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.