Tissue‐Engineered Tracheal Replacement in a Child: A 4‐Year Follow‐Up Study

In 2010, a tissue‐engineered trachea was transplanted into a 10‐year‐old child using a decellularized deceased donor trachea repopulated with the recipient's respiratory epithelium and mesenchymal stromal cells. We report the child's clinical progress, tracheal epithelialization and costs over the 4 years. A chronology of events was derived from clinical notes and costs determined using reference costs per procedure. Serial tracheoscopy images, lung function tests and anti‐HLA blood samples were compared. Epithelial morphology and T cell, Ki67 and cleaved caspase 3 activity were examined. Computational fluid dynamic simulations determined flow, velocity and airway pressure drops. After the first year following transplantation, the number of interventions fell and the child is currently clinically well and continues in education. Endoscopy demonstrated a complete mucosal lining at 15 months, despite retention of a stent. Histocytology indicates a differentiated respiratory layer and no abnormal immune activity. Computational fluid dynamic analysis demonstrated increased velocity and pressure drops around a distal tracheal narrowing. Cross‐sectional area analysis showed restriction of growth within an area of in‐stent stenosis. This report demonstrates the long‐term viability of a decellularized tissue‐engineered trachea within a child. Further research is needed to develop bioengineered pediatric tracheal replacements with lower morbidity, better biomechanics and lower costs.

In 2010, a tissue-engineered trachea was transplanted into a 10-year-old child using a decellularized deceased donor trachea repopulated with the recipient's respiratory epithelium and mesenchymal stromal cells. We report the child's clinical progress, tracheal epithelialization and costs over the 4 years. A chronology of events was derived from clinical notes and costs determined using reference costs per procedure. Serial tracheoscopy images, lung function tests and anti-HLA blood samples were compared. Epithelial morphology and T cell, Ki67 and cleaved caspase 3 activity were examined. Computational fluid dynamic simulations determined flow, velocity and airway pressure drops. After the first year following transplantation, the number of interventions fell and the child is currently clinically well and continues in education. Endoscopy demonstrated a complete mucosal lining at 15 months, despite retention of a stent. Histocytology indicates a differentiated respiratory layer and no abnormal immune activity. Computational fluid dynamic analysis demonstrated increased velocity and pressure drops around a distal tracheal narrowing. Cross-sectional area analysis showed restriction of growth within an area of in-stent stenosis. This report demonstrates the long-term viability of a decellularized tissue-engineered trachea within a child. Further research is needed to develop bioengineered pediatric tracheal replacements with lower morbidity, better biomechanics and lower costs.

Introduction
The application of tissue engineering to restore a segment of airway in those with long-segment tracheal stenosis resistant to treatment is now realistic. Such therapies remain highly controversial, however, due to uncertainty regarding long-term benefit, quality of life and comparative cost. A 5-year follow-up of the first tissue-engineered airway in an adult reported normal lung function and a good quality of life long-term albeit with the need for serial insertions of airway stents (1,2). Approaches based on synthetic scaffolds have also been reported, though only with short-term follow-up to date (3).
In 2012, we reported a 2-year follow-up of the first tissueengineered tracheal transplant in a child using a decellularized deceased donor trachea repopulated with the patient's own respiratory epithelial cells and mesenchymal stromal cells (4). While this demonstrated the validity of this approach in reconstructing an airway over a 2-year period, a number of key questions remain unanswered. The trachea continues to grow up to adulthood and it is still not known whether a transplanted engineered airway has capacity for growth (5). The long-term outcome is also unclear in terms of how the graft remodels over time, whether it continues to support regenerated epithelium and if it continues to be unchallenged by the immune system. An evaluation of the quality of life of the recipient, a chronology of events and estimation of cost is necessary to evaluate the potential for a wider application of this technology and its place within a healthcare system. We address these questions by reporting observations of this child's clinical progress and tracheal epithelialization 4 years after implantation.

Materials and Methods
Clinical data were collated from the patient notes documenting his long-term care at Great Ormond Street Hospital (GOSH). The Peds QL parent-proxy assessment tool was used as a validated method of analyzing health-related quality of life in children with chronic disease and associated learning disabilities (6). Belgium -Technologielaan 15, Leuven, Belgium). Cross-sectional area of the trachea was calculated following methods previously described (7). Tracheal airflow was calculated using a validated method through computational fluid dynamics (8,9).
Endoscopy images of the transplanted trachea were obtained during microlaryngobronchoscopy performed at 15 days, 6, 15 and 42 months following transplantation using a 15-mm zero degree Hopkins Rod and image capture (Karl Storz, Tuttlingen, Germany).
Biopsy samples were routinely processed following formalin fixation, followed by paraffin wax embedding and cutting of 4-mm sections onto glass slides for hematoxylin and eosin (H&E) staining and immunostaining using an automated immunostainer (Leica Bond-Max, Leica, Wetzlar, Germany). Samples of cilia were obtained during routine flexible bronchoscopy by brushing the trachea with a 2-mm cytology brush (Olympus Endotherapy, Olympus, Skinjuku-ku, Tokyo) (10). Samples were analyzed using a digital high-speed video camera (MotionPro X4, IDT, CA) at a frame rate of 500 frames s-1 to allow assessment of ciliary beat frequency and pattern.
The costing for the clinical items were obtained using a combination of NHS reference costs (2010-2011 period) per procedure appropriate for a male child and typical costs for complex tracheal patients provided by GOSH. The laboratory costs were estimated from data gathered from the good manufacturing practice (GMP) laboratories where further grafts were constructed to treat two later patients (data not shown).

Chronology of events
Following transplantation at GOSH in March 2010, the child returned to the intensive care unit (ICU; unit providing level 2 and 3 care) where he remained an inpatient for 8 days, before being discharged from critical care (11). He required 25 procedures postoperatively, mainly to clear secretions and granulation tissue ( Figure 1). The graft itself was malacic in the initial period necessitating the insertion of two bioabsorbable tracheal stents. On four occasions during the initial period admission to the ICU was needed for respiratory support.
The child was discharged home in August 2010, but required a number of return visits to GOSH to address retained secretions, granulation and a malacic segment in the distal transplanted trachea, necessitating the insertion of further stents, one bioabsorbable and two self-expanding nitinol. There were two further admissions to ICU during the first year follow up period and one ICU admission during the second year follow up period.

4-Year Review of a Tissue-Engineered Trachea
By 6 months, the airway was sufficiently stable to allow the child to return to school and the need for repeated interventions declined up to late 2013 when an infection and stenosis within the tracheal stents and un-transplanted left main bronchus necessitated intervention. Following balloon dilatation of the trachea and left main bronchus, the child made a good recovery and has now returned to full-time education.
A Peds QL 4 Á 0 parent proxy quality of life assessment was completed in May 2014. Scores were 50, 60, 55 and 35 for physical, emotional, social and school subscales, respectively, with a total score of 50 out of 100. Since transplantation, the child has grown 15 cm in height to 168 cm and gained 21 kg in weight to 58 kg. Figure 2 shows the cross-sectional area comparison derived from the 2010, 2013 and 2014 CT scans.

Assessment of the transplanted trachea
Comparison of the 2010 tracheal geometry to a normal trachea demonstrates the formation of high velocity region in its distal segment ( Figure 3A) (12). In 2013, the velocity in this region increased further and formed a jet 40 mm below the glottis. By 2014, the jet formed more proximally (circa 17 mm subglottically). The effect of these changes in flow pattern is seen in Figure 3B. A section of the excised homograft trachea exhibited normal ciliated respiratory type epithelium ( Figure 6A). A biopsy of the transplanted trachea 1-month following the procedure showed granulation tissue only ( Figure 6B). At  42 months, a biopsy of the proximal transplant showed a complete epithelial layer with a mix of squamoid and respiratory type epithelium with scanty ciliated cells ( Figure 6C). An immunostained section using CD3 demonstrated no evidence of rejection or lymphocyte-associated epithelial damage and normal submucosal T cell density ( Figure 6D). Staining for Ki67 was normal and cleaved caspase 3 was negative in both pre and posttransplant specimens. Serial serological examination to date has shown no evidence of anti-donor HLA antibodies.
Brushings on routine endoscopy in April 2014 demonstrated ciliated epithelial cells. Ciliary beat frequency was within the normal range at 10.7 Hz (95% confidence    Figure 7). The laboratory costs of preparing the tissueengineered trachea were estimated to be US$15 600.

Discussion
The use of a tissue-engineered tracheal transplant is currently a potential treatment of last resort. In the reported case, all conventional therapies had failed and the technique was used on compassionate grounds in an urgent setting. This intervention has not only preserved life for more than 4 years, but has enabled the child to mature, continue education and be free of medical intervention for long periods. Indeed, the timing and frequency of interventions between the 1st and 4th years following transplantation compare favorably to children suffering  from recurrent tracheal stenosis or those with metallic tracheal stents treated in our institution.
The Peds QL score is consistent with a child suffering from a complex chronic disease (13). A low physical subscale mainly accounted for this and his co-existing conditions, including spastic diplegia, make it difficult to determine the contribution of his airway disease alone to this score.
The emergent nature of our case necessitated the use of intraoperative native airway epithelial patches to reepithelialize the graft (4). The absence of a safe and effective method of monitoring epithelial fate in vivo in humans prohibits observations of the fate of such epithelium. A number of preclinical studies have indicated re-epithelialization occurs from migration of cells from the wound edge following tracheal transplantation (14,15), tracheal replacement with aortic grafts (16,17) or synthetic material (18). While this might question the need for reepithelizing grafts prior to transplantation, evidence exists supporting the role of epithelium in reducing postoperative stenosis and it is probable that transplanted epithelia act as a biological dressing as re-epithelization occurs from the wound edge (19).
Alternative protocols for epithelizing tracheal grafts use cadaveric tracheal cartilage prevascularized within radial forearm fascia and lined with autologous buccal mucosa (20,21). While this approach has reported some success, it is limited by the need for prolonged periods of immunosuppression and the delivery of squamous rather than ciliated epithelium within the airway. The emergent circumstances of our case meant this approach was unsuitable as there was insufficient time for prevascularisation. It is hoped with advances in cell expansion techniques and biomaterials that it will be possible to deliver a fully differentiated autologous respiratory epithelial sheet as has been reported with buccal and corneal epithelium (22,23). This, in combination with a prevascularised decellularized tracheal scaffold, would allow for the delivery of a vascularized epithelized tracheal graft without immunosuppression.
A cross-sectional centerline analysis suggested growth at the proximal and distal portions of the trachea over time.
Cross-sectional area is restricted at 15-45 mm below the first tracheal ring in all scans corresponding to an area of instent stenosis. It is not possible to determine whether the transplanted trachea would have grown had in-stent stenosis been avoided. Whether the transplanted section includes viable tracheal cartilage can also not be determined with routine investigations. However, a complete mucosal layer, dynamic movement of the airway and demonstrable tracheal rings on endoscopy indicate the transplanted trachea is functioning as a cartilaginous frame. Of note, recurrent stenosis has been reported in the other example of a decellularized airway transplant (1).
We also report, for the first time to our knowledge, the use of computational fluid dynamic simulations within a reconstructed trachea. This validated technique provides detailed information on how distinct geometrical features correlate with airflow distribution and provides information

4-Year
Review of a Tissue-Engineered Trachea on pressure (7,8,12). Our results demonstrate an increasing pressure drop at the distal segment of the transplanted trachea due to the jet shown in Figure 3 and highlighted the area most in need of intervention with a corresponding improvement in symptomatology following targeted treatment. We hypothesize that similar results can be derived from high-field-strength magnetic resonance imaging using these techniques to provide accurate, personalized and noninvasive planning, and follow-up following large airway surgery, including transplantation.
The total cost of this tissue-engineered tracheal transplant is greater than conventional forms of airway management; however, our approach is only indicated for a small subset of children with airway stenosis and the calculated costs herein are unlikely to be prohibitive within a modern healthcare system. Our observations and interpretations of the problems the child faced in this early period have fed back into substantially improved tissue-engineering protocols, and an advanced GMP process which will lead to substantial, iterative, reductions in hospital stay, complications and, thus, costs.
To our knowledge, this is the first long-term follow-up report of a child receiving a tissue-engineered trachea. Further research is required to develop bioengineered pediatric tracheal replacements with lower morbidity, better biomechanics and at a cost acceptable to healthcare providers. Future clinical trials of such constructs should include comprehensive assessments of airway physiology, biology, quality of life, and health economics.