INTRODUCTION
Using conventional techniques such as end to end trachea anastomosis, it is not possible to reconstruct a functioning trachea that had lost more than half of its length as a result of stenosis, infection, cancer or congenital anomalies. Although trachea transplantation is technically possible, its clinical applications are limited because of two-stage surgery and the need for a life-long immune suppression use [
1]. Tissue engineering that is used in many cases of organ failure has recently started to come into question for trachea reconstruction as well [
2].
The choice of scaffold on which the cells will be seeded is as important as the choice of stem cells in tissue engineering. Cadaveric trachea is a good option for reconstruction because of its three-dimensional structure, biomechanical properties, flexibility, air-tightness and endurance to collapse [
3]. Decellularized trachea is the best scaffold up to day, due to its non-immunological extracellular matrix (ECM) that does not carry major histocompatibility complex class I (MHC-I) and class II (MHC-II) and its pro-angiogenic properties [
4]. At the same time, retain matrices provide mechanical and chemical signals to aid stem cell differentiation [
5] and regeneration without additional biological additives [
6].
Effective purification of an organ from its cells depends on the origin of the would-be decellularized tissue and the method to be used. Cartilage tissue decellularization is equally challenging. The dense ECM makes full decellularization difficult due to limitations in diffusion [
7]. The tissue is often mechanically disrupted to increase the efficacy of chemical decellularization; thereupon mechanical properties of the matrix are deteriorated [
8]. The dense nature of the cartilage also restricts cell migration into the matrix [
9]. On the other hand, physically devitalized cartilage particles do not exhibit a chondrogenic response to the extent that chemically decellularized cartilage and have greater down regulation of collagen [
10].
The previous studies mostly employed detergent enzymatic method (DEM) and its modifications for the purpose of maintaining the structural integrity of trachea [
1,
11-
13] in contrast to chemical fixation, cryopreservation, or lyophilization [
12]. In this study, we investigated the effects of lyophilization and DEM combinations on the structure, composition and biocompatibility of tracheal matrix for the purpose of preventing the effects of detergent on the matrix by reducing duration of exposure to chemicals and for shortening decellularization time. And we tried to decide which chemicals can be combined with lyophilization method.
DISCUSSION
Tissue engineering studies generally involve cells that make up the relevant tissue, the structure that these cells will attach to (scaffold-matrix) and growth factors that control cellular activities [
15]. As the mesenchymal stem cells are multipotent, they are able to differentiate into adipocytes, chondrocytes, osteocytes, and endothelial cells [
16]. Thus, a failed organ can be reconstructed in vivo with tissue engineering by using the mesenchymal stem cells of the patient and placing them to the failed area on a proper tissue scaffold.
An ideal scaffold needs to be three-dimensional and biocompatible and also requires proper mechanical and physical properties that allow cellular attachment, reproduction and differentiation [
9]. Decellularization of trachea results in loss of all cellular and nuclear materials (becomes nonimmunogenic and the recipient does not receive any immune-suppressive medication) without losing its supportive scaffold properties. During the process, preserving structural contents such as ECM and basal membrain (BM) provides attachment and proliferation of cells and protects the scaffold’s morphological and biomechanical properties. Also, preserving basic fibroblast growth factor and transforming growth factor-β [
17] which affect angiogenesis in ECM increases chondrogenesis (differentiation signal for MSCs into adult cartilage) and provides a living and functional trachea [
4,
9,
18].
Optimal effectiveness in decellularization can be achieved by removing maximum amount of cellular components with minimum damage and loss of ECM [
14]. Decellularization methods can be categorized into physical, chemical, and enzymatic techniques, as well as their combinations. The decellularization method to be used depends on the properties of the tissue [
14]. Physical methods such as thermal shock, ultrasonic, and mechanical breaking cause break-up of cell membrane and remove cellular components from the tissue with agitation and perfusion [
14,
19]. In chemical methods, detergents, solvents, acidic or alkaline solutions or ionic solutions are used [
14]. Decellularization of complex tissues is usually done by short-term use of many different chemicals to increase the effectiveness and to avoid the negative effects of the chemicals on tissue by decreasing contact period. Ionic solutions work by breaking up the cells as in physical methods. Detergents are the most used materials in chemical decellularization and work by dissolving cell membranes [
19,
20]. Detergents are categorized as ionic, non-ionic and zwitterionic. Non-ionic detergents such as Triton X-100 have very mild affects on protein structure. Ionic detergents such as SDS and sodium deoxycholate affect protein structure severely, but they also cause loss of matrix. Zwitterionic detergents such as 3-[(3cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) have an effect range between ionic and non-ionic detergents. The use of different concentrations of these detergents in combination and the use of these detergents in different techniques make the comparison of different studies fairly difficult. Alkaline and acidic solutions such as peracetic acid are not sufficient for decellularization of complex tissues, but they are useful in sterilization [
14,
21]. Several enzymes such as trypsin (a protease), DNase (a nuclease), lipase and a-galactosidase are used frequently for decellularization. However, prolonged use of trypsin to break cell-matrix interactions can cause loss of collagen and thus loss of mechanical strength [
22].
For decellularization of trachea, DEM and its modifications are the most used methods up to day, as they are believed to protect tissue-matrix integration and biomechanical properties and provide best decellularization [
1,
11,
12,
22,
23]. Remlinger et al. [
13] used xenogenic decellularization for trachea in the study in 2010. However, even the studies that employ DEM differ among each other and there is no consensus on how many cycles are required for best decellularization while scaffold would be less damaged. Zang et al. [
1] reported five cycles of DEM were sufficient for decellularization and matrix preservation, whereas Jungebluth et al. [
12] reported this to be 17 cycles in a pig experiment and 25 cycles have been recommended for human tracheas by Baiguera et al. [
11]. The length of periods for which the chemicals are used affects ECM negatively [
11]. Although ionic detergents in trachea decellularization are very successful in removing cellular components, they affect ECM integration and damage the natural structure [
1,
12,
13,
19]. Besides, the detergents used for decellularization may also have an effect on the compatibility of tracheal ECM and the cells to be seeded [
1].
In our previous study, we have decellularized rabbit tracheas using DEM and transplanted the allogenic tracheas seeded with adipose-derived mesenchymal stem cells onto the rabbits and managed to observe consequences of DEM in vivo [
24]. In that study, narrowing of trachea was observed due to fibrosis. Therefore, we designed a study to investigate the combinations of lyophilization and DEM to prevent the excessive effects of chemicals and physical methods.
Lyophilization or freeze-drying is a method in which water content of a material is snap-frozen first, followed by removal of ice-crystals by sublimation initially (primary drying) and then by desorption (secondary drying) [
25]. Intracellular ice crystal formation, osmotic dehydration and mechanical forces during rapid freezing cause disruption of cellular membranes fragmentation of genetic material and cell lysis [
19]. However, the risk of damaging biomolecules and the ECM as well, and the requirement of another process to remove cellular debris are the disadvantages [
20]. Addition of lyoprotectant such as sucrose or trehalose may eliminate the adverse effects on scaffold ultrastructure [
26]. During rehydration after freeze-drying, tissue tends to absorb fluids more. Thus, this absorption force might be the reason to use this method to promote the better infusion of other decellularization agents [
27]. Therefore in our study we thought that combining this method with DEM will assist cell lysis and removal of cellular debris while minimizing the amounts of chemical agents required for effective decellularization and their toxic effect.
The first study that combined physical and chemical decellularization methods (DEM and lyophilization combination) for cartilage decellularization was performed by Sutherland et al. [
10] in 2015 on articular cartilage. The author showed that freezing, lyophilization, and cryo-grinding had no significant effect on GAG and DNA content in the tissue. Chemical decellularization and cryo-grinding (lyophilization) provided 86% reduction in DNA content and 55% reduction in GAG content. It was also demonstrated that the combined method has a better chondrogenic potential compared to physical methods and suggested that partial reduction in GAG content might be beneficial to create a less dense matrix that allows for cell infiltration and migration [
7]. Indeed, retention of GAG within the matrix is beneficial for chondroinduction based on previous studies citing that GAGs such as chondroitin sulfate and aggrecan may have chondroinductive effects in vitro [
10,
28].
In the histopathological analysis of decellularized rabbit tracheas, epithelial structure and many cellular components in the primary mesenchymal zone were visible in group 1 in which trachea was lyophilized only. It is possible that the cells were not removed from the environment after lyophilization although degenerated since the tracheas were not treated with detergents and enzymes. However, this group was one of the groups with the lowest DNA content. Similarly in the histopathological analysis of groups 4 and 6, primary mesenchymal zone remnants were observed. Group 6 showed more DNA content than other groups, but the content was low enough to satisfy decellularization criteria. In the SEM investigations, cellular remnants were observed on the surfaces of groups 1, 3, 4, and 5. BM and matrix were found to be preserved in all groups (collagen fibers were loose in group 6) in the light microscope analysis and although matrix was preserved in all groups, collagen structure in mesenchymal zone was found to be loose in groups 1, 4, and 6 in SEM analysis. Since very small areas can be imaged in histopathological analysis, it is not very accurate to discuss cellular contents; however, these analyses are valuable to observe general morphological structure, cell depletion and the preservation of the framework.
On the other hand, DNA content was low enough to satisfy decellularization criteria of Gilbert [
14] in all groups, but only group 3 had GAG content similar to untreated rabbit trachea. GAG is the main substance of tracheal cartilage and combined with its water storage capacity, provides the trachea with its mechanical strength to resist compressive forces [
29]. GAG loss also explained the early reduction in compressive strength after decellularization. Decellularized trachea showed decreased stiffness and tensile strength compared with native tracheas. In this study, none of the groups had a different tensile strength than the normal trachea but the rigidities of groups 1, 3, and 5 were higher than untreated rabbit trachea. Chemicals used in this decellularization process are inherently damaging to cells. Therefore, matrix scaffolds with high concentrations of residual chemicals are likely to be toxic to cells [
1]. Glucose consumption and lactic acid production levels of all groups (after stem cell seeding) were similar to the only cell-containing medium in this study. This revealed that stem cells seeded on all decellularized trachea groups were able to survive.
We investigated the effects of combined decellularization techniques on the structure, composition, mechanical properties and biocompatibility of tracheal matrix in vitro. According to the results, lyophilization+deoxycholic acid+de-oxyribonuclease method is the best technique with good decellularization with protected matrix and it takes only 3 days to perform the procedure. A limitation to this study is the lack of comparison between combined methods and DEM because we prioritized investigation of these methods to make sure they work properly. Furthermore, a future study was designed to compare the combined decellularization method with DEM for human trachea.