Figures
- FIGURE 1
Different replacement strategies investigated for tracheal replacement: a) 3D printing/bioprinting, b) casting, c) decellularisation and d) electrospinning. Images adapted from [59, 60, 70, 86] with permission.
Tables
- TABLE 1
Epidemiology of tracheal defects
Pathology Epidemiology Reference Traumatic tracheobronchial injuries Cause of 2.5–3.2% of trauma-associated deaths
Present in 0.5–2.0% of trauma patients showing chest/neck injuries
<1% of elective orotracheal intubations
0.05–0.37% of endotracheal intubations[5–8] Tracheal tumours Tracheal malignancies present in <1% of people
Primary tracheal carcinomas present in <1% people each year[9, 10] TBM Overall epidemiology is unknown, but TBM is present in 23% of COPD patients, 44% of patients with chronic bronchitis and 1% of all patients undergoing bronchoscopy [11] Post-intubation tracheal stenosis 6–21% of patients that underwent intubation [12, 13] Oesophageal atresia <1% of births [14] Tracheal agenesis <1% of births [15] Tracheo–oesophageal fistulas <1% of births [16] COPD: chronic obstructive pulmonary disease; TBM: tracheobronchomalacia.
- TABLE 2
Mechanical properties of tracheal cartilage, trachealis muscle and whole trachea
Specimen type Test type Modulus Reference Tracheal cartilage Human Tensile 16.92 MPa [27] Human Tensile 1–15 MPa [30] Porcine Tensile 5.62 MPa [31] Compression 1.36 MPa Ovine Tensile 10.6 MPa [32] Trachealis muscle Human Tensile 5.7–364 kPa [24] Human Tensile 0.84–34.15 kPa [33] Whole trachea Human Tensile 12–20 MPa [27] Human Compression 0.16–2 MPa [34] Porcine Compression 1.10 MPa [31] Ovine Tensile 1.5 MPa [35] Rabbit Compression 1.72 MPa [36] Rabbit Three-point bending 0.214 MPa [37] - TABLE 3
Outcomes of pre-clinical animal studies of tissue engineering tracheal replacement attempts
Species, sample size Material Cells Experiment details Outcomes Ref. Dog, n=3 PP mesh with radiated minced dermal layer Keratinocytes Keratinocytes were cultured on a patch of the composite prosthesis which was then implanted subcutaneously for 1 week, then implanted into a 2×3 cm section within the trachea. Tracheas remained in animals for duration of study (5 months) with some reports of stenosis and initially some inflammation which resolved over time. [72] Dog, n=10 Porous copolymer of PLGA scaffold, mounted on a prosthesis framework, made of PP mesh reinforced with PP rings Keratinocytes Implanted in the peritoneal cavity for 1 week; wrapped in greater omentum to vascularise prosthesis. Complete surgical resection and replacement of a thoracic tracheal segment (5 cm) was then performed using the prosthesis. Still viable at 2 months, no signs of stenosis (one dog omitted due to graft infection in peritoneum). [65] Dog, n=18 PP mesh tube reinforced with a PP spiral and coated with 1% collagen solution which was then freeze dried Autologous PB vs BM aspirate vs BM MSCs Scaffolds were soaked in either aspirate or cell suspensions. Prosthetic placed within 5 cm tracheal defect. Animals were followed up to 12 months. Stenosis occurred in three dogs from PB group. Nine of the 12 dogs in the BM aspirate and MSC groups had an epithelial lining cover age of more than 50%. [73] Dog, n=5 Nitinol frame coated in 3% freeze-dried collagen NA Placed in omentum prior to implantation for 3 weeks. 2 cm trachea section was removed and tracheal prosthesis was sutured in with a 5 mm overlap. Assessed up to 24 months, four out of five dogs survived post operatively. Airway stenosis was not present. Epithelialisation was present; however, vascularisation of graft was limited. [74] Goat, n=10 3D-printed PCL Autologous articular chondrocytes Scaffolds were seeded with 1.2×108 chondrocytes in type I collagen gel and cultured for 1 week. 3.5 cm section of trachea was removed and either grafted back as control or scaffold was implanted. Initial granulation and stenosis were observed. Mean survival was 60 days, which was significantly longer than controls that quickly necrotised. [75] Mouse, n=10 Non-woven PGA mesh Bovine chondrocytes The cell-seeded mesh was wrapped around a silastic stent which was then implanted into nude mice for 4 weeks to generate cartilage prior to implantation in cervical defect. Bovine chondrocytes generated cartilage. However, only one animal survived the procedure, lasting for 1 week. Lack of vascularisation was cited as a problem. [76] Porcine, n=5 Decellularised tracheal matrix Autologus MSC derived chondrocytes and epithelial cells Decellularised trachea was seeded with either cell type or both cell types and implanted into a 6 cm defect. Animals were followed up to 60 days. Only the group that received both cell types survived to day 60. Other groups suffered from infection or developed stenosis. [77] Rabbit, n=6 Freeze-dried PCL collagen sponge Chondrocytes Scaffold was cultured within an in vitro bioreactor for 8 weeks to develop collagen content and expand chondrocytes, prior to implantation in a 1 cm defect. Mean survival of 52 days. At 28 days showed evidence of granulation tissue covering graft, leading to eventual stenosis. [78] Rabbit, n=10 PP mesh Epithelial graft Hairless epithelial graft taken from the ear combined with PP mesh and lateral thoracic fascia tubed around a silicone catheter was implanted into 2 cm defect. Followed up to max 4 weeks. 70% survival at week 4. Epithelial grafts survived in all animals and had fibro vascular connections to fascia. [79] Rat, n=5 Collagen sponge with PP mesh Gingival fibroblasts and ASC Cells were seeded onto collagen sponge in collagen solution, which was allowed to gel forming stratified graft. A tracheal defect of 3×6 mm was induced and graft implanted. Pseudostratified ciliated epithelium containing goblet cells was generated at day 14. Combination of both cell types gave best results. [73] Rat, n=3 Scaffold-free Human chondrocytes, fibroblasts, HUVECs and hMSCs Different cell aggregates were combined and cultured to give fibrous and cartilaginous layers. Cultured for 35 days then implanted into 7×1.7 mm defect. Followed up on day 7 and day 35. Vascularisation and epithelialisation were found to have increased; no cartilage formation was observed. [80] Sheep, n=8 Electrospun blend of PET and PU reinforced with medical-grade PC rings Autologous BM-MNC BM-MNC were enriched by gravity-filtered size exclusion then vacuum seeded onto scaffold. A 5 cm segment of native trachea was isolated and resected with seeded graft. 60% of sheep survived up to 4 months. Graft stenosis present in all, encapsulation and infection occurred, no re-epithelialisation but granulation was observed. [23] Rabbit, n=20 3D-printed PCL Chondrocytes Investigation of two groups 1) scaffolds cultured in chondrocyte suspension for 2 weeks and 2) scaffolds cultured in chondrocyte suspension for 4 weeks. Implanted into a 1.6 cm defect. Sufficient mechanical properties – no collapse. Higher survival rate in group 2 than 1, but all animals died within 10 weeks. Most common cause of death in both groups was granulation tissue (75%, 15/20 animals). No re-epithelialisation occurred. [2] Rabbit, n=4 3D-printed PCL NA Four-axis FDM 3D-printed scaffolds were investigated for tracheal replacement over conventional 3D-printing techniques for more dimensionally accurate scaffolds. Implanted into 1 cm defects. All animals survived until designated time points of 4 and 8 weeks. No signs of severe narrowing were observed. Four-axis scaffolds demonstrated less inflammation and better mucosal regeneration than conventional scaffolds, with ciliated epithelium observed on the lumen. [81] Porcine, n=7 3D-printed PCL and decellularised bovine dermal collagen matrix NA 3D-printed tracheal rings were sutured to a decellularised collagen matrix to obtain composite grafts to allow for better integration with surrounding tissue and provide a structural base for cellular infiltration and growth. Implanted into 4 cm defect. Five out of seven survived until the 3-month end point. Causes of death were cited as pneumonia and airway stenosis due to granulation tissue and secretions. At 3 months, grafts appeared vascularised with ciliated epithelium; however, granulation tissue was present in all remaining animals. [64] Rabbit, n=12 3D-bioprinted scaffold of PCL and sodium alginate hydrogel Bone marrow-derived MSCs and rabbit epithelial cells Multi-layered scaffold of PCL and sodium alginate hydrogel. Two layers of PCL used for providing structural support and three layers of hydrogel contained 1×107 cells/10 mL of either MSC or epithelial cells. Implanted into a 1 cm defect. 12-week study. All animals exhibited no distress or graft failure until sacrifice at 12 weeks. Ciliated epithelial mucosa was observed fully covering scaffolds in all groups. Neo-vascularisation was abundant in all groups. [82] Rabbit, n=7 3D-printed PCL and alginate/collagen type 1 hydrogel laden with chondrocytes Chondrocytes Dual-headed 3D printer used to form scaffold of PCL and alginate/collagen type 1 hydrogel containing chondrocytes. 2 cm defect. 3- or 6-week study. One animal died during surgical procedure due to an airway blood clot. Remaining animals suffered from respiratory distress. Examination showed large rate of stenosis at 83.4%. [83] Rabbit, n=16 3D-printed PLLA Chondrocytes 3D-printed scaffolds were soaked in chondrocyte/hydrogel mixture and cultured for 3 days. Scaffolds were pre-vascularised for two weeks prior to implantation. Two in vivo groups were investigated: no pre-vascularisation and 2 weeks of pre-vascularisation. Control group suffered from serious morbidities and died on average 17+/−7 days post-op, whereas in experimental group six out of eight animals survived until the end point, 2 months. Tracheal stenosis was mainly observed in the control group. [84] Rat, n=27 Collagen/P(LLA–CL) fibre electrospun scaffold RTEC and rat RTC Bilayered scaffold fabricated using electrospinning. Contained dense fibres on the inner layer and porous yarns on the outer layer. Three groups investigated: PV, CS and bare scaffold. For cell seeding, RTECs were injected into inner lumen three times and then seeded with RTC on the outside surface also three times. Cultured for 7 days. Higher immunological indicators found in bare and CS scaffolds, similar levels found in PV scaffolds to control. In-growing capillaries found in PV and CS scaffold after 1–2 week post implant. Simple ciliated columnar epithelium and continuous cartilage cell layer found in PV scaffolds, with only flat epithelium and irregular chondrocytes in CS. No epithelial cells and abundant inflammatory cells in submucosa of bare scaffold. [70] Porcine, n=6 Decellularised trachea NA Decellularisation was performed using the HHP technique. Implanted into 1.5 cm defect. Scaffolds maintained shape, with no inflammation or granulation. Tracheal stenosis and narrowing were mild. [62] Rabbit, n=20 Composite graft of indirect 3D-printed PCL and tmdECM hydrogel/hTMSC sheets hTMSC 1 cm defect. Indirect 3D-printed PCL scaffold was reinforced with silicone rings and then the tmdECM placed on the lumen followed by hTMSC sheet. Cultured for 3 days. Two groups investigated: 1) scaffold with hTMSC sheet and collagen and 2) scaffold with hTMSC sheets and tmdECM hydrogel. Mild stenosis observed in both groups at 1 month, only at anastomosis site not in middle, but became more severe in group 1 at 2 months compared to group 2. Thin epithelial layer formed above granulation tissue at anastomosis site in group 1, whereas lumen was completely covered in epithelial tissue at 2 months in group 2. [85] PP: polypropylene; PB: peripheral blood; BM: bone marrow; MSC: mesenchymal stem cell; NA: not applicable; PCL: polycaprolactone; PGA: polyglycolic acid; PLGA: D,L-lactide-co-glycolide; ASC: adipose-derived stem cells; HUVEC: human umbilical vein endothelial cell; hMSC: human mesenchymal stem cell; PET: polyethylene terephthalate; PU: polyurethane; PC: polycarbonate; BM-MNC: bone marrow mononuclear cell; FDM: fused deposition modelling; PLLA: poly(L-lactic acid); P(LLA–CL): poly(L-lactide-co-caprolactone); RTEC: rat tracheal epithelial cell; RTC: rat tracheal chondrocyte; PV: pre-vascularised; CS: cell-seeded; HHP: high hydrostatic pressure; tmdECM: tracheal mucosa derived decellularised extracellular matrix; hTMSC: human inferior turbinate mesenchymal stromal cell.
- TABLE 4
Translation of non-tissue engineering (TE) and TE approaches for tracheal restoration and regeneration in human clinical trials
Patient(s) details Technology Material(s) Cells Procedure details Outcome Ref. Non-TE approaches 43-year-old female Autograft Forearm freeflap supported with an Ultraflex stent NA A freeflap was harvested and wrapped around a stent after implantation in a 6 cm tracheal defect arising for a carcinoma. Died 16 months after the procedure from pre-existing conditions. [131] 63-year-old female Autograft Forearm freeflap with an external mesh support NA Radial forearm fasciocutaneous flap with a Hemashield vascular graft and PolyMax resorbable mesh. Patient remains symptom-free at 6 months and has returned to normal activities. Bronchoscopy at 6 months showed slight migration but healed flap with no obstruction. [132] 16 patients (37–68 years) Autograft Cartilage rib and fascial skin pad NA Segments of the patient's own cartilage were inserted within a skinpad harvested from the forearm. Construct was wrapped around silicone tube for suturing and implanted within the defect. Three deaths following surgical procedure due to lung infections, acute respiratory distress syndrome and myocardial infarction. Long-term follow-up showed a 65% survival rate. [129, 133] 21-year-old male Allotransplantation Donor cadaveric trachea NA or autologous patient cells Patient suffering from extensive tracheal stenosis. A cadaveric trachea with intact blood supply was implanted heterotopically into the sternocleidomastoid muscle for 3 weeks. It was then implanted orthopically into the trachea defect with a vascularised muscular section of the sternocleidomastoid. Integration of the trachea graft and proper function for 9 weeks without evidence of rejection, ischaemia or infection. [124] 24-year-old female Allotransplantation Donor cadaveric trachea NA Patient suffering from tracheal stenosis and third stage respiratory insufficiency. The allograft was implanted and wrapped with omentum and the patient was placed on immunosuppressive therapy. Uneventful postoperative course with signs of graft rejection, necrosis, bacterial and viral infection which lead to the need of silicon endoprosthesis. Signs for rejection reduced and 1-year follow-up suggested patient was still alive with a restored tracheal lumen. [125] Four patients (17–64 years; three males, one female) Allotransplantation Human cadaveric trachea Recipient's buccal mucosa and/or ingrown recipient cells Patients suffering from tracheal stenosis or tracheal chondrosarcoma. Decellularised tracheas were implanted in the forearm and grafted with buccal mucosa or wrapped in forearm fascia to improve vascularisation. Tracheal necrosis after withdrawal of immunosuppression and poor vascularisation around the grafts leading to partial loss of the allotransplant in three patients while additional approaches for recipient cell repopulation of the construct in one patient allowed a vascularised allotransplant and normal airways 6 months after transplantation. [128] 68-year-old male Stent-supported aortic autograft Aorta autograft and silicone Dumon stent NA Patient presenting tracheal squamous cell carcinoma. A 7 cm abdominal aorta autograft was harvested and replaced with a Dracon graft. The aortic graft was implanted coupled with a silicone Dumon stent to avoid aortic wall injury. Granulation tissue formation led to acute respiratory distress syndrome which was treated by introduction of an additional tracheal stent. Stent was removed due to migration albeit no airway collapse was detected. Pneumonia, respiratory distress and pneumothorax lead to death of patient due to septic shock. [120] Six patients (17–52 years; five males, one female) Stent-supported aortic allograft Fresh or cryopreserved aortic grafts and silicone stent NA Patients suffering from mucoepidermoid and adenoid cystic carcinoma. Aortic allografts were wrapped with well vascularised pectoral muscle or thymopericardial fat flaps and implanted using a silicone stent. Complete resection achieved in 83% of patients. Major morbidity, fistulas and uneventful outcomes. All grafts showed adequate vascularisation and four patients are disease-free. [122] 78-year-old male Stent-supported aortic allograft Cryopreserved aortic graft and custom-made nitinol stent NA Patient suffering from extensive bronchopulmonary malignant tumour pre-treated with chemotherapy. Lung cancer was resected, and a stent-supported graft was implanted. Well-functioning re-implanted lobe found 1 year post procedure. Patient recovered baseline activity with satisfying health quality of life. [121] 20 patients (24–79 years; 13 males, seven females) Stent-supported aortic allograft Cryopreserved aortic graft and custom-made nitinol stent NA Patients presenting proximal lung tumours and malignant or benign lesions of the trachea and bronchi. Radial tumour resections were performed and the stent-supported graft was implanted and covered circumferentially with a local muscle flap. Patient follow-up of 90 days detecting a 5% mortality rate. No adverse effect of the surgical technique used showing a 76.5% survival at a median follow-up of 3 years and 11 months. Regeneration of cartilage and respiratory epithelium observed. [123] TE approaches 26-year-old male Decellularisation Decellularised porcine jejunum containing autologous cell population Recipient's mvECs and skMCs Patient suffering from extensive tracheal and oesophageal defect. Porcine cell-free vascularised scaffolds were obtained through a decellularisation process and seeded to ensure re-endothelialisation with recipient's cell prior to implantation into the 5×2 cm defect. The construct was characterised prior to implantation to ensure safety and maximum performance. The postoperative period was uneventful and the transplanted bioengineered construct was fully integrated presenting a fully functional respiratory epithelium on the lining of the airway without tissue scar formation or tissue dedifferentiation. [134] 12-year-old male Decellularisation Human decellularised trachea containing autologous stem-cell populations Recipient's CD34 or CD45wk haematopoietic stem cells and CD73+, CD90+, CD105+, CD117+ or CD45+ MSCs Patient born with long-segmental tracheal stenosis and pulmonary sling. A decellularised cadaveric trachea was saturated with the recipient's stem cell suspension and implanted coupled with a PDO stent. The construct was saturated with hrEPO, G-CSF and TGF-β. The graft was vascularised 1 week following surgery, but epithelium restoration was not seen until 1 year post-implantation. Biomechanical strength was not appropriate until 18 months. Functional airway found at 2 years follow-up. Patient free from medical intervention in a 4-year follow-up study. [135, 136] 15-year-old female Decellularisation Human decellularised trachea containing autologous stem-cell populations Recipient's BM-MSCs and nasal-derived epithelial cells Patient presenting critical tracheal stenosis. A decellularised cadaveric trachea was pre-seeded in vitro in a custom-made bioreactor before implantation. Patency of the graft and intact anastomoses following implantation. Tracheal graft narrowing was observed 13 days after surgery leading to ventilator compromise and respiratory arrest, which finally resulted in cerebral hypoxic injury and oedema. [137] 78-year-old female TE scaffold Marlex mesh tube covered with a collagen type I and III (porcine dermal atelocollagen) sponge NA Patient showing right lobe thyroid gland tumour and consequent tracheal invasion. The construct was implanted and injected with autologous venous blood for air-tightness, water-tightness and endogenous factors release. The bioengineered construct supported the epithelial growth 2 months after surgery, proper epithelialisation was observed 7 months later and was completely covered with respiratory epithelium 20 months after surgery. Epithelialisation continued to cover the trachea for 2 years showing no complication. [138] Four patients (59–78 years; two male, two female) TE scaffold Marlex mesh tube covered with a collagen type I and III (porcine dermal atelocollagen) sponge NA Patients with history of tracheotomy, stenosis and thyroid cancer. The construct was implanted and injected with autologous venous blood. Observation period of 8–34 months showed sufficient epithelialisation of the construct without obstructions. [139] Three patients (39–71 years; one male, two female) TE scaffold Marlex (PP) mesh tube covered with a collagen type I and III (porcine dermal atelocollagen) sponge NA Patients suffering from stenosis of the trachea and/or cricoids caused by endotracheal intubations. Two-stage operation for the resection of the stenotic regions and implantation of the construct followed by delivery of venous blood and b-FGF to the cartilage defect. All patients were able to breath after implantation and showed no discomfort in daily activities. Enough air space in the trachea was observed 6 months after implantation. [140] Infant (2 months old) 3DP PCL NA Patient suffering from TBM. A custom-designed and custom-fabricated resorbable 3DP airway splint was implanted and predicted to be fully resorbed within 3 years. 21 days after the procedure ventilator support was ended and patient was discharged. 1 year after surgery a patent left mainstem bronchus was observed and no unforeseen problems related to the splint arose. [141] Three infants (3–16 months; three males) 3DP PCL NA Patients suffering from TBM. 3DP splints were customised for each patients needs using CT imaging and CAD modelling software. At the time of publication, all patients did not show signs of airway disease showing continued growth of the primary airways. [78] 46-year-old female 3DP PCL NA Patient with TBM. 3DP splint designed using CT imaging and modelling software was implanted wrapped in an artificial pleural patch. 2 weeks after surgery the patient was discharged from hospital with increased tracheal inner diameter and cross-sectional area. Breathing and physical strength improvement in a 3-month follow-up. No adverse reaction or toxicity was found. [142] Young female 3DP PEKK NA Patient suffering from acquired TBM. A PEKK splint was design using CT imaging and modelling software. PEKK was chosen over PCL for its superior properties and suitability for manufacturing. Restoration of tracheal patency and patient remains asymptomatic, no further complications or hospitalisations were recorded. [143] 15 patients (3–25 months; six male, nine female) 3DP PCL NA Patients suffering from TBM and airway erosion. 3DP split designed using CT imaging and modelling software. A total of 29 splints (10 tracheal, 12 left bronchus and seven right bronchus) were implanted with the help of PP sutures. No patients required splint removal or re-operation. Three mortalities were recorded. At 8.5-month follow-up, 11 patients were living at home. [77] NA: not applicable; mvEC: microvascular endothelial cell; skMC: skeletal muscle cell; MSC: mesenchymal stem cells; PDO: polydioxanone; hrEPO: human recombinant erythropoietin; G-CSF: granulocyte-colony stimulating factor; TGF-β: transforming growth factor-β; BM-MNC: bone marrow mononuclear cell; PP: polypropylene; b-FGF: basic fibroblast growth factor; PCL: polycaprolactone; TBM: tracheobronchomalacia; 3DP: 3D printing-2; CT: computed tomography: CAD: computer-aided design; PEKK: polyetherketoneketone.
Vol 30 Issue 162
Table of Contents
Development and clinical translation of tubular constructs for tracheal tissue engineering: a review
