Tissue‐engineered tracheal implants: Advancements, challenges, and clinical considerations

Abstract Restoration of extensive tracheal damage remains a significant challenge in respiratory medicine, particularly in instances stemming from conditions like infection, congenital anomalies, or stenosis. The trachea, an essential element of the lower respiratory tract, constitutes a fibrocartilaginous tube spanning approximately 10–12 cm in length. It is characterized by 18 ± 2 tracheal cartilages distributed anterolaterally with the dynamic trachealis muscle located posteriorly. While tracheotomy is a common approach for patients with short‐length defects, situations requiring replacement arise when the extent of lesion exceeds 1/2 of the length in adults (or 1/3 in children). Tissue engineering (TE) holds promise in developing biocompatible airway grafts for addressing challenges in tracheal regeneration. Despite the potential, the extensive clinical application of tissue‐engineered tracheal substitutes encounters obstacles, including insufficient revascularization, inadequate re‐epithelialization, suboptimal mechanical properties, and insufficient durability. These limitations have led to limited success in implementing tissue‐engineered tracheal implants in clinical settings. This review provides a comprehensive exploration of historical attempts and lessons learned in the field of tracheal TE, contextualizing the clinical prerequisites and vital criteria for effective tracheal grafts. The manufacturing approaches employed in TE, along with the clinical application of both tissue‐engineered and non‐tissue‐engineered approaches for tracheal reconstruction, are discussed in detail. By offering a holistic view on TE substitutes and their implications for the clinical management of long‐segment tracheal lesions, this review aims to contribute to the understanding and advancement of strategies in this critical area of respiratory medicine.


Translational Impact Statement
This review provides a nuanced exploration of historical attempts and lessons learned in tracheal tissue engineering.Addressing critical challenges such as insufficient revascularization and suboptimal mechanical properties, our work offers innovative insights into manufacturing approaches and clinical applications for airway reconstruction.Emphasizing the need for longterm follow-up assessments, our research aims to revolutionize the clinical approach to longsegment tracheal lesions, contributing valuable advancements to the broader field of tissue engineering.

| INTRODUCTION
Tracheal afflictions characterized by discontinuity, structural weakening, or constriction pose a substantial threat to life.Pathologies affecting the conducting segment of the respiratory tract manifest through diverse clinical conditions, presenting a spectrum of challenges for effective intervention.Notably, the realm of medical intervention encounters a formidable gap in addressing prolonged and extensive tracheal defects. 1 Presently, thoracic surgical procedures, encompassing tracheotomy and primary end-to-end anastomosis, serve as the conventional recourse for managing short-length tracheal damage.However, cautionary measures are warranted as these interventions are deemed unsuitable for defects surpassing 6-cm in adults (or 2-cm in children). 2,3en confronted with such scenarios, patients find themselves navigating treatment modalities like tracheal reconstruction, slide tracheoplasty, and innovative artificial substitutes.Nevertheless, the efficacy of these solutions is compromised by complications such as granulation formation and gradual implant weakening over time, posing critical challenges to their sustained performance. 4,5Consequently, an imperative has emerged for the investigation of the strategies for facilitating the development of tracheal-like structures.
Given the pressing clinical demand and ongoing dynamic initiatives, this comprehensive review aims to shed light on historical efforts in tracheal replacement.Furthermore, it aims to delineate how the insights garnered from past experiences have profoundly influenced and guided the current trajectory of research efforts in tissue engineering (TE).The narrative unfolds by providing a contextual understanding of clinical imperatives, offering insights into pertinent epidemiological considerations, and elucidating the physiological intricacies of the functional trachea.In doing so, it outlines the design prerequisites for an efficacious tracheal substitute.Subsequently, an overview of contemporary reconstruction strategies is presented, followed by a synthesis of the pivotal lessons derived from clinical trials involving diverse technological interventions.

| CLINICAL IMPERATIVES IN TRACHEAL REPLACEMENT
][8][9][10][11] Malignant tumors invading the trachea often necessitate lesion resection and one-stage structure reconstruction, encompassing primary Tracheal or bronchus malignancies such as adenoid cystic carcinoma and squamous cell carcinoma. 12,13TBM, an underreported condition characterized by tracheal cartilage softening, poses another pathological mechanism leading to airway damage. 1 Moreover, prolonged intubation in intensive care unit patients can result in laryngotracheal injuries resulting from tracheotomies and endotracheal intubation procedures.Post-intubation airway stenosis occurs in approximately 5%-20% of intubated cases, and recent evidence suggests a potential rise in tracheal stenosis cases among patients recovering from coronavirus disease 2019 who required mechanical ventilation. 15In the T A B L E 1 Epidemiological landscape of tracheal aberrations.

Clinical pathology Epidemiology References
[18] While tracheal stenosis in adults is typically addressed through resection and anastomosis, this approach is less feasible in infants and children, especially when the lesion involves exceeding 50% of the total trachea length. 2 Attempts to provide support using various stents, comprising biodegradable alternatives and Palmaz stents, have achieved limited success. 19The reported incidence of tracheal pathologies may appear relatively low; Nevertheless, factors like pre-hospital mortality and symptoms resembling other common diseases contribute to the underestimation of their true prevalence. 9,13This diagnostic challenge often leads to elevated morbidity and mortality rates, underscoring the critical clinical need for advancements in tracheal regeneration.
Addressing the multifaceted etiology of tracheal pathologies necessitates a thorough consideration of demographic and physiological variables impacting tracheal morphology.Extensive research underscores significant sexual dimorphism in tracheal dimensions, with males consistently exhibiting larger diameters and greater cartilaginous thickness than females.Noteworthy ethnic disparities have also been identified, particularly narrower tracheal dimensions observed among individuals of Asian descent compared to Caucasians and African Americans.Additionally, advancing age introduces distinct alterations in tracheal structure, characterized by decreased cartilaginous density and increased susceptibility to collapse. 1,5,6ese nuanced demographic insights underscore the imperative for tailored approaches in tracheal TE.Careful consideration of sexspecific variations in tracheal dimensions holds promise in optimizing surgical outcomes and minimizing postoperative complications.Similarly, integration of ethnic disparities in tracheal anatomy informs the development of precision interventions tailored to diverse patient cohorts.Moreover, addressing age-related changes in tracheal biomechanics is crucial for ensuring the resilience and durability of tissueengineered grafts, particularly in geriatric populations. 3Hence, it is paramount that future research endeavors prioritize the investigation of demographic determinants in tracheal TE, thereby advancing the field towards more effective and individualized therapeutic modalities.

| TRACHEAL REPLACEMENT: A COMPREHENSIVE REVIEW OF THE ATTRIBUTES AND DESIGN PREREQUISITES FOR GRAFTS
To engineer tracheal substitutes that faithfully replicate the intricate biological and anatomical characteristics of the trachea, an in-depth understanding of its complex structure, cellular functions, mechanical properties, and vascularization is imperative.Despite its seemingly straightforward appearance, the trachea conceals a sophisticated composition with challenging features that demand meticulous replication.Functionally, the trachea acts as the conduit connecting the upper and lower respiratory tracts, facilitating the ventilation, humidification, and mucociliary clearance of inspired air.The structural framework of trachea consists of 18 ± 2 hyaline cartilaginous rings formed by the annular ligament and trachealis muscle, it requires precise replication to maintain patency and resist forces during respiration. 20e airway mucous membrane, located in the innermost layer of wall, plays a crucial role in mucociliary clearance and serves as the initial barrier against pathogens.Any proposed alternative substitute construct should be highly secure, facilitating the potential regeneration of pseudostratified respiratory epithelium to mitigate the risk of infections. 21The fibrocartilaginous layer in the tracheobronchial area, composed of the cricoid anchored by the trachealis muscle, necessitates a framework for tracheal reconstruction that demonstrates lateral stability and longitudinal flexibility.This scaffold should withstand the various forces encountered during normal respiration, coughing, or other physiological processes. 2Mechanical properties, including resistance to cough-induced fractures, are critical for graft durability and preventing airway lumen collapse. 3deling the intricate mechanical forces acting on the structure of cervical trachea becomes more complex due to abnormal situations such as coughing, forced respiration, and sneezing.Tracheal cartilage exhibits biphasic material behavior, while the trachealis muscle demonstrates hyperelastic characteristics. 22Varied testing methods and specimen types in existing studies pose challenges in understanding native tracheal mechanical properties for potential replacement device design. 23scularization is a paramount consideration for the success of tracheal grafts.The highly segmental blood supply of the native trachea presents challenges for revascularization in replacement scenarios.Past failures involving solid polymer tubes underscore the significance of an effective tracheal blood supply, emphasizing the need for grafts that support revascularization to ensure optimal performance. 24,25e ideal tracheal substitute should exhibit lateral rigidity and longitudinal flexibility.Additionally, it must be biocompatible, nonimmunogenic, non-carcinogenic, non-toxic, and resistant to erosion, dislocation, and stenosis over time. 26Developing tracheal tissue regeneration approaches necessitates creating a suitable 3D environment for respiratory epithelium and hyaline cartilage formation, fostering a vascularized plexus to prevent necrotic tissue, and enabling immune cell infiltration to avoid infections during the regeneration process.Various strategies have been employed to address this significant challenge, with historical significance and promising contemporary approaches discussed in detail in this review, incorporating the latest research findings and advancements.

| TRACHEAL RECONSTRUCTION APPROACHES
The pioneering efforts in airway reconstruction trace back to the late 19th century, with early groundbreaking attempts documented between 1886 and 1899. 27,28The initial human application of primary anastomosis took place in 1886, marking a significant milestone. 29bsequent explorations involved diverse grafting materials, such as autogenous skin, 30 fascial tissue, 31 and costal cartilage, 32 along with the introduction of solid materials in prostheses. 33Tubular tracheal substitutes have been the focus of extensive investigation, employing various strategies such as cadaveric tissue flaps, 34 implanted bioprostheses, 35 tubes derived from intestinal tissue 36,37 and different types of aortic grafts. 38The outcomes, successes, and limita-  41,42 Nevertheless, the explored techniques exhibit substantial promise and have the potential to establish the gold standard in tissue-engineered tracheal constructs.

| 3D printing technology
The application of 3D printing in medical research has gained traction due to its capability for rapid fabrication of customized substitutes for structural reconstruction, allowing modulation of properties such as architecture, mechanical characteristics, and degradation rate. 43is technology enables the creation of intricate, patient-specific, multi-layered designs tailored to individual tracheal features. 44lycaprolactone (PCL), renowned for its exceptional mechanical properties, controllable in vivo degradation, and long-term stability, has been a predominant material in 3D printing TE applications.
Notably, 3D-printed PCL stents have obtained approval from the United States Food and Drug Administration for emergent use in pediatric operation to address benign lesions. 45,46Consequently, PCL has been widely employed in tracheotomy research, producing substitutes that exhibit remarkable resistance to compressive stresses and assist cartilage regeneration. 47Despite these advancements, PCL scaffolds face challenges in pre-clinical studies, triggering an inflammatory reaction that leads to the granulation formation and subsequent lumen stenosis. 48Explorations into novel materials for 3D printing, such as polyurethane (PU), have demonstrated adequate mechanical support and facilitated cartilage formation. 49Continual endeavors in 3D printing of tracheal scaffolds, employing diverse materials and distinctive tubular designs reveal promising initial outcomes, although limited by single-cell type technologies, deficiency of mechanical characterization, and inadequate revascularization. 50e emergence of 3D printing in clinical study has sparked the exploration of direct cell-laden biomaterial printing, often referred to as bioprinting.This procedure reveals potential for creating stented grafts that can support various types of cells, ensuring reproducibility and customization for individual patients. 51However, challenges arise in bioprinting with cell-loaded bioinks, as low bioink melting points can lead to suboptimal biomechanical properties, resulting in the formation of weak structures. 52To address this challenge, researchers have employed dual-headed 3D printers to fabricate structurally robust scaffolds.This entails the fusion of thermoplastic polymers with hydrogels, such as alginate, and the combination of collagen type I hydrogel with PCL. 53Instances of PCL combined with sodium alginate hydrogels have shown success in animal models, exhibiting re-vascularization, ciliary epithelial regeneration, and cartilage formation. 54Bioprinting holds promise for tracheal substitutes, but addressing challenges such as maintaining cell viability, appropriate temperature profiles, and withstanding mechanical stress during bioink extrusion requires further research. 55

| Electrospinning technology
Electrospinning stands out as a versatile and indispensable technique applicable to both synthetic and natural polymers, allowing the rapid fabrication of customizable multi-layered 3D constructs.This method utilizes high voltage to propel the selected polymer onto a collector plate, leading to the formation of nanofibrous structures. 56Its widespread adoption in respiratory TE arises from its unique capability to generate fibrous scaffolds closely resembling the size-scale of native respiratory extracellular matrix using various materials such as PCL, PU, polyethylene terephthalate (PET), and polylactic acid (PLA). 57,580][81] Additionally, material strength assessments through electrospinning have demonstrated a spectrum ranging from mimicking the physical characteristics of the native airway to surpassing them. 82,83While electrospinning exhibits substantial promise as a TE approach for airway reconstruction, it is crucial to acknowledge the limited availability of long-term animal compared to traditional methods [77]  studies in this domain.Further exploration and comprehensive investigations are warranted to establish the enduring viability and success of electrospun tracheal substitutes.

| Decellularized tracheal graft
Decellularized tracheal grafts (DTGs) have emerged as a promising TE strategy for tracheal replacement, leveraging allograft advantages such as an ideal extracellular matrix and an intact airtight structure for cellular adhesion as well as proliferation.The decellularization process involves multiple cycles of detergents and enzymes applied to harvested donor tracheal tissue over an extended period, aiming to eliminate genetic material and prevent immune reactions. 75,78While some animal studies have shown encouraging results with observed revascularization and reepithelialization in specific graft areas, many attempts have faced challenges like trachea stenosis or collapse due to immunoreaction. 84Existing techniques struggle to achieve total removal of cellular debris or genetic material, risking adverse host responses in vivo.Striking a balance is crucial, as complete removal may compromise the airway structure's integrity as a matrix for cell proliferation and compromise physical characteristics. 85Despite the promise of DTGs approaches in providing a scaffold for cellular growth, their extensive processing timelines and donor-recipient matching constraints limit their widespread applicability. 86However, the inherent architecture of DTGs provides a solid basis for tracheal regeneration, surpassing synthetic materials in terms of mechanical integrity.
When integrated with other TE methodologies, this approach shows promise, as indicated by promising preliminary results. 87,88

| Casting technology
Casting methodologies have emerged as pivotal techniques in the realm of TE, offering precise control over scaffold geometries within 3D structures and minimizing inherent variability. 89In TE applications, casting commonly involves tailored hydrogel blends, providing a high degree of customization in terms of biochemical compositions, architectural features, and mechanical properties. 90Notably, hydrogels have demonstrated their efficacy in promoting cellular infiltration and vascularization, showcasing successful applications in diverse areas such as skin wound healing, 91 bone regeneration, 92 and abdominal wall reconstruction. 93amples encompass patterned 2-hydroxyethyl methacrylate hydrogels, closely mimicking the mechanical properties of native airway, 94 and type I collagen, hydrogels like fibrin and agarose, which have demonstrated efficacy in supporting the growth of cultured respiratory ciliated epithelium and vascular venation. 95While the use of hydrogels as artificial prosthetics is presently constrained, preliminary investigations in line with graft requirements have yielded promising results.

| ADVANCEMENTS IN CLINICAL TRANSLATION 6.1 | Pre-clinical evaluation of TE scaffolds
Animal studies evaluating TE tracheal substitutes have shown some success (Table 2).Many of these initiatives, however, encounter limitations due to short animal follow-up periods, typically lasting 1-3 months. 62,70,71Initial efforts often resulted in severe inflammatory reaction, leading to serious stenosis, granulation formation, or transplant failure through infection. 49Ensuring airtightness and fostering vascularization are pivotal factors that substantially contribute to the survival prospects of the graft.These objectives can be accomplished through pre-implantation strategies. 66,72Encasing the construct in omentum not only provides a vascular network but also serves as a tissue source to maintain and seal airtightness, creating a barrier against bacterial colonization.Pre-transplantation in the abdominal cavity facilitates omentum adherence to the substitute and the integration of the vessel. 62Pre-vascularization before transplantation has demonstrated a higher animal survival rate, lower stenosis rates, and improved the re-epithelization efficiency. 72Furthermore, when combined with a cell-seeded construct, this procedure improves both cell survival and graft integration.Pre-seeding of constructs has been observed to increase survival rates, and the integration of prevascularization and pre-seeding with chondrocytes has demonstrated a significant reduction in tracheal stenosis. 70Furthermore, the inclusion of pre-seeded epithelial cells has proven effective in enhancing graft acceptance and overall transplant success when combined with mesenchymal stem cells (MSCs).This combination resulted in complete epithelial coverage of the scaffold, with no indications of distress or transplant failure observed in all cases for a duration of up to 3 months. 77In the most extended preclinical study to date, conducted in a canine model with a 2-year follow-up, a collagen-coated nitinol frame was utilized. 62In this study, the implant was initially enveloped in omentum and implanted into the abdominal cavity for 3 weeks.
Subsequently, it was implanted following a 2-cm cervical tracheal resection, reporting an 80% survival rate over 1.5 years.Histological examination revealed stable epithelialization in a non-stratified monolayer with no present secretory glands or muscular regeneration, although lumen collapse was not observed. 62While other studies using a similar procedure reported comparable results after 2 months, unfortunately, longer-term follow-ups for these studies were not reported. 65Several additional studies in canines have been conducted with follow-ups for up to 1 year. 60,61inical efforts of implanted synthetic TE substitutes have sparked controversy and resulted in fatalities. 96To understand the causes of these failures, subsequent large animal models have been utilized in pre-clinical studies.As emphasized previously, the absence of a functional epithelium poses a substantial risk of inflammation, infection, or anastomotic fistula.Persistent inflammation observed in all cases led to lumen collapse, particularly at the proximal and distal ends of the prosthesis. 96Grafts seeded with MSCs exhibited a delayed onset of respiratory distress, emphasizing the significance of establishing a functional epithelium before implantation.While primarily conducted over the short term, these in vivo studies have underscored significant challenges that need addressing for the development of a viable airway substitute.The inclusion of a pre-seeded layer seems to mitigate bacterial colonization, and pre-vascularization enhances graft survival.However, concerns persist, particularly regarding the presence of granulation tissue in some instances.

| Clinical evaluation of TE scaffolds
Although only a limited number of TE strategies for tracheal replacement have progressed to clinical applications, noteworthy advancements have been observed (Table 3).The initial documented success occurred in 2005, 100 featuring a Marlex prosthesis coated with type I and II collagen.The implantation of this prosthesis demonstrated successful formation of the airway epithelium.Histological examination unveiled the successful integration of the graft into surrounding tissues, leading to the satisfactory regeneration of airway epithelium.
Two years postoperatively, optimal epithelial growth was observed, confirming the successful integration of the implanted substitute.
Mechanical studies further validated the patency of the airway. 100bsequently, this approach was applied to a broader patient cohort, showcasing a well-epithelialized lumen without apparent airway obstructions.This technology holds promise for treating the benign and malignant diseases of the airway. 101The investigation also delved into the potential synergy of this technology with growth factor delivery to enhance graft performance and achieve optimal regeneration universally.The hypothesis entailed the addition of basic fibroblast growth factor (bFGF) to the cartilage defect, with the goal of enhancing vascular formation and preserving tracheal patency.Nevertheless, the use of bFGF might be restricted in cases involving malignant tumor populations due to an elevated risk of recurrence. 102 2010, the inaugural successful surgical implantation of DTGs took place, involving the transplantation of a decellularized cervical tracheal graft seeded with buccal mucosa. 109,110Although this approach was extended to a cohort of four patients, graft poor graft vascularization and necrosis still posed challenges in the clinical trial's outcome. 101In a different series of attempts, cadaveric tracheas underwent decellularization and pre-seeding with the recipient's MSCs and growth factors before implantation.The objective was to ensure substitute re-vascularization and promote chondrogenesis.
The results showed varying outcomes, including tracheal narrowing.
However, a particular DTG successfully regenerated epithelium around the damaged section without complications during a 4-year follow-up. 98,99While some TE approaches have shown promise, further extensive follow-up period research works involving a larger cohort are imperative to establish this technology as a viable alternative for tracheal reconstruction.
As previously mentioned, 3D printing technology has emerged as a potential approach for addressing airway lesions in pediatric patients, with recent FDA approval for 3D-printed airway splints. 1 The application of a 3D-printed splint to treat a serious patient of TBM was first published in the beginning of the 20th century 103 and has since been extended to a broader population, showing low general mortalities. 104,105However, the utilization of 3D-printed tracheal splints has primarily been limited to pediatric populations with TBM, leveraging guided airway growth during early developmental stages to facilitate natural resolution of TBM.Apart from cases involving pediatric cases, there is only one documented instance of using a 3Dprinted airway splint in an adult, demonstrating sustained airway patency 3 months post-procedure.Ongoing clinical trials are also exploring custom-made airway stents.These stents are meticulously crafted using patient-specific sacrificial molds 3D-printed from CT scanning, resulting in personalized tracheal prosthetics made from silicone elastomer.Several patients in clinical applications have reported an improved quality of life with no observed complications.While this personalized approach shows promise, long-term results are still pending.Despite early successful attempts to use 3D printing for tracheal stenosis and malignancies, there is a crucial need to extend follow-up studies to assess splint resorption.Additionally, establishing optimal designs and validated manufacturing processes is vital to ensure safety and efficacy in future clinical application attempts. 117e first step in the successful development of a TE tracheal substitute involves a thorough characterization of the proposed construct.This encompasses the assessment of material performance, degradation, mechanical strength, and flexibility.Moreover, the manufacturing process ideally allows for the production of individualized constructs tailored to specific patient needs, incorporating antibacterial, antiproliferative, antitussive, and non-migrating properties. 2,3A limitation in tissue-engineered substitutes is the absence of consistent and relevant data on airway biomechanics, standardized tests for evaluating mechanical properties, and a diverse range of mechanical values for potential grafts. 118Therefore, there is a need to establish a standardized procedure for evaluating mechanical strength to better comprehend the requirements of TE material.
This standardized procedure should also account for variations in airway biomechanics among patients of diverse genders or age groups. 116Various scaffolds have been devised to support epithelialization in grafts, as a mature pseudostratified epithelial lining is crucial to prevent tissue outgrowth, granulation, and stenosis. 119Various strategies have been explored in both laboratory settings and living organisms.However, the absence of prolonged evaluations and the occurrence of granulation and inflammatory reactions after transplantation have impeded further progress.This underscores the need for enhanced TE approaches that facilitate comprehensive and functional re-epithelialization early in the graft development process. 117Vascularization of TE scaffolds remains a critical challenge that must be addressed for stents to progress into clinical applications.It is widely acknowledged that vascularization is imperative to achieve a functional substitute, as the absence of vascular formation inevitably leads to poor outcomes, no benefit for the patient, and high morbidity. 120though several procedures reveal promise in implant revascularization, further long-term assessment of patients and adequate trials are necessary to demonstrate the suitability of these strategies for airway reconstruction. 100,101 summary, the restricted TE strategies for tracheal reconstruc-  121,122 The mismatch in mechanical properties, rigidity causing erosion of adjacent blood vessels, and the inability to integrate within surrounding tissue resulted in dislodgement during coughing and airway obstruction. 123Other solid prostheses, including polyethylene (PE), 124 stainless-steel wire, 125 silicone stent, 126 and tantalum, 127 have also been explored as alternatives in tracheal reconstruction.
Recognizing issues with solid prostheses, research turned to porous alternatives to facilitate better tissue formation and structure regeneration.A notable development in porous prostheses occurred with the successful development of Marlex mesh prosthesis, made from highdensity PE and polypropylene.In an animal study, it demonstrated adequate graft survival and patency during the 16-months follow-up. 128wever, its human application was disappointing, marked by erosion of surrounding blood vessels and lumen collapse. 129The Marlex prosthesis highlighted the significance of respiratory epithelium growth on the inner lumen, serving as a protective barrier against inhaled foreign bodies. 130Other attempts at porous prostheses included PE, silicone, dermal grafts, 66 wire mesh, 131 and biomedical engineering tubes, but these yielded similarly unsatisfactory results. 132rrent treatment for tracheobronchial complications often involves commercially available stents, such as the silicone-based self-expanding PolyFlex™, 133 the Ultraflex™, 134 Gold Studded Stents ® and Novatech's silicone Dumon ® . 135Clinical application of silicone stents has generally improved symptoms and quality of life.Despite being well-tolerated in most cases, long-term follow-ups have revealed significant drawbacks, including granulation tissue obstruction, stent migration, excessive immune response, infections, mucus retention, and lumen collapse, as documented in researche with extended follow-up periods. 136,137

| Aortic allograft
Allograft implantation for long-segment tracheal lesions has mainly concentrated on two donor tissue sources: tracheas and aortas.
Evaluations of the viability of fresh and cryopreserved stented aortic grafts as a potential substitute for tracheal reconstruction have verified newly formed cartilage and respiratory epithelium regeneration in many animal models.However, some pre-clinical studies have reported unsatisfied cartilage regeneration. 138Despite these promising findings in preclinical models, clinical attempts to utilize stented aortic grafts as donor tissues for airway replacement have raised notable concerns.In a 2006 case, a 68-year-old male experienced severe complications and ultimately succumbed to the procedure involving aortic allograft transplantation. 112In another instance, a 78-year-old male was supported with a stent after the implantation of a cryopreserved and stented aortic allograft.However, this case exhibited no cartilage formation, accompanied by a decrease in forced expiratory volume. 114Similar concerns were observed in a clinical cohort study involving six patients who received cryopreserved descending aortas that were silicone-stented and subcutaneously implanted for prevascularization.Long-term follow-ups revealed minor complications, including stent migration, blood vessel erosion, and even tracheoesophageal fistulas in some cases.Furthermore, biopsied tissues showed no evidence of newly formed cartilage or respiratory epithelium regeneration. 139e TRITON- Furthermore, 28.6% of patients achieved stent-free survival, with a 5-year survival rate of 75%. 116

| Tracheal graft
The inaugural human tracheal transplantation, conducted in 1979, demonstrated no graft rejection or major complications during a 2-month follow-up. 109In 1990, another early success involved a onestage primary tracheal replacement with a silicone stent and extensive immunosuppressive therapy, with no reported issues in a follow-up duration of 2 years. 110Despite these early achievements, subsequent attempts at trachea transplantation encountered limited clinical success. 140Recent advancements by Delaere et al. 141 introduced a twostep procedure, involving the revascularization of the tracheal graft in the patient's forearm before implantation. 142Although tissue necrosis occurred in some cohort studies, the technology remains promising. 140lograft transplantation, providing a structurally sound analogue, exhibits variable success.Successful cases often involve long-term immunosuppressive therapy, which may be unsuitable for malignant cases.Allografts, lacking adequate mechanical properties and cartilage regeneration, usually necessitate stent support. 140However, stents bring new challenges, including anastomotic fistula, erosion of adjacent tissue and blood vessels, infection, and granulation formation.Poor re-vascularization of grafts requires implantation into a second surgical site, heightening hospitalization risks and infection. 140spite limited success with traditional organ implantation, past attempts provide insights into the requirements for successful tracheal grafts, steering current research toward overcoming these challenges using TE strategies.
Clinical exploration of tracheal transplantation with autologous tissue involves a two-step operation procedure.Tracheal autografts involve harvesting cartilage from ribs, encasing it within a forearmderived fascial skin pad, and wrapping it around a temporary silicone scaffold for transplantation.Preserving the radial artery and skin pad veins is vital for post-transplant conduit revascularization.This technique eliminates the need for immunosuppressants, as no synthetic materials are introduced.143However, challenges exist: autografts lack a respiratory epithelial layer and depend on healthy cartilage and respiratory function, posing risks like secretion obstruction and cartilage fracture leading to implant failure.143,1447 | CONCLUSION Over the past decade, TE strategies have emerged for tracheal tissue regeneration, but their clinical application has been limited and smallscale.Challenges such as poor mechanical properties, insufficient vascularization, and inadequate re-epithelialization have hindered graft performance.Long-term follow-up assessments in both pre-clinical and clinical studies are lacking, essential for evaluating feasibility.A successful airway reconstruction construct must have the necessary mechanical properties and support the formation of vascular capillaries and respiratory epithelium to prevent complications like granulation formation, contamination, tissue necrosis, fistula, and lumen collapse.TE in airway reconstruction, highlighted by promising prototypes and clinical trials, provides insights into essential properties for a successful tracheal substitute.Addressing challenges in mechanical properties, epithelialization, and vascularization could revolutionize the clinical approach to long-segment tracheal lesions.
Animal studies on tissue-engineered tracheal replacement approaches.
T A B L E 2 (Continued) T A B L E 2 (Continued) Transitioning TE and non-TE strategies for tracheal reconstruction and regeneration in human clinical trials.
1 study (Clinicaltrials.govIdentifier: NCT04263129), led by Martinod's group from October 2009 to October 2021, involved 35 patients undergoing trachea or bronchus reconstruction for both benign and malignant lesions.The overall hospital mortality rate was 2.9%.With a median follow-up of 2.5 years, 77.1% of patients remained in good health condition, with no reported deaths directly attributed to the implanted grafts.Scaffold-related granulomas requiring bronchoscopic treatment occurred in 52.9% of patients.