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Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, PennsylvaniaMcGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
Corresponding author. McGowan Institute for Regenerative Medicine, 450 Technology Drive, Suite 300, Bridgeside Point II (BSP2), Pittsburgh, PA 15219. Tel.: +412 624-5252; fax: +412 624-5256.
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PennsylvaniaDepartment of Surgery, University of Pittsburgh, UPMC Presbyterian Hospital F1281, Pittsburgh, PennsylvaniaDepartment of Bioengineering, University of Pittsburgh, Benedum Hall, Pittsburgh, Pennsylvania
Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement.
Materials and methods
Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition.
Results
Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels.
Conclusions
ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.
Large airway defects, specifically tracheal defects, pose a substantial problem for surgeons in both pediatric and adult populations. Current treatment options include resection of the damaged trachea with attempted repair by end-to-end anastomosis. Autografts and allografts have been largely unsuccessful due to donor and size limitations. Synthetic constructs intended for segmental tracheal replacement induce a robust inflammatory response with subsequent fibrosis and stricture. Allogeneic and xenogeneic extracellular matrix (ECM)-based scaffolds offer a potential alternative approach.
The ECM represents the secreted product of the resident cells of each tissue and is therefore ideally suited to support cell homeostasis and growth. ECM can be harvested by decellularization of tissue by chemical, mechanical, and enzymatic means, leaving behind the structural components (e.g., collagen, laminin, and fibronectin) as well as embedded signaling molecules and other bioactive components (e.g., growth factors and matrix-bound nanovesicles). Whereas the process of decellularization removes cells and cell components from tissues that could be immunogenic, allogeneic, and xenogeneic ECM can be implanted without immune-mediated rejection, and can potentially serve as a bioscaffold for host tissue regrowth. Acellular ECM bioscaffolds have been successfully used in a wide variety of preclinical and clinical applications including skeletal muscle reconstruction,
Esophageal preservation in five male patients after endoscopic inner-layer circumferential resection in the setting of superficial cancer: a regenerative medicine approach with a biologic scaffold.
For most clinical applications, ECM-based bioscaffolds become populated by host cells that proliferate, differentiate, and spatially organize after implantation during the remodeling process. However, this approach is ineffective for tubular structures that require a functional surface epithelium immediately at the time of implantation such as the trachea. Airway epithelium is composed of both ciliated and secretory epithelial cells which together provide a protective layer of mucus that is continually mobilized through the airway (the “mucociliary ladder”). In previous studies that have used acellular bioscaffolds for tracheal repair (i.e., without the mature ciliated respiratory epithelium to provide mucociliary clearance), mucus obstruction occurs, resulting in unacceptable complications such as malacia and stricture.
Thus, an approach that provides a mature epithelium that is functional at the time of implantation is required.
On harvest and in vitro culture, primary airway epithelial cells “dedifferentiate” and adopt a proliferative phenotype, losing their ciliated and mucus-producing phenotypes. Specific culture conditions in specialized media and at an air-liquid interface (ALI) are required to stimulate the rematuration process. ECM hydrogels are currently marketed as in vitro cell culture substrates.
These hydrogels may allow for preimplantation creation of a functional mucosal epithelium.
In the present study, ECM-based materials derived from two different source tissues were evaluated for their ability to support proliferation and differentiation of functional airway epithelial cells. The ECM harvested from various tissues has site-specific composition and organization and therefore it is logical that tracheal-derived ECM would be ideal for upper airway epithelial cell growth. However, it has been shown that tissue-specific ECM materials are not necessarily required or even preferred for site specific tissue reconstruction.
For example, urinary bladder matrix (UBM), an FDA-approved ECM-based material that is commercially available, has been successfully used to repair not only urinary bladder
The objective of the present study is to compare airway epithelial cell growth on homologous (tracheal) and heterologous (urinary bladder) ECM substrates.
Materials and methods
Overview of experimental design
Tracheal ECM was isolated from porcine tracheas. The porcine tracheas used were purchased from Animal Biotech Industries, Inc. The tracheas were subjected to a decellularization protocol that consisted of osmotic shock with sterile deionized water and exposure to a detergent solution to induce cell lysis and enzymatic solutions to cleave remnant proteins and double-stranded DNA (dsDNA). Tracheas were subjected to cyclical pressure changes between room atmosphere and 94% vacuum (6.325 kPA absolute pressure) during these washes in a custom-designed apparatus. Decellularization was confirmed using criteria that included quantification of residual DNA.
The ability of the resulting tracheal ECM scaffold to support bronchial epithelial cells in vitro was then determined. Primary human bronchial epithelial cells (HBECs) were seeded to the luminal side of a sheet of tracheal ECM bioscaffold. After 1 d in proliferation media, the cells were brought to an air liquid interface in differentiation media. When the intact decellularized trachea was determined to be cytocompatible, the soft tissue (i.e., noncartilaginous) luminal aspect of the tracheal ECM bioscaffolds was separated, lyophilized, powdered and pepsin solubilized to form a tracheal ECM hydrogel. The ability of tracheal ECM hydrogel to support HBEC differentiation in vitro was determined using the same cell culture conditions that were used for the intact tracheal bioscaffolds. Cell morphology and differentiation were determined by histology, histochemistry and immunolabeling. Scaffolds and hydrogels derived from urinary bladder were used as a comparative heterologous ECM.
Production of acellular ECM derived from urinary bladder
The harvesting and preparation of ECM derived from porcine urinary bladder was conducted as previously described.
Briefly, porcine urinary bladders were harvested and the tunica serosa, tunica muscularis externa, tunica submucosa, and majority of the tunica muscularis mucosa were mechanically removed. The remaining tissue was soaked in a 1.0 N saline solution to dissociate the urothelial cells from the luminal surface of the tunica mucosa leaving the basement membrane and the subjacent lamina propria which in combination is referred to as the UBM.
The remaining UBM-ECM material consists of an approximately 50-μm thick sheet of extracellular matrix. UBM-ECM scaffolds were lyophilized using an FTS Systems Bulk Freeze Dryer (Model 8-54). UBM-ECM was used both as a lyophilized sheet (“UBM scaffold”) and as a hydrogel (“UBM hydrogel”) after solubilization as previously described.
with minor modifications. Briefly, tracheas were soaked in sterile deionized water (DI) with 30 min of negative pressure vacuum cycling (15 cycles, −0.95 kPa max vacuum) then immersed overnight in fresh sterile DI water. This process was repeated daily with a detergent solution of 0.25% Triton X-100 and 0.25% sodium deoxycholate in phosphate-buffered saline (PBS), DI, and an enzymatic solution of 2000 KU/mL DNase for 10 d. Cyclical pressure changes are theorized to allow infiltration of the solutions improving removal of cellular debris.
The decellularized tracheas were then placed in a solution of 15% peracetic acid and 4% ethanol for approximately 18 h. Tracheal ECM scaffolds were lyophilized using an FTS Systems Bulk Freeze Dryer (Model 8-54). Decellularization was confirmed by the same methods and criteria used for UBM-ECM.
Esophageal preservation in five male patients after endoscopic inner-layer circumferential resection in the setting of superficial cancer: a regenerative medicine approach with a biologic scaffold.
This decellularized trachea served as a bioscaffold (“tracheal scaffold”) as well as the source of tracheal soft tissue ECM to formulate into a hydrogel (“tracheal hydrogel”). Tracheal soft tissue was excised from the luminal surface of the decellularized tracheal scaffold, lyophilized, and milled to obtain powdered tracheal soft tissue ECM to form a hydrogel.
Histology
UBM-ECM, and tracheal ECM were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained by 4′,6-diamidino-2-phenylindole, hematoxylin and eosin (H&E), Masson's trichrome and Safranin O.
DNA quantification and fragment length analysis
To assess total DNA content of the UBM-ECM and tracheal ECM, the scaffolds were cut into thin strips and digested with Proteinase K at 37°C for up to 72 h or until no visible scaffold material remained. Supernatants were purified with Phenol-Chloroform-Isoamyl alcohol (25:24:1) followed by centrifugation at 10,000 × G for 10 min. Aqueous layers were removed, and ethanol precipitated at −20°C for at least 8 h to isolate any DNA present. Samples were dehydrated in a vacuum manifold and rehydrated in 1X Tris-EDTA buffer. DNA content was quantified using the PicoGreen DNA assay (Invitrogen) after manufacturer's instructions. To determine DNA fragment size, samples were separated by electrophoresis on a 3% LMP agarose gel with ethidium bromide at 60V for 1 h stained and visualized with ultraviolet transillumination.
Hydrogel formation
Hydrogels were manufactured by established protocols.
Briefly, lyophilized porcine UBM and tracheal ECM scaffolds were milled to a powder form and solubilized with 1 mg/mL pepsin in 0.01 N HCl for 72 h at room temperature. Gelation occurred upon neutralization of pH and salt content with NaOH and 10X PBS at 4°C followed by warming to 37°C, and the concentration of the gel was adjusted by dilution with 1X PBS to 8 mg/mLl.
In vitro culture of human bronchial epithelial cells
Primary HBECs were obtained from a 43-year-old female and 20-year-old male from the Cystic Fibrosis Research Center Lung Cell and Tissue Core Facility (University of Pittsburgh) as previously described. The cells were procured from excess lung tissue from explanted human lungs after lung transplantation at the University of Pittsburgh Medical Center under written consent and an approval by the institutional review board protocol REN18110058/IRB970946.
Transforming growth factor-β1 selectively recruits microRNAs to the RNA-induced silencing complex and degrades CFTR mRNA under permissive conditions in human bronchial epithelial cells.
Cells were seeded on either decellularized and lyophilized UBM or tracheal scaffolds or UBM and tracheal hydrogels at high density (50,000 cells/cm2). For the first 24 h, cells were cultured submerged in proliferation medium (Lifeline BronchiaLife Medium), followed by culture at an ALI in differentiation medium (LifeLine Air-Liquid Interface Differentiation Medium) for 3-21 d to facilitate differentiation into mature ciliated and goblet cell phenotypes
(Fig. 2A). The culture medium was replaced every 2-3 d.
A pilot study (n = 1) was conducted to demonstrate if there is a difference in cell proliferation of HBEC on UBM and trachea hydrogels. Cells were seeded on tissue culture plastic, UBM hydrogels (8 mg/mL), and trachea hydrogels (8 mg/mL) at 12,000 cells/well in a 96-well plate. Metabolic activity was assayed by alamar blue as an indicator of cell number on days 2, 4, and 6.
Cell characterization
Formalin-fixed, paraffin-embedded cell-seeded scaffolds and hydrogels were processed for histology, histochemistry, and immunolabeling. Cell-seeded ECM scaffolds were evaluated at day 7 and 21, and cell-seeded hydrogels were evaluated by histology at day 4 and 19 for cell morphology and formation of a confluent monolayer. Assessment included the presence/absence of mucin using Alcian blue which identifies airway goblet cells. The presence of acetylated tubulin was determined to identify cilia. The primary antibody was mouse monoclonal antiacetylated tubulin antibody (1:500) (Sigma T7451) with horseradish peroxidase–conjugated goat antimouse secondary antibody (1:200) (Dako P0447). The presence of the basement membrane was determined by immunolabeling for collagen IV. The primary antibody was rabbit anti-Coll IV (1:200) (ab6586) with horseradish peroxidase–conjugated goat antirabbit secondary antibody (1:200) (Sigma A0545).
Results
Porcine tracheas can be effectively decellularized
The use of a negative pressure chamber combined with osmotic shock, detergents, and enzymes effectively decellularized porcine trachea (Fig. 1A and B). The analysis of decellularized trachea stained with H&E and 4′,6-diamidino-2-phenylindole showed an absence of cells and intact nuclei (Fig. 1C). PicoGreen analysis showed 193 ng dsDNA/mg ECM in the decellularized trachea and 11,717 ng dsDNA/mg ECM in the native trachea tissue (Fig. 1E). This decellularization method resulted in a 98% reduction of dsDNA relative to native trachea tissue. Gel electrophoresis showed that residual dsDNA fragments were less than 200 bp in length (Fig. 1F), consistent with published criteria for sufficient decellularization.
Masson's trichrome staining showed a lack of nuclei and only collagenous connective tissue remaining (Fig. 1H). Safranin O revealed minimal glycosaminoglycans both in native and decellularized tissue (Fig. 1I and J, respectively).
Fig. 1Trachea decellularization. Porcine trachea (A) is bisected and decellularized (“Decell”) (B). Decellularization was verified by H&E staining of native trachea (C) compared with decellularized trachea (D) and with assessment of dsDNA content using PicoGreen (E) and gel electrophoresis (F). Components of the mucosal layer are also shown using Masson's trichrome staining in native trachea tissue and decellularized trachea tissue (G and H, respectively). Glycosaminoglycan by Safranin O staining is shown in native trachea tissue and decellularized trachea tissue (I and J). (Color version of figure is available online.)
Both tracheal and UBM scaffolds support the proliferation of HBEC and formation of a confluent layer
HBEC grew on both tracheal and UBM scaffolds. By day 7, the cells formed a confluent monolayer on both substrates and maintained this monolayer through 21 d (Fig. 2).
Fig. 2Air-liquid interface for HBEC in vitro culture and histology of HBEC on ECM scaffolds. To induce maturation of HBEC, cells are submerged in proliferation medium on day 1, followed by culture at ALI in differentiation medium for up to 21 d to facilitate differentiation into mature HBEC phenotypes (A). HBEC growth and maintenance of a confluent layer on the luminal aspect of UBM scaffolds at 7 (B) and 21 (C) days and tracheal scaffolds at 7 (D) and 21 (E) days (H&E stain). Arrows denote cell layer. (Color version of figure is available online.)
Both tracheal and UBM hydrogels support proliferation of HBEC
At day 4, cells formed a confluent monolayer on both ECM substrates. By 19 d, cells on the tracheal hydrogel showed a pseudostratified columnar morphology similar to the native airway epithelium (Fig. 3D). Cells on UBM hydrogels continued to maintain a confluent monolayer (Fig. 3C). In a pilot study, an alamar blue assay was used to quantify HBEC growth on UBM compared with trachea gel and showed growth was not significantly different between tracheal and UBM hydrogels at any time point (Supplementary Material, Figure A).
Fig. 3Histology of HBEC on ECM hydrogels. HBEC maintenance of a confluent layer on UBM hydrogels at 4 (A) and 19 (C) days and differentiation into a pseudostratified cell layer on tracheal hydrogels at 4 (B) and 19 (D) days (H&E stain). (Color version of figure is available online.)
Tracheal and UBM hydrogels support differentiation of HBEC
HBEC showed positive staining for Alcian blue when grown both on tracheal and UBM hydrogels, indicative of the presence of goblet cells. Positive staining was noted as early as 4 d and remained until at least 19 d (Fig. 4).
Fig. 4Histochemistry of HBEC on ECM hydrogels. HBEC differentiation into goblet cells on day 4 and 19 on UBM (A and C) and on tracheal (B and D) hydrogels was assessed histologically using Alcian blue staining. (Color version of figure is available online.)
HBEC grown on both UBM and tracheal hydrogels stained positive for acetylated tubulin, seen in mature cilia of respiratory epithelium by day 19
Cells seeded on tracheal hydrogels showed finger-like projections, consistent with the presence of cilia (Fig. 5).
Fig. 5Immunolabeling of HBEC on ECM hydrogels to evaluate for cilia. The ciliated bronchial epithelial cells in native trachea (A) stain for acetylated tubulin and are no longer seen on decellularized trachea (B). HBEC stained positive for acetylated tubulin on UBM (C) and tracheal (D) hydrogels seen at day 19. Arrows denote presence of cilia. (Color version of figure is available online.)
Tracheal and UBM hydrogels support formation of a basement membrane by HBEC
By day 14, both UBM and tracheal hydrogels showed a continuous intact basement membrane at the base of HBEC monolayer (Fig. 6).
Fig. 6Immunolabeling of collagen IV on ECM hydrogels to evaluate basement membrane deposition. The basement membrane of native trachea (A) is not present after decellularization (B). Basement membrane deposition by HBEC at 14 d seen on UBM (C) and tracheal (D) hydrogels (collagen IV stain). Arrows denote collagen IV deposition. (Color version of figure is available online.)
Prior studies have attempted to use decellularized bioscaffolds for tracheal transplantation, however, such attempts have been unsuccessful due to the need for a functional respiratory epithelium immediatelyon implantation. The mature respiratory epithelium is critical for mucociliary clearance to avoid complications of mucus obstruction including stenosis and infection. The present study investigated the use of ECM-based hydrogels as a substrate for functional respiratory epithelium.
Porcine tracheas were successfully decellularized using a modified version of a previously published method.
The process used a combination of cyclic negative pressure and osmotic shock, detergents, and enzymes and resulted in a 98% reduction relative to native trachea tissue. The thoroughness of cell remnant removal and residual dsDNA fragments that were less than 200 bp in length has been directly correlated with less adverse host reactions.
The ECM tested was derived from decellularized porcine tissue. ECM is secreted from the resident cell population of each tissue and organ, and it is therefore plausible that site-specific cells would proliferate and differentiate best on homologous ECM. Previous studies, however, have shown that ECM harvested from tissues other than the tissue of origin can often support cell and tissue growth equally as well as heterologous ECM.
For example, in one study of ECM-based esophageal repair, similar constructive remodeling was observed after in vivo implantation of ECM scaffolds derived from esophagus, urinary bladder, or small intestine submucosa. However, esophageal ECM was shown to induce greater migration of esophageal stem cells and better support esophageal organoid formation than heterologous ECM, suggesting that for some applications ECM tissue source may be an important variable.
Therefore, the present study compared heterologous (UBM) with homologous (trachea-derived) ECM to determine which substrate would be preferred for development and delivery of a functional respiratory epithelium in engineered tracheal tissue. The trachea-based hydrogels used the soft tissue from the luminal aspect of the trachea as this is the anatomic site of bronchial epithelial cells. UBM was chosen as a substrate for comparison as it supports epithelial growth, is an FDA-approved ECM material, and is more easily produced than tracheal ECM.
The results of the present study showed that both UBM and tracheal ECM support HBEC growth, but the hydrogel forms performed better than the decellularized 2-dimensional tissue forms. Criteria used to evaluate the suitability of a substrate for development of a mature airway epithelium include organization of cells into a multicellular monolayer, differentiation of cells, and deposition of a basement membrane. All three criteria were met by UBM and tracheal hydrogels confirming that both substrates can be used for cell growth and differentiation. H&E staining demonstrated pseudostratified columnar epithelial morphology similar to native trachea after cell culture in ALI by 19 d. Differentiation was assessed at 19 d by expression of acetylated tubulin, a marker for ciliated cells, and positive Alcian blue staining indicating goblet cell development. Epithelial cell layers on both substrates stained positive for collagen IV beneath their basilar surface by 14 d consistent with deposition of a basement membrane. No notable differences were noted between urinary bladder and tracheal ECM hydrogels, and our results suggest both substrates are equally suitable for the development of a mature respiratory epithelium.
The ability of ECM scaffolds to affect cell behavior and encourage tissue remodeling is attributed to the ECM components such as cytokines and chemokines, cryptic peptides, and matrix-bound nanovesicles
that become accessible to infiltrating host cells after implantation and degradation. This process is mimicked when the ECM bioscaffold is enzymatically digested into a hydrogel, which may explain why HBEC preferred hydrogels to decellularized tissue bioscaffolds as a substrate.
Limitations of the present study include a lack of a quantitative measurement of cell proliferation and differentiation. Further investigation into the characteristics of the hydrogel such as protein composition would help better understand the individual ECM components that influence cell behavior. In addition, the functional analysis of the motile cilia would further confirm that the respiratory epithelium is mature and ready for implantation.
Conclusions and future directions
Both tracheal- and urinary bladder-derived ECM hydrogels support growth and differentiation of bronchial epithelial cells. Therefore, when considering segmental tracheal replacement, either ECM hydrogel can be used as a substrate to promote a mature respiratory epithelium. Additional future directions include identifying the components within the ECM responsible for supporting a functional respiratory epithelium. Studies are currently exploring the role these components play in the ability of ECM to promote constructive remodeling.
Next steps will be to use the mature respiratory epithelium to epithelialize a decellularized tracheal bioscaffold, possibly with a luminal coating of ECM hydrogel, for airway replacement. This strategy will address the need for immediate functionality at the time of surgical implantation.
Acknowledgment
Funding sources: AKR was supported by the Ruth L. Kirschstein National Service Research Award [5T32 HL129949-2 A1]. Additional funding for the work was supported by the National Institutes of Health [NIH R01 HL136494-01A1 Matricellular Signaling in Engineered Tracheal Transplantation] as well as the Cystic Fibrosis Foundation Research Development Program.
Declarations of interest: None.
Author’s contributions: S.F.B. conceived of the idea with A.K.R. contributing to study design. W.A.D., J.R., L.Z., S.J., and A.K.R. were involved in acquisition of the data with analysis and interpretation by all authors. A.K.R. drafted the manuscript with critical revisions by S.F.B. and W.A.D. and consultation with M.M., L.Z., J.R., and S.J.
Esophageal preservation in five male patients after endoscopic inner-layer circumferential resection in the setting of superficial cancer: a regenerative medicine approach with a biologic scaffold.
Transforming growth factor-β1 selectively recruits microRNAs to the RNA-induced silencing complex and degrades CFTR mRNA under permissive conditions in human bronchial epithelial cells.
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