Tracheal and main bronchi dysfunctions represent an unmet medical need in respiratory medicine. Indeed, congenital malformations, malignancies, inflammations, infections, or even traumatic events, including postoperative complications, can alter tracheal and main bronchi structure and function, heavily affecting patients’ life. Nowadays, this issue represents an extraordinary leading-edge topic. Indeed, as recently stated by the European Laryngological Society, the heightened number of long-term intubations and the huge tracheostomy rate in critically ill COVID-19 patients might shortly determine an unprecedented increase in laryngotracheal granulomas, stenosis, malacia, tracheal necrosis, tracheo-oesophageal and trachea-innominate fistulae (Alturk et al., 2020; Mattioli et al., 2021; Piazza et al., 2021). The first-choice treatment for managing short tracheal structural alterations is based on tracheal resection and reconstruction by end-to-end anastomosis. However, surgery is inapplicable in longer tracheal injuries exceeding 5 cm in adults and 3 cm in children, and only palliative care can be offered to these patients (Delaere et al., 2019).

To date, a variety of approaches has been proposed and clinically adopted to replace long-segment tracheal defects and reconstruct this vital organ. However, despite the great efforts, the limited results clearly pointed out that a final solution for restoring a functional respiratory system is extremely challenging and far from being addressed (Grillo, 2002; Soriano et al., 2021).

In the last century, a plethora of non-TE approaches has been proposed and applied in small series or even single patients to treat long-circumferential tracheal defects. Based on common methodologies, these attempts can be further clustered into three main categories: synthetic prostheses, allotransplantations, and autologous tissue reconstruction. Table 1 summarizes some of the main non-TE approaches tested so far and still considered as potential tracheal reconstruction strategies.

Tissue engineering (TE) approaches rely on the combination of cells with an appropriate scaffold for the treatment of significant tissue defects. Indeed, in this interdisciplinary field, principles of biomaterial engineering, genetics, cell biology and clinical science are combined to develop a biological graft to maintain, restore or improve tissues or whole organs (Shafiee and Atala, 2017; Vranckx and Hondt, 2017). This solution can simultaneously address different problems, such as donor shortage (often fatal for patients in critical status, struggling with long waiting lists), immunosuppression therapy and donor site morbidity. In fact, TE strategies take advantage of the body’s regenerative potential and, by in vitro expansion, allow to obtain enough cells to regenerate extensive body areas from a small biopsy (Gallico, 1985; Corradini et al., 2016). Thus, this field represents a promising strategy for expanding the current reconstructive armamentarium to treat severe unmet medical needs (Vranckx and Hondt, 2017).

Between 2008 and 2017, TE attempts for windpipe and main bronchi’s replacement have been tested in compassionate cases, and the trachea has been acclaimed by both the scientific community and mass media as the first tissue-engineered organ (Fountain, 2012). However, this field was impaired by a history of scientific and ethical misconduct (The Lancet, 2018; The Lancet Editors, 2018; Jungebluth et al., 2019), which has led to a general sense of mistrust concerning airway TE potential.

To date, three main strategies have been proposed in tracheal TE, which involves the use of allogenic decellularized human cadaveric donor tissue, synthetic patient-tailored scaffolds, or an “in vivo bioreactor” strategy. Table 1 summarizes some of the main experiences in this field.

Failures and controversies raised by the clinical application of several tracheal substitutes have led to a renewed interest in preclinical studies based on animal models. According to Niermeyer et al., 73% of articles focused on tracheal reconstruction and published between 2015 and 2020 involved an in vivo preclinical model. This reveals the need for a more in-depth preliminary analysis of the tracheal substitutes before embarking on new clinical applications (Niermeyer et al., 2020).

This paragraph focuses on the latest in vivo preclinical TE attempts, since they stand as one of the most promising approaches to face hurdles that still hinder this field. Indeed, articles concerning tissue-engineered tracheal grafts (TETGs) aim to improve graft integration within the recipient, mimick complex native trachea biomechanics and prevent host adverse responses (Greaney & Niklason, 2021). To give an idea of the possible future clinical applications, a few non-exhaustive examples belonging to the different categories of the current TE approaches are listed below and summarized in Table 2.

Concerning the use of synthetic material, several methodologies have been investigated to produce tubular scaffold suitable for tracheal replacement. In 2011, a casting-based manufacturing process was used to create a tracheal-shaped substitute starting from mixtures of rat fibroblast/MSCs and collagen hydrogels. The stiffness of the resulting bioartificial trachea was compared to the one of the native trachea showing no differences. However, when the former was transplanted in a rat model, only three of the nine treated animals survived the implantation, dying within 48 h after surgery. The author suggests that these results could be related to the absence of epithelial cells on the inner layer of the transplanted bioartificial trachea (Naito et al., 2011).

Some years later, Pepper et al. employed a polyethylene terephthalate (PET) and polyurethane (PU) scaffold combined with polycarbonate rings. This structure was seeded with BM-MNCs and transplanted in eight sheep to replace a 5 cm long tracheal defect. Despite promising mechanical tests, all animals showed graft stenosis associated with granulation tissue. Overall, this attempt remarked the centrality of the epithelialization and neovascularization, in the absence of which the outcomes are poor (Pepper et al., 2019).

Using a mouse model, another group compared synthetic non-resorbable PET/PU vs. resorbable poly(l-lactide-co-ε-caprolactone)/Polyglycolic acid (PLCL/PGA) scaffolds. Even in this study, graft’s stenosis was revealed in both conditions, with no signs of respiratory epithelization in the central part of the grafts (Dharmadhikari et al., 2019).

Several groups also investigated synthetic scaffolds based on polycaprolactone (PCL), given its easy moldability through 3D printers (Gao et al., 2017; Rehmani et al., 2017). To mimic the tracheal structure and mechanical profile, Lee and colleagues used a bellows-designed PCL scaffold reinforced with collagen, silicon rings and seeded with human turbinate mesenchymal stromal cells (hTMSC) sheets (Lee et al., 2018). After implantation in rabbits, the PCL was successfully incorporated within the adjacent tissue and lined by airway epithelium. However, respiratory distress and mild granulation process were observed in all animals. Additionally, the higher levels of interleukin-2 and interferon gamma detected in treated animals, compared to the baseline values, suggested a possible acute rejection (Lee et al., 2018). Two years later, another group used both electrospinning and 3D printing techniques to generate a PCL synthetic scaffold for tracheal reconstruction in a rabbit model. In this study, human bronchial epithelial cells (hBECs) were used to populate the inner layer of the PCL scaffold, while the outer layer was repopulated by either induced pluripotent stem cells-derived mesenchymal stem cells (iPSC-MSCs) or induced pluripotent stem cells-derived chondrocytes (iPSC-Chds). A regenerated epithelium was observed in both conditions at the study endpoint (4 weeks). However, in the iPSC-MSCs group, the epithelium was fully specialized and better organized than in the iPSC-Chds group, suggesting a paracrine effect of iPSC-MSCs in promoting the re-epithelialization process (Kim et al., 2020). To overcome the aforementioned problems related to synthetic tracheal TE grafts, other groups tried to reduce the immunogenicity of the grafts, increasing their survival prospects through pre-implantation vascularization strategies (Soriano et al., 2021). For example, Zhao et al. seeded smooth muscle cells onto a PGA-nitinol stent scaffold to allow the deposition of a collagenous matrix and increase the scaffold angiogenetic properties. The resulting construct was then decellularized, preserving the new extracellular matrix, and implanted in rabbits and nonhuman primates. No graft collapse was observed, while a columnar respiratory epithelium lined the construct lumen. However, mid-grafts stenosis was present (Zhao et al., 2016). In the preclinical study performed by Sakaguchi and colleagues, 80% of dogs transplanted with a pre-vascularized collagen-coated nitinol scaffold survived for more than 18 months. No signs of stenosis were reported, while a monolayer of ciliated cells covered the graft’s lumen. These promising results may be due to the pre-vascularization obtained through the graft’s heterotopic implantation into the omentum (Sakaguchi et al., 2018). Similar beneficial effects of the pre-vascularization process were observed in a rabbit preclinical study where a 3D printed PLLA (poly L-lactic acid) scaffold seeded with autologous chondrocytes was used to reconstruct a 1.5 cm long tracheal segment. Indeed, the engineered pre-vascularized trachea appeared integrated within the adjacent tissues and covered by respiratory epithelium. Besides, no signs of stenosis were observed with only sporadic granulation tissue formation (Gao et al., 2019).

Besides synthetic scaffolds, decellularized grafts have been employed in preclinical settings. However, when used without the cellular component, they behaved similarly to the polyhedral oligomeric silsesquioxane–poly (carbonate-urea)urethane (POSS-PCU) synthetic scaffolds, mainly developing stenosis (Maughan et al., 2017). Otherwise, when recellularized with epithelial cells and MSC-derived chondrocytes, they become functional and well-tolerated, without signs of rejection or airway collapse (Go et al., 2010). These results remark the need for the cellular component to promote graft re-epithelialization and structural stability essential for preventing host inflammatory response and graft failure.

Finally, new frontiers may arise from scaffold-free constructs. Indeed, through a novel Bio-3D printing system, many groups developed artificial tracheas assembling spheroids composed of various cell types, such as chondrocytes, MSCs and endothelial cells (Taniguchi et al., 2018; Machino et al., 2019). In these studies, to obtain a structure that recapitulates the tracheal shape, the multicellular spheroids were first placed into needle arrays and then underwent a maturation phase inside a bioreactor. Finally, the artificial scaffold-free constructs were implanted in rat models to evaluate the graft performance. At the end of the studies, these artificial tracheas showed signs of chondrogenesis, vasculogenesis, and an epithelium lining the lumen. However, the need for a permanent stent to avoid graft collapse and the presence of granulation tissue represent some limitations of this innovative technique (Taniguchi et al., 2018; Machino et al., 2019).

Tracheal and main bronchi dysfunctions represent an unmet and growing medical need, especially in the case of wide circumferential structural alterations where available surgical strategies are ineffective or inapplicable. The long-term clinical outcomes of reconstructive approaches tested so far clearly point out the difficulty of restoring a functional trachea (Grillo, 2002; Soriano et al., 2021).

Since the first challenge of any tracheal replacement’s attempt is to reproduce or mimic the native tracheal structural properties, a suitable tracheal substitute must fulfil specific biomechanical requirements. Indeed, it has to be airtight and possess longitudinal flexibility and lateral rigidity to withstand forces arising from respiration, coughing, neck movements and pressure created by the adjacent oesophagus (Grillo, 2002; Boazak and Auguste, 2018). Former approaches not fulfilling these requirements resulted in airway collapse, strictures, graft migration, or haemorrhages which, altogether, stand as the primary cause (68%) of graft-related mortality (Greaney and Niklason, 2021). Consequently, a preliminary assessment of the tracheal substitute’s suitability needs to be performed to reduce avoidable life-threatening adverse events on the patients. Indeed, several in vivo preclinical studies have been carried out to evaluate biochemical properties of decellularized (Zhao et al., 2016), synthetic (Dharmadhikari et al., 2019) or scaffold-free constructs (Machino et al., 2019). However, since the first requirement for an adequate tracheal substitute is to mimic the tracheal’s native biomechanical properties, the definition of standard approaches and biomechanical tests would be extremely helpful to obtain comparable results among different studies and further improve this field (Martínez-Hernández et al., 2021).

Another limitation for a successful reconstructive approach is the lack of studies on the biocompatibility between the tracheal substitute and the host. As previously described, this problem has been reported to trigger acute rejection, granulation tissue formation and graft necrosis, especially in the case of synthetic prostheses (Neville et al., 1976, Neville et al., 1990; Toomes et al., 1985), bioprosthetic substitutes (Hoffman et al., 2001) and tracheal allograft (Levashov et al., 1993; Delaere et al., 2010; Delaere et al., 2012). Before implantation, specific biocompatibility studies should be performed to ensure that the tracheal substitute is made of safe, non-immunogenic material, well-tolerated by the recipient and able to integrate within the body. In order to avoid the aforementioned rejection problem, particularly marked in the case of allograft, also autologous tissues have been clinically applied (Olias et al., 2005; Spaggiari et al., 2005; Fabre et al., 2013, Fabre et al., 2015; Zhang and Liu, 2015; Mercier et al., 2018). However, even this procedure presented some problems, such as donor-site morbidity (Udelsman et al., 2018). In this scenario, TE represents a unique opportunity to rebuild extensive body surfaces, combining cells extracted from a small autologous biopsy with an appropriate scaffold (Shafiee and Atala, 2017; Vranckx and Hondt, 2017).

Regardless of the reconstructive approach, one of the common hurdles in tracheal replacement is graft epithelialization. Indeed, airway functions are critically dependent on the respiratory epithelium, and its absence triggers several complications, including mucous stagnation, infections, graft stenosis, granulation tissue formation, chondromalacia and fibrogenic reaction (Heijink et al., 2014; Paternoster and Vranckx, 2021). Therefore, any tracheal replacement attempt should be aimed at restoring a functional and well specialized respiratory epithelium (Davis and Wypych, 2021). Several aspects must be considered to ensure the presence of a continuous, self-renewing and specialized respiratory epithelium lining the lumen of the tracheal substitute. First, due to the respiratory tissue’s high complexity and cellular heterogeneity, epithelial cells or sheets derived from other autologous districts (i.e., skin or oral mucosa) cannot be used for airway functional recovery (Tan et al., 2017; Udelsman et al., 2018). Second, spontaneous post-surgery reepithelization of the tracheal substitute cannot be taken for granted. In clinical and preclinical studies, several groups relied on the migration of epithelial cells from the recipient’s wound edges (Martinod et al., 2017, Martinod et al., 2018; Rehmani et al., 2017), with some successful epithelialization only on short-sized scaffolds (Zhao et al., 2016; Lee et al., 2018; Sakaguchi et al., 2018). Indeed, local epithelial cells can only cover a few millimeteres of the graft, while the central part of the tracheal substitute is often left uncovered, leading to the aforementioned adverse consequences of an absent epithelium (Delaere and Van Raemdonck, 2019). Therefore, a precise and rational reepithelization strategy is mandatory, especially for tracheal substitutes of larger dimensions. On this note, in TE approaches, the culture conditions used for the in vitro expansion phase must preserve the proliferative and differentiative potential of the airway epithelial stem cells, preventing their exhaustion. Indeed, the experience gained from regenerative approaches of other human epithelial tissues clearly pointed out that a specific number of stem cells is strictly required to allow the permanent engraftment, renewal, and restoration of the epithelium (Pellegrini et al., 2011; Maurizi et al., 2021). In order to carefully monitor these aspects and have a clear overview of all the process variables, extensive cellular characterization must be performed. This comes through the identification of population-specific molecular markers to be adopted as quality controls during each step of the reconstructive procedure.

To date, there is a huge gap between basic-research knowledge on airway epithelial cells’ biology and the application of these insights to TE translational approaches. Indeed, despite the huge work carried out to understand the airway epithelial cells physiology and heterogeneity (Garcıá et al., 2019; Basil et al., 2020; Goldfarbmuren et al., 2020; Zaragosi et al., 2020; Davis and Wypych, 2021), the few groups that clinically employed airway epithelial cells for tracheal TE approaches did not exploit this knowledge, reporting only limited or inadequate cellular characterization (Macchiarini et al., 2008; Elliott et al., 2017).

In order to bridge this gap, preclinical studies—especially those based on in vitro models—can be extremely useful to precisely evaluate all the interactions among the different components of the bioengineered construct. Indeed, once in contact with the scaffold, colonizing cells should retain their ability to proliferate and differentiate into their respective specialized cell types, a crucial aspect for re-establishing the system’s physiology. Moreover, in the case of TE approaches encompassing cells derived from different tissues (i.e., epithelial cells, chondrocytes, endothelial cells, neural cells etc.), their mutual interaction must be carefully studied to exclude possible acute or chronic cytopathic effects.

Another common issue with all the tracheal reconstructive approaches is the vascularization of the tracheal substitute. Till now, this aspect has deeply hampered the outcomes of treated patients in several clinical studies (Delaere et al., 2012; Delaere et al., 2019). Indeed, efficient and rapid restoration of the blood supply is mandatory to sustain graft survival, allow its integration within the surrounding tissue, avoid necrosis or contamination, and support new cartilage and epithelium regeneration. Some approaches tried to overcome this matter by wrapping tracheal substitutes in a highly vascularized tissue, frequently transposed omentum (Levashov et al., 1993; Elliott et al., 2012; Menna et al., 2021). Although these efforts have been initially interpreted as successful, long-term follow-ups demonstrated that, in most cases, this strategy only temporarily delays the inevitable consequences of wound breakdown at the anastomoses (Delaere and Van Raemdonck, 2014). Alternatively, tracheal substitutes can be in vivo vascularized through heterotopic implantation into the recipient’s forearm, followed by orthotopic repositioning. Still, such strategies’ long-time requirements and invasiveness have strongly limited their application. Once more, a possible solution may arise from in vitro or in vivo preclinical studies investigating the formation of a vascular network through interaction with endothelial cells or from stimulating the revascularization process through the delivery of pro-angiogenic factors (Lovett et al., 2009).

Alongside vascularization, restoring a functional innervation system within the tracheal substitute should be considered. Indeed, the airway epithelium works conjointly with the immune and nervous systems to guarantee respiratory homeostasis (Davis and Wypych, 2021). So far, this aspect has been neglected, as no significant clinical studies have encompassed the inclusion of this component. Thus, it remains a crucial issue to be addressed in the future.

Finally, another critical point to be mentioned regards the legislative side. Since their proposal in 2001 (Directive 2001/83/EC later expanded in REGULATION (EC) No 1394/2007; https://www.ema.europa.eu/en/human-regulatory/overview/advanced-therapies/legal-framework-advanced-therapies), GMP have further complicated the development of novel tissue-engineered advanced therapeutic medicinal products (ATMPs). These regulations have proven themselves critical to achieving safer and standardized advanced therapies (Aiuti et al., 2017; Hirsch et al., 2017; Ram-Liebig et al., 2017; Barbagli et al., 2018; Pellegrini et al., 2018; Kueckelhaus et al., 2021). Nevertheless, the resulting complexity and high expenses in the manufacturing process strongly discouraged researchers from exploring the TE area of study. These obstacles have led to the predilection of non-TE approaches, which instead fall under less stringent legislation.

To conclude, tracheal reconstruction stands as a huge challenge and has yet to be achieved. In this review, we summarized the main lessons learned from the clinical and most recent in vivo preclinical studies, as well as the new frontiers of tracheal TE. We have highlighted the most common issues that still hinders tracheal reconstruction. In our opinion, a strong correlation between basic science and translational medicine is mandatory; careful and extensive preclinical studies are crucial to tackling all the described aspects. Before embarking on new clinical applications, more work on the preclinical side should be done to prevent patients’ exposure to avoidable life-threatening consequences. Only by addressing these points reconstructive approaches can become a turning point in long-circumferential tracheal defects management and affirm themselves as a milestone in regenerative medicine.