Tracheal Grafts Research Articles

Microvascular Regeneration and Perfusion of Partially Decellularized Tracheal Grafts.

A critical barrier to successful tracheal transplantation is poor vascularization. Despite its importance, little is known about microvascular regeneration in tissue-engineered grafts. We have demonstrated that partially decellularized tracheal grafts (PDTG) support neotissue formation including new submucosal microvasculature (CD31+). However, the perfusion of this neovasculature is unknown. In this study, we used a mouse model of tracheal replacement to measure the microvascular regeneration and perfusion of PDTG. PDTG and syngeneic tracheal grafts (STG, surgical control) (n = 5 for each group) were orthotopically transplanted into C5BL/6 J mice. We quantified vascularity of STG and PDTG samples at 1 and 3 months with conventional histology (N = 3 ~ 10/group). At 1, 3, and 6 months, animals were injected with fluorescein isothiocyanate (FITC) tomato lectin into the left ventricle. After perfusion, tracheas were fixed, harvested, mounted, stained for CD31 expression, and imaged with resonant scanning confocal microscopy. Percent CD31+, FITC area was compared between groups and endpoints compared with native trachea. Microvascular intersections were quantified using Sholl analysis. Functional microvasculature was seen in both groups. Although percent vascularization (CD31) in PDTG was restored by 3 months, microvascular pattern in PDTG displayed a unique morphology compared with control. Surgery alone appeared to globally change microvascular pattern and perfusion. PDTG demonstrated equivalent perfusion to surgical control by 6 months. Sholl analysis revealed a reduction of microvessel intersectionality that persisted in PDTG and was not seen in surgical or native controls. PDTG exhibited microvascular regeneration. Perfusion was present in PDTG, improved, and persisted over long-term time points. NA Laryngoscope, 2024.

Improved Composite Hydrogel for Bioengineered Tracheal Graft Demonstrates Effective Early Angiogenesis.

Background/Objectives: Collagen-agarose hydrogel blends currently used in tracheal graft bioengineering contain relatively high concentrations of collagen to withstand mechanical stresses associated with native trachea function (e.g., breathing). Unfortunately, the high collagen content restricts effective cell infiltration into the hydrogel. In this study, we created an improved hydrogel blend with lower concentrations of collagen (<5 mg/mL) and characterized its capacity for fibroblast invasion and angiogenesis. Methods: Four collagen-agarose hydrogel blends were created: 1 mg/mL type 1 collagen (T1C) and 0.25% agarose, 1 mg/mL T1C and 0.125% agarose, 2 mg/mL T1C and 0.25% agarose, and 2 mg/mL T1C and 0.125% agarose. The hydrogel surface was seeded with fibroblasts, while both endothelial cells and fibroblasts (3:1 ratio) were mixed within the hydrogel matrix. We assessed early angiogenesis by observing fibroblast migration and endothelial cell morphology (elongation and branching) at 7 days. In addition, we performed immunostaining for alpha-smooth muscle actin (aSMA) and explored the gene expression of various angiogenic markers (including vascular endothelial growth factor; VEGF). Results: Gels with lower agarose concentrations (0.125%) with 1 or 2 mg/mL T1C were more effective in allowing early attachment and migration of surface-applied fibroblasts compared to gels with higher (0.25%) agarose concentrations. The low-agarose gels also allowed cells to quickly adopt a spread morphology and self-assemble into elongated structures indicative of early angiogenesis, while demonstrating positive immunostaining for aSMA and increased gene expression of VEGF by day 7. Conclusions: Hydrogel blends with collagen and low agarose concentrations may be effective in allowing early cellular infiltration and angiogenesis, making such gels a suitable cell substrate for use in the development of composite bioengineered tracheal grafts. The collagen-agarose hydrogel blend is meant to be cast around a three-dimensional (3D) printed polycaprolactone support structure and wrapped in porcine small intestine submucosa ECM to create an off-the-shelf bioengineered tracheal implant.

Long-Term Survival and Regeneration Following Transplantation of 3D-Printed Biodegradable PCL Tracheal Grafts in Large-Scale Porcine Models.

Polycaprolactone (PCL) implants in large animals show great promise for tracheal transplantation. However, the longest survival time achieved to date is only about three weeks. To meet clinical application standards, it is essential to extend the survival time and ensure the complete integration and functionality of the implant. Our study investigates the use of three-dimensional (3D)-printed, biodegradable, PCL-based tracheal grafts for large-scale porcine tracheal transplantation, assessing the feasibility and early structural integrity crucial for long-term survival experiments. A biodegradable PCL tracheal graft was fabricated using a BIOX bioprinter and transplanted into large-scale porcine models. The grafts, measuring 20 × 20 × 1.5 mm, were implanted following a 2 cm circumferential resection of the porcine trachea. The experiment design was traditionally implanted in eight porcines to replace four-ring tracheal segments, only two of which survived more than three months. Data were collected on the graft construction and clinical outcomes. The 3D-printed biosynthetic grafts replicated the native organ with high fidelity. The implantations were successful, without immediate complications. At two weeks, bronchoscopy revealed significant granulation tissue around the anastomosis, which was managed with laser ablation. The presence of neocartilage, neoglands, and partial epithelialization near the anastomosis was verified in the final pathology findings. Our study demonstrates in situ regenerative tissue growth with intact cartilage following transplantation, marked by neotissue formation on the graft's exterior. The 90-day survival milestone was achieved due to innovative surgical strategies, reinforced with strap muscle attached to the distal trachea. Further improvements in graft design and granulation tissue management are essential to optimize outcomes.

Decellularized Wharton's Jelly and Amniotic Membrane Demonstrate Potential Therapeutic Implants in Tracheal Defects in Rabbits.

Tracheal grafts have been investigated for over a century, aiming to replace various lesions. However, tracheal reconstruction surgery remains a challenge, primarily due to anatomical considerations, intraoperative airway management, the technical complexity of reconstruction, and the potential postoperative morbidity and mortality. Due to research development, the amniotic membrane (AM) and Wharton's Jelly (WJ) arise as alternatives within the new set of therapeutic alternatives. These structures hold significant therapeutic potential for tracheal defects. This study analyzed the capacity of tracheal tissue regeneration after 60 days of decellularized WJ and AM implantation in rabbits submitted to conventional tracheostomy. An in vivo experimental study was carried out using thirty rabbits separated into three groups (Control, AM, and WJ) (n = 10). The analyses were performed 60 days after surgery through immunohistochemistry. Different immunomarkers related to scar regeneration, such as aggrecan, TGF-β1, and α-SMA, were analyzed. However, they highlighted no significant difference between the groups. Collagen type I, III, and Aggrecan also showed no significant difference between the groups. Both scaffolds appeared to be excellent frameworks for tissue engineering, presenting biocompatibility and a desirable microenvironment for cell survival; however, they did not display histopathological benefits in trachea tissue regeneration.

Proteomic Analysis of Surgery-induced Stress Post-Tracheal Transplantation Highlights Changes in Matrisome.

Investigate the impact of Surgery-induced stress (SIS) on the normal airway repair process after airway reconstruction using a mouse microsurgery model, mass spectrometry (MS), and bioinformatic analysis. Tracheal tissue from non-surgical (N = 3) and syngeneic tracheal grafts at 3 months post-replacement (N = 3) were assessed using mass spectrometry. Statistical analysis was done using MASCOT via Proteome Discoverer™. Proteins were categorized into total, dysregulated, suppressed, and evoked proteins in response to SIS. Dysregulated proteins were identified using cut-off values of -1 <log2FoldChange >1 and t-test (p value <0.05). Enriched pathways were determined using STRING and Metascape. At the three-month post-operation mark, we noted a significant increase in submucosal cellular infiltration (14343 ± 1286 cells/mm2, p = 0.0003), despite reduced overall thickness (30 ± 3 μm, p = 0.01), compared to Native (4578 ± 723 cells/mm2; 42 ± 6 μm). Matrisome composition remained preserved, with proteomic analysis identifying 193 commonly abundant proteins, encompassing 7.2% collagens, 34.2% Extracellular matrix (ECM) glycoproteins, 6.2% proteoglycans, 33.2% ECM regulators, 14.5% Extracellular matrix-affiliated, and 4.7% secreted factors. Additionally, our analysis unveiled a unique proteomic signature of 217 "Surgery-evoked proteins" associated with SIS, revealing intricate connections among neutrophils, ECM remodeling, and vascularization through matrix metalloproteinase-9 interaction. Our study demonstrated the impact of SIS on the extracellular matrix, particularly MMP9, after airway reconstruction. The novel identification of MMP9 prompts further investigation into its potential role in repair. NA Laryngoscope, 134:4052-4059, 2024.

Tissue-engineered tracheal implants: Advancements, challenges, and clinical considerations.

Restoration of extensive tracheal damage remains a significant challenge in respiratory medicine, particularly in instances stemming from conditions like infection, congenital anomalies, or stenosis. The trachea, an essential element of the lower respiratory tract, constitutes a fibrocartilaginous tube spanning approximately 10-12 cm in length. It is characterized by 18 ± 2 tracheal cartilages distributed anterolaterally with the dynamic trachealis muscle located posteriorly. While tracheotomy is a common approach for patients with short-length defects, situations requiring replacement arise when the extent of lesion exceeds 1/2 of the length in adults (or 1/3 in children). Tissue engineering (TE) holds promise in developing biocompatible airway grafts for addressing challenges in tracheal regeneration. Despite the potential, the extensive clinical application of tissue-engineered tracheal substitutes encounters obstacles, including insufficient revascularization, inadequate re-epithelialization, suboptimal mechanical properties, and insufficient durability. These limitations have led to limited success in implementing tissue-engineered tracheal implants in clinical settings. This review provides a comprehensive exploration of historical attempts and lessons learned in the field of tracheal TE, contextualizing the clinical prerequisites and vital criteria for effective tracheal grafts. The manufacturing approaches employed in TE, along with the clinical application of both tissue-engineered and non-tissue-engineered approaches for tracheal reconstruction, are discussed in detail. By offering a holistic view on TE substitutes and their implications for the clinical management of long-segment tracheal lesions, this review aims to contribute to the understanding and advancement of strategies in this critical area of respiratory medicine.

Dynamic flow for efficient partial decellularization of tracheal grafts: A preliminary rabbit study.

Bioengineered tracheal grafts are a potential solution for the repair of long-segment tracheal defects. A recent advancement is partially decellularized tracheal grafts (PDTGs) which enable regeneration of host epithelium and retain viable donor chondrocytes for hypothesized benefits to mechanical properties. We propose a novel and tunable 3D-printed bioreactor for creating large animal PDTG that brings this technology closer to the bedside. Conventional agitated immersion with surfactant and enzymatic activity was used to partially decellularize New Zealand white rabbit (Oryctolagus cuniculus) tracheal segments (n = 3). In parallel, tracheal segments (n = 3) were decellularized in the bioreactor with continuous extraluminal flow of medium and alternating intraluminal flow of surfactant and medium. Unprocessed tracheal segments (n = 3) were also collected as a control. The grafts were assessed using the H&E stain, tissue DNA content, live/dead assay, Masson's trichrome stain, and mechanical testing. Conventional processing required 10 h to achieve decellularization of the epithelium and submucosa with poor chondrocyte viability and mechanical strength. Using the bioreactor reduced processing time by 6 h and resulted in chondrocyte viability and mechanical strength similar to that of native trachea. Large animal PDTG created using our novel 3D printed bioreactor is a promising approach to efficiently produce tracheal grafts. The bioreactor offers flexibility and adjustability favorable to creating PDTG for clinical research and use. Future research includes optimizing flow conditions and transplantation to assess post-implant regeneration and mechanical properties. NA.

Regeneration Process of an Autologous Tissue-Engineered Trachea (aTET) in a Rat Patch Tracheoplasty Model.

The treatment of long-tracheal lesion is difficult because there are currently no viable grafts for tracheal replacement. To solve this problem, we have developed an autologous Tissue-Engineered Trachea (aTET), which is made up of collagenous tissues and cartilage-like structures derived from rat chondrocytes. This graft induced successful long-term survival in a small-animal experiment in our previous study. In this study, we investigated the regeneration process of an aTET to attain reproducible success. We prepared an aTET by using a specially designed mold and performed patch tracheoplasty with an aTET. We assigned twenty-seven rats to three groups according to the three types of patch grafts used: aTET patches (the aTET group), fresh tracheal autograft patches (the Ag group), or polylactic acid and polycaprolactone copolymer sheets (the PPc group). In each group, gross and histological evaluations were performed at 1 month (n = 3), 3 months (n = 3), and 6 months (n = 3) after implantation. We obtained high survival rates in all groups, but only the PPc group attained thick tracheal walls with granular tissues and no tracheal regeneration. On the other hand, the aTET and Ag groups reproducibly achieved complete tracheal regeneration in 6 months. So, an aTET could be a promising candidate for tracheal regeneration grafts.

Airway disease decreases the therapeutic potential of epithelial stem cells

BackgorundTissue-engineered tracheal grafts (TETG) can be recellularized by the host or pre-seeded with host-derived cells. However, the impact of airway disease on the recellularization process is unknown.MethodsIn this study, we determined if airway disease alters the regenerative potential of the human tracheobronchial epithelium (hTBE) obtained by brushing the tracheal mucosa during clinically-indicated bronchoscopy from 48 pediatric and six adult patients.ResultsOur findings revealed that basal cell recovery and frequency did not vary by age or region. At passage 1, all samples produced enough cells to cellularize a 3.5 by 0.5 cm2 graft scaffold at low cell density (~ 7000 cells/cm2), and 43.75% could cellularize a scaffold at high cell density (~ 100,000 cells/cm2). At passage 2, all samples produced the number of cells required for both recellularization models. Further evaluation revealed that six pediatric samples (11%) and three (50%) adult samples contained basal cells with a squamous basal phenotype. These cells did not form a polarized epithelium or produce differentiated secretory or ciliated cells. In the pediatric population, the squamous basal cell phenotype was associated with degree of prematurity (< 28 weeks, 64% vs. 13%, p = 0.02), significant pulmonary history (83% vs. 34%, p = 0.02), specifically with bronchopulmonary dysplasia (67% vs. 19%, p = 0.01), and patients who underwent previous tracheostomy (67% vs. 23%, p = 0.03).ConclusionsIn summary, screening high-risk pediatric or adult population based on clinical risk factors and laboratory findings could define appropriate candidates for airway reconstruction with tracheal scaffolds.Level of evidence. Level III Cohort study.Graphical

Assessing the Biocompatibility and Regeneration of Electrospun-Nanofiber Composite Tracheal Grafts.

Composite tracheal grafts (CTG) combining decellularized scaffolds with external biomaterial support have been shown to support host-derived neotissue formation. In this study, we examine the biocompatibility, graft epithelialization, vascularization, and patency of three prototype CTG using a mouse microsurgical model. Tracheal replacement, regenerative medicine, biocompatible airway splints, animal model. CTG electrospun splints made by combining partially decellularized tracheal grafts (PDTG) with polyglycolic acid (PGA), poly(lactide-co-ε-caprolactone) (PLCL), or PLCL/PGA were orthotopically implanted in mice (N = 10/group). Tracheas were explanted two weeks post-implantation. Micro-Computed Tomography was conducted to assess for graft patency, and histological analysis was used to assess for epithelialization and neovascularization. Most animals (greater than 80%) survived until the planned endpoint and did not exhibit respiratory symptoms. MicroCT confirmed the preservation of graft patency. Grossly, the PDTG component of CTG remained intact. Examining the electrospun component of CTG, PGA degraded significantly, while PLCL+PDTG and PLCL/PGA + PDTG maintained their structure. Microvasculature was observed across the surface of CTG and infiltrating the pores. There were no signs of excessive cellular infiltration or encapsulation. Graft microvasculature and epithelium appear similar in all groups, suggesting that CTG did not hinder endothelialization and epithelialization. We found that all electrospun nanofiber CTGs are biocompatible and did not affect graft patency, endothelialization and epithelialization. Future directions will explore methods to accelerate graft regeneration of CTG. N/A Laryngoscope, 134:1155-1162, 2024.

Anastomotic stenosis of bioengineered trachea grafts is driven by transforming growth factor β1-induced signaling, proinflammatory macrophages, and delayed epithelialization

ObjectiveAnastomotic stenosis caused by hypertrophic granulation tissue often develops in response to orthotopically implanted bioengineered tracheal grafts. To determine mechanisms responsible for the development and persistence of this granulation tissue, we looked for changes in gene expression from tissue specimens from the graft-native interface. MethodsRNA was isolated from paraffin-embedded tissue samples of the anastomotic sites of orthotopically implanted bioengineered tracheal grafts of 9 animals. Tissue samples were binned into 3 groups based on degree of stenosis: no stenosis (<5%), mild stenosis (25%-50%), and moderate and severe stenosis (≥75%). Sections of healthy trachea tissue were used as control. The expression levels of ∼200 genes related to wound healing, plus several endogenous controls, were measured with a pathway-focused predesigned primer array. ResultsExpression of ARG2, IL4, RPL13 A, TGFBR3, and EGFR decreased, whereas expression of RUNX2 was increased in stenotic wounds compared with nonstenotic tissue. Based on the cell types present in the trachea and wound healing, this expression profile indicates a lack of M2 anti-inflammatory macrophages, absent epithelial cells, and transforming growth factor β1-induced signaling. ConclusionsThese findings represent a significant step for tracheal tissue engineering by identifying several key mechanisms present in stenotic granulation tissue. Further research must be conducted to determine what modifications of the graft substrate and which coadministered therapeutics can be used to prevent the development of hypertrophic granulation tissue.

Regeneration of tracheal neotissue in partially decellularized scaffolds

Extensive tracheal injury or disease can be life-threatening but there is currently no standard of care. Regenerative medicine offers a potential solution to long-segment tracheal defects through the creation of scaffolds that support the generation of healthy neotissue. We developed decellularized tracheal grafts (PDTG) by removing the cells of the epithelium and lamina propria while preserving donor cartilage. We previously demonstrated that PDTG support regeneration of host-derived neotissue. Here, we use a combination of microsurgical, immunofluorescent, and transcriptomic approaches to compare PDTG neotissue with the native airway and surgical controls. We report that PDTG neotissue is composed of native tracheal cell types and that the neoepithelium and microvasculature persisted for at least 6 months. Vascular perfusion of PDTG was established within 2 weeks and the graft recruited multipotential airway stem cells that exhibit normal proliferation and differentiation. Hence, PDTG neotissue recapitulates the structure and function of the host trachea and has the potential to regenerate.

Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia.

Tracheal grafts may be necessary to bridge long-segment defects after curative resection for airway obstructions. Bioengineered grafts have emerged as an appealing option, given the possibilities of altering the histologic and cellular profile of the conduit. We previously designed a bioreactor capable of luminally decellularizing and recellularizing a ferret trachea with surface airway epithelia (SAE) basal cells (BCs), and we sought to assess the fate of these grafts when transplanted in an orthotopic fashion. As adjuncts to the procedure, we investigated the use of a vascular endothelial growth factor (VEGF)-laden hydrogel and of immunosuppression (IS) in graft revascularization and viability. IS was shown to limit early graft revascularization, but this effect could be counteracted with VEGF supplementation. Submucosal gland (SMG) loss was shown to be inevitable regardless of the revascularization strategy. Lastly, the bioengineered tracheas survived one month after transplant with differentiation of our implanted BCs that then transitioned into a recipient-derived functional epithelium. The work presented in this manuscript has important implications for future cellular and regenerative therapies.

Long-Term Chondrocyte Retention in Partially Decellularized Tracheal Grafts.

Decellularized tracheal grafts possess the biological cues necessary for tissue regeneration. However, conventional decellularization approaches to target the removal of all cell populations including chondrocytes lead to a loss of mechanical support. We have created a partially decellularized tracheal graft (PDTG) that preserves donor chondrocytes and the mechanical properties of the trachea. In this study, we measured PDTG chondrocyte retention with a murine microsurgical model. Murine in vivo time-point study. Research Institute affiliated with Tertiary Pediatric Hospital. PDTG was created using a sodium dodecyl sulfate protocol. Partially decellularized and syngeneic grafts were orthotopically implanted into female C57BL/6J mice. Grafts were recovered at 1, 3, and 6 months postimplant. Pre- and postimplant grafts were processed and analyzed via quantitative immunofluorescence. Chondrocytes (SOX9+, DAPI+) present in the host and graft cartilage was evaluated using ImageJ. Partial decellularization resulted in the maintenance of gross tracheal architecture with the removal of epithelial and submucosal structures on histology. All grafts demonstrated SOX9+ chondrocytes throughout the study time points. Chondrocytes in PDTG were lower at 6 months compared to preimplant and syngeneic controls. PDTG retained donor graft chondrocytes at all time points. However, PDTG exhibits a reduction in chondrocytes at 6 months. The impact of these histologic changes on cartilage extracellular matrix regeneration and repair remains unclear.

Optimization of Chondrocyte Viability in Partially Decellularized Tracheal Grafts.

Advancements in tissue-engineered tracheal replacement (TETR) show promise for the use of partially decellularized tracheal grafts (PDTG) to address critical gaps in airway management and reconstruction. In this study, aiming to leverage the immunoprivileged nature of cartilage to preserve tracheal biomechanics, we optimize PDTG for retention of native chondrocytes. Comparison in vivo murine study. Research Institute affiliated with Tertiary Pediatric Hospital. PDTG were created per a shortened decellularization protocol using sodium dodecyl sulfate, then biobanked via cryopreservation technique. Decellularization efficiency was characterized by DNA assay and histology. Viability and apoptosis of chondrocytes in preimplanted PDTG and biobanked native trachea (control) was assessed with live/dead and apoptosis assays. PDTG (N = 5) and native trachea (N = 6) were orthotopically implanted in syngeneic recipients for 1-month. At the endpoint, microcomputed tomography (micro-CT) was employed to interrogate graft patency and radiodensity in vivo. Vascularization and epithelialization were qualitatively analyzed using histology images following explant. PDTG exhibited complete decellularization of all extra-cartilaginous cells and reduced DNA content compared to control. Chondrocyte viability and nonapoptotic cell populations were improved utilizing biobanking and shorter decellularization time. All grafts remained patent. Evaluation of graft radiodensity at 1 month revealed elevation of Hounsfield units in both PDTG and native compared to host, with PDTG showing higher radiodensity than native. PDTG supported complete epithelialization and functional reendothelialization 1-month postimplantation. Optimizing PDTG chondrocyte viability is a key component to successful tracheal replacement. Ongoing research seeks to evaluate the acute and chronic immunogenicity of PDTG.

Comparison of the biological properties between 3D-printed and decellularized tracheal grafts

This study sought to characterize the differences between the 3D-printed and decellularized tracheal grafts, providing the basis for the synthesis of the more reasonable and effective tissue-engineered trachea. We compared the biomechanical properties and biocompatibility of the 3D-printed tracheal graft and decellularized tracheal graft in vitro and evaluated the biocompatibility, immune rejection and inflammation of the two materials through in vivo implantation experiments. Compared with the decellularized tracheal graft, the 3D-printed tracheal graft was associated with obviously higher biomechanical properties. The results demonstrated enhanced growth of BMSCs in the decellularized tracheal graft compared to the 3D-printed one when co-culture with two tracheal graft groups. Moreover, the CCK-8 assay demonstrated significant cell proliferation on the decellularized tracheal graft. Serum IgG and IgM measured in vivo by implantation testing indicated that the 3D-Printed tracheal graft exhibited the most significant inflammatory response. HE staining indicated that the inflammatory response in the 3D-printed tracheal graft consisted mainly of eosinophils, while little inflammatory cell infiltrates were observed in the decellularized tracheal graft. CD68 immunohistochemical analysis indicated that the infiltration of macrophages was not significant in both tracheal grafts. Our findings suggest that the biomechanical properties of the 3D-printed tracheal grafts are better than the decellularized tracheal grafts. Nonetheless, the decellularized tracheal graft exhibited better biocompatibility than the 3D-printed tracheal graft.

Biomimetic in situ tracheal microvascularization for segmental tracheal reconstruction in one-step.

Formation of functional and perfusable vascular network is critical to ensure the long-term survival and functionality of the engineered tissue tracheae after transplantation. However, the greatest challenge in tracheal-replacement therapy is the promotion of tissue regeneration by rapid graft vascularization. Traditional prevascularization methods for tracheal grafts typically utilize omentum or muscle flap wrapping, which requires a second operation; vascularized segment tracheal orthotopic transplantation in one step remains difficult. This study proposes a method to construct a tissue-engineered tracheal graft, which directly forms the microvascular network after orthotopic transplantation in vivo. The focus of this study was the preparation of a hybrid tracheal graft that is non-immunogenic, has good biomechanical properties, supports cell proliferation, and quickly vascularizes. The results showed that vacuum-assisted decellularized trachea-polycaprolactone hybrid scaffold could match most of the above requirements as closely as possible. Furthermore, endothelial progenitor cells (EPCs) were extracted and used as vascularized seed cells and seeded on the surfaces of hybrid grafts before and during the tracheal orthotopic transplantation. The results showed that the microvascularized tracheal grafts formed maintained the survival of the recipient, showing a satisfactory therapeutic outcome. This is the first study to utilize EPCs for microvascular construction of long-segment trachea in one-step; the approach represents a promising method for microvascular tracheal reconstruction.

Partial decellularization eliminates immunogenicity in tracheal allografts.

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long-segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T-cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation involves implanting an organ that was originally in contact with the environment, thus not being sterile from the outset. While the decellularization protocol (through detergent exposition [2% sodium dodecyl sulfate], continuous stirring, and osmotic shocks) is conducted in line with aseptic measures, it does not provide sterilization. Therefore, one of the main challenges is ensuring sterility prior to in vivo implantation. Although there are established gamma radiation sterilization protocols for inorganic materials, there are no such measures for organic materials. Additionally, the protocols in place for inorganic materials cannot be applied to organic materials, as the established radiation dose (25 kGy) would completely destroy the implant. This paper studies the effect of an escalated radiation dose in a decellularized rabbit trachea. We maintained the dose range (kGy) and tested escalated doses until finding the minimal dose at which sterilization is achieved. After determining the dose, we studied effects of it on the organ, both histologically and biomechanically. We determined that while 0.5 kGy did not achieve sterility, doses of both 1 kGy and 2 kGy did, with 1 kGy, therefore, being the minimal dose necessary to achieve sterilization. Microscopic studies showed no relevant changes compared to non-sterilized organs. Axial biomechanical characteristics were not altered at all, and only a slight reduction in the force per unit of length that the organ can radially tolerate was observed. We can therefore conclude that 1 kGy achieves complete sterilization of decellularized rabbit trachea with a minimal, if any, effects on the organ.

De-Epithelialization Protocol with Tapered Sodium Dodecyl Sulfate Concentrations Enhances Short-Term Chondrocyte Survival in Porcine Chimeric Tracheal Allografts

Background: Tracheal transplantation is indicated in cases where injury exceeds 50% of the organ in adults and 30% in children. However, transplantation is not yet considered a viable treatment option partly due to high morbidity and mortality associated with graft rejection. Recently, decellularization (decell) has been explored as a technique for creating bioengineered tracheal grafts. However, risk of post-operative stenosis increases due to the death of chondrocytes, which are critical to maintain the biochemical and mechanical integrity of tracheal cartilage. In this project, we propose a new de-epithelialization protocol that adequately removes epithelial, mucosal, and submucosal cells while maintaining a greater proportion of viable chondrocytes. Methods: The trachea of adult male outbred Yorkshire pigs were extracted, decontaminated, and decellularized according to the original and new protocols before incubation at 37 °C in DMEM for 10 days. Chondrocyte viability was quantified immediately following post-decellularization and on days 1, 4, 7, and 10. Histology was performed pre-decellularization, post-decellularization, and post-incubation. Results: The new protocol showed a significant (p &lt; 0.05) increase in chondrocyte viability up to four days after de-ep when compared to the original protocol. We also found that the new protocol preserves ECM composition to a similar degree as the original protocol. When scaffolds created using the new protocol were re-epithelialized, cell growth curves were near identical to published data from the original protocol. Conclusion: While not without limitations, our new protocol may be used to engineer chimeric tracheal allografts without the need for cartilage regeneration.