Tracheal Replacement Research Articles

Aortic allografts: final destination?—a summary of clinical tracheal substitutes

The patient population in desperate need for an airway substitute are individuals with long segment tracheal defects that are considered, technically, inoperable. Regardless of the underlying etiology, benign or malignant growing processes, this patient category enters a palliative setting or require tracheal transplantation. Different airway substitutes have been categorized by Grillo as follows; tracheal transplantation, autogenous tissue, non-viable tissue, tissue-engineering and foreign materials. These fields have been explored in the past in animal models and in clinical patients. Research on airway replacement has been exposed to a level of controversies in the past years. The field has been turbulent and apocryphal. In particular, the area of tissue-engineering using stem cells has suffered from a major set-back leaving scientists, clinicians and ethical committees skeptical. Recently, a hopeful study emerged using aortic allografts as tracheal substitutes in patients with airway defects. The initial results seem promising and reliable. The developments of the field at this point seem striking and hopeful. The focus of this review is to shed light on developments in the field of aortic allografts as substitute for tracheal replacement.

3D Bioprinted Artificial Trachea with Epithelial Cells and Chondrogenic-Differentiated Bone Marrow-Derived Mesenchymal Stem Cells.

Tracheal resection has limited applicability. Although various tracheal replacement strategies were performed using artificial prosthesis, synthetic stents and tissue transplantation, the best method in tracheal reconstruction remains to be identified. Recent advances in tissue engineering enabled 3D bioprinting using various biocompatible materials including living cells, thereby making the product clinically applicable. Moreover, clinical interest in mesenchymal stem cell has dramatically increased. Here, rabbit bone marrow-derived mesenchymal stem cells (bMSC) and rabbit respiratory epithelial cells were cultured. The chondrogenic differentiation level of bMSC cultured in regular media (MSC) and that in chondrogenic media (d-MSC) were compared. Dual cell-containing artificial trachea were manufactured using a 3D bioprinting method with epithelial cells and undifferentiated bMSC (MSC group, n = 6) or with epithelial cells and chondrogenic-differentiated bMSC (d-MSC group, n = 6). d-MSC showed a relatively higher level of glycosaminoglycan (GAG) accumulation and chondrogenic marker gene expression than MSC in vitro. Neo-epithelialization and neo-vascularization were observed in all groups in vivo but neo-cartilage formation was only noted in d-MSC. The epithelial cells in the 3D bioprinted artificial trachea were effective in respiratory epithelium regeneration. Chondrogenic-differentiated bMSC had more neo-cartilage formation potential in a short period. Nevertheless, the cartilage formation was observed only in a localized area.

A reassessment of tracheal substitutes-a systematic review.

Tracheal substitutes remain an active area of research. For rare patients with large or complex defects that cannot be repaired primarily, replacement of the airway may represent the only treatment option. The present systematic review aims to assess the clinical successes and setbacks of current methods of airway replacement. Systematic review using Medline and PubMed from 01 January 2000 to 01 October 2017 focusing on clinical translation of circumferential or near circumferential (>270°) tracheal substitutes. Studies were identified using key phrases including terms such as "tracheal replacement", "tracheal regeneration", "tracheal transplant", "tracheal tissue engineering", and "tracheal substitution". Animal or non-clinical studies were excluded. Reviews were included if they contained clinical updates. Twenty-one studies were included in assessment comprising a mix of case reports, case studies, and a single review with clinical updates on prior studies. Since 2001, 41 patients have undergone a reported circumferential or near circumferential tracheal substitution through four underlying methodologies including allotransplantation, autologous tissue reconstruction, bioprosthetic reconstruction, and tissue engineered reconstruction. Each modality has unique advantages and disadvantages with varying success in clinical application. The need for tracheal substitution remains a difficult clinical problem without an ideal prosthetic or graft material. While various modalities have had limited clinical success, further laboratory work is necessary before tracheal substitutes can become widely adopted, especially in the case of tissue engineered conduits, which have been setback by premature clinical translation.

A Modular Strategy to Engineer Complex Tissues and Organs.

Currently, there are no synthetic or biologic materials suitable for long‐term treatment of large tracheal defects. A successful tracheal replacement must (1) have radial rigidity to prevent airway collapse during respiration, (2) contain an immunoprotective respiratory epithelium, and (3) integrate with the host vasculature to support epithelium viability. Herein, biopolymer microspheres are used to deliver chondrogenic growth factors to human mesenchymal stem cells (hMSCs) seeded in a custom mold that self‐assemble into cartilage rings, which can be fused into tubes. These rings and tubes can be fabricated with tunable wall thicknesses and lumen diameters with promising mechanical properties for airway collapse prevention. Epithelialized cartilage is developed by establishing a spatially defined composite tissue composed of human epithelial cells on the surface of an hMSC‐derived cartilage sheet. Prevascular rings comprised of human umbilical vein endothelial cells and hMSCs are fused with cartilage rings to form prevascular–cartilage composite tubes, which are then coated with human epithelial cells, forming a tri‐tissue construct. When prevascular– cartilage tubes are implanted subcutaneously in mice, the prevascular structures anastomose with host vasculature, demonstrated by their ability to be perfused. This microparticle–cell self‐assembly strategy is promising for engineering complex tissues such as a multi‐tissue composite trachea.

Tracheal replacement.

Tracheal reconstruction is one of the greatest challenges in thoracic surgery when direct end-to-end anastomosis is impossible or after this procedure has failed. The main indications for tracheal reconstruction include malignant tumours (squamous cell carcinoma, adenoid cystic carcinoma), tracheoesophageal fistula, trauma, unsuccessful surgical results for benign diseases and congenital stenosis. Tracheal substitutes can be classified into five types: 1) synthetic prosthesis; 2) allografts; 3) tracheal transplantation; 4) tissue engineering; and 5) autologous tissue composite. The ideal tracheal substitute is still unclear, but some techniques have shown promising clinical results. This article reviews the advantages and limitations of each technique used over the past few decades in clinical practice. The main limitation seems to be the capacity for tracheal tissue regeneration. The physiopathology behind this has yet to be fully understood. Research on stem cells sparked much interest and was thought to be a revolutionary technique; however, the poor long-term results of this approach highlight that there is a long way to go in this research field. Currently, an autologous tissue composite, with or without a tracheal allograft, is the only long-term working solution for every aetiology, despite its technical complexity and setbacks.

Scaffold-free trachea regeneration by tissue engineering with bio-3D printing.

Currently, most of the artificial airway organs still require scaffolds; however, such scaffolds exhibit several limitations. Alternatively, the use of an autologous artificial trachea without foreign materials and immunosuppressants may solve these issues and constitute a preferred tool. The rationale of this study was to develop a new scaffold-free approach for an artificial trachea using bio-3D printing technology. Here, we assessed the circumferential tracheal replacement using scaffold-free trachea-like grafts generated from isolated cells in an inbred animal model. Chondrocytes and mesenchymal stem cells were isolated from F344 rats. Rat lung microvessel endothelial cells were purchased. Our bio-3D printer generates spheroids consisting of several types of cells to create 3D structures. The bio-3D-printed artificial trachea from spheroids was matured in a bioreactor and transplanted into F344 rats as a tracheal graft under general anaesthesia. The mechanical strength of the artificial trachea was measured, and histological and immunohistochemical examinations were performed. Tracheal transplantation was performed in 9 rats, which were followed up postoperatively for 23 days. The average tensile strength of artificial tracheas before transplantation was 526.3 ± 125.7 mN. The bio-3D-printed scaffold-free artificial trachea had sufficient strength to transplant into the trachea with silicone stents that were used to prevent collapse of the artificial trachea and to support the graft until sufficient blood supply was obtained. Chondrogenesis and vasculogenesis were observed histologically. The scaffold-free isogenic artificial tracheas produced by a bio-3D printer could be utilized as tracheal grafts in rats.

Advances in tracheal transplantation

The length of tracheal defect or stenosis exceeded 5 cm could not be treated by simple resection and end-to-end anastomosis of the remaining trachea. Various ways of tracheal replacement had appeared sequentially, such as radial forearm free flap with cartilage grafts, tracheal tissue-engineering and tracheal allotransplantation. Among these methods, tracheal allotransplantation displayed a better long-term result. In this review, we are focused on recent advances in tracheal allotransplantation, particularly on revascularization and reepithelialization of graft, as well as on the application of immunosuppressive agents.

Tissue engineering of a composite trachea construct using autologous rabbit chondrocytes.

The repair of large tracheal segmental defects remains an unsolved problem. The goal of this study is to apply tissue engineering principles for the fabrication of large segmental trachea replacements. Engineered tracheal replacements composed of autologous cells (neotracheas) were tested in a New Zealand White rabbit model. Neotracheas were formed in the rabbit neck by wrapping a silicone tube with consecutive layers of skin epithelium, platysma muscle, and an engineered cartilage sheet and allowing the construct to mature for 8-12weeks. In total, 28 rabbits were implanted and the neotracheas assessed for tissue morphology. In 11 cases, neotracheas deemed sufficiently strong were used to repair segmental tracheal defects. Initially, the success rate of producing structurally sound neotracheas was impeded by physical disruption of the cartilage sheets during animal handling, but by the end of the study, 15 of 18 neotracheas (83.3%) were structurally sound. Of the 15 structurally sound neotracheas, 11 were used for segmental reconstruction and were left in place for up to 21days. Histological examination showed the presence of variable amounts of viable epithelium, a vascularized platysma flap, and a layer of safranin O-positive cartilage along with evidence of endochondral ossification. Rabbits that had undergone segmental reconstruction showed good tracheal integration, had a viable epithelium with vascular support, and the cartilage was sufficiently strong to maintain a lumen when palpated. The results demonstrated that viable, trilayered, scaffold-free neotracheas could be constructed from autologous cells and could be integrated into native trachea to repair a segmental defect.

Serial Analysis of Tracheal Restenosis After 3D-Printed Scaffold Implantation: Recruited Inflammatory Cells and Associated Tissue Changes.

Tracheal restenosis is a major obstacle to successful tracheal replacement, and remains the greatest challenge in tracheal regeneration. However, there have been no detailed investigations of restenosis. The present study was performed to analyze the serial changes in recruited inflammatory cells and associated histological changes after tracheal scaffold implantation. Asymmetrically porous scaffolds, which successfully prevented tracheal stenosis in a partial trachea defect model, designed with a tubular shape by electrospinning and reinforced by 3D-printing to reconstruct 2-cm circumferential tracheal defect. Serial rigid bronchoscopy, micro-computed tomography, and histology [H&E, Masson's Trichrome, IHC against α-smooth muscle actin (α-SMA)] were performed 1, 4, and 8weeks after transplantation. Progressive stenosis developed especially at the site of anastomosis. Neutrophils were the main inflammatory cells recruited in the early stage, while macrophage infiltration increased with time. Recruitment of fibroblasts peaked at 4weeks and deposition of α-SMA increased from 4weeks and was maintained through 8weeks. During the first 8weeks post-transplantation, neutrophils and macrophages played significant roles in restenosis of the trachea. Antagonists to these would be ideal targets to reduce restenosis and thus play a pivotal role in successful tracheal regeneration.

In-vivo characterization of a 3D hybrid scaffold based on PCL/decellularized aorta for tracheal tissue engineering

IntroductionAs common treatments for long tracheal stenosis are associated with several limitations, tracheal tissue engineering is considered as an alternative treatment. Aim of studyThis study aimed at preparing a hybrid scaffold, based on biologic and synthetic materials for tracheal tissue engineering. Materials and methodsThree electrospun polycaprolactone (PCL) scaffolds, namely E1 (pure PCL), E2 (collagen-coated PCL) and E3 (PCL blended with collagen) were prepared. Allogeneic aorta was harvested and decellularized. A biodegradable PCL stent was fabricated and inserted into the aorta to prevent its collapse. ResultScaffold characterization results revealed that the 2-h swelling ratio of E2 was significantly higher than those of E1 and E3. In the first 3months, E2 and E3 exhibited almost equal degradabilities (significantly higher than that of E1). Moreover, tensile strengths of all samples were comparable with those of human trachea. Using rabbit's adipose-derived mesenchymal stem cells (AMSCs) and primary chondrocytes, E3 exhibited the highest levels of GAG release within 21days as well as collagen II and aggrecan expression.Fot the next step, AMSC-chondrocyte co-culture seeded scaffold was sutured to the acellular aorta, implanted into rabbits' muscle, and finally harvested after 4weeks of follow up. ConclusionHarvested structures were totally viable due to the angiogenesis created by the muscle. H&E and alcian blue staining results revealed the presence of chondrocytes in the structure and GAG in the produced extracellular matrix.Since tracheal replacement using biologic and synthetic scaffolds usually results in tracheal collapse or granulation formation, a hybrid construct may provide the required rigidity and biocompatibility for the substitute.

Tissue-Engineered Tracheal Regeneration: Current Status

Tracheal regeneration is very challenging clinical demand because the trachea is not a simple windpipe, but a multilayered, complex structure. The tissue-engineering technique is widely accepted as promising strategy in tracheal regeneration. For successful regeneration, a substitute for trachea should provide not only appropriate laterally rigidity and longitudinally flexibility for sustaining the luminal shape of the trachea, but also favorable environment for respiratory ciliated epithelium, smooth muscle and blood vessel cells to regenerate. To date, a variety of materials such as polyglycolic acid, poly (lactic-co-glycolic acid), polycarprolactone, nanocomposite polymers and many naturally-derived scaffolds have been investigated. With these investigations, several clinical attempt of tracheal replacement with artificial trachea have been tried, but clinical outcome has not been quite satisfying. This article reviews the regeneration of C-shaped cartilage, respiratory ciliated epithelium and neovascularization of artificial trachea, together with the difficulties, plausible options and future perspectives. Key words: Regenerative medicine ㆍ Scaffold ㆍ Stem cells ㆍ Tissue engineering ㆍ Trachea

Transplantation of the decellularized tracheal allograft in animal model (rabbit)

It has been difficult to perform tracheal allotransplantation without immunosuppression. To determine whether decellularized trachea can be used in tracheal replacement, we evaluated the viability of decellularized tracheal allografts in a rabbit model of immunosuppressant-free transplantation. Half allograft (Group 1, n=7) was harvested from adult New Zealand white rabbits, subjected to a detergent-enzymatic method (containing sodium deoxycholate/DNase lavations) of decellularization for as many cycles as needed, and the other half was stored in phosphate-buffered saline at 4°C as a control (Group 2, n=7). Bioengineered and control tracheas were then implanted in 14 age-matched rabbits. In Group 1 (decellularized), all rabbits survived, whereas in Group 2(control), all rabbits died of airway obstruction between 20 days and 45 days after operation. Histologically, the decellularized allografts displayed complete regeneration of epithelium and cartilage, but the fresh allografts showed inflammatory changes, no epithelium, and no cartilage. Complete regeneration of epithelium and cartilage tracheal rings occurred after the implantation of decellularized tracheal allografts without immunosuppression. We demonstrate that the decellularized process reduces the allogeneic response to the trachea. Therefore, we believe that the decellularized tracheal allograft is an excellent choice for tracheal replacement. To our knowledge, this is the first study to observe the long-term (1 year) prognosis of this transplanted trachea.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.

Development of tracheal reconstruction methods from scaffold engineering to injectable matrix

Backgrand: For patients with long segment of tracheal stenosis, resection and replacement is necessary. Tracheal reconstruction associated with complications such as stenosis, insufficiency of blood supply and surgical complication. Methods: In this study, we prepared collagen-chitosan scaffold and sheep's decellularized trachea plus culture medium with nanocup contain growth factors for chondrocyte and fibroblast-epithelial cell culture in rotary bioreactor. After attachment of cells, engineered trachea put into the omentum. Peritoneal stem cell interact with the epithelial and chondrocytes with attendance of growth factors released from nanocup. The bioengineered trachea with omental pedicle transposed from behind the sternum and transplanted in the position of resected trachea. In third experiment, we introduce the in situ tracheal repair technique with injectable matrix for reconstruction of long-segmental stenosis of trachea in a 29-year-old woman. Results: Severe tortuosity in the first experiment and mild stenosis was seen in the second experiment. In third experiment, normal shape in tracheal diameter was seen at injection sites. The interior portion of the trachea at virtual computed tomography (CT) scan and bronchoscopy were similarity to normal after four month fallowing up of injection. Conclusions: Severe tortuosity, mild stenosis in whole length of trachea in this study and disadvantage of trachea transplantation include open surgery, fatal consequences of anastomosis leakage, and rupture besides large mediastinal vessels; hence, we decided to introduce the novel in situ tracheal repair technique. Injectable bioresorbable scaffolds may be used as a temporary scaffolding for transplanted cells and thereby allow the cells to secrete extracellular matrix of their own to enable, in the long term, a complete and natural tissue replacement.

Evaluation of Immunosuppressive Therapy Use for Tracheal Transplantation with Trachea-Mimetic Bellows Scaffolds in a Rabbit Model.

The objective of this study was to evaluate the use of immunosuppressive therapy with high-dose cyclosporine, high-dose azathioprine, and a combination of low-dose cyclosporine and azathioprine after tracheal reconstruction by using a trachea-mimetic graft of polycaprolactone (PCL) bellows-type scaffold in a rabbit model. Twenty-four healthy New Zealand white rabbits were used in the study. All underwent circumferential tracheal replacement using tissue-engineered tracheal graft, prepared from PCL bellows scaffold reinforced with silicone ring, collagen hydrogel, and human turbinate mesenchymal stromal cell (hTMSC) sheets. The control group (Group 1) received no medication. The three experimental groups were given daily cyclosporine intramuscular doses of 10 mg/kg (Group 2), azathioprine oral doses of 5 mg/kg (Group 3), and azathioprine oral doses of 2.5 mg/kg plus cyclosporine intramuscular doses of 5 mg/kg (Group 4) for 4 weeks or until death. Group 1 had longer survival times compared to Group 2 or Group 3. Each group except for Group 1 experienced decreases in amount of nutrition and weight loss. In addition, compared with the other groups, Group 2 had significantly increased serum interleukin-2 and interferon-γ levels 7 days after transplantation. The results of this study showed that the administration of cyclosporine and/or azathioprine after tracheal transplantation had no beneficial effects. Furthermore, the administration of cyclosporine had side effects, including extreme weight loss, respiratory distress, and diarrhea. Therefore, cyclosporine and azathioprine avoidance may be recommended for tracheal reconstruction using a native trachea-mimetic graft of PCL bellows-type scaffold in a rabbit model.

Structural Integrity of a Simple Method to Repair Disrupted Tracheal Tube Pilot Balloon Assemblies.

An intact pilot balloon assembly is crucial to the proper function of a cuffed tracheal tube. Disruption of the pilot balloon, transection of the inflation line, or valve incompetence results in cuff deflation, which may lead to inadequate ventilation and aspiration of secretions. Such failures typically result in tracheal tube replacement, but this may be a safety risk if a difficult reintubation is anticipated. We recently encountered such a patient who remained intubated postoperatively and in whom the inflation line was transected, causing a large leak. We describe a method to reconstitute the inflation line and report on the structural integrity of the repair. We hypothesized that the repaired assembly would maintain cuff pressure not statistically different from an intact device, but that the inflation line would be weaker. The distal (tapered) portion of a 22-gauge intravenous (IV) catheter was partially inserted into the severed end of the inflation line. A new pilot balloon was cut from an intact tracheal tube with the tubing attached, the end of which had been dilated using a 22-gauge IV catheter. The new tubing was then guided over the protruding portion of the catheter, creating an internal stent. We measured the drop in cuff pressure after 8 hours in an artificial trachea for repaired and intact tracheal tubes. We tested the integrity of the repaired segments, underwater, to high-pressure inflation. We measured the static tensile strength of the inflation line from intact and repaired tracheal tubes. Data are presented as the mean ± standard error. Differences were assessed using the unpaired, 2-sided Student t test, with P < .05 required to claim statistical significance. Eight-hour interval measurements in 10 intact versus 10 repaired tracheal tubes demonstrated no significant difference in pressure drop (mean difference = 0.5 cm H2O; 95% confidence interval, -2.2 to 1.2 cm H2O; P = .54). There was no visible air leak from 10 repaired inflation line segments when the cuff was inflated to 120 mm Hg. The force needed to break the repaired inflation line was lower than for the intact tubing (n = 7 of each; mean difference = -21.9 N; 95% confidence interval, -25.7 to -18.1 N; P < 10). Repairs to tracheal tubes from various manufacturers with inner diameters ranging from 3.0 to 8.0 mm were successful. Repairing a disrupted pilot balloon assembly using an IV catheter as a stent inside the inflation line is an effective temporizing measure in situations where ventilation is impaired and where tracheal tube replacement may present an excessive patient risk.

An Investigation of Topics and Trends of Tracheal Replacement Studies Using Co-Occurrence Analysis

This study evaluated tracheal regeneration studies using scientometric and co-occurrence analysis to identify the most important topics and assess their trends over time. To provide the adequate search options, PubMed, Scopus, and Web of Science (WOS) were used to cover various categories such as keywords, countries, organizations, and authors. Search results were obtained by employing Bibexcel. Co-occurrence analysis was applied to evaluate the publications. Finally, scientific maps, author's network, and country contributions were depicted using VOSviewer and NetDraw. Furthermore, the first 25 countries and 130 of the most productive authors were determined. Regarding the trend analysis, 10 co-occurrence terms out of highly frequent words were examined at 5-year intervals. Our findings indicated that the field of trachea regeneration has tested different approaches over the time. In total, 65 countries have contributed to scientific progress both in experimental and clinical fields. Special keywords such as tissue engineering and different types of stem cells have been increasingly used since 1995. Studies have addressed topics such as angiogenesis, decellularization methods, extracellular matrix, and mechanical properties since 2011. These findings will offer evidence-based information about the current status and trends of tracheal replacement research topics over time, as well as countries' contributions.

Engineered Tissue-Stent Biocomposites as Tracheal Replacements.

Here we report the creation of a novel tracheal construct in the form of an engineered, acellular tissue-stent biocomposite trachea (TSBT). Allogeneic or xenogeneic smooth muscle cells are cultured on polyglycolic acid polymer-metal stent scaffold leading to the formation of a tissue comprising cells, their deposited collagenous matrix, and the stent material. Thorough decellularization then produces a final acellular tubular construct. Engineered TSBTs were tested as end-to-end tracheal replacements in 11 rats and 3 nonhuman primates. Over a period of 8 weeks, no instances of airway perforation, infection, stent migration, or erosion were observed. Histological analyses reveal that the patent implants remodel adaptively with native host cells, including formation of connective tissue in the tracheal wall and formation of a confluent, columnar epithelium in the graft lumen, although some instances of airway stenosis were observed. Overall, TSBTs resisted collapse and compression that often limit the function of other decellularized tracheal replacements, and additionally do not require any cells from the intended recipient. Such engineered TSBTs represent a model for future efforts in tracheal regeneration.

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Treatment of large tracheal defects after resection: Laryngotracheal release and tracheal replacement.

Resection with direct tracheal or laryngotracheal anastomosis is the standard procedure employed for treatment of benign stenosis or occasionally primary or secondary tracheal malignancy. Literature review. A tracheal anastomosis usually heals without complications provided that the ends being joined are adequately supplied with blood, an atraumatic suturing technique is used, and the anastomosis does not become infected. It is especially important that the anastomosis is not subjected to tension. Various techniques of laryngeal and tracheal release serve to reduce the tension on the anastomosis by mobilizing and reducing the distance between the two segments to be approximated. These techniques can be used in different combinations depending on situation encountered during surgery. In cases where more than 50% of the tracheal length must be excised, prosthetic replacements, autologous tissue transfer and allografts are required. All present various problems. The use of tissue-engineering techniques utilizing autologous stem cells has opened new perspectives for tracheal replacement. Such procedures are still in an experimental state.