives: Our group has had good results in tracheal mucosal regeneration using a collagen vitrigel-sponge scaffold in an animal model. In this study, the effectiveness of this scaffold with the application of basic fibroblast growth factor (b-FGF) was investigated., Methods: A collagen vitrigel-sponge scaffold was fabricated with simultaneous addition of b-FGF. Three types of collagen vitrigel-sponge scaffolds were made: no b-FGF, 10 ng of b-FGF, and 100 ng of b-FGF. At 3, 5, 7, and 14 days after implantation in rats, the tracheas were removed and histologically evaluated. The regeneration of mucosal epithelium and the subepithelial layer was evaluated., Results: Mucosal epithelium, including pseudostratified epithelium and ciliated cells, regenerated earlier in the scaffolds when b-FGF was applied than when b-FGF was not applied. Regeneration of the subepithelial layer, infiltration of inflammatory cells and fibroblasts, and angiogenesis were promoted earlier in the scaffolds with b-FGF application., Conclusions: Our technique for tracheal reconstruction using collagen vitrigel-sponge scaffolds with b-FGF application affords a feasible approach for accelerating the regeneration of the intraluminal surface and subepithelial layer of tracheal tissue.

Background: Treatment for adult subglottic stenosis is technically demanding and no therapeutic algorithm exists. We performed the present meta-analysis of treatment for this condition in an attempt to compare efficacy on the basis of type of procedure., Methods: We identified 24 eligible retrospective studies reporting the therapeutic results for inclusion criteria. Meta-analysis was performed by combining the results of the reported success rates; success is defined as a condition requiring no further treatment. The relative risk was used as a summary statistic., Results: Pooled success rates of laryngotracheal resection and anastomosis (12 articles) and laryngoplasty with or without grafting (7 articles) were 95% and 76%, respectively, using a random-effects model. Success rates of endoscopic dilatation and laser resection (6 articles) varied between 40% and 82%. Meta-regression analysis showed a significant difference in the success rates between laryngotracheal reconstruction and laryngoplasty and between laryngotracheal reconstruction and an endoscopic procedure. When the indication for endoscopic management was a lesion size less than 1 cm, the results were significantly better., Conclusions: The success rate of laryngotracheal reconstruction is significantly higher than that of laryngoplasty or endoscopic intervention; however, endoscopic intervention is worth trying for lesions smaller than 1 cm without framework destruction., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Since the 1980s, tissue engineering has become one of the major areas of endeavor in medical research, applying the principles of biology and engineering to the development of functional substitutes for damaged tissue. Using this technology, various attempts have been made to create and apply a tissue-engineered prosthetic trachea, or airway. In addition to the conventional tissue engineering approach, a new substantially different concept has been advocated in Japan since 2000. This is "in situ tissue engineering," where a tissue is created not in vitro but in vivo, exploiting the potential of the living body for wound healing. An artificial trachea created by in situ tissue engineering has already been applied in human patients for reconstruction of airway defects, and promising results have been obtained. This article reviews recent progress in the relatively new field of airway reconstruction employing tissue engineering., (© The Japanese Association for Thoracic Surgery 2011)

Objectives: Our previous studies focused on basic research and the clinical applications of an artificial trachea. However, the prefabricated artificial trachea cannot be utilized for pediatric airways, because the tracheal frame needs to expand as the child develops. The purpose of this study was to evaluate the potential of induced pluripotent stem (iPS) cells for the regeneration of the tracheal wall., Methods: We cultured iPS cells in a 3-dimensional (3-D) scaffold in chondrocyte differentiation medium (bioengineered scaffold model), and the results were compared with those in a 3-D scaffold without iPS cells (control scaffold model). The 3-D scaffolds were implanted into tracheal defects in 8 nude rats. After 4 weeks, the regenerated tissue was histologically examined., Results: Implanted iPS cells were confirmed to exist in all 5 rats implanted with bioengineered scaffolds. Cartilage-like tissue was observed in the regenerated tracheal wall in 2 of the 5 rats in the bioengineered scaffold model, but in none of the 3 rats in the control scaffold model., Conclusions: Implanted iPS cells were confirmed to exist in the bioengineered scaffold. Cartilage-like tissue was regenerated in the tracheal defect. This study demonstrated the potential of iPS cells in the regeneration of the tracheal wall.

In some types of tracheal disease, tracheal resection is required. For patients with tracheal resection, artificial grafts, made from collagen sponge with a spiral polypropylene stent and mesh, have been clinically used by our group. However, epithelial regeneration was confirmed to be slow. In the present study, we investigated the potential of gingival fibroblasts (GFBs) and adipose-derived stem cells (ASCs) as autologous transplanted cells in combination with artificial graft for tracheal epithelial regeneration. In in vitro co-culturing with tracheal epithelial cells, GFBs stimulated epithelial cell differentiation and reconstruction of a pseudostratified epithelium. ASCs stimulated epithelial cell proliferation and reconstruction of a multi-layered epithelium. Subsequently, we prepared three kinds of bioengineered scaffolds from GFBs and/or ASCs and implanted them into rat tracheal defects. The bioengineered scaffolds containing GFBs were covered with tracheal epithelial cells after 1 week, and highly ciliated epithelium was formed after 2 weeks of transplantation. The bioengineered scaffold containing ASCs induced thick epithelium, and then pseudostratified epithelium containing goblet cells was formed. Furthermore, the application of both GFBs and ASCs had synergistic effects on tracheal epithelial regeneration, suggesting that bioengineered scaffolds containing GFBs and ASCs are useful for hastening tracheal epithelial regeneration., ((c) 2010 Elsevier Ltd. All rights reserved.)

Objective: We have developed a prosthesis that includes a collagen layer for tracheobronchial reconstruction and applied it in a canine model. In previous studies luminal epithelization remained partial or rather slow because of the early disintegration of the collagen layer. We have improved this type of prosthesis by coating the luminal surface with a biodegradable polymer, which serves to protect the collagen layer. The effect of the polymer coating on the epithelization of the luminal surface of the prosthesis was examined., Methods: The main frame consisted of a polypropylene mesh tube, measuring 15 mm in inner diameter and 30 mm in length, with reinforcing rings. Collagen extracted from porcine skin was conjugated to this frame. The luminal surface was coated with a polymer, poly (L-lactic-acid-co-epsilon-caprolactone). In 5 beagle dogs the left main bronchus was replaced with this prosthesis, periodic bronchoscopic observations were conducted, and microscopic evaluations were performed., Results: All dogs survived until they were killed, except for 1 animal in which pneumonia developed, and this animal died at 13 months after replacement. None of the dogs showed adverse complications caused by the prosthesis. Bronchoscopic observations revealed that the polymer remained on the luminal surface for 2 weeks. The luminal surface in 4 dogs was completely covered with ciliated columnar epithelium or nonciliated squamous epithelium, and 90% epithelization was achieved in 1 dog., Conclusions: The biodegradable polymer coating protected the collagen layer and promoted better epithelization. This improved epithelization on the luminal surface could therefore potentially increase the success rates in airway replacement with artificial prostheses., (Copyright 2010 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Objectives: Although our group has had mostly successful results with clinical application of a tracheal prosthesis, delayed epithelial regeneration remains a problem. In our previous studies using rats, it was demonstrated that tracheal fibroblasts accelerated proliferation and differentiation of the tracheal epithelium in vitro and in vivo. The purpose of this study was to evaluate the effects of fibroblasts on epithelial regeneration in larger tracheal defects in rabbits., Methods: We developed a bioengineered scaffold, the luminal surface of which was coated with fibroblasts. This scaffold was implanted into tracheal defects in 12 rabbits (bioengineered group), and scaffolds without fibroblasts were implanted in 12 rabbits (control group). The regenerated epithelium was histologically examined by light microscopy, scanning electron microscopy, and immunohistochemical studies., Results: In the bioengineered group, a stratified squamous epithelium was observed on the surface 7 days after transplantation. However, in the control group, the scaffolds were exposed. Fourteen days after implantation, a columnar ciliated epithelium was observed in the bioengineered group. The average thickness of the regenerated epithelium in the bioengineered group was significantly greater than that in the control group., Conclusions: This study indicated that fibroblasts had a stimulatory effect that hastened regeneration of the epithelium in large tracheal defects.

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.

Objectives: The objective of the present study was to demonstrate the efficacy of the clinical application of in situ tissue engineering using a scaffolding technique for laryngeal and tracheal tissue., Methods: We have developed a tissue scaffold made from a Marlex mesh tube covered by collagen sponge. Based on successful animal experimental studies, in situ tissue engineering with a scaffold implant was applied to repair the larynx and trachea in 4 patients., Results: In 1 patient with subglottic stenosis, the thyroid cartilage, cricoid cartilage, and cervical trachea with scarring and granulation were resected and reconstructed by use of the scaffold. In 3 patients with thyroid cancer, the trachea and cricoid cartilage with tumor invasion were resected and the scaffold was implanted into the defect. Postoperative endoscopy during the observation period of 8 to 34 months showed a well-epithelialized airway lumen without any obstruction., Conclusions: Our current technique of in situ tissue engineering using a scaffold shows great potential for use in the regeneration of airway defects.

Objectives: The purpose of the current study was to demonstrate the efficacy of in situ tissue engineering of the cricoid and trachea in a canine model., Methods: Marlex mesh tube reinforced with polypropylene threads and covered by collagen sponge was used as a tissue scaffold for airway regeneration in 9 beagle dogs. The anterior half of the cricoid cartilage was resected in 5 dogs, whereas the cricoid cartilage and cervical trachea were simultaneously resected in 4 dogs. The tissue scaffold was implanted into the resultant defect., Results: Endoscopic examination showed no airway obstruction for a postoperative period of 3 to 40 months in all dogs. Granulation tissue was observed in 2 dogs, and slight mesh exposure in 1 dog, although all were asymptomatic. Light microscopy and electron microscopy showed the endolaryngeal and endotracheal lumen to be covered by ciliated epithelium. According to strain-force measurement, the framework was firmly supported by regenerated tissue, as well as the normal cricoid and trachea., Conclusions: Our current tissue scaffold provides a rigid framework for the airway, and the collagen coating invites tissue regrowth around the tube. This study presents the possibility of successful reconstruction of the cricoid and trachea with epithelial regeneration by means of in situ tissue engineering.

Objectives: Our group applied a tracheal prosthesis, which was composed of polypropylene as the frame and collagenous sponge as the scaffold, to the first human case and had successful results. The objective of this study was to find a way to acquire more rapid re-epithelialization with fibroblasts on this tracheal prosthesis., Methods: Tracheal epithelial cells, which were isolated from the trachea of rats, were suspended in a collagenous gel. The collagenous gel with fibroblasts was layered on a collagenous sponge. The grafts of this "bioengineered trachea" were implanted into tracheal defects of rats, and the regenerated epithelium on the grafts was histologically examined., Results: Seven days after implantation, stratified squamous epithelium covered almost all of the surface of the gel, and some of the implanted fibroblasts in the gel were lined up just below the epithelium. Fourteen days after implantation, columnar and cuboidal ciliated epithelium covered almost all of the surface of the defects, and the implanted fibroblasts had disappeared. The numbers of regenerated epithelial cells at 14 days after implantation were larger than those of control models without fibroblasts, with statistical significance., Conclusions: The results suggested that the grafts of bioengineered trachea composed of collagenous sponge and collagenous gel with tracheal fibroblasts accelerated epithelial differentiation and proliferation in vivo.

The tracheal epithelium maintains the health of the respiratory tract through mucociliary clearance and regulation of ion and water balance. When the trachea is surgically removed, artificial grafts have been clinically used by our group to regenerate the trachea. In such cases, the tracheal epithelium needs 2 months for functional regeneration. Previous study has shown that fibroblasts facilitate tracheal epithelial regeneration. In this study, heterotopic fibroblasts originating from the dermis, nasal, and gingival mucosa were cocultured with tracheal epithelial cells to evaluate their potential as autologous transplanted cells for tracheal epithelial regeneration. The epithelia induced by the heterotopic fibroblasts showed differences in structure, cilia development, mucin secretion, and expression of ion and water channels. These results indicated that nasal fibroblasts could not induce mature tracheal epithelium and that dermal fibroblasts induced epidermis-like epithelium. Only the gingival fibroblasts (GFBs) could induce morphologically and functionally normalized tracheal epithelium comparable to the epithelium induced by tracheal fibroblasts. Epithelial cell proliferation and migration were also upregulated by GFBs. These results indicate that GFBs are useful as autologous transplant cells for tracheal epithelial regeneration.

Objectives: The aims of this study are to investigate the efficiency of a tissue engineering approach to partial tracheal reconstruction and to improve epithelialization of the reconstructed trachea. The trachea must be resected in some cases of cancer or trauma. Various restructuring techniques are used, with no consensus on the best approach. Two problems that arise when treating tracheal defects by conventional techniques are an inability to regenerate ciliated epithelium at the reconstructed site and having to perform multiple procedures to achieve the desired repair. This study is designed to address these problems., Study Design: Preliminary, an animal experiment., Methods: Surgery was performed on five adult beagles under anesthesia. After the making of a longitudinal cervical skin incision, the trachea was exposed and a circular defect created. A polypropylene and collagen scaffold preclotted with peripheral blood was inserted to the defect site. Postoperatively, the site was evaluated fiberscopically, histologically, and radiographically., Results: All dogs did well postoperatively. Fiberscopic examination showed that the implanted scaffolds were completely covered with regenerated mucosa with capillaries in all cases. Histologic data showed ciliated epithelium regenerated at the operated site from 1 month postoperatively. Newly formed cartilage was detected in the specimens from 8 to 12 postoperative months. Computed tomography images revealed the fine luminal contour of the regenerated site., Conclusions: Good epithelial regeneration was observed after repair of a round tracheal resection using a simple tissue engineering technique, making the technique a good substitute for conventional approaches to tracheal reconstruction in patients with cancer or trauma.

Several artificial grafts for covering deficient trachea have been produced through tissue engineering. Recently, our group clinically used an artificial trachea made from collagen sponge for patients with noncircumferential tracheal resection. However, the slowness of epithelial regeneration on the surface of the artificial trachea was confirmed as one particular problem. In this study, we co-cultured tracheal epithelial cells with fibroblasts and examined effects of fibroblasts on epithelial regeneration in vitro. Fibroblasts activated epithelial cell proliferation and migration. In co-culture with fibroblasts, epithelial cells reconstructed pseudostratified epithelium, which was composed of ciliated, goblet, and basal cells. Furthermore, a basement membrane was reconstructed between epithelial cells and fibroblasts, and integrin beta4 was also observed there. Fibroblasts rapidly increased mucin secretion by epithelial cells. These results indicate that stimulatory effects of fibroblasts on epithelial cell migration, proliferation, and differentiation would reduce the time required for covering of epithelial cells on the defect of luminal surface and hasten regeneration of morphologically and functionally normalized epithelium involving the reconstruction of basement membrane.

Objectives: The slowness of epithelialization on the artificial trachea that has been successfully used in humans is a problem. The purpose of this study was to develop a way to regenerate the epithelium on the surface of this artificial trachea., Methods: In an in vitro study, isolated rat tracheal epithelial cells were seeded on a collagenous gel that was stratified on a collagenous sponge. Histologic and immunohistochemical examinations were made. In an in vivo study, we transplanted grafts with green fluorescent protein-positive tracheal epithelial cells onto the tracheal defects of normal rats. At 3, 7, 14, and 30 days after the operation, histologic and immunohistochemical examinations were made., Results: In the in vitro study, the 3 layers--the epithelium, gel, and sponge--could be observed. The epithelium expressed cytokeratin 14, cytokeratin 18, and occludin. In the in vivo study, the artificial trachea was covered with epithelium at 3 days after operation, and then the epithelium differentiated from single- or double-stratified squamous epithelium into columnar ciliated epithelium. Green fluorescent protein-positive cells were found 3 days after operation., Conclusions: We believe that the method used in our experiment is an effective way to regenerate the epithelium on the surface of an artificial trachea. With further experimentation, this method should be suitable for clinical application.

Objectives: The objective of the present study was to demonstrate regenerative medicine of the tracheal tissue by using an in situ tissue engineering technique for airway reconstruction., Methods: Based on the previous successful experimental animal studies, the current regenerative technique was applied to repair of the trachea of a 78-year-old woman with thyroid cancer. A Marlex mesh tube covered by collagen sponge was used as a tissue scaffold. The operative intervention included right hemithyroidectomy, resection of the trachea, and tracheoplasty using the scaffold. The right half of three rings of the trachea was resected, and the scaffold material was sutured to the defect of the trachea., Results: After 2 weeks, the mesh collagen structure of the artificial material could be seen with endoscopy in most of the implanted area. The artificial material was covered with epithelial growth after 2 months. Epithelialization continued to cover the artificial material completely for 2 years without any complications., Conclusions: The current regenerative technique avoided tracheotomy, a second operation, and deformity. Good epithelialization has been observed on the tracheal luminal surface without any complications for 2 years. Although long-term observation is required, regenerative medicine of the tracheal tissue appears feasible for airway reconstruction.

Objectives: We investigated whether bone morphogenetic protein 2, released slowly from a gelatin sponge, could induce cartilage regeneration in a canine model of tracheomalacia and evaluated the long-term results., Methods: A 1 x 5-cm gap was made in the anterior cervical trachea by removing 5-cm long strips of 10 sequential cartilagines. In the control group (n = 5), the gaps were left untreated. In the gelatin sponge group (n = 5), a gelatin sponge soaked in a buffer solution was implanted in each defect. In the bone morphogenetic protein group (n = 5), a gelatin sponge soaked in a buffer solution containing 12 microg bone morphogenetic protein 2 was implanted in each defect., Results: Tracheomalacia was observed in the control and gelatin sponge groups but not in the bone morphogenetic protein group. No regenerated cartilage was detected in the control or gelatin sponge groups, even 6 months after surgery. In contrast, regenerated cartilage, which had developed from the host perichondrium, was observed around the stumps of the resected cartilagines in the bone morphogenetic protein group. This regenerated cartilage maintained the integrity of the internal lumen for longer than 6 months. A compressive fracture test revealed that the tracheal cartilage in the bone morphogenetic protein group was significantly more stable than that in the gelatin sponge and control groups (P =.0015 and P =.0001, respectively)., Conclusions: In this canine model of tracheomalacia, cartilage regeneration was induced around the stumps of tracheal cartilagines by bone morphogenetic protein 2 released slowly from a gelatin sponge. This regenerated cartilage was not reabsorbed for longer than 6 months and was strong enough to maintain the integrity of the internal lumen of the trachea.

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 degrees C for 48 h, but the rinsing step was omitted. In group 3 (n = 6), the grafts were treated at 4 degrees C for 48 h, rinsed thoroughly with physiologic saline, and stored at 4 degrees 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.

Background: Planar cell polarity (PCP) coordinates the patterning and orientation of cells and their structures along tissue planes, and although its acquisition during the formation of airway epithelium has been described, the mechanisms for its maintenance and reconstruction are poorly understood. We aimed to clarify whether ambient environment change by orthotropic autologous transplantation affected PCP at the cellular level.Methods: We performed orthotropic autologous transplantation by inverting tracheal segments in rats, and then performed morphological evaluation by microscopy. The PCP of the tracheal epithelium was assessed over time by analyzing the directions of mucociliary transport and ciliary beat, the positional relationship between the basal body and basal foot, and the bias of Vang-like protein 1 (Vangl1) at 2, 4, and 6 months postoperatively.Results: After 2 months, the directions of mucociliary transport and ciliary beat were preserved toward the lung in the inverted tracheal segments. The positional relationship between the basal body and the basal foot, and the bias of Vangl1, also indicated preservation of PCP in the inverted tracheal segments. Similar results were obtained at 6 months.Conclusion: The PCP of ciliated epithelium was preserved in reversed trachea, even after long-term observation. [ABSTRACT FROM AUTHOR]

Conclusion: This new regenerative therapy shows great potential for the treatment of stenosis of the trachea and/or cricoids (STC). Objectives: To estimate the potential of tissue-engineered artificial trachea (AT) for treatment of STC in clinical applications. We previously reported that AT was a useful material for implantation into a tracheal defect after resection of cancer. There are many causes of stenosis of the respiratory tract and STC is particularly difficult to treat. Methods: The AT was a spiral stent composed of Marlex mesh made of polypropylene and covered with collagen sponge made from porcine skin. Three patients with STC were treated by this tissue-engineering method. All of them suffered from STC caused by long endotracheal intubations. They underwent a two-stage operation. In the first operation, after resection of the stenotic regions, the edge of the tracheal cartilage was sutured to the edge of the skin. The tracheal lumen was exposed and a T-shaped cannula was inserted into the large tracheostoma. At 3 weeks to 2 months after the first operation, the trachea and skin were separated. The trimmed AT with venous blood and basic fibroblast growth factor (b-FGF) was then implanted into the cartilage defect. Results: Postoperatively, all patients were able to breathe easily and had no discomfort in their daily activities. Six months after the second operation, we observed enough air space in the trachea and cricoid by computed tomography (CT) imaging and fiber endoscopy. [ABSTRACT FROM AUTHOR]