3D-Bioprinting for Lung and Tracheal Bioengineering: An Overview

1. Introduction

In our respiratory system, the lungs play a crucial role in the physiological function of respiration, involving blood oxygenation and gas exchange [1]. In the human body, the lungs are divided into distal and proximal regions, each with distinct anatomical structures and physiological functions. The distal region of the lung consists of bronchioles, alveolar ducts, alveolar sacs, and alveoli, which are responsible for gaseous exchange, airway collapse, and immune defense [2]. It is also lined by various cell types including a continual layer of airway epithelial cells, alveolar type I, and alveolar type II cells [3]. The proximal region of the lung comprises the two-thirds volume of the lungs, consisting of alveolar spaces, conducting airways, and small airways lined by respiratory epithelium or respiratory mucosa [4]. This epithelium consists of ciliated pseudostratified columnar epithelial cells, basal cells, goblet cells, and various secretory cells [5]. The primary functions of this region include airway conduction, mucociliary clearance to remove debris and pathogens, filtration, and air humidification [6]. Among all these components in the proximal region, the trachea is the most important conducting airway, which facilitates air passage from the nose to the lungs. It is a semiflexible, cartilaginous, and tube-like structure ranging from 10 to 14 cm long and 1.5–2 cm wide [7]. The trachea comprises 18 to 22 D-shaped cartilaginous rings, with C-shaped cartilage forming the anterior and lateral walls. The trachea is vascularized by several segmental arteries that supply blood to the trachea through its lateral wall. Within the tracheal wall, tracheal arteries further divide into anterior and posterior branches, linking with parallel arteries from the other side. The subclavian artery’s tracheoesophageal branches supply blood to the cervical trachea, while the thoracic trachea gets its blood supply from bronchial arteries branching directly from the aorta [8]. This intricate blood supply is crucial for the trachea’s function and healing, making it a critical factor in tracheal surgeries. One of the primary challenges of tracheal reconstruction is the difficulty of re-establishing this essential blood supply, as inadequate new blood vessel formation often leads to graft failure.

In a healthy lung, cell turnover occurs gradually. However, when the lung is damaged, lung stem cells show significant self-renewal, proliferation, and differentiation to maintain cellular balance [9]. After major injuries such as surgical lung lobe removal, extensive repair occurs to compensate and create new alveoli. A group of adult stem cells (ASCs) are involved in repairing and regenerating the lungs in response to injury [1]. Airway basal stem cells (ABSCs) are the primary adult stem cells found in the airway surface. They are characterized by markers Tp⁶³⁺, Krt⁵⁺, PDPN⁺, NGFR⁺, and Krt¹⁴⁺. There are different sub-populations within ABSCs, such as “hillock” cells (marked by Krt¹³⁺ cell markers), which are involved in forming squamous epithelium [10]. Another variant of basal stem cells, Trp⁶³⁺ and Krt⁵⁺ cells known as distal alveolar stem cells (DASCs), have been identified as a group of cells capable of replacing injured alveolar cells [11]. Some researchers suggest that a set of Trp⁶³⁺ and Krt⁵⁺ cells, termed lineage-negative epithelial precursor (LNEP) cells, are responsible for regenerating alveoli damaged by bleomycin [12]. Multipotent bronchoalveolar stem cells (BASCs) marked by CC¹⁰⁺ and SPC⁺, located at the bronchioalveolar duct junction, can differentiate into various cell types including bronchiolar epithelium, type 2 (AT2) and type 1 (AT1) cells, aiding in distal airway repair. Pulmonary neuroendocrine cells (PNECs), concentrated at airway bifurcations, act as progenitors during wound or damage-induced healing. LGR⁶⁺ cells also demonstrate the ability to differentiate into multiple mature cell types within the bronchioalveolar region and contribute to tissue regeneration [1]. AT2 cells marked by Sftpc⁺ are specialized stem cells found in distal lung tissues capable of replacing damaged AT1 cells (marked by HOPX⁺ and AQP⁵⁺). Secretory (club) cells also possess the ability to self-renew, differentiate into ciliated cells, as well as de-differentiate into basal cells (Figure 1) [1, 13, 14, 15].

Because stem cells can self-renew, proliferate, and differentiate into various cell lineages, they hold great potential for regenerative medicine [16]. Although the majority of lung regeneration research is conducted using mouse models, it is recognized that the lungs of humans and mice differ significantly biologically. Nonetheless, current research on the human lung suggests that key transcriptional and signaling pathways, as well as cell lineages, are quite comparable [17, 18, 19, 20]. Huang et al. [21] have generated lung progenitor cells from hESC/iPSCs. These cells can differentiate into airway epithelial cells and produce definitive endoderm by enhancing differentiation. After development in vitro and in vivo in immunodeficient mice, the lung progenitors were able to turn into various pulmonary epithelial/progenitor cells, including basal cells, club (Clara) cells, goblet cells, and ciliated cells. The progress in understanding how ESCs and iPSCs can be directed to differentiate in the pulmonary area holds significant therapeutic promise [21]. Recently, chronic respiratory disorders had a significant impact on millions of individuals worldwide. Lung transplantation can be used as a treatment option for patients with end-stage chronic respiratory diseases. However, there are several significant challenges associated with lung transplantation including a shortage of donor lungs, the risk of organ rejection, and post-transplant infections. These challenges highlight the need for advancements in lung regeneration techniques, which could offer alternative treatment options and improve outcomes for patients with chronic respiratory diseases [22, 23]. Recently, tissue engineering approaches have gained immense attention, particularly in the field of airway bioengineering. 3D bioprinting is one such approach that has shown promise in mimicking intricate airway tissue constructs [24]. Despite several attempts, creating a successful 3D-bioprinted airway tissue substitute for airway bioengineering has been challenging due to various factors. This chapter aims to highlight the recent advancements in regenerative medicine and airway bioengineering, the level of customizations achieved in mimicking the physical aspects of airway bioengineering, cellular assembly, and the current gaps for further development toward clinical application (Figure 2).

2. Airway bioengineering

Currently, chronic respiratory disorders continue to cause considerable health burden worldwide. Organ transplantation is the only option for individuals with end-stage airway disorders. However, organ transplantation remains a challenge due to various factors. To address this unmet clinical need, various tissue engineering strategies have been used to create a functional and transplantable tissue or organ. Airway tissue engineering aims to create a fully functional airway biomimetic construct, thus eliminating the requirement of organ transplantation. Additionally, the generation of physiologically relevant artificial tissue constructs for evaluating novel drugs and identifying clinical solutions for chronic respiratory disorders [25]. The prospect of producing lung tissue ex vivo for transplantation is also being investigated via an increasing variety of tissue engineering techniques [26].

Various approaches have been used in airway tissue engineering including cells, scaffolds, growth factors, and fabrication techniques for the creation of fully functional physio-mimetic airway construct [27]. The choice of an ideal scaffold is one of the most controversial debates in airway tissue engineering. An ideal scaffold should be biocompatible, bioactive, non-immunogenic, and non-toxic which imitates the internal environment while maintaining the mechanical properties of the lungs and trachea [23]. Scaffolds can be created from a variety of polymeric materials. These include decellularized matrix, gelatin, collagen, silk fibroin, pluronic F-127, poly-lactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), and poly-lactic-glycolic acid (PLGA). These materials can be used individually or in combination with one another [28]. In a preliminary study, Delaere et al. implanted a recellularized decellularized tracheal scaffold which was pre-treated with epithelial stem cells into the recipient’s forearm. After 4 months of implantation, the scaffold transformed into a fully vascularized and regenerated tissue, which could be used to treat tracheal defects [29]. Park et al. in [30] created a 3D ring-shaped scaffold using indirect 3D-printing. The scaffold was made of polycaprolactone (PCL) based silicone bellow and coated with a layer of decellularized tracheal mucosal matrix and human inferior turbinate mesenchymal stromal cell (hTMSC) sheets. They implanted this tracheal graft into rabbits and found successful healing with complete re-epithelization after 2 months [30]. Growth factor selection is also crucial for the regeneration of the airway tissues. Various growth factors including platelet-derived growth factors, epithelial growth factors, basic fibroblast growth factors (bFGFs), vascular epithelial growth factors (VEGF), granulocyte colony-stimulating factors, transforming growth factors (TGF), insulin-like growth factors have been used for tracheal tissue engineering [23]. Apart from selecting biomaterials and growth factors, the choice of fabrication techniques is also crucial for airway bioengineering. Decellularization, electrospinning, and 3D bioprinting are the most commonly used techniques for airway bioengineering [31, 32, 33].

The most widely used technique for creating functional airway replacements over the past decade has involved the use of decellularized scaffolds derived from rodents, birds, pigs, and humans [34]. Decellularization is the process of removing cells from tissue, which leaves behind the proteins of the extracellular matrix that maintain the mechanical and bioactive properties of the tissue [35]. Butler et al. found that using a vacuum-assisted technique for decellularization improves the quality of tracheal grafts for therapeutic use. Acellular grafts have been clinically studied for human tracheal bioengineering [36]. In a study conducted by Shin et al., a decellularized scaffold seeded with chondrocytes was implanted in a rabbit model, resulting in the regeneration of respiratory tissue and neo-cartilage with a low inflammatory response [37].

Currently, most studies recommend 3D bioprinting over other traditional scaffold-fabricating techniques because it has a better ability to mimic and create intricate tissue structures by placing cells, biomaterials, and growth factors in a precise, layer-by-layer manner [38]. With 3D bioprinting, we can achieve high precision and control over factors like porosity, pore size, and overall structure. Additionally, we can accurately place growth factors, drugs, and other biochemicals alongside cells, while maintaining proper control over scaffold fabrication [39, 40, 41]. In 2015, Horvath and colleagues used 3D bioprinting to print a gelatinous protein mixture that contained epithelial and endothelial cells. This resulted in homogeneous and thinner cell layers compared to manual approaches, which are necessary for creating an ideal air-blood barrier. This study represented a significant advancement in validating the printed airway tissues, even though the air-blood barrier system obtained was not suitable for implantation. Nonetheless, it shows great promise for pharmaceutical and in vitro toxicology evaluations using basic lung studies (Figure 3) [42].

3. 3D-bioprinting

The field of bioengineering has expanded over the years to include the creation of new techniques for tissue regeneration, repair, and replacement. This also involves constructing in vitro tissue models to test and screen medications, as well as to understand different disease mechanisms and tissue development. Despite advancements in bioengineering, there is still a lack of intricate tissue models and tissue-tissue interfaces for testing and discovering various drugs. Additionally, there is a shortage of donor tissues available for transplantation or tissue regeneration [40]. These issues, including tissue or organ shortages, challenges with tissue models, and tissue-tissue interface, can be addressed with tissue fabrication technologies [43]. Biofabrication is the creation of a structurally organized, biologically active product using automated methods that involve live cells, biomolecules, and cell aggregates such as biomaterials, microtissues, or hybrid cell materials. This is achieved through bioprinting or bioassembly, followed by a tissue maturation process [43]. In the traditional tissue fabrication method, cells and macromolecules are placed into a scaffold with a porous structure that mimics the characteristics of the extracellular matrix. This method has successfully been used to engineer various tissue constructs, such as cartilage, bone, and skin. However, it has limitations in reproducing the natural structure of tissues and organizing different types of cells in a specific pattern [41]. Moreover, it lacks versatility and reproducibility [43]. These issues can be addressed through three-dimensional (3D) bioprinting, also known as additive manufacturing or rapid prototyping [41]. Using 3D bioprinting, we can create intricate tissue structures by precisely arranging biomaterials and cells layer by layer [40]. The precision and ability of 3D bioprinters to deposit biomaterials accurately, while better mimicking the complex structure of tissues through heterogeneous placement of cells and vasculatures, give them an advantage over traditional scaffold-based methods. In 3D bioprinting, we can achieve high precision and control over porosity, macro-morphology, and pore size, as well as precise placement of growth factors, drugs, DNA, proteins, and other biochemicals alongside cells. This allows for proper control over the fabrication of the scaffold [39, 40, 41]. By coordinating bioink deposition and motorized stage movement, 3D bioprinting creates a 3D tissue construct with pre-programmed geometries and structures made of living cells and biomaterials [43]. Different bioprinting strategies have been developed over the years, each with its own applications, limitations, and advantages. The commonly used bioprinting techniques include stereolithography-based bioprinting [44], inkjet bioprinting [45], extrusion-based bioprinting [46], and laser-based bioprinting [47].

3D bioprinting consists of three main components: the bioink, the bioprinter, and the associated bioprinting procedures. These procedures involve creating a 3D model, depositing the cell-laden bioink, and then incubating the bioprinted organ for maturation [48]. The process of 3D bioprinting involves three main steps: preprocessing, processing, and post-processing. During preprocessing, the targeted organ or tissue is imaged using ultrasound, CT, and MRI to create a 3D model, which is then converted into a file compatible with a 3D bioprinter. In the processing stage, human cell lines are cultured and suspended in bioink that mimics the targeted tissue’s properties. This cell-laden bioink is then precisely deposited layer by layer according to the 3D model to create the living 3D organ or tissue. Finally, in the post-processing stage, the bioprinted organ or tissue is incubated in a bioreactor to mature before being used for drug testing, transplantation, or as a disease model [39, 40, 41].

The three approaches used in 3D bioprinting are mini tissue building blocks, biomimicry, and autonomous self-assembly [49].

4. 3D-bioprinting for lung bioengineering

Efforts have been made to replicate the structure and functions of the alveoli, aiding the study of gas exchange interactions, development of pulmonary disease models, and drug testing and toxicology studies. The major challenge in achieving this is the difficulty in mimicking the complex cell composition in a thin layer and providing a supporting microenvironment. Additionally, it is difficult to mimic the elasticity of the tissues and the gas permeability across the membrane [50]. The distal lung presents the challenge of incorporating different cell types at specific locations to form functional membranes, alongside their complicated geometries. To address this issue, 3D bioprinting approaches have been used. These techniques are necessary to recreate the 3D microenvironment and mimic the organ’s natural system. For studies of the air-blood barrier system, 3D bioprinting is needed to control cell deposition at the micro or nanoparticulate levels. 3D bioprinted models can help to reduce the need for animal testing by mimicking in vivo conditions [42]. Recent advances have improved the control and resolution of printing strategies to enhance cell viability during and after printing. There have also been significant developments in optimizing the mechanical properties of bioinks and printed structures. 3D bioprinting is also necessary for creating organoids and models of lung disease or lung cancer for studying their development and testing drugs (Figure 4) [50].

For bioprinting organ models, the isolation of the respective cell progenitors is required. Often, the isolation of progenitors of the distal lung is challenging due to the presence of proximal cell progenitors that contaminate the sample. This leads to a decrease in efficacy and a decreased level of cell differentiation into the desired types. The bronchioalveolar stem cells are the progenitor cells for the distal lung. These are essential for the repair of damaged epithelial cells. To overcome the contamination of proximal progenitor cells, Alsafadi et al. devised a 3D-printed guide to isolate progenitor cells from individual mice without contamination. This 3D lobe divider (3DLD) has been successfully used in mouse models for the isolation of proximal and distal cell progenitors. This can be used for the isolation of cells for organoid culture [51].

Grigoryan et al. [52] successfully created 3D alveoli-like structures using stereolithography. The structures were printed using a biocompatible bioink and were tested for their normal functions. The printed design included blood vessels that transported oxygenated blood, and the alveoli were filled with gases like oxygen and nitrogen. The researchers observed gas exchange between the 3D structure and the blood, and the system also replicated the rhythmic movement of pulmonary tissue. This technique shows promise for studying air permeability mechanisms in the lungs. However, the 3D-printed structures were larger than natural alveoli and required a supporting block. Additionally, the cells were not seeded in the structure, except for the red blood cells that carried the oxygenated blood. Researchers suggested that the model could be modified to incorporate cells [52]. In 2023, da Rosa et al. developed a 3D bioprinted in vitro lung model using Wharton’s jelly mesenchymal stem cells (WJ-MSC). They isolated and characterized the stem cells using flow cytometry, then differentiated them to form pulmonary cells in spheroids. The resulting cells included alveolar type I cells, alveolar type II cells, ciliated cells, and goblet cells, which were confirmed using histochemistry. The cells were then mixed with sodium alginate and gelatin to create bioink and printed using an extrusion-based bioprinting technique. The 3D structure was analyzed for the expression of markers and cell viability. This study presented a promising new model for in vitro studies using 3D printing techniques (Figure 5) [53].

Researchers recently developed a 3D alveolar barrier model using inkjet-based bioprinting. The model replicated the structure and function of the pulmonary system, consisting of alveolar type I cells, alveolar type II cells, lung fibroblasts, and pulmonary microvascular endothelial cells. This model aimed to simulate the physiological properties of the alveolar barrier, such as its shape, thickness, and function. Previous studies had successfully mimicked the function of the alveolar barrier but struggled with replicating the size and shape of the alveolar structures. This new model successfully established the necessary cell–cell and cell-matrix interactions to maintain tissue function. It featured a 3D structured monolayer design, resembling the physiological barrier, and was compared against a non-structured 3D alveolar barrier model. The final membrane was uniform and approximately 10 μm thick. The model also demonstrated interactions between endothelial and fibroblast cells, crucial for lung development. The immunohistological analysis confirmed the expression of barrier proteins and surfactants by the cells. This model has potential applications in studying influenza A pathogenesis, as well as serving as a platform for research on other respiratory diseases and drug screening (Figure 6) [54].

5. 3D-bioprinting for tracheal bioengineering

The lung is divided into two main parts: the airway and the vasculature. The airways form the core of the pulmonary system and consist of a branched network that runs from the trachea to the terminal bronchioles. The tracheobronchial tree branches dichotomously, extending from the trachea (generation 0) to the terminal bronchioles (generation 23) [55]. These 23 generations of airways have a descending hierarchy of diameter, ranging from 1.5 cm at the trachea to 0.5 mm at the respiratory bronchioles [38]. On average, the trachea is 10–13 cm long. It is made up of 16–20 C-shaped cartilaginous rings located at the front and connected at the back by trachealis muscles. Each ring is about 4 mm tall [56]. The windpipe is lined with a ciliated pseudostratified columnar epithelium consisting of glandular, mucin-producing goblet cells, which moisten and protect the airway [57].

The anatomical features of segmental blood supply and the risk of infection due to continuous exposure to the outer environment pose a serious challenge in reconstructing the trachea. High mortality rates of tracheal stenosis have made the reconstruction of the trachea a great concern. Several attempts have been made to repair the tracheal segments using various bioengineered artificial tissue substitutes. However, none of them have provided an ultimate solution due to multiple issues such as the formation of granulation tissue at the anastomosis site, flattening and softening of the tissue framework, and the absence of functional respiratory epithelium [58].

Recent advancements in 3D bioprinting have shown promise in reconstructing the trachea. Cells and scaffolds are the most crucial aspects for tissue regeneration while meeting the prerequisites for scaffold design and fabrication [59]. To ensure new epithelialization, vascularization, and cartilage formation, an engineered/bioprinted scaffold should exhibit proper biomechanical characteristics and functionality post-implantation. The scaffold must be bioresorbable, meaning it should degrade at a rate that matches the rate of new tissue regeneration. If the degradation is too fast, it may lead to tracheomalacia, while prolonged degradation may result in a foreign body response, stenosis, and scar formation [60]. It has been reported that a PCL scaffold integrated with alginate and type 1 collagen, seeded with chondrocytes, enhances the formation of fibrous tissue post-implantation [61]. The presence of MSCs on tracheal scaffolds has been reported to enhance the development of new tissue upon implantation. Huo et al. [62] developed a strategy for 3D bioprinting of a cartilage-vascularized fibrous tissue-integrated trachea, demonstrating successful regeneration and mechanical function [62]. Park et al. [63] introduced a two-step extrusion-based 3D bioprinting method to create a clinically relevant size trachea-mimetic cellular construct, leading to successful tracheal cartilage formation in a mouse model [63]. Rehmani et al. [64] used 3D bioprinting to create bioengineered tracheal grafts for the reconstruction of anterior tracheal defects in a large-animal model, showing successful tracheal reconstruction and graft incorporation [64]. Shieh et al. [65] investigated the potential of 3D printing for creating customized external airway splints for tracheomalacia, showcasing the possibilities for personalized medical devices in tracheal applications [65]. These studies collectively highlight the potential of 3D bioprinting for tracheal reconstruction, with a focus on tissue-specific bioinks, clinically relevant sizes, and personalized medical devices. A study conducted by Park et al. used 3D-printed PCL-collagen-based scaffolds seeded with human turbinate MSC have been used to regenerate the tracheal epithelium in rabbit models. They observed highly ciliated and mature epithelium on the 3D-printed grafts implanted in rabbit models [66]. In another study, Bae et al. [67] used 3D-bioprinted PCL-alginate-based grafts seeded with MSCs, along with epithelial cells, and observed neo-epithelialization, neo-vascularization, and neo-cartilage formation in rabbit models [63]. Among all available methods of bioprinting, extrusion, stereolithography, and organoid printing were widely adopted. However, considering limitations such as the risk of infection, irritation, and reduced biocompatibility degeneration over time, scaffold-free initiatives have been developed by coupling 3D bioprinting with spheroids of aggregated cells (Figure 7) [69, 70].

6. Challenges and future perspectives

In recent years, 3D bioprinting has emerged as a promising approach for engineering functional tissues and organs. It increases the possibility of creating complex macro- and microscale architecture using multiple cell types. This novel approach holds the potential to address the shortage of donor organs for transplantation. Despite its promises, 3D bioprinting of the respiratory system comes with several challenges that need to be addressed to achieve viable and functional tissue constructs. One of the major challenges is to mimic the complex intricate architecture of the respiratory system. The lung consists of branching airways, alveoli, blood vessels, and intricate tissue organization that contribute to its functioning. Its structure, mechanical properties, dynamic environment, and other key physiological factors make the construction of its biomimetic difficult. Yet another challenge is to achieve the features necessary for pulmonary function, such as microscale resolution and connectivity in both airways and vascular networks [50]. Achieving uniform thickness across the 3D-bioprinted lung tissue is another significant challenge due to the heterogeneity of the structure [71]. However, efforts have been made to address these challenges. One study developed a human alveolar lung-on-a-chip model using inkjet-based bioprinting. They created a micron-thick, three-layered tissue that successfully maintained the structure of the lung model [72].

The complex globular structure of alveoli poses a significant challenge for accurately replicating its shape and function through 3D bioprinting. Current 3D bioprinting techniques struggle to imitate the elasticity of the bronchi and alveoli structures due to the complex microstructure in the respiratory system. This elasticity is important as these structures change in size during breathing [73]. It is also quite challenging to replicate the air permeability mechanism of the lungs using 3D-bioprinting techniques due to the complex structure of the respiratory system. Additionally, accurately replicating the size of alveoli adds to this challenge [74]. Current models have difficulty with these features. However, a recent study focused on constructing alveolar models based on degradable hydrogel microspheres and successfully created an in vitro 3-dimensional endothelial alveolar model with a multicellular composition and vesicle-like structure [75]. Another study reported the fabrication of a human air-blood tissue barrier analog, enabling the creation of thinner and homogenous cell layers necessary for an optimal air-blood tissue barrier [42]. The accurate mimicry of cell–cell and cell-matrix interactions in the lungs poses significant challenges, as these interactions are essential for the proper functioning of the system. Current technologies have limitations in recreating these interactions, prompting researchers to explore alternative strategies. One approach involves using lung ECM-derived hydrogels as model systems to mimic native lung physiology [76].

In bioengineering, researchers often use a polymer scaffold as a framework to create the desired shape and structure of an organ. Matching the degradation rate of the scaffold with the production of a new matrix is crucial for tissue development and functionality. If the scaffold degrades too quickly, the newly formed tissue may not have enough support to survive and integrate properly. Conversely, if the scaffold degrades slowly, it may hinder the growth and development of the new tissue. Some researchers are investigating alternative approaches that avoid using synthetic scaffolds, focusing instead on aggregating cells together to allow them to form a desired tissue structure on their own. These approaches aim to eliminate the challenges and limitations associated with scaffold degradation [50].

Creating airways from different types of cells and maintaining the structure of the lungs presents significant challenges. The cells needed for this process can come from donors or the host’s cells. Keeping the air passages open and ensuring a proper blood supply to the entire lung structure poses further challenges. Lungs are vulnerable to damage and infection due to their constant exposure to the environment [39]. While there have been advancements in 3D bioprinting techniques, each method has its limitations. Traditional fabrication methods struggle to recreate thin, multilayered structures and the spatial arrangement of various cell types [72]. Inkjet printers can cause stress on cells due to the size of the nozzles, affecting cell viability and functionality [77]. Laser-based 3D bioprinting is complex and expensive, and while it offers high precision, it has limitations in producing large-scale constructs [78].

In extrusion-based 3D bioprinting, the standard setup slows down printing speed, negatively impacting cell viability, differentiation, and matrix formation. Increasing printing speed or using larger nozzles could improve speed but might compromise printing quality. Therefore, there is a need to improve extrusion-based 3D bioprinting to shorten printing time for large clinical constructs [63]. Advances in bioink formulation and cell biology show promise for successful bioprinting of complex organ systems. Some researchers have successfully developed 3D lung models using certain types of stem cells and hydrogels, offering a way to mimic native lung physiology and study the interaction between cells and the lung matrix [53, 79, 80].

7. Conclusion

3D bioprinting has revolutionized the field of lung and tracheal bioengineering by offering an innovative approach to replicating the intricate structures of these vital organs. This technology uses advanced printing techniques to layer biocompatible materials, living cells, and growth factors, creating tissue constructs that closely mimic the natural architecture of lung and tracheal tissues. One of the significant advantages of 3D bioprinting is its ability to customize and fabricate complex geometries with high precision, which is essential for replicating the alveolar sacs, bronchioles, and tracheal rings. These advancements offer a promising solution to the shortage of donor organs and the limitations of traditional tissue engineering methods.

The integration of stem cells and bioactive molecules within the bioprinted constructs has shown potential in promoting tissue growth, regeneration, and vascularization, which is essential for the long-term viability and functionality of the engineered tissues. Recent studies have demonstrated partially successful bioprinting of tracheal segments and alveolar structures, highlighting the potential of this technology in addressing respiratory diseases such as chronic obstructive pulmonary disease (COPD), lung cancer, and tracheal stenosis. However, challenges remain, including ensuring mechanical stability, biocompatibility, and integration of bioprinted tissues with the host body. Despite these challenges, ongoing research and interdisciplinary collaboration drive significant progress. Innovations in biomaterials, cell sourcing, and bioprinting techniques continue to enhance the quality and functionality of bioprinted lung and tracheal tissues. Moreover, advances in computational modeling and imaging technologies are aiding in designing and optimizing bioprinted constructs, ensuring better clinical outcomes. As the technology matures, it promises to provide viable alternatives to organ transplants and enable personalized treatments tailored to individual needs. Ultimately, 3D bioprinting for lung and tracheal bioengineering represents a transformative step forward in regenerative medicine, potentially significantly improving patient outcomes and addressing the growing demand for organ replacements.