Advancements in lung regeneration: from bench to bedside

Abstract

Lung disorders rank among the leading causes of mortality worldwide, presenting a critical challenge in healthcare. The underlying limited regenerative capacity of the lung makes this an unmet clinical need, often necessitating lung transplantation for patients with severe lung disease. However, the lack of viable donor organs underscores the urgent need for alternative therapeutic strategies. Addressing this challenge requires a comprehensive understanding of the structure of lung tissue and the complexities inherent in its regeneration. In this review, we explore recent breakthroughs in lung regenerative medicine, highlighting innovative approaches aimed at tackling lung and tracheal diseases. From stem cell and cell-based therapies to the utilization of biological and synthetic materials, as well as the strategic deployment of growth factors, a diverse array of strategies is being explored to rejuvenate lung function. By critical analysis of in vitro, preclinical, and clinical studies, this review aims to provide a comprehensive overview of the emerging technology of lung regeneration research, shedding light on promising avenues for future therapeutic interventions.

Introduction

Tissue engineering in the respiratory system is complex and requires more research, although marginal achievements have been obtained. Lung tissue engineering possesses various important challenges, such as mechanobiology, heterogenicity, immunogenicity, and vascularization of cells, and addressing all of them in a chosen scaffold is the most significant difficulty. Nowadays, new scaffolds and biomaterials are necessary to maintain lung tissue regeneration while preferably supplying protective properties against the aggression of microbes and inflammation because of the lethality and morbidity of pulmonary disorders [1,2,3,4]. Recently, pulmonary diseases have become a major problem worldwide. Abundant people have suffered from lung disorders in the United States, and the same rates have been distributed in Asia and Europe. Many environmental elements involving bacterial and viral (the latest, SARS-CoV-2) infections, air pollution, and tobacco use, similar to genetic elements, and give structural changes in the lungs and overreactions of the immune system engaging in chronic and acute diseases, including cancer, fibrosis, and obstruction which are presented deficiency of respiratory system with occurrence, especially in elderly [1, 5]. Current treatment alternatives for replacing injured organs are dependent on receiving a new lung from the same species due to the limited ability of the human body for regeneration. Also, restrictions in the treatment contain morbidity of the donor site and scarcity. These conditions promote covering the requirement for biological alternatives, and regenerative medicine and tissue engineering have worked on them. Research and works in this scientific area have developed the definition of regenerative medicine as the junction of stem cells, advanced material science, developmental biology, physics, and clinical translation to complex tissue and organ systems regeneration [4, 6, 7]. In lung regenerative medicine, numerous therapeutic agents and cellular factors, including cytokines like IL-33 and IL-22, injury-activated alveolar progenitors (IAAPs), trefoil factors, gene therapy, the microbiome, and platelet-rich plasma (PRP), play vital roles in healing and repair processes. IL-33 and IL-22 are critical cytokines in regulating inflammation and tissue repair. IL-33 is associated with immune responses that promote repair in lung tissue, while IL-22 aids in epithelial regeneration and protection. Trefoil factors, small peptides released in response to injury, enhance mucosal healing and support lung tissue recovery. Injury-activated alveolar progenitors (IAAPs) are resident cells in the lung that become activated after injury, facilitating repair and cellular regeneration. These cells, along with gene therapy approaches, hold significant promise for treating chronic lung diseases, as they enable precise targeting of defective genes or signaling pathways involved in lung pathology. The microbiome, which plays a vital role in immune modulation, also contributes to lung health and regeneration, influencing inflammatory responses and repair mechanisms. Platelet-rich plasma (PRP) therapy, derived from concentrated platelets in the blood, is a growing regenerative strategy due to its rich content of growth factors and cytokines that promote tissue healing. By leveraging these factors, regenerative medicine aims to create novel, multi-targeted approaches to repairing damaged lung tissue and improving recovery from lung diseases [2, 4, 5, 7,8,9,10,11,12,13,14,15]. To improve the results of treatments for pulmonary disease, tissue engineering developments are being advanced that aim to regenerate the entire organ, its different parts, and components. The general idea is to find alternatives to tissue engineering that can be grafted in the body for a long duration and, therefore, promote the lung tissue’s action and structure. Three fundamental pillars of tissue engineering approaches contain stimuli, cells, and biomaterial scaffolds. Scaffolds activate physical signals for 3D adhesion cell growth, which can occur in vivo. Also, stimulatory signals may be required to activate their action [1, 4, 7, 9, 16]. Transplantation of the lung can be distinguished as a final option in the treatment of various lung disorders, like bronchopulmonary dysplasia, pulmonary fibrosis, pulmonary hypertension, end-stage lung, and chronic lung disease. The survival rate in lung transplantation after five years is approximately 50%. Several end-stage patients will expire without finding an appropriate lung donor in that period. Eminently, donor organ shortage has been a major challenge in the transplantation of the lung. Two of the most important approaches to tissue engineering are recellularization and decellularization, which provide operative lung replacements and will conquer the limitations of the donor’s lung. Decellularization has been drawn to be attractive due to its ability to generate an entire ECM in lung engineering development. Decellularization aims to provide an appropriate scaffold of lung tissue that involves a suitable framework for the regenerated lung tissue action [4, 5, 7]. Therefore, the current approaches to lung regenerative medicine are discussed in this review article, such as various types of scaffolds, cell therapies, and supportive factors that are effective in different respiratory diseases.

The application of biological and synthetic scaffolds for respiratory medicine

An alternative treatment in regard to end-state lung disorder is lung transplantation, although it is kept restricted through waiting-list mortality and donor organ shortage. Peripheral defects of the long-segment trachea pose a significant challenge for reconstruction in surgeries subsequent to traumatic injury, malignancy, or airway stenosis. Transplant recipients often cause diabetes, malignancy, hypertension, renal failure, and other consequent diseases of immunosuppression. In contrast, the recipients have to live with lifelong immunosuppression, try to substitute the trachea with scaffolds and synthetic prosthetics, and experience complications such as inflammation, stricture, infection, mucous build-up, and stenosis eventually. Lung/trachea tissue engineering or bioartificial suggests the capacity for long-period graft survival with no requiring for long-duration therapy by immunosuppressive agents [17]. Some factors, such as scaffold material, design, and fabrication, have played key roles in human organ development. Also, 3D scaffolds maintain the mechanical structure of the aimed organs, stimulating negligible inflammation or toxicity. Scaffolds are also divided as natural or synthetic compounds based on the construction method of the scaffolds that exerted their advantages in clinical studies for forming bioartificial organs involving practical pulmonary tissues [4]. Scaffolds of engineered tissue are planned to conduct as an endorsing structure, retaining the airway open, preparing a pattern for the proliferation of the stem cells, imitating the mechanical and biological action of the normal ECM, and stabilizing and regenerating the injured site. Scaffolds should be nontoxic, which is the most important feature because of the consequent rigidity to prevent collapsing, while they should be flexible to incorporate the natural movement of necks. The airflow through the airways builds a susceptibility to airborne infections like viral or bacterial. Cilia prepare a mechanism and a barrier for particulate expulsion in native tissue, and vascularization is required at the interface of air tissue to imitate the function. Porous scaffolds permit interactions between cells and polymers so the host connective tissues can interpose with each other. Other than the concerns about the type of cells, longevity, immunogenicity, and function, they must be biocompatible. A wide range of complex practical and structural compounds are used in ECM scaffold production, and they are particularly structured and appropriate for the designed organ or tissue. Molecules like laminin, collagen, fibronectin, and elastin are amid these compounds [18]. These scaffolds no longer need any direct anastomosis, and they are formed in 2D-structures. Nevertheless, these cells need nutrients and oxygen. Currently, ECM scaffolds are usually utilized for the reorganization of tissues for clinical objectives or investigational involving lung bioengineering [19]. Although synthetic materials have low biocompatibility, they have been drawn to attention as candidates for use as scaffolds in tissue engineering. Moreover, their acute activation of thrombus is related to low biocompatibility, so it should be improved to lysis and prevent blood clotting. However, synthetic materials involving degradable polymeric materials are being extensively examined to form scaffolds with porous or fibrous geometry. In respiratory system tissue engineering, biodegradability and elasticity are effective properties [4, 20]. Furthermore, when the cells were seeded scaffolds formed in a bioreactor or in tissue culture to regenerate into whole practical tissues, it has been named an in vitro product of engineered tissue. On the other hand, when the cells were seeded or placed in acellular scaffolds within the living body to reorganize new tissues, that is named in vivo tissue engineering [21].

Trachea scaffolds

The trachea, commonly called a windpipe, is a flexible cartilaginous tube. It links the nasal cavity to the lungs and is considered a ventilating structure for the respiration process in humans. By damaging the trachea and when the functioning ceases, respiratory disorders begin. Trachea diseases can cause substantial complications, drastically decreasing patients’ quality of life. These are different, but they include tracheoesophageal fistulae, stenosis, tracheal carcinoma, tracheobronchial injury, malacia, and harmful compound ingestion. Available therapeutic approaches are restricted. Recently, a suitable treatment has been the resection of the trachea with an initial reorganization to restore the connection. Other treatment methods involve aortopexy, slide tracheoplasty, and tracheal stenting, although they have different levels of success. Currently, no clinical options are available for people who suffer from airway diseases of the long segment. Alternative possibilities are being progressed for trachea substituents involving prosthetics, allografts, and autografts or a mix of these options, despite limitations concluding infection, inflammation, unsuitable sizing, and lack of effective imitation of the physiological effects in the original tissue. To date, scientists have focused on using tracheal scaffolds based on an appropriate cadaveric allotransplant or synthetic polymer. For proper operative implant engineering, the physiology of the trachea must be found. The trachea consists of smooth muscles and connective tissue layers endorsed via U-formed hyaline cartilage rings that keep the tube open. The inside coverage consists of different kinds of cells, like brush, ciliated, pseudostratified columnar epithelium, and secretory cells. The connective hollow tube progresses from the diverticulum of the respiratory system. The tracheal cartilage mesenchymal rudiments existed in the eighth week. Over the next two weeks, the cartilage progresses into fibroblastic tissues, and smooth muscle is aligned with the trachea. Scaffolds can be composed of synthetic materials (biodegradable or non-biodegradable) or biological as one of the decellularized allotransplants. Synthetic scaffolds have been examined in different forms, including gelatin sponge, PLGA, polyethylene glycol-based hydrogel, PCL, Marlex mesh, polyester-urethane, and other modified versions. The therapeutic alternatives for children are restricted to the increment of complications because of the difference in the trachea size and alterations over time due to growth. However, several innovative treatments might be used in adults [22]. The scaffolds that don’t have an immunological response would cancel the possibility of the transplant rejection risks, which is one of the requirements for a long life after transplantation. Also, degradable polymers must be biologically safe and degrade at suitable rates for tissue regeneration to prevent structural trachea collapse. Polymeric implants of tissue engineering are preferred because they do not elicit a significant response from the immune system, which may usually be presented in allografts. Despite this, transplant tracheal tissue engineering has been conducted clinically in a few cases. Scaffolds utilized in airway tissue engineering have three areas of focus, namely, non-biodegradable synthetic, biodegradable scaffold, and decellularized tissue scaffold [21] (Table 1).

Table 1 Different types of trachea scaffolds in regenerative medicine

In vivo studies

There are two classes of materials that are used in tracheal regeneration practices: (1) natural and (2) synthetic. Natural scaffolds are originated from native biological material, but synthetic scaffolds are humanmade and synthetized. Various polymeric materials, like polylactic acid, PGA, silk, PCL, decellularized matrix, gelatin, and collagen, are used to produce the synthetic scaffolds. Ruszymah et al. (2005) produced a hydrogel-based cell-seeded scaffold, which was coated with PGA and was used to cover the PGA mesh. Unfortunately, it failed in 2 months [23]. The bio-artificial dacron was implanted in the trachea of mice, and after four weeks, an epithelial cell sheet covered it. After examination, regenerated epithelial lining and neo-vascularization on the luminal surface of the scaffold were proved [24]. Luo et al. (2013) used a silicon stent implemented with the chondrocytes-seeded nonwoven PGA mesh in rabbits. According to the results, the trachea was fully regenerated after eight weeks [25]. Tsao et al. (2014) utilized a stem cells-seeded PLGA scaffold using a solvent-casting method to regenerate tissue. Unfortunately, this newly designed PLGA-fabricated scaffold deformed after one month [26]. Clark et al. fabricated a trachea scaffold with bone marrow-mononuclear cell-seeded PET/PU with high porous characteristics by implying a vacuum seeding system right before implantation, and then, they used it in the trachea of sheep. Still, the scaffold had a delayed stenosis after six weeks [27]. Also, to enhance the biochemical feature of this modified PET/PU, scientists designed a trachea scaffold with fiber-coated polycarbonate rings. Then, bone marrow-devised mononuclear cells were implemented in this designed scaffold under special vacuum conditions for use in the sheep model [28]. Dharmadhikari et al. showed that re-epithelialization can be supported by synthetic scaffolds in orthotopic patch implantation. However, epithelialization is limited to segmental synthetic scaffolds, and cell seeding does not influence it [29]. Gao et al. made a chondrocytes-preseeded 3D PCL scaffold, examined it in vivo, and figured out that the cartilaginous tissue was regenerated [30]. In one work, scientists who designed 3D-printed tracheal scaffolds using a new modeling method with four-axis fused deposition figured out that it had an interconnected porous structure like normal tissue. This modified 3D PCL scaffold was implemented in the trachea of the rabbit model and resulted in faster healing and suitable biomechanical stability of the regenerated tissue [31]. Li et al. reported a negative result after implanting the PCL tube with good mechanical properties for resisting, and finally, it collapsed. Moreover, to suppress the growth of the granulation tissue, a PCL was modified by genipin-crosslinked gelatin. Compared to the standard PCL tube, this modified form had a better survival time in the animal model. Genipin can effectively decrease the formation of granulation tissue at the tracheal anastomoses [32]. Defect in respiratory mucosa causes infection and obstruction, which require the functional reclamation of the respiratory epithelium. A site-specific ECM can recover the function of the organ by enhancing the migration of cells. Research has announced that decellularized trachea can be a good ECM, although there are many reports about mucosal-architecture destruction and incomplete cell removal from cartilage. Jung et al. reported a new scaffold, xenogenic acellular respiratory mucosa matrix, with high biocompatibility [33]. Nomoto et al. (2006) seeded tracheal epithelial cells into the mentioned scaffold for more rapid tissue repairment. They implemented this scaffold in rats and reported a total mucosal tissue regeneration association within two weeks [9]. For improving the speed of tissue healing, different cell types, like adipose stem cells, blood cells, MSCs, epithelial, and fibroblast cells, were used for synthetizing a spongy and collagen-PP scaffold. Animal studies showed that the synthesized scaffolds had graft rejection because of the lack of vessel formation [8, 34, 35]. Weidenbecher et al. (2007) used a newly manufactured Hyalograft scaffold based on C-ECM hyaluronic acid. This seeded scaffold with autologous auricular chondrocytes showed cartilage reconstruction, both in vivo and bioreactor, but in the human body, the scaffold showed degradation of neo-cartilage and inflammation [36]. The porcine decellularized cadaveric trachea was seeded with tracheal stem cells, and epithelial was used in the tracheal defect in the pig model. This scaffold could fully regenerate tissue with no immune responses [37]. A new modified respiratory structure was synthetized by Heikal et al. (2010) with nasal fibroblast and epithelial in polymerized fibrin. Afterward, they implemented it into a titanium mesh. This structure was used in sheep for the regeneration of the tracheal defect, and according to the results, it could regenerate an immature-form of the epithelium similar to the normal tissue [38]. Özpolat et al. (2013) manufactured a freeze-dried gelatin scaffold, which was implemented with bFGF and seeded with MSCs. They used it in the rat model, and according to the results, chondrogenesis with better epithelial tissue regeneration was seen at the tracheal defect [39]. In another study, scientists used HUC (human umbilical cord) serum in a PCL/collagen scaffold. This produced scaffold was utilized in a tracheal defect model in vivo. The results showed a complete cartilaginous tissue repairment with no inflammation [40]. In one study, scientists fabricated aPLGA–collagen scaffold, and it showed good mechanical strength as an engineered trachea, but the formed cartilage was poor [41]. In another study, scientists produced an allogenic porcine decellularized cartilage scaffold seeded with cells, which was then used in vivo. According to the results, it had low inflammation [42]. One of them most important proteins of the ECM is a laminin that acts in the differentiation and cell migration. Lee et al. (2015) constructed a PCL porous membrane scaffold which was coated with laminin and examined its effects with or without MSC for in vivo test. The results confirmed that MSC and laminin had synergistic effect in mucosalization, and also, in tissue regeneration after one week [43]. Park et al. (2015) produced a PCL–gelatin scaffold implemented by TGF using 3D printing technique, that finally, results showed a good mechanical characteristic similar to that of native tissue along with suitable tissue regeneration [44]. In a study, a decellularized aorta three-layer tubular-shape scaffold was constructed out of PCL alone, or with collagen by Ghorbani et al. (2017). They seeded it with chondrocytes and adipose-derived MSC and implemented it in tracheal tissue of rabbit model for 1 month. Results showed a good cell adhesion and biocompatibility with a suitable mechanical characteristic [45]. Indirect printing approach is another technique for making a silicon 3D ring-shaped matrix. In one study, they used this silicon ring surface which was covered with a layer of stratifying tracheal mucosa that was implemented with decellularized ECM. Animal study in rabbits showed a satisfying tracheal defect regeneration [46]. Wu et al. (2018) designed a trilayered scaffold, implemented with collagen fibers, PLGA + Silk, electrospun PLCL. They finally seeded them with endothelium and smooth muscle cells of human. After 10 weeks, results showed a positive proliferation, cellular infiltration, and biodegradability [47]. First, this matrix was recellularized by chondrocyte stem cells with expression of the SOX9⁺ factor. This matrix is used in the trachea and could successfully regenerate it [48].

In vitro studies

Researchers for designing a suitable bioengineered tracheal implant performed comprehensive analyses of distinctive biomaterials. They showed that a combination of collagen vitrigel membrane and silk has better mechanical properties in addition to a suitable cellular differentiation for creating a mature epithelium tracheal tissue [49]. O’Leary et al. have shown that a CHyA-B scaffold could assist the differentiation of Calu-3 cells by enhancing the ciliation, the formation of intercellular tight junction (TJ), and mucin expression. For creating a submucosal tissue of the airways, a co-culture of Calu-3 cells/Wi38 lung fibroblasts was made on the scaffold, and finally, they validated the use of CHyA-B for supporting co-culture [50]. Poon (2018) produced some micro-engineered scaffolds with distinct biophysical and biochemical properties, like stiffness and topography, and then he worked on their effect on biofunctional tracheal repair. He also found that a matrix with polydimethylsiloxane and alveolar-mimetic curvature has better differentiation markers expression and cell viability in comparison with the collagen vitrigel membrane. Some biomaterials with biomimetic function cause a proper lung epithelial progenitor cell (EPC) differentiation in the respiratory tract [2]. In distal parts of the respiratory tract and near the airway, BMP4 and BMP7, respectively, are expressed during lung development. The proximal epithelium and trachea construction are stimulated by BMP7 [51]. A limitation of the decellularization technique is the detachment of cells from the normal tissue matrix. LMT was used to solve this issue because it caused a better matrix detachment of the cell, and better porosity was observed in the matrix with no cells. A tubular LMT-treated decellularized matrix is similar to the native form with intact ECM biocomponents, which, overall, results in better tracheal tissue regeneration and tissue adhesion [52]. Recently, Abdul Samat et al. figured out that all blended compounds were not miscible, although their mechanical properties and morphology showed better. Furthermore, blended material has great compatibility with live tissue, as well as an appropriate degradation rate and absorption, which makes its usage suitable in additive manufacturing for medical purposes [53]. Recent findings showed that ECM hydrogels worked better in helping the differentiation and growth of HBEC than decellularized ECM bioscaffolds. Also, they had the potential to promote the mature epithelium of the respiratory system [54].

Clinical studies

Omori et al. [55] used a tissue-engineered tracheal scaffold in 78-year-old patients with thyroid cancer for the first time. They made a spongy collagen scaffold that was ring-like in shape and coated it with collagen gel to intimate the normal trachea. Therefore, they substituted the right half of the damaged tracheae with the scaffold. After two months, the tracheae started to form an epithelium-like tissue for two years with no problem. However, within five months, only a slow epithelization was shown in the regenerated tissue [55]. Although xenogeneic tissues are either a source of grafting or an important choice for tissue transplantation, they may transmit disease, and some graft rejection problems make their usage limited. In one study, the scientists fabricated a stem cells-seeded decellularized cadaveric human tracheal scaffold. After four months, this scaffold was fully vascularized and regenerated [56]. Dr. Waddell used a cadaveric graft, which consisted of a human trachea, and he could develop suitable techniques for decellularization and recellularization. Finally, tracheal fabrication made using bioengineered methods was totally changed by the process used. Then, appropriate doses of growth factors were used in the patient to improve chondrocytes proliferation and vascularization. The tracheal transplant with engineered tissue regenerated completely in 15 months, but for dilation, it needed to be for 18 months [57].

Lung scaffolds

The lung develops through a series of signaling processes and is considered a complex and special organ. It is susceptible to some harmful agents, such as bacterial and viral pathogens, environmental irritants, and toxic chemicals. Other agents that may damage the lung are barotrauma, mechanical trauma, low and high oxygen concentrations, and ionizing radiation that may cause both chronic and acute injuries to respiratory vasculature and epithelium. The lung can regenerate itself using different processes that are involved in lung development, too [58]. Regeneration refers to maintaining lung functioning after damage through some steps. If the damage persists, the number of these processes, like fibrogenesis or inflammation, can be pathogenic. However, they may be essential for the normal functioning of the lungs in the short-term. Tissues from PCs can be derived from dedifferentiated cells or blood circulation. Lung regeneration needs precise and arranged signaling processes that act in different mechanisms like proliferation, differentiation, and migration of various cells, just like lung development [11]. Chronic lung diseases, like COPD, are the third most fatal disease worldwide. Other chronic lung diseases, like pulmonary arterial hypertension or interstitial lung disease [59], and some genetic disorders, like cystic fibrosis (CF) and α1-antitrypsin disorder, are deteriorating diseases that have no cure. Lung transplantation is the only way to help patients with the end-stage lung. Lung transplantation has two main drawbacks, which are a high possibility of rejection and a low number of suitable donor lungs. The five-year survival rate for lung transplantation is 50%, which means even after transplantation, 50% of the patients would die. Regardless of significant efforts that have been made to enhance operative care, the mean survival rate has remained unchanged since 1990. These patients need new approaches to lung tissue engineering benefits from some acellular tissues like skin [60]. Ideally, the cells that were harvested from the recipient should be implemented in a scaffold using the ex vivo technique, which means they should grow in a bioreactor until maturation, and eventually, they could be transplanted. In one study, an acellular scaffold, which was derived from a decellularized normal lung, was used in vitro models [61]. So, recent in vivo, in vitro, and clinical studies related to lung scaffolds in animals and humans have been summarized to conduct the last correlated results to lung tissue engineering.

In vitro studies

There are numerous types of decellularization processes. One of them is performed using detergents. Li et al. found that cell adhesion can be improved by using the decellularized lung scaffold, which originated from the dextrose/SLES/Triton X-100. In general, the SLES detergent alleviated the damage to ECM better than SDS. Moreover, perfusion with dextrose could protect the scaffold better, mainly collagen [62]. Pouliot et al. showed that 12 h of pig lung decellularized ECM pepsin-digested provides an ideal balance between physical ECM hydrogel characteristics and effects on lung cells. So, they can be used as decellularized functional cells in lung tissue engineering [63]. Teoh et al. showed that POSS-PCL had good mechanical characteristics, and the foam elastomer of POSS-PCL and the solid elastomer could positively make a relationship between the cartilaginous part of the human trachea and U-shaped rings. The bronchial and tracheal scaffolds are promising engineered tissues [64]. Huang et al. showed that a new biomaterial ink, which is made of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized bacterial cellulose (OBC) nanofibrils and regenerated silk fibroin (SF), was produced to be used as a scaffold and OBC nanofibrils oriented together with print lines. In one week, the epithelial phenotype stayed unchanged. This characteristic makes them a promising scaffold in respiratory tissue engineering [65]. Wu et al. showed some networks produced with a reversely 3D printed porous structure, which was impregnated with a modified hydrogel with collagen nanofibrous, can be successfully used in synthetic human tracheobronchial epithelium developing. hBEpiCs could attach to a special monolayer on a scaffold and grow on it. They are co-cultured with hBM-MSCs or hBFs, with significant improvement in adherens junctions, well-constructed intercellular TJ, mucin ciliation, and expression. These multi-layered 3D scaffolds have a top differentiated epithelium monolayer, with hBM-MSCs or hBFs that are increasing on a nanohybrid scaffold. They made airway-like tissue, which could be used as a scaffold for supporting cellular organization and co-culture and for trachea defect regeneration [66]. Azimi et al. have reported that the composite scaffolds had the potential to create fibers and alveolar walls. Eventually, scientists found that it had a suitable piezoelectricity characteristic that is seen more in collagen and elastic proteins. All of these characteristics made ZnO/P(VDF-TrFE) fiber meshes a promising choice for pulmonary regeneration. Additionally, this mesh hampered the biofilm formation by P. aeruginosa and S. aureus. This scaffold could also inhibit S. aureus adhesion and S. aureus and P. aeruginosa infection, regardless of the scaffold kind [1]. Ban et al. showed that bFGF and a modified colloidal crystal scaffold can help the proliferation of mouse and human lung and bronchial epithelial cells [67]. In another study, Ghaedi et al. showed that various types of cells (ESCs, MSCs, primary airway, alveolar, immortalized airway, and IPSC) were cellular remobilization and inhabitance in appropriate decellularized samples. They retained the 3D structure, ECM proteins, and expression of differentiated lung epithelial cells, like CFTR, P63, T1-α, SPB, SPD, and CCSP [51]. Tebyanian et al. showed that various decellularization processes with immunogenic properties could make a big difference in the physiological, mechanical, and biochemical properties of artificial lungs. Parenchymal cells are in contact with biochemical agents in decellularized organs, and physical methods are performed to disturb the cell membrane. and finally, the cells would more easily be removed from the would be easier. The composition of the scaffolds can be influenced by each one of the steps in the preparation process [68]. Sun et al. examined a new drug efficiency for screening lung cancer by mimicking lung structure. According to the results, they found that the mimicked lung alveoli response was similar to real lung carcinoma after gemcitabine treatment [69]. Soriano et al. showed that a 3D scaffold has promising epithelial barrier formation in vitro, and it kept its characteristics in co-culture conditions. Barrier integrity had another way of recovery than in epithelial cultures like CHyA-B and Transwell^®. The pro-inflammatory cytokines secreted from the basolateral side were higher in cells grown in a 3D scaffold than in 2D. Furthermore, 3D epithelial cultures with bleomycin had higher cytotoxicity and cytokine levels. CHyA-B scaffolds helped bronchial epithelial cells differentiation and grow better in a 3D co-culture model. It supported them with distinctive resistances compared to the Transwell^®. So, this work demonstrated the need to upgrade in vitro tests with high-tech models [70]. Also, in another study, Falcones et al. reported that mesenchymal stromal cells showed good characteristics like viability. According to the contraction tests, the cell-matrix interaction existed in the bioprinted scaffolds. Two characteristics of the cells, the length of the focal adhesion and its capacity, in hydrogel scaffold were improved. The expression of the CXCR4 receptor was increased 20-fold in the 3D-cultured cells, too, which was better than in plastic. Cytokine secretion was decreased in a 3D scaffold in vitro. Additionally, the shape of these cells from 3D culture was so distinctive from 2D cultures [71]. Thus, these decellularization protocols could maintain a 3D structure and the ECM. Many reports show successful decellularization in humans and animals, such as rats, pigs, and mice. Also, the ex vivo study of Guenhart et al. targeted lung cell replacement in extracorporeal organs, which they found possible [72].

In vivo studies

Weymann et al. showed an increasing in airway compliance, but biomechanical integrity was maintained unchanged in comparison to normal tissue. Both porcine lungs and trachea can be decellularized to produce a 3D acellular scaffold that preserves structural integrity and ECM for respiratory tissue engineering [17]. Wang et al. showed that endogenous alveolar vascular endothelial and stem cells seeded into the 3D structure porous scaffold (collagen) very early. With low levels of fibrosis and inflammation, some functional structures similar to alveoli and microvascular were produced, so finally, the injured lung had a morphology similar to normal tissue. In total, this functional scaffold could help the damaged angiogenesis and respiratory structures [74]. Shirani et al. revealed that one of the promising approaches to preparing a lung scaffold is cross-linking the lung ECM with biochemical composition, microstructure, and mechanical characteristics that are similar to the normal lung. This study showed that the cross-linking agent is also crucial. The modified lung, which was cross-linked with EDC/NHS, had better characteristics like improved proliferation and attachment of lung MSCs, but the immune response was like a non-cross-linked one both in vivo and in vitro [3]. Zhou et al. reseeded decellularized porcine lung scaffolds with epithelial and endothelial cells. They repopulated the surface of the scaffold with epithelial PCs; then, they fabricated a live and effective scaffold with the ability to exchange gas [74] (Table 2).

Table 2 Different types of lung scaffolds in regenerative medicine

EVs therapy in lung regeneration

Intercellular communication is mediated by EVs, which are lipid bilayer particles filled with proteins and nucleic acids. MSCs secrete EVs that are an alternative to live cells, offering distinct biological and logistical advantages. Since EVs have variable content, they can mimic MSCs’ ability to mitigate inflammatory processes and reduce lung damage by mimicking their effects. Additionally, it has been shown that the composition of EVs in MSCs secretomes is greatly influenced by their environments, and this has proven to be a promising strategy for enhancing their therapeutic efficacy. Cell confluence, the number of cellular passages, oxygen availability, and priming EVs biogenesis biology with cytokines, heparin, and serum content can affect both the quality and quantity of EVs [75]. AdSC-EVs have been demonstrated to facilitate ALI through mechanisms such as immune modulation of macrophages and endothelial protection. COVID-19-associated ARDS (CARDS) is reported in approximately 15–30% of patients hospitalized with COVID-19. Alveolar macrophages secrete less inflammatory cytokines when ADSC-EVs are administered, while anti-inflammatory cytokines are produced more readily. EVs also play a significant role in mediating these therapeutic effects when internalized by recipient alveolar macrophages by transferring mitochondrial components derived from donor cells. ATP generation and mitochondrial oxidative phosphorylation (OXPHOS) are increased as a result of this internalization [76]. It has been shown that EVs cause cell dysfunction in the pulmonary epithelium, endothelium, macrophages, and neutrophils, resulting in inflammation and injury that can lead to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Inactivated or damaged lung cells can secrete EVs containing factors associated with their injury phenotypes, which can be used to diagnose lung injuries and predict their progression. As a result, these EV markers can also be used for therapeutic intervention to attenuate lung injury and prevent the development of ALI/ARDS. MSC-EV shows promise in reducing or even curing ALI/ARDS through multiple mechanisms that impact inflammation, dysregulation, and apoptosis in different types of pulmonary cells [12]. According to Van der Koog et al. (2024), secretomes produced by mesenchymal cells, in particular EVs, might possess regenerative properties. According to their findings, EV- or SF-enriched concentrates prevented and improved PCLS emphysema induced by elastase, leading to reduced lung injury and upregulation of alveolar epithelial markers, indicating potential parenchymal regeneration. EV- and SF-enriched lung fibroblast concentrations attenuated elastase-induced lung tissue destruction, improved lung function, and enhanced alveolar epithelial cell gene expression in vivo. By restoring parenchymal tethering, alveolar repair not only facilitates gas exchange but also ensures optimal airway function. Conclusion: EV- and SF-enriched lung fibroblast concentrates are both therapeutically effective for lung injury and emphysema [14]. Saneh et al. (2024) evaluated the efficacy of EVs generated from hiPSCs and their alveolar progeny (diPSCs) in preventing hyperoxic injury in preterm lung explants. As a result of hyperoxia exposure, fetal lung explants enlarged their airspaces, increased their MLI, and upregulated their anti-oxidant Prdx5 and Nfe2l2, while their expression of VEGFa decreased. Improvements in parenchymal histology were achieved with hiPSC-EVs. The in vitro model did not reveal obvious changes in the vasculature structure. A positive correlation was observed between diPSC-EVs and VEGFa and antioxidant genes, suggesting that these cells were pro-angiogenic and cytoprotective. As a result of EV proteomic analyses, potential pathways influencing lung regeneration have been uncovered. The researchers concluded that hiPSC- and diPSC-EVs have the potential to attenuate the effects of prematurity and oxygen exposure on lung changes. By reducing the global burden of BPD, they provided a novel cell-free approach to the prevention and/or treatment of the disease [77].

Cells and stem cells in lung regenerative medicine

There has been various progress in lung regenerative medicine, such as in cells and stem cells. Stem cells have been known as multipotent capacity function in differentiation to multiple cells and can develop tissue regeneration, tissue homeostasis, and growth. The complex tissue and organs can be created by company stem cells and cells in co-culture [78]. Recently, many studies have used stem cells as seed cells. 3D culture and the use of bioreactors can expand the scale of stem cell culture, which can simulate the microenvironment in vivo better when combining biomaterials, co-culture, gene delivery, or cytokines to improve tissue differentiation. In addition, in situ tissue regeneration techniques can achieve a tissue for engineering in vivo [79]. Therapies based on cells and stem cells are considered one of the regenerative medicine applications [10]. hIPSCs can be derived from tissue-specific stem cells so that they can be used in regenerative medicine, including the respiratory system. For instance, Hawkins et al. (2021) reported that hIPSC could be directly differentiated into airway basal cells (ABCs) [80]. Here are some of the recent studies in regard to the respiratory system:

Respiratory development using cells/stem cells

hIPSCs

HIPSCs can be derived from stem cells, and this would help regenerative medicine. Hawkins et al. (2021) showed that human hIPSCs differentiated into airway basal cells (ABCs). They reached this success with a dual fluorescent reporter system (TP63tdTomato, NKX2-1GFP). After the first emergence, they purified them as immature NKX2-1GFP⁺ lung stem cells, and consequently, they performed a TP63 program while making a pattern for proximal airway epithelium. ABCs and their differentiated progeny model the problems in some genetic and acquired airway diseases, such as ciliary defects in primary ciliary dyskinesia, chloride channel dysfunction in cystic fibrosis, and the mucus metaplasia of asthma [80]. In another study, Fields et al. (2020) produced a hIPSC line cell from a healthy mature male donor. Integration-free Sendai Virus could reprogram peripheral blood mononuclear cells (PBMCs) [81].

Mouse and humans Sox9+ progenitors and basal cells (BC)

Nichane et al. (2017) isolated a special multipotent embryonic respiratory progenitor called mouse Sox9⁺ and expanded them in 3D cultures for the long term. Cultured Sox9⁺ progenitors could be transcript in vivo, and they could make both alveolar and airway cells in vitro. Also, they differentiated into all important alveolar and airway lineages after being transplanted into injured adult mouse lungs [82]. LSPC were identified with regenerative capacity, and their usage in reconstructing effective organs or tissue is considered an important hope for curing such illnesses (Fig. 1). Ma et al. (2018) showed that an uncommon number of Sox9⁺ BCs exists in the airway that can reproduce the mature normal lung. They used the bronchoscopic brush to collect human Sox9⁺ BCs. Human Sox9⁺ BCs can be differentiated into bronchiolar and alveolar epithelium after transplanting into a damaged mouse lung, like the normal system. After manipulating the lung microenvironment with Pirfenidone, which suppresses TGF-β signaling, the transplantation efficiency can be further boosted. The repairment of the lung tissue and its functioning was assessed in less than 1-year-old patients after cell transplantation. According to the results, a new functional lung could be reproduced by a special transplantation method of LSPC called orthotopic, which is useful in future therapeutic strategies [83].

Fig. 1

Mesenchymal stem cells (MSC) can be found in the lungs, and the vascular system is composed of large to smallest blood vessels. The first layer of a three-layered structure is the intima, which has contact with the lumen. The second layer is tunica media, where vascular smooth muscle cells exist and encircle the vascular lumen and create many layers. Finally, it is the outermost adventitia, which interfaces between the vessel wall and around tissues, which is called perivascular space. The perivascular space is not circular compared to the adventitia. It is shown that lung-resident MSCs (LR-MSCs; blue) exist mainly in the wall of the large lung vessels in the adventitia. In this niche, there are other kinds of stem cells, too: HSCs (shown by green cells) and EPCs (indicated by yellow cells). Different MSCs and their relative progenitors reside between endothelial and epithelial cells, and the interstitium space (mesenchyme progenitor cells (PC) is shown by light blue cells) [135]

Organoids

Nowadays, organotypic structures are being used in biomedical research. The consistent organotypic cultures, which are called organoids, are established from varying tissues, such as epithelial tissue. Organoids are self-organizing structures that imitate the biochemistry and physiology of the natural tissue at both morphologic and molecular levels. So, it can be a good structure for studying the development, organogenesis, and stem niches. Some protocols have been used to produce an organoid that mimics lung structure and function. Still, most methods referenced to human cells had complex protocols, and the differentiation potential or physiological function was not sufficient. Like previous studies, Sachs et al. (2019) reported that primary human airway cells, which were obtained by bronchoalveolar lavages or biopsies (cryopreserved or fresh), differentiate into a hollow spherical organoid that is so much like the tissue organization of the real human airways [84]. Although these airway organoids (AO) lost their proliferative and differentiation potential, they could be passaged for at least one year with consistent features. Sachs et al. proved some methods for manipulating the lung organoids and effectively modeling numerous lung pathologies. AOs derived from cystic fibrosis patients had lower swelling when challenged with cAMP‐inducing agents like forskolin [85]. It is proved that the epithelium of the lung is derived from the endodermal germ layer, and through complex processes of signaling events, it will turn into bronchioles, bronchi, and alveoli. Miller et al. (2019) described a protocol that undergoes many of these steps to differentiate hPSCs into ventral-anterior foregut spheroids. Then, they will differentiate into bud tip progenitors and normal lung organoids. The final normal lung organoid will be similar to normal bronchioles or bronchi, which are encompassed by lung MSC and some cellular markers related to the alveoli. The bud tip progenitor organoids include a number of highly multipotent and proliferative cells with strong differentiation features both in vivo and in vitro. The two hPSC-derived models used in this study have been identified within human fetal tissue. So, these kinds of progenitor organoids are useful for investigating epithelial changes, although they can also be utilized to model epithelial tissue in lung development [86]. Also, in another study, it was reported that those LBOs produced from hPSCs contain pulmonary endoderm and mesoderm, and they will develop into airways and alveolar structures in Matrigel 3D culture. Developing a new lung from hPSCs in a 3D culture would give us more details about human development, and it will help us with disease modeling, regenerative medicine, and drug discovery. According to the analysis of the features and expressions, it is proved that the developed structures existed until two-thirds of the human gestation. Syncytial virus infection in vitro obstructed the lower airways and bronchiolitis, which led to detachment, swelling, and shedding of cells, similar to that of the normal lung. Mutation in Hermansky-Pudlak syndrome type-1 (HPS1) leads to intractable pulmonary fibrosis, the early-onset form. Finally, it causes an accumulation of MSCs and ECM, so this model is suggested for use in fibrotic disease in vitro. Because LBOs refabricate lung development, they are considered a promising choice for lung disease modeling [87]. An overview of how to use organoids is illustrated in Fig. 2.

Fig. 2

Some strategies for creating airway organoids/spheroids. (a) Spheroids can be produced from different organs like nasal polyps or cells from nasal brushing/curettage. They can be grown in liquid media. (b) Differentiated or stem/PCs retrieved from bronchial brushing, nasal or bronchoalveolar lavage (BAL). After induction, they may form spheroids in matrigel. Such spheroids can be generated through 3 methods: [1] After forming the 2D ALI cultures, spheroids will be formed from them; [2] By adding self-renewal cues; [3] The conditioned reprogramming culture (CRC) [136]

Ex vivo organ repair/ basal epithelial stem cell

Ultimately, bioengineered organs can be used for transplantation of end-stage organs, with no risk of rejection. Gilpin et al. (2016) isolated a population of proliferative KRT5(+) TP63(+) basal epithelial stem cells for the first time from normal human lungs. Its expansion capacity was shown in a typical 2D culture. Moreover, they reseeded acellular rat scaffold ex vivo and proved that primary pulmonary endothelial cells were continually proliferated. So, they reseeded the normal lung scaffold under similar body conditions to assess their regenerative capacity and clinical scalability. The sustained metabolic activity and the cell viability were confirmed by analysis of the regenerated tissue for seven days. Based on the tissue analysis, an extensive recellularization was shown with regular morphology. Also, the phenotype of the basal epithelial cells had no change. The recellularized lung structures showed rudimentary gas exchange capacity and dynamic compliance. According to the results, the usage of normal airway stem cells was proved in the field of lung regeneration [88].

Disease treatment with cells

Thoracic malignancies

Radiotherapy is an efficient way for thoracic malignant tumors, although it may damage the pulmonary system and cause pulmonary failure in some of the patients. Extracellular vesicles derived from MSCs or other sources are a new choice for the treatment of lung diseases without using cells (Fig. 3). Xudan lei et al. (2021) investigated whether MSC-derived EVs (MSC-EVs) could attenuate lung injuries caused by radiation. In this regard, they exposed a 15 Gy radiation to the thoracic of mice and examined the protective effects of MSC-EVs on vascular permeability, endothelial cell damage, fibrosis, and inflammation. They found that MSC-EVs helped lower inflammation and fibrosis. Moreover, MSC-EVs decreased the levels of radiation-induced DNA damage by lowering ATM/ P21/P53 signaling. Finally, it was confirmed that the downregulation of ataxia telangiectasia mutated (ATM) was regulated by miR-214-3p, which was enriched in MSC-EVs. Moreover, the secretory phenotype development was hampered by MSC-EVs, and the radiation-induced injury of endothelial cells was attenuated [89].

Fig. 3

Formation and composition of EVs. (A) Its different shapes are from exosomes (with sizes of up to 150 nm in diameter) and microvesicles (up to 1 nm diameter) to apoptotic bodies (up to 5 μm). EV formations begin from endosomes. They will be transformed into multivesicular bodies (MVBs). They will be fused and degraded with lysosomes, or they will combine with the plasma membrane and turn into intraluminal vesicles, which finally will be released as exosomes. (B) The molecular composition of EVs. They may include HSP proteins, signaling proteins, nucleic acids, enzymes, chaperones, and transcription factors inside them, and immune-interacting molecules, tetraspanins, lipid rafts, and vesicle-trafficking proteins on their surface [137]

Bronchopulmonary dysplasia (BPD)

BPD is considered an important chronic lung disease that occurs in premature infants, with high rates of mortality and morbidity. MSC transplantation has been proposed as a candidate therapeutic method to improve BPD symptoms. Small extracellular vesicles have been conducted as functional therapeutic components of MSCs, which possess an important role in the betterment of a BPD mice model. You et al. (2020) have shown that hucMSC-sEVs could be efficiently absorbed after intratracheal use via lung tissue and continue to exist for 72 h as a minimum. These findings demonstrated that hucMSC-sEVs rehabilitated lung function and structure of the alveolar along with alleviated pulmonary hypertension in a BPD rat model. Hence, hucMSC-sEVs refined angiogenesis and alveolarization through preserving pulmonary vascular endothelial cells (PVECs) and lung type II alveolar epithelial cells (TIIAECs), that are correlated with PTEN/Akt signaling pathway in a BPD rat model [15].

Coronavirus disease (COVID-19)

SARS-CoV-2 is one of the members of the coronaviridae that recently affected the world and led to respiratory diseases. SARS-CoV-2 is a significant factor in cytokine storm and secretion of inflammatory and pro-inflammatory cytokines after the infection. Also, these cytokines are responsible for the induction of pulmonary injury via inflammation-causing ARDS (acute respiratory distress syndrome) and even mortality. MSCs are suitable therapeutic approaches in the ARDS treatment lines that decrease lung damage and inhibit inflammation through their immunomodulatory effects. Also, MSCs can prevent apoptosis and regenerate lung cells. A scientist suggested the administration of MSCs-derived exosomes due to similar restorative and immunomodulatory activities of exosomes to MSCs, and these derived exosomes do not have any related problems to injection and cell maintenance. For assessment of this idea, MSCs could be derived from normal tissues. Afterward, it is possible to produce exosomes from supernatants. Then, the exosome must be examined in a COVID-19 transgenic animal model via injection. Finally, various analyses such as histological assessment, investigation of lung activity, cell and viral load analysis, and inflammatory proteins and cytokines investigation can be assessed. COVID-19 does not have any precise therapeutic approaches.

Moreover, accepted approaches to treatment can inhibit cytokine storm, inflammation, and ARDS and facilitate the finding of the novel supportive treatment method based on the mentioned effects [90]. MSCs have recently been recommended to save alveolar epithelial cells and alleviate the CRS (cytokine release syndrome) through releasing abundant types of factors; it could be helpful for ARDS patients induced by COVID-19 [15]. Human MSCs have been slightly recognized for their ability to adhere to surfaces of plastic culture and their normal phenotype basis of markers expressed after in vitro growth. MSCs identification is also regarded as the combined expression of CD73, CD90, and CD105, while without expression of HLA-DR, CD14, CD34, and CD45 (Fig. 4A). In addition, it is required that MSCs differentiate into three different lineage cells involving osteocytes, chondrocytes, and adipocytes, following inductive conditions of cultures. hBM-MSCs are a special population of cells with high differentiation characteristics. Also, these types of cells attach to plastic, have morphology like fibroblast, and undergo extensive and clonal proliferation accepted by CFU-F. MSCs can usually express different further markers of surface involving CD271, CD146, CD140b, CD73, CD10, and the disialoganglioside GD2. Furthermore, they can express pluripotency markers, Oct4, like nanog, SSEA-4 that determine various cell populations in the bone marrow. MSCs have the potential to become differentiated into epithelial cells, and two important processes called EMT (mesenchymal-epithelial transition) and MET (epithelial-mesenchymal transition) have happened during embryo or tumor development (Fig. 4). Also, MSCs exert antimicrobial effects and deliver intrapulmonary cells in an ALI induced by LPS in vivo, alleviating inflammation and improving survival. Several regenerative activities are mediated via exosome secretion, and Mesenchymal stem cells provide a remarkable concentration of exosomes in comparison with other types of cells [91].

Fig. 4

General features of MSCs and their function. (A) The conventional phenotype for identification of MSCs contains the expression of CD90, CD73, and CD105, while without expression of HLA-DR, CD14, CD34, and CD45. MSCs can differentiate in vitro into chondrocytes, adipocytes, and osteoblasts when they are isolated once. (B) The biological activities of MSCs in COVID-19 pneumonia (systemic disease) involve antiviral and immunomodulatory function, differentiation of cells, mesenchymal-epithelial transition (MET), and angiogenesis, which is due to therapeutic activity and the paracrine secretion of soluble factors (so-called secretome) [91]

Pulmonary emphysema (PE)

An important component of COPD (chronic obstructive pulmonary disease) is PE, and its progression is attributed to impairment of pulmonary regeneration, loss of alveolar structure, remodeling of the abnormal lung, anti-proteolytic and proteolytic function imbalance, apoptosis of lung epithelial cells, and heavy inflammatory response. To improve respiratory injury with higher effectiveness in cell therapy and lung tissue engineering, pre-differentiating transplanted cells into more limited lineage cells could increase their capacity, both anatomically or functionally, to be used in injured lung tissues. Lan et al. (2019) proposed the investigation of the regenerative capacity of alveolar epithelium in an emphysema model by utilizing precursors of lung epithelial that had already been differentiated from AFMSCs (amniotic fluid mesenchymal stem cells). Moreover, differentiation of eGFP-expressing AFMSCs to LEPLCs (lung epithelial progenitor-like cells) was performed under mSAGM (modified small airway growth media) for seven days of induction. AFMSCs that are differentiated were implemented intratracheally into PPE (porcine pancreatic elastase)-induced PE in mice, and at the end of 2 weeks, related fibrotic, inflammatory, and PE indices and pathological alterations were analyzed after using PPE at 6 weeks. Consequently, predifferentiated AFMSCs transplantation by intratracheal injection demonstrated reverse elastase-induced PE and improved alveolar regeneration in the PPE-induced mice model [92].

Pulmonary fibrosis (PF)

The clinical usage of novel drugs has been able to hamper the deterioration of the disease and develop the quality of life in subjects with PF. However, the injured lung tissues do not improve via the administration of these compounds. Therefore, therapeutic therapy, such as lung tissue engineering or stem cell therapy, is imperative. Also, the clinical administration of MSCs therapy in humans with IPF (idiopathic pulmonary fibrosis) has been implied to be safe. It appears that combined treatment with pharmaceutical compounds and MSCs transplantation could have further advantages. Nevertheless, the appropriate study design for its effectiveness does not exist yet. Also, Chuang et al. (2018) recommended that MSCs transplantation in combination with drug administration involving pirfenidone (TGF-β) and nintedanib (PDGF/VEGF/FGF), two FDA (Food and Drug Administration)-approved pharmaceutical compounds in therapeutic approaches of IPF, for synergistic activities [93].

ALI/ARDS

One of the leading causes of ARF (acute respiratory failure) is ALI/ARDS, which could be reduced via hCMSCs (human chorionic villi-derived MSCs) in the airway injury model. Additionally, Liraglutide as a GLP-1 (glucagon-like peptide 1) agonist presents promoting proliferation and anti-inflammatory properties. Feng et al. (2020) have suggested investigating the potential combination therapy with liraglutide and hCMSCs on ALI. They evaluated the dose-dependent and time-dependent manner of Ang-1, SPC, FGF, and GLP-1R with LPS by western blot and qRT-PCR. They determined the combined therapy of MSCs and liraglutide properties on symptoms of the ALI animal model through different analyses. As regards outcomes, it is elucidated that the combined therapy with liraglutide and hCMSCs could be an appropriate scheme for treating ALI (Table 3) [94]. Based on the abovementioned, the imminent new requirements for MSCs therapies, and the recent restrictions on their production in vitro, scientists are faced with the demanding to enforce innovative strategies that enhance their proliferation in vitro and production of MSCs-derived secretome. The extent to which MSC exosomes can be utilized for cell-free regenerative medicine depends on the ideal option of the optimum MSC, which is determined via donor parameters and the origin of tissue for every use, setting suitable standards for MSC in vitro development and the assessed of practical effectiveness examinations of the acquired products (Fig. 5) [95].

Table 3 The cells/stem cells used in respiratory development and disease treatment

Fig. 5

Strategies of MSC production on the basis of MSC sources, the system utilized for in vitro proliferation, final products for ARDS treatment, and culture cell conditions [95]

Growth factors

Already, growth factors have been known as peptides that change the rate of cell proliferation, but this simple definition is no longer true. Possibly, growth factors are likely to be thought of as a protein family that can change the behavior of the cells and impact the organization of tissues. Generally, these strength cytokines act in a microenvironment in which activity is managed through protein binding, receptor expression, and the existence of other factors. There are several approaches to finding out growth factors in the lung, although each has its restrictions. Clinical examinations are usually conducted in humans after the onset of the disorder, and the early stages of the disease are not reported. BALF can include cytokines from the interstitium of the lung, but epithelial barrier permeability will change the associations, and it is complicated to normalize. In addition, animal cells may have different phenotypes at various ages and various stages of lung disease. Growth factors and grow factors receptors included in immature lung progression and growth may change responses in the duration of lung damage and repair [96]. For controlled stem cell reprogramming in lung tissue engineering, a better understanding of the signaling pathways seems to be essential. The adjusted amounts of molecules in the signal, such as FGF [97], BMP4 ([98], Notch [99], retinoic acid [100], HGF (hepatocyte growth factor) [101], and TGF-β [102] pathways show their significant effects on lung regeneration. Therefore, some of the most utilized growth factors in lung tissue engineering or lung diseases in recent years have been described.

FGF

The Discovery of types of FGF has significantly expanded since the initial finding of FGF1 and FGF2 occurred, as well as the recent FGF gene family containing 22 members. FGFs possessed critical functions in the progress and support of several organs and tissues isolated from three known germ layers such as endoderm (for instance, pancreas, lung, liver, gastrointestinal), mesoderm (such as bone, cartilage, adipose tissue, striated and smooth muscle), and ectoderm (as well as skin and brain). For example, cardiac FGF2 and FGF1 derived from mesoderm affects lung development, specifically at embryonic day 8 in the endoderm of the ventral foregut. Mesenchyme-derived FGF10 conducts on the opposed epithelium immediately after the lung domain specification. It expresses FGFR (FGF receptor)-2b to stimulate branching morphogenesis, a repetitive procedure that determines the pseudo glandular stage of lung progression, and FGF9 is engaged in the progression of pulmonary vascularization [103]. CAFs (Cancer-associated fibroblasts) have been commonly recognized to stimulate tumorigenesis by different mechanisms. For instance, FGF/FGFR-dependent lung carcinogenesis has been explained. Hegab AE et al. (2019) have prepared a lung adenocarcinoma mice model by inducing overexpression of FGF9 in alveolar cells. FGF9 cytokine results in the quick development and growth of different tumor nodules in adult lungs, which are similar to adenocarcinoma. Also, the findings of this research have revealed that released FGF2 by CAF provides the progression of tumor cells. CAFs overexpressed Mmp7, TGF-β, FGF2, and FGF9, integrated more collagen, and released inflammatory cytokines, which are vital in recruiting cells. FGFR blocker was not adequate to eradicate the tumor, although it drastically inhibited the tumor and its stromal cells, possibly because of the emergence of alternative mechanisms (maintenance /resistance) [104]. Yu P et al. (2017) have investigated the FGF activity in vascular progression. Lymph and blood vessels are directly engaged in the maintenance of fluid homeostasis and oxygenation of tissues. The gathering of vascular connections includes proliferation, migration, and sprouting of endothelial cells. There is negligible information about FGF’s effect on metabolism regulation and vascular progression, which are dependent on VEGF. However, knowledge about this cytokine and related metabolism regulation and development of vascularization is considerable. FGFR signaling was identified as an important adjuster in vascular progression through the expression of FGF, which controls the c-MYC and then modulates the HK2 expression. Without FGF signals, levels of HK2 have reduced inputs, leading to the decrement of glycolysis, which causes impaired migration and proliferation of the endothelial cell. Generally, a crucial part of the endothelial glycolysis modulation is dependent on FGF in adult vascular development and growth, which can be pivotal in the development of the lung [105]. Tang Q et al. (2020) evaluated FGF2’s effects on LPS-induced lung damage and its signaling and mechanism of action in vivo and in vitro. They used LPS to induce the ALI/ARDS mice model. Also, BEAS-2B was used to assess the FGF2 activity on the respiratory function, as well as related signaling and the mechanism in vitro. Levels of FGF2 decrease oxidative stress in lung tissue in mice models. The outcomes of the in vitro examination showed that FGF2 efficiently decreased levels of inflammation and BEAS-2B cell apoptosis in the LPS-induced model. Moreover, the activity of the PI13K/Akt signaling pathway was increased in vitro [13]. The signaling of FGF has a major effect on the regeneration, development, and homeostasis of respiratory tissues. Rabata A et al. (2020) analyzed the role of the FGF ligands alone and the interaction between EGF (epithelial growth factor), FGF signaling, and WNT mechanisms in differentiation and morphogenesis of lung epithelial cells in 3D cell culture of mice model ex vivo. The result showed that FGF signaling stimulates lung sphere formation from LPSCs. Immunohistochemical and ultrastructural assessments demonstrated that LSPCs generated more differentiated pulmonary cell descendants. Furthermore, FGF10, FGF9, FGF7, and FGF2 promote the formation of lung organoids in a 3D ECM model. FGF9 implied the ability to reduce and maintain LSPC survival/initial divisions. FGF10 and FGF7 provided greater organoids and provoked branching of organoids with more frequency in comparison with FGF9 or FGF2. Higher concentrations of FGF and FGF2 have elevated both formations of lung sphere and lung organoid efficiencies by increasing the affinity and stability of FGFRs. These findings showed that the FGF signaling level is a major factor in the differentiation and survival of LPSC and the morphogenesis of lung epithelial cells. EGF signaling has a significant function as a supportive factor in lung organoid formation induced by FGF. The assessment of the cell-type composition and architecture of tissue showed cells with alveolar-like regions that express different cell markers in the lung organoids. Also, FGF ligands revealed variations in the development of different types of epithelial cells, and FGF9 indicated a promoter effect on proximal cell kinds containing basal and ciliated cells. FGF10 and FGF7 differentiated the distal lineages of the lung [106]. The FGFR/FGF-related signaling pathways control a wide range of biological procedures, including wound healing, embryogenesis, tissue homeostasis, cancer, and angiogenesis. It presents these modulatory activities through regulating differentiation, migration, survival, proliferation, and aim cell metabolism [107]. KGF (Keratinocyte growth factor) is one of the stromal paracrine mitogens isolated from the FGF family. KGF is released via dermal fibroblasts and particularly stimulates the proliferation of keratinocytes [108]. KGFR is a tyrosine kinase receptor that is localized mainly on epithelial cells (ECs), and these types of receptors possess remarkable action in the differentiation and proliferation of epithelial cells, development of embryonic cells, epithelial wound restoration, development and formation of tumors, and immunity [109]. Wang J et al. (2020) evaluated the KGF effect on the restoration, apoptosis, intracellular adhesion, proliferation, and secretion of inflammatory cytokines of in vitro 16HBE 14o-bronchial ECs model. The level of expression of both KGF and its receptor was reported in cultured cells by Western blotting and RT-qPCR. The expression of molecular adhesion and apoptosis was analyzed using KGF, and outcomes revealed that KGF and KGFR remarkably were higher in composed cells from patients with asthma. Moreover, administration of KGF could restrict IFN-γ- and TNF-α-induced apoptosis in vitro. Overall, KGFR and KGF demonstrated their capacity to support bronchial ECs in asthma by suppressing EC apoptosis while increasing the migration and proliferation of ECs [110]. In another study, Tenghaho S et al. (2019) investigated the KGF2 role in 45 rat ALI models induced by oleic acid. The findings of the study showed that KGF2 might alleviate ALI symptoms in an induced rat model by keeping the barriers of pulmonary microvascular endothelial cells, which is a functional preventive measure of ALI [111].

Retinoic acid (RA)

RA is a vitamin A bioactive metabolite with a well-known regulatory effect in the respiratory system development. RA signaling is composed of the development of the diaphragm, lungs, airways, and trachea during the duration of embryogenesis. RA maintains an influence on the health of the respiratory system during postnatal life. RA activity disruption prepares phenotypes dramatically during the development of embryonic cells in vivo and human diseases containing CDH (congenital diaphragmatic hernia), tracheomalacia, lung agenesis or hypoplasia, and tracheoesophageal fistula. Many methods of experiments have been employed to propose RA signaling pathways in the duration of embryonic lung formation [112]. RA exerts its capacity to improve the action of the endothelial and epithelial barrier, development, and even inhibit injury imposed via inflammation on these barriers by modulating the activity of immune cells. Another study by Callaghan et al. (2020) has sought to detect whether RA could increase the function of the baseline barrier and mitigate barrier leak induced by TNF-α in human bronchial ECs model in vitro. As a result, RA elevates the function of the baseline barrier of 16HBE cell layers implied via approximately 90% increment of TER (transepithelial electrical resistance) and about 20% reduction in the flux of 14 C-mannitol. Synchronously, a 70% rise in claudin-4 induced by RA demonstrates that RA impacts the TJ complex. RA efficiently reduced the 16HBE barrier leakage induced by TNF-α, alleviated almost 60% of the 14 C-mannitol leakage induced by TNF-α, and 80% of the leakage to 14 C-inulin. In contrast, the leakage related to the IL-6 was decreased by it [113]. Apart from the development of the respiratory system, scientists have been analyzing the antibacterial activity of RA in vivo since 1989. Currently, the ATRA (all-trans-retinoic acid) antibacterial properties have been indicated. Also, these effects revealed the capacity of ATRA to modulate the response of the immune system to infection by promoting expression of the pivotal gene, activation of monocytes, and autophagy induction, which causes its usage as an HDT (host-directed therapy). One of the intriguing roots of administration in TB (tuberculosis) treatment is inhalation. Thus, the researchers have provided ATRA-MP (ATRA-loaded microparticles) within the appropriate size for inhalation use. O’Connor G et al. (2019) designed an ATRA control release for TB treatment with inhalation administration afterward, evidenced that this process suggests improving HDT aiming, holding antibacterial activity, and betterment of pulmonary pathology in comparison with ATRA solution [114].

TGF-β

TGF-β is a protected pleiotropic agent that controls abundant biological procedures containing regeneration of tissue, tumorigenesis, and responses of the immune system. TGF-β is also required for homeostasis and organogenesis of the lung tissue as documented in mice model studies, which are genetically designed. Moreover, TGF-β is very important in epithelial and mesenchymal cell interactions in the duration of branching morphogenesis of the lung and development of alveolar structures. The activation and expression of the three ligand isoforms of TGF-β in the respiratory system, particularly in the lungs, are temporarily and modulated via different pathways and mechanisms. The structure of the lung is susceptible to pathogen and environmental stimuli and is prone to allergic response, carcinogenesis, and inflammation [102]. Differentiation of fibroblast to myofibroblast induced by TGF-β supports generated in COPD and idiopathic PF, although the capacity of fibroblast to contribute to remodeling lung epithelial cells remains elusive. NJ-Blichfeldt et al. (2019) examined pretreated primary hFB (human lung fibroblasts) with recombinant human TGF-β to stimulate the differentiation of myofibroblasts, then co-cultured the cells with ECs of mice lung adhesion molecule-positive cells (EpCAM+) to assess their ability for helping the formation of epithelial organoids in vitro. Complementing organoid culture media recombinant FGF7 or HGF encouraged the formation of organoids when employing pretreated fibroblast with TGF-β. Furthermore, induced differentiation of myofibroblast by TGF-β leads to inclined β-catenin/Wnt pathway and weakened fibroblast function to endorse epithelial regeneration probably via several mechanisms involving regulation of secreted growth factor [115].

Growth hormone-releasing hormone (GHRH)

Although many tissues, including the lung, produce localized GHRH, it is initially released from the hypothalamus. This hormone promotes the secretion of growth hormone (GH) via the pituitary and modulates GH and IGF-1 production. GHRH-R is expressed in human lung tissues, showing that growth hormone or GHRH might play a part in the development, repair, and growth of lungs. Antagonists of GHRH-R, such as synthetic peptides, show the growth-suppressing activity of carcinogenesis in lung cells in vivo and in vitro; moreover, they possess antioxidative, pro-apoptotic, and anti-inflammatory properties. For instance, MIA-602 is an antagonist of GHRH-R that reduces both fibrosis and inflammation in the ALI induced by bleomycin in vivo [116]. Uddin et al. (2019) also evaluated the GHRH and its agonistic and antagonistic analogs in pivotal intracellular pathways that modulate the permeability of endothelial cells. The outcomes proved the documented anti-inflammatory function of antagonists of GHRH in the vasculature. Furthermore, GHRH has exerted its ability to be used in the treatment of severe respiratory abnormalities as well as ARDS [117].

VEGF

The ECs have a major part in angiogenesis progression, which leads to cell remodeling, adhesion, migration, invasion, survival, and proliferation; angiogenic agents like growth factors, proteolytic enzymes (metalloproteinases), proliferation, cell adhesion molecules, vasoactive peptides, cytokines, and activator of plasminogen attach to their ECs receptors and activate the signaling pathways such as EGFR pathway, PI3K/AKT pathway and mTOR, which launch the angiogenesis process. Cytokines usually promote angiogenesis involving indirect and direct proangiogenic markers, which direct proangiogenic markers including HGF, FGF2, and VEGF. In addition, indirect proangiogenic biomarkers contain TGF-β, IL-8, IL-6, and PDGF. FGF2 and VEGF are the most powerful angiogenesis stimulator, which promotes the proliferation and migration of ECs in vessels for the promotion of balance in balance new vessels. VEGF is secreted in hypoxic situations with a HIF-1α (hypoxia-inducible factor) effect and leads to bronchial cell inflammation and alterations that cause the development of different respiratory diseases, like COPD, pulmonary hypertension, PF, lung cancer, and asthma. In addition, VEGF and FGF2 overexpression have significant interactions with lung diseases [118]. For example, Mender L et al. (2018) demonstrated in an SCLC mice model that a combination of anti-VEGF and anti-PD-L1 aimed therapy improves the result of treatment due to their synergistic effects in comparison with individual therapy with anti-PD-L1 and anti-VEGF. SCLC constitutes the most aggressive neoplasm in the pulmonary zone and is usually determined with limited survival at the last stage despite combination therapy with chemotherapies. The outcomes recommended that combined therapy with anti-PD-L1 and anti-VEGF can be used as a promising strategy for treating patients with SCLC (Table 4) [59].

Table 4 Growth factors used in respiratory development and disease treatment

Bioreactor technologies for pulmonary engineering

The systems of ex vivo culture are necessary for complete lung tissue engineering, which involves complicated bioreactors aimed to reproduce, as far as possible, the circumstances that existed in the chest. Most whole-lung reactors attempt to prepare passive venous drainage, pulsatile arterial perfusion, control of dynamic ventilator, and assembly and recirculate the trans-pleural effluent. In all conditions, these systems must be designed and engineered to preserve sterility at all stages of setup culture and must be constructed to authorize the required interventions in experiments. Due to the importance of monitoring, nutrient regulation, pH, water, and gas levels in the process, levels of dissolved gases have a significant function on the phenotype and development of cells. Mechanical ventilator pressure impacts the integrity of the barrier non-linearly. At the same time, pulsatile perfusion in various settings has been revealed to possess effective functions on both endothelial action and recruitment of microvascular tissues in the lung. These systems are gradually developed and used in the surgery relating to the ex vivo lung. One of the recent findings enables noninvasive real-time calculation of barrier resistance of capillary alveolar tissues, and it would be referred to as an adoption of some tools to be critical for precise whole-organ barrier function estimation [5].

Conclusion

Stem cells have great potential in the future, and they can make tissue engineering and regeneration more advanced. Those therapies based on stem cells that help damaged or diseased tissues are considered in the field of tissue engineering or regenerative medicine. Stem cells both act as an assisting cell and a source for a multipotent differentiation population in a co-culture, which improves the damaged tissue. These cells can be used to generate new structures like organoids, and they are used in treating diseases like PE, PF, COPD, etc. Emerging therapeutic strategies in lung regenerative medicine are expanding to harness molecular and cellular mechanisms that enhance tissue repair and regeneration. IL-33 and IL-22 show promise as modulators of inflammation and immune responses, critical in re-establishing lung homeostasis. Trefoil factors, with their mucosal protective properties, combined with IAAPs, represent potential avenues for repairing alveolar damage. Furthermore, gene therapy targeting lung-specific pathways could offer a means to modulate these factors, potentially reversing chronic lung conditions precisely. Integrating microbiome-centered therapies may also reshape host-microbial interactions, strengthening immune resilience and supporting tissue repair processes. PRP, with its growth factor-rich profile, has shown regenerative potential in other tissues, and its adaptation to the lung may accelerate healing. Future research integrating these approaches could unlock synergistic effects, providing a comprehensive regenerative toolkit for treating lung injuries and chronic diseases. Furthermore, they are used to improve lung conditions in COVID-19 patients with high respiratory involvement. Other than that, they are used accompanied by scaffolds. Scaffolds are appropriate approaches for the replacement of defects in the trachea and lung tissues. Scaffolds are normally divided into three groups: non-biodegradable synthetic, biodegradable scaffold, or decellularized tissue scaffolds, which are safe biologically and degrade at good rates regarding tissue regeneration to prevent structural trachea collapse, although scaffolds require more development due to their importance in children and adequate investigation is not provided yet. Children have grown, and the flexibility of the hollow tube is important. Furthermore, more clinical studies are needed to solve the problems in the transplantation of the trachea and lungs in patients, especially children. Growth factors are essential for both tissue development and amendment. There are many growth factors relating to the respiratory system, but the most important one is FGF. It consists of my subunits and which one of them plays a crucial role in development. They can be used either alone or with other elements, such as hydrogels, scaffolds, and cell cultures.

Future direction

Stem cells and scaffolds have been used in different areas, either for developmental or healing purposes. Different conditions benefit from stem cell therapy accompanied by scaffolds, growth factors, or alone. They have the potential to produce a new lung in vivo, but in real live objects, it needs to thrive far more. Moreover, it has some drawbacks; for example, after seeding some cells in the damaged lung, it is not supported for a long time, and after some years, that patient will die. In the future, the authors think that there will be a lot of work to increase this crucial limitation of using stem cells. It can be solved by testing different growth factors, different scaffolds, and even nanomaterials, which help through this challenge.