Segmental Tracheal Defects Research Articles

Implante 3D de copoliamida associada à elastômero termoplástico (PCTPE) para reparação traqueal de coelhos (Oryctolagus cuniculus): estudo preliminar

Abstract Large segmental tracheal defects can pose a serious clinical challenge owing to the lack of suitable substitutes for reconstructive surgery. Polymeric biomaterials are widely used in medicine. However, the implantation of biomaterials triggers a series of biological events, and material biocompatibility is of paramount importance in regenerative medicine. The objective of this study was to evaluate the use of a copolyamide associated with thermoplastic elastomer (PCTPE) in 3D-printed prostheses for repairing partial tracheal defects in rabbits. Sixteen male New Zealand White rabbits were used, and partial tracheal defects were created in the ventral region from the 4th to the 8th tracheal ring. The animals were subdivided into groups (n=4) based on the time of euthanasia, scheduled at seven days (G7), 15 days (G15), 30 days (G30), and 60 days (G60). Histopathological analysis with hematoxylin and eosin staining revealed that the 3D implant of PCTPE exhibited a foreign body reaction, and inflammation persisted for up to 60 days. Histochemistry with picrosirius red revealed a tendency for a greater amount of type I collagen to accumulate in the early stages of inflammation, whereas type III collagen was predominant in later evaluation periods. These findings suggest an exacerbated and inadequate tissue-material interface reaction and perpetuation of the inflammatory process.

Photocrosslinked natural hydrogel composed of hyaluronic acid and gelatin enhances cartilage regeneration of decellularized trachea matrix

Repair of long segmental trachea defects is always a great challenge in the clinic. The key to solving this problem is to develop an ideal trachea substitute with biological function. Using of a decellularized trachea matrix based on laser micropore technique (LDTM) demonstrated the possibility of preparing ideal trachea substitutes with tubular shape and satisfactory cartilage regeneration for tissue-engineered trachea regeneration. However, as a result of the very low cell adhesion of LDTM, an overly high concentration of seeding cell is required, which greatly restricts its clinical translation. To address this issue, the current study proposed a novel strategy using a photocrosslinked natural hydrogel (PNH) carrier to enhance cell retention efficiency and improve tracheal cartilage regeneration. Our results demonstrated that PNH underwent a rapid liquid-solid phase conversion under ultraviolet light. Moreover, the photo-generated aldehyde groups in PNH could rapidly react with inherent amino groups on LDTM surfaces to form imine bonds, which efficiently immobilized the cell-PNH composite to the surfaces of LDTM and/or maintained the composite in the LDTM micropores. Therefore, PNH significantly enhanced cell-seeding efficiency and achieved both stable cell retention and homogenous cell distribution throughout the LDTM. Moreover, PNH exhibited excellent biocompatibility and low cytotoxicity, and provided a natural three-dimensional biomimetic microenvironment to efficiently promote chondrocyte survival and proliferation, extracellular matrix production, and cartilage regeneration. Most importantly, at a relatively low cell-seeding concentration, homogeneous tubular cartilage was successfully regenerated with an accurate tracheal shape, sufficient mechanical strength, good elasticity, typical lacuna structure, and cartilage-specific extracellular matrix deposition. Our findings establish a versatile and efficient cell-seeding strategy for regeneration of various tissue and provide a satisfactory trachea substitute for repair and functional reconstruction of long segmental tracheal defects.

De-Epithelialized Heterotopic Tracheal Allografts without Immunosuppressants in Dogs: Long-Term Results for Cartilage Viability and Structural Integrity.

Reconstruction of long segmental tracheal defects is difficult because no ideal tracheal substitutes are currently available. Tracheal allotransplantation maintains cartilage and epithelium viability but requires immunosuppression because of epithelial immunogenicity. We aimed to obtain an epithelium-decellularized allograft that maintains cartilage viability and to evaluate long-term outcomes of such allografts implanted on dog backs without immunosuppressants. Twenty-five tracheas harvested from mongrel dogs were used to explore the period of epithelium decellularization by combined use of 1% sodium dodecyl sulfate and an organ preservation solution and to assess the chondrocyte viability and immunogenicity of the tracheas after decellularization. Sixteen epithelium-decellularized tracheal allografts and 10 fresh tracheal segments (6 cm long) were implanted in 26 beagles for durations of 10 days and 1, 3, 6, and 12 months. Macroscopic and microscopic examinations were used to evaluate the morphology, viability, and immune rejection of the allografts. Safranin-O staining was used to detect glycosaminoglycans. The epithelium disappeared after 24 hours of decellularization. At 72 hours, almost no nuclei remained in the mucosa, while the mean survival rate of chondrocytes was 88.1%. Histological analysis demonstrated that the allograft retained intact tracheal rings and viable cartilage after heterotopic implantation for 1 year, with no immunological rejection. There were no significant differences in the glycosaminoglycan contents among the implanted epithelium-decellularized allografts. Epithelium-decellularized tracheal allografts with chondrocyte viability can be achieved by combined use of a detergent and organ preservation solution, which showed satisfactory cartilage viability and structural integrity after long-term heterotopic transplantation. Further studies on orthotopic transplantation are needed to assess the feasibility of allografts in reconstructing long segmental tracheal defects.

Biomimetic Trachea Regeneration Using a Modular Ring Strategy Based on Poly(Sebacoyl Diglyceride)/Polycaprolactone for Segmental Trachea Defect Repair

AbstractThe lack of a biomimetic tracheal substitute featured with cartilage ring/vascularized connective tissue alternate structure (CVCAS) has significantly retarded a clinical breakthrough of tissue‐engineered trachea in repairing extensive tracheal lesions. Herein, a novel modular strategy based on cartilage rings and biomaterial rings is used to generate a biomimetic trachea with CVCAS. Poly(sebacoyl diglyceride) and polycaprolactone (PCL) (PSeD/PCL) are blended to fabricate a porous scaffold with satisfactory mechanical properties, biodegradability, and biocompatibility. A cartilage ring is engineered in vitro using ring‐shaped PSeD/PCL scaffold loaded with chondrocytes and cartilage tube is generated in vivo through the fusion of multiple modular cartilage rings. More importantly, biomimetic tracheas with CVCAS are successfully fabricated in vivo through alternately stacked cartilage rings and PSeD/PCL rings on a silicon tube. Notably, the engineered biomimetic trachea exhibits not only a bionic structure but also superior mechanical properties compared with the native rabbit trachea. Finally, segmental tracheal defects in rabbits are repaired by transplanting the biomimetic trachea with pedicled muscular flaps. The biomimetic trachea establishes stable blood supply and provided sufficient mechanical strength and biological function similar to those of native trachea. This work represents the first successful regeneration of biomimetic trachea with CVCAS and a promising approach for clinical application.

Long-segmental tracheal reconstruction in rabbits with pedicled Tissue-engineered trachea based on a 3D-printed scaffold

Long-segmental tracheal defects constitute an intractable clinical problem, due to the lack of satisfactory tracheal substitutes for surgical reconstruction. Tissue engineered artificial substitutes could represent a promising approach to tackle this challenge. In our current study, tissue-engineered trachea, based on a 3D-printed poly (l-lactic acid) (PLLA) scaffold with similar morphology to the native trachea of rabbits, was used for segmental tracheal reconstruction. The 3D-printed scaffolds were seeded with chondrocytes obtained from autologous auricula, dynamically pre-cultured in vitro for 2 weeks, and pre-vascularized in vivo for another 2 weeks to generate an integrated segmental trachea organoid unit. Then, segmental tracheal defects in rabbits were restored by transplanting the engineered tracheal substitute with pedicled muscular flaps. We found that the combination of in vitro pre-culture and in vivo pre-vascularization successfully generated a segmental tracheal substitute with bionic structure and mechanical properties similar to the native trachea of rabbits. Moreover, the stable blood supply provided by the pedicled muscular flaps facilitated the survival of chondrocytes and accelerated epithelialization, thereby improving the survival rate. The segmental trachea substitute engineered by a 3D-printed scaffold, in vitro pre-culture, and in vivo pre-vascularization enhanced survival in an early stage post-operation, presenting a promising approach for surgical reconstruction of long segmental tracheal defects. Statement of SignificanceWe found that the combination of in vitro pre-culture and in vivo pre-vascularization successfully generated a segmental tracheal substitute with bionic structure and mechanical properties similar to the native trachea of rabbits. Moreover, the stable blood supply provided by the pedicled muscular flaps facilitated the survival of chondrocytes and accelerated epithelialization, thereby improving the survival rate of the rabbits. The segmental trachea substitute engineered by a 3D-printed scaffold, in vitro pre-culture, and in vivo pre-vascularization enhanced survival in an early stage post-operation, presenting a promising approach for surgical reconstruction of long segmental tracheal defects.

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Aortic allografts: final destination?—a summary of clinical tracheal substitutes

The patient population in desperate need for an airway substitute are individuals with long segment tracheal defects that are considered, technically, inoperable. Regardless of the underlying etiology, benign or malignant growing processes, this patient category enters a palliative setting or require tracheal transplantation. Different airway substitutes have been categorized by Grillo as follows; tracheal transplantation, autogenous tissue, non-viable tissue, tissue-engineering and foreign materials. These fields have been explored in the past in animal models and in clinical patients. Research on airway replacement has been exposed to a level of controversies in the past years. The field has been turbulent and apocryphal. In particular, the area of tissue-engineering using stem cells has suffered from a major set-back leaving scientists, clinicians and ethical committees skeptical. Recently, a hopeful study emerged using aortic allografts as tracheal substitutes in patients with airway defects. The initial results seem promising and reliable. The developments of the field at this point seem striking and hopeful. The focus of this review is to shed light on developments in the field of aortic allografts as substitute for tracheal replacement.

Tissue engineering of a composite trachea construct using autologous rabbit chondrocytes.

The repair of large tracheal segmental defects remains an unsolved problem. The goal of this study is to apply tissue engineering principles for the fabrication of large segmental trachea replacements. Engineered tracheal replacements composed of autologous cells (neotracheas) were tested in a New Zealand White rabbit model. Neotracheas were formed in the rabbit neck by wrapping a silicone tube with consecutive layers of skin epithelium, platysma muscle, and an engineered cartilage sheet and allowing the construct to mature for 8-12weeks. In total, 28 rabbits were implanted and the neotracheas assessed for tissue morphology. In 11 cases, neotracheas deemed sufficiently strong were used to repair segmental tracheal defects. Initially, the success rate of producing structurally sound neotracheas was impeded by physical disruption of the cartilage sheets during animal handling, but by the end of the study, 15 of 18 neotracheas (83.3%) were structurally sound. Of the 15 structurally sound neotracheas, 11 were used for segmental reconstruction and were left in place for up to 21days. Histological examination showed the presence of variable amounts of viable epithelium, a vascularized platysma flap, and a layer of safranin O-positive cartilage along with evidence of endochondral ossification. Rabbits that had undergone segmental reconstruction showed good tracheal integration, had a viable epithelium with vascular support, and the cartilage was sufficiently strong to maintain a lumen when palpated. The results demonstrated that viable, trilayered, scaffold-free neotracheas could be constructed from autologous cells and could be integrated into native trachea to repair a segmental defect.

Tissue-engineered trachea from a 3D-printed scaffold enhances whole-segment tracheal repair

Long segmental repair of trachea stenosis is an intractable condition in the clinic. The reconstruction of an artificial substitute by tissue engineering is a promising approach to solve this unmet clinical need. 3D printing technology provides an infinite possibility for engineering a trachea. Here, we 3D printed a biodegradable reticular polycaprolactone (PCL) scaffold with similar morphology to the whole segment of rabbits’ native trachea. The 3D-printed scaffold was suspended in culture with chondrocytes for 2 (Group I) or 4 (Group II) weeks, respectively. This in vitro suspension produced a more successful reconstruction of a tissue-engineered trachea (TET), which enhanced the overall support function of the replaced tracheal segment. After implantation of the chondrocyte-treated scaffold into the subcutaneous tissue of nude mice, the TET presented properties of mature cartilage tissue. To further evaluate the feasibility of repairing whole segment tracheal defects, replacement surgery of rabbits’ native trachea by TET was performed. Following postoperative care, mean survival time in Group I was 14.38 ± 5.42 days, and in Group II was 22.58 ± 16.10 days, with the longest survival time being 10 weeks in Group II. In conclusion, we demonstrate the feasibility of repairing whole segment tracheal defects with 3D printed TET.

Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique.

Lacking ideal biodegradable scaffolds greatly restricts development of tissue-engineered trachea. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. By laser micropore technique (LMT), the current study efficiently enhanced the porosity and decellularized efficacy of DTM. The LMT-treated DTM basically retained the original tubular shape with mild matrix damage. After chondrocyte seeding followed by in vitro culture and in vivo implantation, the constructs formed mature tubular cartilage with matrix content and mechanical strength similar to native trachea. The current study provides an ideal scaffold and a promising strategy for cartilage regeneration and functional reconstruction of trachea.

Prefabrication and Prelamination Strategies for the Reconstruction of Complex Defects of Trachea and Larynx

Complex tracheal and laryngeal defects can be reconstructed using prelamination and prefabrication techniques. Three clinical situations are described in detail in the article. In short segment restenosis defects within scarred surroundings, we restore the fibrocartilaginous defect with a radial forearm fascia flap prelaminated with buccal mucosa or cartilage. This provides a newly vascularized inner lining to the tracheal defect and restores the tubular convexity. For long segment defects we need a technique that can withstand respiratory forces. We use a heterotopic prefabrication strategy to vascularize a tracheal allograft wrapped in forearm fascia. Chimerism is created by replacing donor respiratory epithelium with buccal mucosa of the recipient. After orthotopic transfer, this chimerism allows immunosuppression to be tapered and stopped when bronchoscopy shows mucosal integrity of the new trachea, since the recipient epithelium replaces the allogeneic inner tracheal lining by means of a chronic rejection process. A distinct situation occurs after resection of a unilateral larynx tumor, which usually results in a total laryngectomy with loss of both vocal cords, since reconstruction of the hemilarynx is considered too complex. First, we prefabricate a nearby four-ring autologous tracheal segment using radial forearm fascia. In a second stage, this orthotopically vascularized trachea restores the laryngeal structure with the aim to conserve one vocal cord and thus speech. Orthotopic and heterotopic prelamination and prefabrication strategies offer efficient and reproducible solutions for the restoration of challenging short and long segment tracheal defects, as well as unilateral laryngeal defects. The series in this review article are based on previous studies and case reports. The level of evidence is III-"Study of nonconsecutive patients, without a universally applied gold standard: case-control study".

Long-term functional reconstruction of segmental tracheal defect by pedicled tissue-engineered trachea in rabbits

Due to lack of satisfactory tracheal substitutes, reconstruction of long segmental tracheal defects (>6 cm) is always a major challenge in trachea surgery. Tissue-engineered trachea (TET) provides a promising approach to address this challenge, but no breakthrough has been achieved yet in repairing segmental tracheal defect. The longest survival time only reached 60 days. The leading reasons for the failure of segmental tracheal defect reconstruction were mainly related to airway stenosis (caused by the overgrowth of granulation tissue), airway collapse (caused by cartilage softening) and mucous impaction (mainly caused by lack of epithelium). To address these problems, the current study proposed an improved strategy, which involved in vitro pre-culture, in vivo maturation, and pre-vascularization of TET grafts as well as the use of silicone stent. The results demonstrated that the two-step strategy of in vitro pre-culture plus in vivo implantation could successfully regenerate tubular cartilage with a mechanical strength similar to native trachea in immunocompetent animals. The use of silicone stents effectively depressed granulation overgrowth, prevented airway stenosis, and thus dramatically enhanced the survival rate at the early stage post-operation. Most importantly, through intramuscular implantation and transplantation with pedicled muscular flap, the TET grafts established stable blood supply, which guaranteed maintenance of tubular cartilage structure and function, accelerated epithelialization of TET grafts, and thus realized long-term functional reconstruction of segmental tracheal defects. The integration of all these improved strategies finally realized long-term survival of animals: 60% of rabbits survived over 6 months. The current improved strategy provided a promising approach for long-term functional reconstruction of long segmental tracheal defect.

Invited Commentary

The surgical reconstruction of segmental tracheal defects that include greater than 50% of tracheal length is challenging at best, with success rates that leave much to be desired. The paper by Tsukada and colleagues in this issue of The Annals of Thoracic Surgery [1] describes a two-stage end-to-end reconstruction technique that takes advantage of the innate wound contraction properties of tracheal tissue. The investigators repair a 50-mm experimentally created defect with a synthetic, resorbable copolymer graft and silicone stent in an adult canine model.

The Chondrocyte: Biology and Clinical Application

Chondrocyte is a unique cell type in articular cartilage tissue and is essential for cartilage formation and functionality. It arises from mesenchymal stem cells (MSCs) and is regulated by a series of cytokine and transcription factor interactions, including the transforming growth factor-beta super family, fibroblast growth factors, and insulin-like growth factor-1. To understand the biomechanisms of the chondrocyte differentiation process, various cellular model systems have been employed, such as primary chondrocyte culture, clonal normal cell lines (HCS-2/8, Ch-1, ATDC5, CFK-2, and RCJ3.1C5.18), and transformed clonal cell lines (T/C-28a2, T/C-28a4, C-28/I2, tsT/AC62, and HPV-16 E6/E7). Additionally, cell culture methods, including conventional monolayer culture, three-dimensional scaffold culture, bioreactor culture, pellet culture, and organ culture, have been established to create stable environments for the expansion, phenotypic maintenance, and subsequent biological study of chondrocytes for clinical application. Knowledge gained through these study systems has allowed for the use of chondrocytes in orthopedics for the treatment of cartilage injury and epiphyseal growth plate defects using tissue-engineering approaches. Furthermore, the potential of chondrocyte implantation for facial reconstruction, the treatment of long segmental tracheal defects, and urinary incontinence and vesicoureteral reflux are being investigated. This review summarizes the present study of chondrocyte biology and the potential uses of this cell in orthopedics and other disciplines.

Differential scanning calorimetric examination of the tracheal cartilage after primary reconstruction with differential suturing techniques

Objective Long segmental tracheal defects often lead to life threatening clinical conditions. Treatment of these lesions still represents an unsolved surgical challenge. The aim of the study was to test the effect of continuous and simple interrupted suturing in the replacement of long segment tracheal defects using polytetrafluoroethylene (PTFE) vascular prosthesis in a rabbit model. Methods 2 cm long segment of the cervical trachea was resected in 20 New Zealand rabbits. The trachea was replaced with reinforced polytetrafluoroethylene vascular graft. The anastomoses were performed telescopically using continuous (group I, n = 10) or simple interrupted sutures (group II, n = 10). Laser Doppler measurements were taken before the resection and following the anastomoses. Length of survivals were noted, the patency and microscopical pattern of the anastomoses were evaluated. Calorimetric examinations were performed to detect possible structural changes in the tracheal cartilage. Results Following the resection local microcirculation decreased by 9 ± 4%. The anastomoses caused a significant decrease of 29% in group I ( p = 0.02) and 13% in group II. The mean survival was 58 ± 17 and 135 ± 25 days, respectively. Calorimetric results showed no change after the resection, but significant shift in melting temperature and calorimetric enthalpy proved the presence of structural changes of the cartilage in group II. Conclusions We saw significant lowering of microcirculation following continuous sutures, while simple interrupted stitches produced only moderate decrease. We found that interrupted suture technique is superior to the continuous technique causing only moderate damage to the tracheal anastomosis.

Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea

Background This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage–derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. Methods Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter × 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. Results The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. Conclusions This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.

Tissue engineered cartilage generated from human trachea using DegraPol scaffold.

To date numerous attempts have been undertaken to conquer the challenging problem of reconstructing long segmental tracheal defects, as yet without lasting success. Recently, employing concepts of tissue engineering in animals, cartilage-like constructs were transplanted in vivo. However, both the feasibility of fabricating tracheal replacements and the use of human tracheal chondrocytes (HTC) for tissue engineering are still under investigation. In this study, we optimized isolation and cultivation techniques for human tracheal cartilage, assessing the feasibility of seeding these cells onto a novel, three-dimensional (3-D) polyester-urethane polymer (DegraPol). Human tracheal cartilage was harvested from the trachea of lung donors, digested in 0.3% collagenase II, and the condrocytes serially passaged every 7-9 days. Cells were also cultivated over agar plate during the total 6-8 weeks expansion phase. Thereafter, chondrocytes were seeded onto DegraPol (pore sizes 150-200 microm) with a seeding density of 2.4 x 10(7)/ml, and chondrocyte-polymer constructs maintained during in vitro static culture. HTC displayed stable proliferation kinetics in monolayer culture with positive expression of collagen type II. Following polymer seeding, both cellular proliferation and extracellular matrix (ECM) production, as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and glycosaminoglycan assays, continued over extended culture. Active growth of HTC on DegraPol was further demonstrated by Alcian blue staining, with the histomorphological appearance of the construct resembling that of native cartilage. Scanning electron microscopy showed chondrocyte growth and ECM synthesis both on the surface and inside the porous scaffold, with a dense cell layer on the surface of the scaffold and a lower cell distribution in the scaffold's interior. The harvested chondrocytes from human trachea cartilage expand well in vitro and possess the ability to form new cartilage-like tissue when seeded onto DegraPol matrix. However, improved culture conditions are needed to permit cellular growth throughout cell-polymer constructs.

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells.

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Ham's F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell-polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudo-stratified columnar epithelium, with a distinct interface between tissue-engineered cartilage and epithelium. Safranin-O staining shows that tissue-engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.

Microvascular transfer of long tracheal autograft segments in the canine model.

The reconstruction of long segment tracheal defects represents an unsolved clinical dilemma. Prior attempts to directly revascularize tracheal segments have been unsuccessful. The objective of this study was to evaluate orthotopic autotransplantation of revascularized long tracheal segments in the canine model. Ten randomly selected mongrel dogs underwent excision, orthotopic reimplantation, and microvascular revascularization of a long segment (8.0 cm) of cervical trachea. The cranial thyroid artery and the internal jugular vein served as the vascular supply for the tracheal segment. The animals were maintained for a period of 30 days during which time graft viability was measured by routine endoscopic assessment and tracheal biopsies. Ex vivo, tracheal autografts were examined grossly for graft healing an d microscopicallyfor histologic architecture. Seven of 10 dogs survived the predetermined 30-day postoperative study period without complications. Postmortem examination demonstrated that 7 dogs had healed tracheal autograft segments with normal histologic architecture, 2 dogs sustained a postoperative wound infection and tracheal dehiscence, and 1 dog sustained a fatal postoperative hematoma. For the first time, we have demonstrated direct revascularization of long segment tracheal autografts in the dog model using the cranial thyroid artery and internal jugular vein as the vascular supply.

Use of a revascularized, tubed costal myoperiosteal graft for repair of circumferential, segmental tracheal defects

Reconstruction of extensive laryngotracheal stenosis continues to pose a significant surgical challenge. Previous work in our laboratory has demonstrated the utility of vascularized perichondrium for reconstruction of cervical tracheal defects in a rabbit model. Because most potential vascularized donor sites in human beings are periosteal, it was important to demonstrate that vascularized periosteum was also useful for laryngotracheal reconstruction in a larger animal model. We therefore performed a 2-stage reconstruction of a circumferential, segmental cervical tracheal defect using a revascularized, tubed myoperiosteal graft in a canine model (n = 8). A rigid, patent tube was produced in 6 animals (75%) after completion of the first stage (7 to 10 weeks). After transfer of the vascularized free graft to the tracheal defect, 5 of 6 animals survived from 4 to 18 weeks. Severe stenosis (>90%) was present in 2 animals, and moderate stenosis (40% to 60%) was present in the remaining 3 animals. One animal was observed for 18 weeks and was found to have a 40% circumferential stenosis at autopsy. Light microscopy revealed exuberant bone proliferation in all specimens. Unrestrained osteogenesis may limit the utility of vascularized periosteum in reconstruction of extensive tracheal defects. (Otolaryngol Head Neck Surg 1999;120:706-12.)