Current Strategies for Tracheal Decellularization: A Systematic Review

The process of decellularization is crucial for producing a substitute for the absent tracheal segment, and the choice of agents and methods significantly influences the outcomes. This paper aims to systematically review the efficacy of diverse tracheal decellularization agents and methods using the PRISMA flowchart. Inclusion criteria encompassed experimental studies published between 2018 and 2023, written in English, and detailing outcomes related to histopathological anatomy, DNA quantification, ECM evaluation, and biomechanical characteristics. Exclusion criteria involved studies related to 3D printing, biomaterials, and partial decellularization. A comprehensive search on PubMed, NCBI, and ScienceDirect yielded 17 relevant literatures. The integration of various agents and methods has proven effective in the process of tracheal decellularization, highlighting the distinct advantages and drawbacks associated with each agent and method.


Introduction
Trachea's abnormality has been a rising problem in recent years, both congenital abnormalities and acquired abnormalities.Congenital defects may vary in degree and forms, including tracheal agenesis, massive tracheo-oesophageal fstula, and/or tracheomalacia.Acquired defects are commonly caused by trauma, be it blunt or sharp.According to literature, the worst and the most common case of traumatic tracheal abnormalities is post intubation tracheal stenosis (PITS), with the prevalence of 6%-21% of intubated patients.Around 10% of mild stenosis patients may remain undetected for more than 10 years.Te newest literature shows that the prevalence of PITS in London is 926 new cases per year [1].
Tere are 2 types of tracheal repair based on the afected segments.In short-segment defect, which is defned as defects in less than half of the total tracheal length in adults and/or less than a third of the total tracheal length in children, the go-to procedure would be either an end-to-end tracheoplasty or a slide tracheoplasty [2].On the other hand, long-segment defect is defned as defects in half or more of the total tracheal length in adults and/or a third or more of the total tracheal length in children.Tere are still no defnitive treatments for long segment defects.Te patient would usually receive temporary palliative care, such as Ttubes or stents, which have a high rehospitalization and infection prevalence [2].
In recent years, the feld of regeneration medicine has been trying to develop a defnitive treatment for longsegment tracheal defects and the most promising method right now would be replacing the afected part.Tracheal grafts can be divided into groups such as autologous, homograft, prosthetic, and combined graft.Each of the groups has their own strengths and weaknesses.In autologous and homografts, the problem lies in fnding a donor, vascularization failure, failure in thriving, and the need for prolonged immunosuppressants.Prosthetic grafts on the other hand may be a solution to some of those problems.However, prosthetic grafts are too infexible and proinfammatory, making them the second-best option for a tracheal graft [3].
In order to fnd a suitable tracheal replacement, graft materials development has been increasing these past few years.Tissue engineering has been used in various ways, such as blood vessels and heart valves.Tissue engineering needs 3 major components, which are scafolds, healthy cells, and a bioreactor.
Scafolds are the base of the new cells.Scafold should have the same mechanical properties of the original organs and should be able to support the cells' adhesion, migration, proliferation, and diferentiation.According to these standards, the best tracheal scafold right now would be a decellularized trachea as it has the same biomechanics, fexibility, and proangiogenic.Te purpose of decellularization is to remove the immunogenic components of the hosts without harming the extracellular matrices (ECMs) since ECMs' main function is to grow, maintain, and regenerate the cells.ECM's main components are collagen, proteoglycan, glycoprotein, and glycosaminoglycan.Tere are 3 methods of decellularization, which are enzymatic, chemical, and physical.Te most popular one is the chemical decellularization method where detergent is used to decellularize the scafold.In tracheal decellularization, the tricky part is the cartilage as it is really thick and will take time for the detergent to penetrate; meanwhile, the ECMs cannot take that much detergent exposure.Tus, this paper is written to evaluate the various decellularization methods and to fnd one that removes the most immunogenic components yet preserve the most ECMs [4,5].
Te objective of this research is to assess the efectiveness of various tracheal decellularization methods in experimental animals' trachea by the deoxyribonucleic acid (DNA) count and histopathological analysis (HPA) examinations.

Materials and Methods
Te inclusion criteria for this research were animal experimental studies published between the years 2018 and 2023, written in English, primarily studying the process of tracheal decellularization, and describing the outcomes of histopathological analysis, DNA, ECM, and biomechanical characteristics.Excluded studies were studies aiming for partial decellularization, having end products of nontracheal grafts, and/or involving 3D printing and biomaterials.Te search was done through PubMed, NCBI, and ScienceDirect using the terms trachea or tracheal and decellularization or decellularization.Te last search was done in September 2023.Te population, intervention, comparison, and outcome (PICO) framework was used as described in Table 1.Te detail of the literature identifcation process was explained using the PRISMA fowchart in Figure 1.

Results and Discussion
A comprehensive search of PubMed, NCBI, and Science-Direct yielded 1044 potentially eligible studies.Of these, 200 duplicates were removed.Upon title and abstract screening, 808 studies were excluded due to being reviews, case reports, editorial comments, not in English, or unrelated to the topic.Two studies were disqualifed due to unavailability of full texts.Subsequently, a meticulous full-text review led to the exclusion of 16 more studies, including those with partial decellularization, improper outcomes, incorrect interventions, and nonexperimental designs.Consequently, a total of 17 studies were selected for inclusion in this review.Te characteristics of the methods, agents used, and outcomes of each included studies are presented inTable 2.

Tracheal Anatomical Properties.
Structurally, the tracheobronchial system can be classifed into the conductive part (cartilage) and the airway part (noncartilage).It is located in the medial side of the body where it extends from the neck and to the thorax; topographically, it starts from vertebrae C5-6 and extends down until T5 where it will branch into 2 bronchi.It connects the larynx and the bronchus, and functionally, it is semifexible, 1.5-2 cm wide, and 10−13 cm long [3,22].
Tracheal structure includes mucosa, submucosa, hyaline cartilage, and adventitial layer.Te mucosal layer includes pseudostratifed columnar epithelium and goblet cells; goblet cells will secrete mucous to trap the debris and dirt for the cilia will sweep them away.Submucosa is the deepest part of the tracheal lumen with the most blood vessels and nerve, and its function is to maintain tracheal structural integrity [3,22].

Tracheal ECM Roles.
Tracheal biomechanical properties come from the ECMs which consist of glycosaminoglycan (GAG), collagen, proteoglycan, and other glycoproteins.Collagen is the main component that gives the trachea its biomechanical properties.Te collagen fbers make the trachea characteristically laterally rigid and longitudinally fexible.In addition, the ECMs have three main functions, which are intercellular signalling via paracrine signalling, intracellular signalling via autocrine signalling, and cellular formation via mechanical pressure [3,22,23].

Tracheal Tissue Engineering.
Tracheal tissue engineering has been on the rise due to the complications of autografts and allografts.Tracheal tissue engineering includes resecting the afected organ and changing it with a scafold that has been seeded with stem cells.Te main components of tracheal tissue engineering are the scafold, cell source, agent, and method [3,22,23].

Tracheal Scafold.
Tere are two main types of tracheal scafolds with their own strength and weaknesses.Te frst is the synthetic tracheal scafold.It is more versatile when it comes to shape and size, but the macro-and microanatomy 2 International Journal of Biomaterials of the scafold is lacking compared to the biological scafold.One of the examples of the synthetic tracheal scafold includes biodegraded molecules from polyglycolic acid and nanocomposite polymer (POSS) covalently bonded to polyurethane (PCU) [24].Te second type is the biological decellularized scaffold.Tis type is more popular and favourable since it supports the cellular adhesion, proliferation, and diferentiation process [25].Te decellularization process is needed in order to lose all the immune-inducing systems within the trachea that can be activated with major histocompatibility complex I and II (MHC-I and MHC-II) [23].Te components of the natural decellularized scaffolds are exactly like the original.Te only downside is that during the decellularization process, there might be some cellular and structural changes due to ECM destruction.Terefore, some researchers are looking for a way to minimize the ECM destruction while optimizing the decellularization process.One of the advantages of using bioscafold is that the patients have no need of taking immunosuppressants since the tracheas are decellularized and seeded with the patient's stem cells [22].
Every decellularization process needs a decellularization agent and every decellularization agent has its own pros and cons.Some of the popular decellularization agents used are mentioned in the following.

Chemical Agent.
Te types of chemical agents commonly used include acids, bases, detergents, hypotonichypertonic solutions, and solvents to lyse and kill cells [26,27].Some of the chemicals used in the decellularization process are as follows.
(1) Acid and Bases.Decellularization methods involving acids and bases catalyse the hydrolytic degradation of biomolecules, cytoplasmic components, and nucleic acids [28].Like detergents, they have the capacity to disrupt the extracellular matrix (ECM) constituents and structures.Acidic compounds either donate hydrogen ions (H+) or form covalent bonds with electron pairs to facilitate hydrolytic degradation.Peracetic acid (PAA), hydrochloric acid, and acetic acid are commonly employed for the decellularization process [29,30].
Peracetic acid and hydrochloric acid are among the acid agents employed in the decellularization process.Peracetic acid functions by disrupting cell membranes and solubilizing cytoplasmic organelles.However, it comes with the drawback of damaging the extracellular matrix (ECM) architecture.On the other hand, hydrochloric acid induces cell lysis, denatures proteins, and catalyses the hydrolytic degradation of biomolecules.Yet, its disadvantage lies in its impact on intracellular molecules, particularly glycosaminoglycans (GAG) [31][32][33].Hence, it is essential to choose suitable acids and concentrations.Peracetic acid (PAA) at 0.1% concentration is considered an optimal treatment for thin tissues, as it minimally afects extracellular matrix (ECM) structures and components [34].
In contrast to acidic compounds, alkaline substances can release hydroxide ions (OH−) and interact with acids to produce salts.Ammonium hydroxide, sodium hydroxide, and sodium sulphide are commonly used bases in decellularization [35].Bases achieve tissue decellularization by denaturing chromosomal DNA and inducing cellular lysis.Particularly, alkaline solutions with a pH exceeding 11 prove efective in eliminating cellular remnants, given the susceptibility of DNA to denaturation [34].Ammonium hydroxide functions by solubilizing cytoplasmic components, disrupting nucleic acids, and catalysing the hydrolytic degradation of biomolecules.However, drawbacks include its impact on the GAG content, collagen, and growth factors, as well as a weakening of the mechanical properties of the scafold.Alkaline solutions with a pH range of 10-12 can cause signifcant harm to collagen fbers, fbronectin, and GAGs.In addition, they may trigger intense host responses and lead to the formation of fbrotic tissues [32,35,36].
(2) Organic Diluent.Te mechanism of action of these agents involves cell membrane lysis.Commonly utilized types include alcohol, acetone, and 1% tributyl phosphate (TBP) for solid tissue decellularization.In the case of acetone and alcohol, they have the capacity to precipitate ECM proteins and infuence ECM ultrastructure.In the decellularization of solid tissues such as tendons, tributyl phosphate is more efective at preserving ECM structure and composition.In addition, TBP demonstrates virucidal efects (inactivating viruses) without afecting coagulation factors [26,27].
(3) Hyper/Hypotonic Fluid.Hypotonic solutions lyse cell membranes by increasing cell volume beyond their limits.Tese agents do not signifcantly impact changes in ECM components.Hypertonic solutions cause cells to lose volume and eventually die.Te drawback of this type is its incapacity to efectively remove residual DNA from cell death.In the process, scafold materials are immersed in hypotonic and/ or hypertonic solutions over several cycles to achieve optimal results [26,27].
(4) Ionic Detergent.Ionic detergent includes sodium dodecyl sulfate (SDS), sodium dodecyl cholate (SDC), Triton X-200, and sodium hypochlorite.Te most popular one right now is the SDS 0.1%.Sodium dodecyl sulfate (SDS), also known as sodium lauryl sulfate (SLS), is a widely recognized anionic surfactant with amphiphilic properties, combining hydrophilic and hydrophobic characteristics.It dissolves lipids but can cause skin and eye irritation.SDS is produced by reacting dodecanol with sulfuric acid and sodium hydroxide, and the chemical reaction produced is as follows (Figure 2) [37,38].SDS 0.1% is a commonly used agent, but various concentrations of SDS (0.01, 0.025, 0.05, 0.075, and 0.1%) have started to be studied in the context of the decellularization process.Like nonionic detergents, this agent can also infuence the ECM structure.Furthermore, this agent can remove growth factors in the ECM [26,27].
(5) Nonionic Detergent.Nonionic detergent will disrupt the cellular structure by destroying the lipid-lipid and lipidprotein bind without destroying the protein-protein compound.Triton X-100 0.5% is the most popular one due to its ability to maintain protein structures and GAG sulfate.Te advantage of these agents is that they do not disrupt the bonds between proteins or sulfated glycosaminoglycans.Teir drawback, however, is their potential to reduce the concentration of laminin/fbronectin in the ECM structure [26,27].

Physical Agent.
Physical agents employ various physical conditions, including temperature, mechanical force, pressure, and electrical currents, to disrupt cell membranes and induce lysis, ultimately resulting in the removal of cells from the scafold matrix.Tese physical methods represent an alternative approach to decellularization and have been explored for their potential benefts in tissue engineering [26,27].
(1) Freezing-Tawing.Decellularization is done by purposefully freezing the intracellular fuid and destroying the cells; the downsides of this method are that it cannot get rid of the genetic materials and ruins the ECMs.Tis agent is usually done with the help of some detergents and/or nuclease to optimize the results [26,27].
(2) Mechanical Pressure.Tese agents are typically employed in organs or tissues with less dense ECM (e.g., the lungs and the liver).Teir mechanism of action involves releasing cells from the tissue or organ through applied pressure.However, one drawback of these agents is their potential to cause structural damage to the ECM.Precisely controlling the applied force is a crucial aspect of using these agents efectively [26,27].
(3) Electroporation.Another term for this agent is nonthermal irreversible electroporation.Its mechanism involves disrupting the potential diference across the cell membrane, thereby interfering with cell permeability and ultimately causing cell death using an electric current.An advantage of this agent is its ability to preserve the biomechanical structure of the ECM.However, a limitation is its suitability for use in small, thin tissues or smaller organs [26,27].International Journal of Biomaterials (4) Immersion and Agitation.Immersion and agitation represent a more suitable approach for small, delicate, and thin organ sections, as well as tissues lacking intrinsic vascular structures [39][40][41].Immersion and agitation involve submerging tissues in decellularization solutions with continuous mechanical agitation, and its efectiveness relies on various parameters such as agitation intensity, decellularization agent, and tissue dimensions [39,42].Te process, following tissue immersion in the agents, facilitates cell rupture, cell detachment from basement membranes, and the elimination of cellular components.Employing immersion and agitation as an optimal physical decellularization method ofers numerous advantages.First, dynamic immersion and agitation achieve a more uniform detergent exposure compared to static decellularization, resulting in better decellularization outcomes with reduced exposure time to aggressive agents [43,44].Second, this method minimally impacts ECM surface structure, collagen integrity, mechanical strength, and GAG content [44][45][46][47][48]. Tird, it is more accessible and easily executed than whole organ perfusion.However, it may infict more tissue damage compared to perfusion due to the limited chemical difusion caused by agitation [34].
(5) Sonication.Sonication operates by generating acoustic cavitation bubbles, inducing shear stress efects, and consequently rupturing the cell membrane.It facilitates agent penetration by emitting vibrations, aiding in the removal of cellular debris.However, the drawback lies in the potential disruption of main structural fbers and adverse efects on vascular tissues with high power or prolonged duration of sonication [49][50][51].

Enzymatic Agent
(1) Trypsin.Te working mechanism of this agent involves breaking the peptide bonds between carboxyl, arginine, and lysine.Several studies using 0.5% and 1% trypsin with exposure times of 48 and 24 hours have been shown to cause ECM damage.Further research on 0.02% trypsin for 1 hour has a less signifcant impact on the ECM structure after decellularization [26,27].
(2) Exo/Endonuclease.Te working mechanism of this agent involves breaking the bonds of RNA and DNA components.Commonly used types of this agent include DNase (0.2-0.5 mg/mL) and RNase (0.2-50 μg/mL).Te application of this agent is often combined with others to remove any remaining DNA/RNA from the decellularized scafold [26,27].
(3) Dispase.Te mechanism of action of this agent involves catalysing primarily collagen IV and fbronectin in the basement membrane, separating it from the epithelial layer.Commonly used types of this agent include 4 mg/mL Dispase II for 45 minutes to remove it.For the removal of hair, fat, and epidermis, Dispase II at 0.24 mg/mL for 3 hours is used [26,27].
(4) Phospholipase A2.Te mechanism of action of this agent involves damaging phospholipid components.Tis agent is typically used in combination with other decellularization agents.Its advantage is in preserving collagen and proteoglycans in the ECM structure although it has a minor impact on GAG composition [26,27].
3.5.4.Compound Agent.Physical, chemical, and enzymatic agents each have their own advantages and disadvantages and work through distinct mechanisms.To make the decellularization process more efective and efcient, the use of combinations of agents has been explored.For example, a combination of physical and chemical methods has led to the development of cryochemical agents for liver decellularization.Another example involves the combination of agitation, alkali, detergent, enzymes, and light-emitting diodes in the decellularization process of tracheal organs [26,27].
3.6.Decellularization Methods.After determining the decellularization agent, the next step would be choosing the method.Te organ's or tissue's characteristics need to be considered in choosing the decellularization method.Some of the examples include the following.

Whole Organ Perfusion.
It is used on large and dense organs or tissues with internal vascularization and usually uses ante-or retrograde perfusion.Tis method uses the organ's own vascular system to distribute the decellularization agent; after distributing the decellularization agent, the dead cells and the remaining decellularization agent will be drained through the veins.Some of the organs that can be decellularized through this method are the muscle, lungs, liver, kidneys, and heart [26,27].

Immersion and Agitation
. Tis method is used for organs without any decent internal vascularization; the tissue would be immersed and agitated in the decellularization agent where the agent will difuse into the cells.Te factors that afect the outcome include the agitation intensity, decellularization agent, and tissue density and size.It usually takes 1-2 hours for thin preparations and 12-72 hours for thick preparations to fnish.DNase and/or RNase are needed to clean out the remaining cellular components.Te downside of this method is that some of the cells could already be destroyed via DNase and/or RNase before even coming into contact with the decellularization agent which will afect the ECM's integrity [26,27].

Pressure Gradient.
Pressure gradient is commonly used on hollow organs.Tis mechanism is similar to immersion and agitation.However, it is more optimized by creating a pressure gradient between the extracellular space and the intracellular space, thus optimizing the difusion process [26,27].
12 International Journal of Biomaterials 3.6.4.Supercritical Fluid.Tis method uses a highly viscous and transportable fuid to kill the cells.Tis mechanism can also preserve the sample and minimalize the lyophilization process [26,27].

Post-Decellularization Evaluation.
Post-decellularization evaluation is used to assess the decellularized organs; some of the parameters being measured are the number of DNA strains left, the toxicity, the cellular immunity, and the ECMs [27].Some of the evaluation methods are discussed as follows.

Histologic and Immunohistochemistry (IHC) Evaluation.
Histologic and IHC evaluations are mainly qualitative testing, yet also may serve as a quantitative examination.Qualitative testing is done by comparing the quality of the sample to the original organ, while quantitative testing is done by counting the number of cellular nuclei.First, the organ would need to be fxated in a parafn block and then cut into smaller pieces.Subsequently, the samples are stained with hematoxylin and eosin for diferentiating the ECMs and the nuclei [20,52].For a more focused ECM examination, the staining used are toluidine blue and safranin O for GAG assessment, Masson's trichrome for collagen, and Van Gieson for elastin [20,53].IHC examination is used to assess the scafold's remaining immunological factors [54,55].Other than that, IHC examinations can also evaluate the vascularization potential of the tissue by using anti-CD-31, anti-vWF, and anti-FGF [54].Te downside of this method is that it takes a long time and depends heavily on the examiner [56].
3.7.2.DNA Quantifcations.DNA quantifcation shows the number of DNA in the decellularized graft as a marker for the graft's immunogenicity ability.Te recipient's immunogenicity tolerance is <50 ng dsDNA/mg of the graft's dry weight; this number is also the marker of a successful decellularization process.Te frst step of DNA quantifcation is to put the sample into an enzymatic solution usually phosphate bufer saline (PBS) for around 24 hours and then the DNA is isolated and examined in a special machine.
Tere are 2 types of DNA quantifer; one of the machine examples is the Quant fuor dsDNA System E2670 Promega that shows the results in DNA/mg scafold's dry weight and a nanophotometer that shows the results in nanogram [21].
3.7.3.GAG Quantifcations.GAG quantifcation is used to measure a method's efectivity in clearing out the cells without destroying the ECMs, thus the need for a control sample for comparison.Te GAG quantifcation is done using a light spectrophotometer [21].

Biomechanics Testing.
Biomechanics testing is also quantitative testing that the decellularized trachea is being compared to the control samples.Te tracheas will be pulled from 2 sides uniaxially.Te data consist of the force given and the increase in length.Te proximal, intermediate, and distal parts of the trachea have diferent biomechanics; thus, testing all 3 of them is recommended [52].
3.7.5.Toxicity Testing.Toxicity testing is done to check on the scafold's toxicity level post-decellularization and sterilizing.Te purpose is to assess the toxicity level of the tissue induced by the decellularization agents and/or the remaining bacteria on the tissue; thus, toxicity testing is usually done alongside a bacterial load examination to fnd out if the problem is in the sterilizing process or the decellularization agents [20,27,57].
In Table 2, the DNA counts and staining results of various tracheal decellularization methods from 2018 to September 2023 are presented.

Conclusions
In conclusion, the selection of a decellularization agent and method should be carefully tailored to the specifc tissue or organ under consideration, as each comes with its own set of advantages and disadvantages.According to the fndings from the reviewed studies, the optimal scafold with the minimal DNA content and preserved extracellular matrices (ECMs) is achieved by combining various agents of physical, chemical, and enzymatic nature.
Nonetheless, it is essential to recognize that the quest for the ultimate decellularization method is an ongoing process.Further experiments and research are imperative to explore and refne the selection of agents and methods, aiming for the development of the most efective, safe, and versatile decellularization protocol.Te study's limitations encompass the need for more extensive investigations across various tissue types, focusing on the efects of decellularization on the seeding process and its potential immunogenic efects on the recipient organ.Future research may involve comparative analyses of diferent decellularization techniques, shedding light on their impacts on tissue biomechanics and immunogenicity, to further advance the feld of tissue engineering.

Figure 1 :
Figure 1: Flowchart of the data gathering process.

Table 1 :
PICO framework description used in this study.

Table 2 :
Table of protocols of tracheal decellularization and their results.
°Cfor 24 h under vacuum.Te scafolds were then washed in sterile distilled water three times for 30 min and incubated for another 24 h in sterile distilled water at 4 °C.Following the wash step, the scafolds were subjected to enzymatic digestion with 2 kU/mL DNAse (Sigma, California, USA) and 4 U/mL RNAse (Biofroxx, Einhausen, Germany) in 1 M NaCl at 37 °C at diferent times (8 h (VAD 8 h group), 16 h (VAD 16 h group), and 24 h (VAD 24 h group)) as experimental groups.
mL deoxyribonuclease (type II from bovine pancreas) and 85 mg/mL ribonuclease (type III A from bovine pancreas).In the fourth stage of the decellularization process, the three protocols difer.Samples were immersed in a 1% solution of either (i) sodium dodecyl sulfate (lauryl sulfate, SDS) identifed as Triton-SDS, (ii) Triton X-100 identifed as Triton-Triton, or (iii) tributyl phosphate (TnBP) identifed as Triton-TnBP.Tis second wash was carried out with 50 mM Trizma's base and antibiotics for 48 h at room temperature.Samples were subsequently rinsed with distilled water and then in a second modifcation of the process, immersed in a 50 mM tris bufer adjusted to pH 9.0 with antibiotics.