Mechanical Evaluation of Tracheal Grafts on Different Scales.

Tissue engineered (or bioengineered) tracheas are alternative options under investigation when the resection with end-to-end anastomosis cannot be performed. One approach to develop bioengineered tracheas is a complex process that involves the use of decellularized tissue scaffolds, followed by recellularization in custom-made tracheal bioreactors. Tracheas withstand pressure variations and their biomechanics are of great importance so that they do not collapse during respiration, although there has been no preferred method of mechanical assay of tracheas among several laboratories over the years. These methods have been performed in segments or whole tracheas and in different species of mammals. This article aims to present some methods used by different research laboratories to evaluate the mechanics of tracheal grafts and presents the importance of the tracheal biomechanics in both macro and micro scales. If bioengineered tracheas become a reality in hospitals in the next few years, the standardization of biomechanical parameters will be necessary for greater consistency of results before transplantations.

Repeated systemic administration of adipose tissue-derived mesenchymal stem cells prevents tracheal obliteration in a murine model of bronchiolitis obliterans.

OBJECTIVE: To investigate the effect of adipose tissue-derived mesenchymal stem cell (ASC) administered either systemically or locally in a murine model of bronchiolitis obliterans. RESULTS: When compared to controls, systemic treatment with 106 ASCs on D0 and a second dose on D7 significantly prevented tracheal obliteration 28 days after heterotopic tracheal transplantation (median of 94 vs. 16%; P < 0.01). A single dose tended towards less stenosis than controls, but did not reach statistical significance (28 vs. 94%; P = 0.054). On the contrary, repeated local injection was incapable of preventing tracheal obliteration when compared to a single injection or controls (37 vs. 71 vs. 87%). Two intravenous doses also tended to be better than two local injections (16 vs. 37%; P = 0.058), and were better than a single local dose (16 vs. 71%; P < 0.01). CONCLUSION: A second dose of ASC, given systemically after 7 days, reduces luminal obliteration in a heterotopic tracheal transplantation model in mice, suggesting that ASC can be used to prevent obliterative bronchiolitis after lung transplantation.

[History and progress of trachea transplantation in Mexico]. / Historia y avances en trasplante de tráquea en México.

Central airway obstruction and particularly tracheal stenosis is a clinical problem where definite resolution is a tracheal resection, evaluating the magnitude, length, and ventilatory compromise of patient. The resectable fragment is limited to 30% of the total length in children, or 6 cm in adults with terminal end anastomosis. The replacement of longer sections through allogeneic transplantation has been disappointing due to the unfeasibility of the organ, rejection of the graft, and the highly complicated surgical procedure. Tissue bioengineering has designed the replacement of functional organs generated in vitro in the short term, with the absence of immunological responses to the graft. This is based on a non-biological matrix where epithelial and mesenchymal cells are planted in such a matrix. In this document, we review the history and development of trachea transplantation in Mexico as well as the application of these new technologies in the context of its world development, which is a reality in other countries as a new alternative in obstructive illness of the airway.

MicroRNA screening and functional study of obliterative bronchiolitis in a rat model simulating lung transplantation.

The aim of this study was to provide the experimental basis for effective prevention and treatment of obliterative bronchiolitis (OB) by studying the changes on the microRNA (miRNA) expression profile after an orthotopic tracheal transplantation (OTT) simulating lung transplantation (LT). The OTT was performed on inbred rats to establish an OB animal model simulating LT, which was confirmed successful through pathological examination after 4 weeks. A miRNA microarray was used to screen for the most significantly differentially expressed miRNA in the OB tissues of donor transplanted trachea and real-time quantitative PCR was then used to validate the reliability of the microarray results. The microarray detection obtained 29 OB-related miRNAs, composed of 15 and 14 significantly up- and down-regulated miRNAs, respectively, among which miR-146a, miR-155, and miR-451, whose function is involved in the immune and inflammatory reactions, were subjected to relative quantitation research. The LT-simulated OTT-induced OB showed significantly differential expressions of multiple miRNAs, among which miR-146a and miR-155 were highly expressed, while miR-451 was lowly expressed, suggesting that these miRNAs may play an important regulatory role in the OB pathological process after LT.

Assessment of the mechanics of a tissue-engineered rat trachea in an image-processing environment.

OBJECTIVES: Despite the recent success regarding the transplantation of tissue-engineered airways, the mechanical properties of these grafts are not well understood. Mechanical assessment of a tissue-engineered airway graft before implantation may be used in the future as a predictor of function. The aim of this preliminary work was to develop a noninvasive image-processing environment for the assessment of airway mechanics. METHOD: Decellularized, recellularized and normal tracheas (groups DECEL, RECEL, and CONTROL, respectively) immersed in Krebs-Henseleit solution were ventilated by a small-animal ventilator connected to a Fleisch pneumotachograph and two pressure transducers (differential and gauge). A camera connected to a stereomicroscope captured images of the pulsation of the trachea before instillation of saline solution and after instillation of Krebs-Henseleit solution, followed by instillation with Krebs-Henseleit with methacholine 0.1 M (protocols A, K and KMCh, respectively). The data were post-processed with computer software and statistical comparisons between groups and protocols were performed. RESULTS: There were statistically significant variations in the image measurements of the medial region of the trachea between the groups (two-way analysis of variance [ANOVA], p<0.01) and of the proximal region between the groups and protocols (two-way ANOVA, p<0.01). CONCLUSIONS: The technique developed in this study is an innovative method for performing a mechanical assessment of engineered tracheal grafts that will enable evaluation of the viscoelastic properties of neo-tracheas prior to transplantation.

The impact of topically applied preservation solutions on the respiratory epithelium of tracheal grafts submitted to cold ischemia: functional and morphological analysis.

OBJECTIVE: Advances in graft reepithelialization and revascularization have renewed interest in airway transplantation. This study aims to determine whether topically applied preservation solutions can ameliorate ischemic injury to tracheal grafts. We analyzed 1) the effects of cold ischemia on the mucociliary clearance of tracheal grafts and 2) the impact of topically applied preservation solutions on the effects of cold ischemia on mucociliary clearance. METHOD: Tracheal segments (n=217) from 109 male Wistar rats were harvested, submerged in low-potassium-dextran-glucose, histidine-tryptophan-ketoglutarate, or saline solution (saline group), and stored at 4°C for 6, 10, 16, or 24 hours. A control group (not submerged) was analyzed immediately after harvesting. In situ mucociliary transport and ciliary beating frequency were measured using a stroboscope. Epithelial integrity, cellular infiltration, and mucus storage were quantified by light microscopy and image analysis software, along with transmission electron microscopy. RESULTS: 1) The effects of cold ischemia: in situ mucociliary transport and ciliary beating frequency were greater in the control group than after cold ischemia. Microscopic analysis results were similar between groups. 2) The effects of preservation solutions: there was no difference between the low-potassium-dextran-glucose, histidine-tryptophan-ketoglutarate, and saline groups in functional or light microscopy analysis. The saline group presented stronger signs of ischemic injury with transmission electron microscopy. CONCLUSIONS: Cold ischemia diminished the mucociliary clearance of the tracheal respiratory epithelium. Topically applied preservation solutions did not ameliorate the injury caused by cold ischemia to the tracheal respiratory epithelium.

The impact of topically applied preservation solutions on the respiratory epithelium of tracheal grafts submitted to cold ischemia: functional and morphological analysis

OBJECTIVE: Advances in graft reepithelialization and revascularization have renewed interest in airway transplantation. This study aims to determine whether topically applied preservation solutions can ameliorate ischemic injury to tracheal grafts. We analyzed 1) the effects of cold ischemia on the mucociliary clearance of tracheal grafts and 2) the impact of topically applied preservation solutions on the effects of cold ischemia on mucociliary clearance. METHOD: Tracheal segments (n=217) from 109 male Wistar rats were harvested, submerged in low-potassium-dextran-glucose, histidine-tryptophan-ketoglutarate, or saline solution (saline group), and stored at 4°C for 6, 10, 16, or 24 hours. A control group (not submerged) was analyzed immediately after harvesting. In situ mucociliary transport and ciliary beating frequency were measured using a stroboscope. Epithelial integrity, cellular infiltration, and mucus storage were quantified by light microscopy and image analysis software, along with transmission electron microscopy. RESULTS: 1) The effects of cold ischemia: in situ mucociliary transport and ciliary beating frequency were greater in the control group than after cold ischemia. Microscopic analysis results were similar between groups. 2) The effects of preservation solutions: there was no difference between the low-potassium-dextran-glucose, histidine-tryptophan-ketoglutarate, and saline groups in functional or light microscopy analysis. The saline group presented stronger signs of ischemic injury with transmission electron microscopy. CONCLUSIONS: Cold ischemia diminished the mucociliary clearance of the tracheal respiratory epithelium. Topically applied preservation solutions did not ameliorate the injury caused by cold ischemia to the tracheal respiratory epithelium. .

Successful circumferential free tracheal transplantation in a large animal model.

BACKGROUND: Long segment tracheobronchial stenoses are associated with high morbi-mortality rates and difficult treatment. Transplantation hasn't proved to be useful yet. Currently, the successful results achieved in small animal models couldn't be satisfactorily accomplished or extrapolated in large mammals. We aimed to evaluate the viability of orthotopic tracheal autoimplantation in an ovine model. METHODS: All animals underwent tracheal transplantation of 4 cm (5-7 rings) of the cervical trachea and were divided randomly in two groups: isolated autoimplantation (Group A/6) and autoimplantation with omental wrapping (Group B/6). Clinical follow up and weekly bronchoscopical examinations were performed. The grafts were macroscopically, histologically, and bacteriologically analyzed. RESULTS: In group A, four animals achieved their planed survival and were sacrificed up to 60 days after transplantation with viable grafts. In group B, only two sheep had successful results. Graft failure with infection, necrosis and severe stenosis was observed in the rest of the animals from both groups. Pseudomonas aeruginose was isolated in all cases. The main complication of the omental pedicle was vascular congestion and peritracheal hemorrhage. CONCLUSIONS: Contrary to the data reported to date, we found that tracheal transplantation is viable in a large mammal like the sheep. The main complication observed in this animal model was graft infection. The use of an omental pedicle with the technique applied worsened the grafts survival. The encouraging results obtained in this investigation justify further research in order to manage graft infection, leading us to establish a suitable large animal model for allotransplantation.

[Inhalation anesthesia during spontaneous ventilation in a patient with a tracheolaryngeal transplant requiring debridement of fibrous tissue obstructing the lumen]. / Anestesia inhalatoria con preservación de la ventilación espontánea en un paciente trasplantado de laringe y tráquea sometido a desbridamiento intraluminal.

Graft stenosis from fibrous granulation tissue is not an uncommon problem in recipients of a transplanted trachea and larynx. We describe the case of a man with a transplanted trachea who was seen for respiratory difficulty and stridor secondary to stenosis. Examination through a rigid bronchoscope and surgical debridement were both performed under anesthesia with sevoflurane alone while the man breathed spontaneously. The outcome of treatment with sequential debridements of fibrotic tissue and autologous stem cell injection was satisfactory and the patient was discharged.

Tracheal transplant with a prefabricated microsurgical flap.

OBJECTIVE: To test the viability of a tracheal autotransplant, with an original technique using a prefabricated flap from a complete tracheal neovascularized segment (CTNVS) of the sternohyoid muscle anastomosed by a microsurgical technique. STUDY DESIGN: An experimental study using dogs as an animal model. METHODS: Ten mongrel dogs weighing 23 to 40 kg were divided into two groups: group I (control), five animals submitted to autotransplant of the CTNVS without a microsurgical vascular anastomosis; and group II, five dogs submitted to autotransplant of the CTNVS with a microsurgical vascular anastomosis. RESULTS: All group I dogs developed respiratory insufficiency and died because of necrosis and stenosis of the autotransplanted CTNVS, whereas all group II dogs completed a minimum period of 90 days of observation without any clinical changes. Macro- and microscopic analysis revealed intact tracheal structures. CONCLUSIONS: The present clinical and morphological findings demonstrate that the CTNVS autotransplant is viable, when a microsurgical vascular anastomosis is used.

Evaluation of propofol as an anesthetic in swine tracheal transplant surgery / Evaluación del propofol como anestésico para cerdos sometidosa trasplante de tráquea

Because the swine have been used as an ideal animal model for different medical investigations, it has been useful to the advance in vital organs transplant field. The trachea transplant is a surgical procedure which requires special conditions in anesthetic depth and muscular relax, for a long period, and in addition, an excellent intra and post-operatory analgesic. The aim of this study was to use a combination of xylacine and ketamine, as premedication and evaluate propofol as a general anesthetic in trachea transplant donor or recipient pigs. All the methodology was under the approval of the Committee of Ethics for the Experimentation with Animals of the University of Antioquia. Ten donors and 10 recipients female Yorkshire pigs having a body weight of about 30 kg were used. Trachea extraction from a donor and its transplantation to a recipient in the same surgical procedure was performed. The average body weight (PP) was 30 ± 2.92 kg for both the groups, the average value were as follows: time of recumbency (TR) 8.25 ± 2:85 min; latency period (PL) 6.05 ± 1.73 min, (for both groups); surgical time (TQ) for donors and recipients was 80 ± 0.02, and 247 ± 0.02 min, respectively; heartbeat rate (FC) 90.34 ± 8.14 bpm, O2 saturation (SO2) 95.47 ± 1.79 %; exhaled PCO2 31.13 ± 1.89 mmHg; temperature (T) for both groups was 37.51 ± 0.74oC. The mean arterial pressure average (PAM) for both group was 65.47 ± 5.94 mmHg; the average time of esternal recumbecency (TRE) for donor female pigs was 16.50 ± 4.09 min, and the average time to stand up (TP) for swine recipients was 30.70 + 3.27 min. These results indicate that Propofol can be considered as a safe anesthetic for use in continuous perfusion. Since it has not an analgesic effect it is strongly recommended to combine it with opioids during anesthetic-surgical procedures; it can be also used with neuromuscular preanesthetics or inhaled anesthetics.

Los cerdos se han utilizado como modelo animal ideal para diversas investigaciones médicas; han sido útiles para el avance en el trasplante de órganos. El trasplante de tráquea es un procedimiento quirúrgico que requiere condiciones especiales en profundidad anestésica y relajación muscular por un período largo, y además, una analgesia intra y del postoperatoria excelente. Nuestra investigación utiliza una combinación de xylacine y ketamina, como premedicación y evaluar el propofol como anestésico general en cerdos donantes y receptores en quienes el trasplante de la tráquea sería hecho. Toda la metodología contó con la aprobación del Comité de Ética para la Experimentación con los Animales de la Universidad de Antioquia. Utilizamos 10 donantes y 10 cerdos raza Yorkshire hembras con un peso corporal de cerca de 30 kilogramos. Se realizó la extracción de la tráquea de un donante y el trasplante a un receptor en el mismo procedimiento quirúrgico. El peso corporal (PP) fue de 30 ± 2.92 kg para todo el grupo, el tiempo de recumbencia (TR) para ambos grupos fue de 8.25 ± 2.85 min, el período de latencia (PL) para ambos grupos fue de 6.05 ± 1.73 min, el promedio de tiempo quirúrgico (TQ) para los donantes fue de 80 min ± 0.02, el TQ de los receptores fue de  247m ± 0.02. La presión arterial media (PAM) para todo el grupo fue de 65.47 ± 5.94 mmHg, el promedio de frecuencia cardiaca (FC) para ambos grupos fue de 90.34 ± 8.14 ppm, el promedio de saturación de oxigeno (SO2) fue de 95.47 ± el 1.79% y el CO2 espirado fue de 31.13 ± 1.89 mmHg y el promedio de la temperatura (t) para ambos grupos fue de  37.51 ± 0.74oC. La tiempo de recumbencia esternal (TRE) para las cerdas donantes fue de 16.50 ± 4.09 min y el tiempo para pararse (TP) para los receptores fue de 30.70 ± 3.27 min. El propofol se puede considerar como anestésico seguro para el uso en la perfusión continua durante la anestesia. Puesto que no tiene un efecto analgésico se recomienda combinarlo con opioides.

Management of larynx and trachea donors.

INTRODUCTION: Laryngeal transplantation is a possibility for patients with irreversible laryngeal disease, such as complex trauma and larynx cancer. The objective of performing this procedure was to solve problems that these patients face with a laryngectomy. The medical literature has reviews about larynx transplantations, but almost nothing about the larynx donor. The following is our experience on management of these donors. MATERIALS AND METHODS: Selection criteria was as follows: (1) 18-50 years old; (2) gender and ABO blood type matched between donor and recipient; (3) No abuse of tobacco, cocaine, and marijuana, (4) tracheal intubation time <3 days; and (5) time in the intensive care unit <7 days. The preservation was simple hypothermia with larynx infusion via the carotid artery with University of Wisconsin solution. RESULTS: Between 2001 and 2006, we managed 25 donors, among whom 12 grafts were discarded. The 13 larynx donors were of average age 27.2 +- 7.9 years and their cause of death was head trauma. Each was of male and 12 were multiorgan donors. Three donors had previous consumption of tobacco and 2 donors of marijuana. There were 2 cases of acute rejection episodes. Graft survival rate at 2 years was 90%. DISCUSSION: These donors may have differences from other multiorgan donors: (1) they do not require strict fluid management; (2) vasoactive agents may be used in higher doses than in organ donors, and (3) the larynx tolerates hemodynamic instability. It was necessary to use some donors who had used addictive substances, showing that some selection criteria may be flexible. There was no conflict between thoracic surgeons and larynx surgeons. The priority always was for life-saving organs. Family consent was sometimes difficult because of the retrieval times and body donor reconstruction. The larynx surgery retrieval demanded an additional 2-5 hours during routine multiorgan donor surgery, and always the family asked about body reconstruction. The body appearance was always preserved.

Manejo de la vía aérea en trasplante de tráquea / Managing of the airway in transplant of trachea

A pesar de los grandes avances en cirugía de resección traqueal y en dispositivos como las endoprótesis, existen pacientes en quienes dichas técnicas no son viables debido al compromiso tan extenso de su estenosis; por lo tanto, su problema no se ha podido resolver alterando la calidad de vida y muchas veces poniendo en riesgo la vida de los pacientes, secundario a una obstrucción de la vía aérea. A continuación, se describe la experiencia del grupo de la Universidad de Antioquia y el Hospital Universitario San Vicente de Paúl, en el manejo de la vía aérea durante el transplante de tráquea realizado a 5 pacientes en la institución, ya que dicho procedimiento se convierte en un reto para el anestesiólogo por compartir el campo quirúrgico con el cirujano. Gracias al entrenamiento previo en cadáveres y en modelos experimentales animales y al trabajo multidisciplinario llevado a cabo, es que se llega a mostrar buenos resultados.

Cryopreserved tracheal grafts: a review of the literature.

Cryopreserved tracheal grafts have been used in several experimental models of long segment replacement. The clinical application of the procedure has been limited due to the fact that contradictory results have been reported. The purpose of this article is to present a review of the literature on tracheal cryopreservation. Despite the fact that most authors indicate that cryopreserved tracheal allografts retain viability and have a low immunological response, though they continue to function after transplantation with good epithelialization and patency, cryopreservation leads to significant damage to cartilage, the degree of which is based on the freezing-storage methods that affect the function and durability of a graft. The long-term storage of cartilage must therefore be investigated in more detail in basic research models of cartilage viability: the evaluation of chondrocyte apoptosis, and the use of different solutions for tracheal cryopreservation other than RPMI-1640, Dulbecco's modified Eagle's, Eurocollins, and TC-199. Furthermore, problems that involve improving the blood supply to the graft after extensive resection and immunosuppression must be resolved before tracheal cryopreservation can become a clinically established method for tracheal grafts.

Heterotopic transplantation of glycerin-preserved trachea: effect of respiratory epithelium desquamation on acute rejection.

An effective preservation method and decreased rejection are essential for tracheal transplantation in the reconstruction of large airway defects. Our objective in the present study was to evaluate the antigenic properties of glycerin-preserved tracheal segments. Sixty-one tracheal segments (2.4 to 3.1 cm) were divided into three groups: autograft (N = 21), fresh allograft (N = 18) and glycerin-preserved allograft (N = 22). Two segments from different groups were implanted into the greater omentum of dogs (N = 31). After 28 days, the segments were harvested and analyzed for mononuclear infiltration score and for the presence of respiratory epithelium. The fresh allograft group presented the highest score for mononuclear infiltration (1.78 +/- 0.43, P < or = 0.001) when compared to the autograft and glycerin-preserved allograft groups. In contrast to the regenerated epithelium observed in autograft segments, all fresh allografts and glycerin-preserved allografts had desquamation of the respiratory mucosa. The low antigenicity observed in glycerin segments was probably the result of denudation of the respiratory epithelium and perhaps due to the decrease of major histocompatibility complex class II antigens.

Heterotopic transplantation of glycerin-preserved trachea: effect of respiratory epithelium desquamation on acute rejection

An effective preservation method and decreased rejection are essential for tracheal transplantation in the reconstruction of large airway defects. Our objective in the present study was to evaluate the antigenic properties of glycerin-preserved tracheal segments. Sixty-one tracheal segments (2.4 to 3.1 cm) were divided into three groups: autograft (N = 21), fresh allograft (N = 18) and glycerin-preserved allograft (N = 22). Two segments from different groups were implanted into the greater omentum of dogs (N = 31). After 28 days, the segments were harvested and analyzed for mononuclear infiltration score and for the presence of respiratory epithelium. The fresh allograft group presented the highest score for mononuclear infiltration (1.78 ± 0.43, P <= 0.001) when compared to the autograft and glycerin-preserved allograft groups. In contrast to the regenerated epithelium observed in autograft segments, all fresh allografts and glycerin-preserved allografts had desquamation of the respiratory mucosa. The low antigenicity observed in glycerin segments was probably the result of denudation of the respiratory epithelium and perhaps due to the decrease of major histocompatibility complex class II antigens.

Viability of a 12-ring complete tracheal segment transferred in the form of a compound flap: an experimental study in dogs.

INTRODUCTION: There is a lack of an ideal substitute for the injured tracheal tissues. Autologous or homologous grafts and prostheses are inadequate because of their unreliable and nonviable nature for the reconstruction of a complete tracheal segment (CTS). OBJECTIVE: We studied the viability of a 12-ring CTS transferred in the form of a compound flap from the sternohyoid muscle (SM) for tracheal reconstruction. METHODS: Sixteen mongrel dogs of both sexes were divided into two groups of eight dogs each. In group I (control), a 12-ring CTS was removed and then reimplanted as an autograft. In group II, an SM flap was used to envelop four rings of the CTS to produce a compound CTS flap neovascularized by the SM (CTSNV). After 21 days, a 12-ring CTSNV, consisting of the four rings previously covered with SM, four rings above, and four rings below (without muscle covering), was sectioned and completely separated from the trachea. The CTS was then reimplanted at its original site, with the SM being the only vascular supply pedicle for the flap. RESULTS: The results were evaluated clinically and by macroscopic and microscopic examination of the surgical specimens. All the animals in group I (control) died or were killed by the first or second week because of necrosis and stenosis of the CTS graft. In group II, all animals were killed after 77 days of follow-up without showing any alteration in the CTSNV. CONCLUSIONS: We conclude that a compound 12-ring CTSNV flap remains viable and can be mobilized from the trachea without the risk of necrosis or stenosis.