Development and clinical translation of tubular constructs for tracheal tissue engineering: a review

Effective restoration of extensive tracheal damage arising from cancer, stenosis, infection or congenital abnormalities remains an unmet clinical need in respiratory medicine. The trachea is a 10–11 cm long fibrocartilaginous tube of the lower respiratory tract, with 16–20 tracheal cartilages anterolaterally and a dynamic trachealis muscle posteriorly. Tracheal resection is commonly offered to patients suffering from short-length tracheal defects, but replacement is required when the trauma exceeds 50% of total length of the trachea in adults and 30% in children. Recently, tissue engineering (TE) has shown promise to fabricate biocompatible tissue-engineered tracheal implants for tracheal replacement and regeneration. However, its widespread use is hampered by inadequate re-epithelialisation, poor mechanical properties, insufficient revascularisation and unsatisfactory durability, leading to little success in the clinical use of tissue-engineered tracheal implants to date. Here, we describe in detail the historical attempts and the lessons learned for tracheal TE approaches by contextualising the clinical needs and essential requirements for a functional tracheal graft. TE manufacturing approaches explored to date and the clinical translation of both TE and non-TE strategies for tracheal regeneration are summarised to fully understand the big picture of tracheal TE and its impact on clinical treatment of extensive tracheal defects.

Current Strategies for Tracheal Replacement: A Review

Airway cancers have been increasing in recent years. Tracheal resection is commonly performed during surgery and is burdened from post-operative complications severely affecting quality of life. Tracheal resection is usually carried out in primary tracheal tumors or other neoplasms of the neck region. Regenerative medicine for tracheal replacement using bio-prosthesis is under current research. In recent years, attempts were made to replace and transplant human cadaver trachea. An effective vascular supply is fundamental for a successful tracheal transplantation. The use of biological scaffolds derived from decellularized tissues has the advantage of a three-dimensional structure based on the native extracellular matrix promoting the perfusion, vascularization, and differentiation of the seeded cell typologies. By appropriately modulating some experimental parameters, it is possible to change the characteristics of the surface. The obtained membranes could theoretically be affixed to a decellularized tissue, but, in practice, it needs to ensure adhesion to the biological substrate and/or glue adhesion with biocompatible glues. It is also known that many of the biocompatible glues can be toxic or poorly tolerated and induce inflammatory phenomena or rejection. In tissue and organ transplants, decellularized tissues must not produce adverse immunological reactions and lead to rejection phenomena; at the same time, the transplant tissue must retain the mechanical properties of the original tissue. This review describes the attempts so far developed and the current lines of research in the field of tracheal replacement.

Vascularization strategies for tissue engineering for tracheal reconstruction

Tissue engineering technology provides effective alternative treatments for tracheal reconstruction. The formation of a functional microvascular network is essential to support cell metabolism and ensure the long-term survival of grafts. Although several tracheal replacement therapy strategies have been developed in the past, the critical significance of the formation of microvascular networks in 3D scaffolds has not attracted sufficient attention. Here, we review key technologies and related factors of microvascular network construction in tissue-engineered trachea and explore optimized preparation processes of vascularized functional tissues for clinical applications.

Scaffold-free tracheal cartilage engineering using roller bottle culture

Resection with primary anastomosis can only repair up to 50% of the adult trachea and up to 30% of the pediatric trachea when damaged. There is a strong clinical need for long-segment tracheal replacements. The goal of this research was to create a seamless, scaffold-free cartilage cylinder for tracheal tissue engineering in vitro. Primary bovine articular chondrocytes were seeded onto tracheal moulds for roller bottle culture and the effect of rotational speed, growth factor supplementation, and chondrocyte layering were investigated. After the 4-week culture period, samples were evaluated biochemically, histologically, and biomechanically. The results indicated that rotation was necessary for full tissue coverage, with slower rotational speeds generating thicker tissue with an improved extracellular matrix, IGF-1 supplementation generating thicker tissue rich in glycosaminoglycans with inferior mechanical properties, and chondrocyte layering producing thinner tissue with increased mechanical properties. Overall, scaffold-free tissue engineering can generate seamless cylindrical cartilage constructs using roller bottle culture for future applications in long-segment tracheal replacement.

Scaffold-free tracheal cartilage engineering using roller bottle culture

Resection with primary anastomosis can only repair up to 50% of the adult trachea and up to 30% of the pediatric trachea when damaged. There is a strong clinical need for long-segment tracheal replacements. The goal of this research was to create a seamless, scaffold-free cartilage cylinder for tracheal tissue engineering in vitro. Primary bovine articular chondrocytes were seeded onto tracheal moulds for roller bottle culture and the effect of rotational speed, growth factor supplementation, and chondrocyte layering were investigated. After the 4-week culture period, samples were evaluated biochemically, histologically, and biomechanically. The results indicated that rotation was necessary for full tissue coverage, with slower rotational speeds generating thicker tissue with an improved extracellular matrix, IGF-1 supplementation generating thicker tissue rich in glycosaminoglycans with inferior mechanical properties, and chondrocyte layering producing thinner tissue with increased mechanical properties. Overall, scaffold-free tissue engineering can generate seamless cylindrical cartilage constructs using roller bottle culture for future applications in long-segment tracheal replacement.

3D Printed Biomimetic PCL Scaffold as Framework Interspersed With Collagen for Long Segment Tracheal Replacement

The rapid development of tissue engineering technology has provided new methods for tracheal replacement. However, none of the previously developed biomimetic tracheas exhibit both the anatomy (separated-ring structure) and mechanical behavior (radial rigidity and longitudinal flexibility) mimicking those of native trachea, which greatly restricts their clinical application. Herein, we proposed a biomimetic scaffold with a separated-ring structure: a polycaprolactone (PCL) scaffold with a ring-hollow alternating structure was three-dimensionally printed as a framework, and collagen sponge was embedded in the hollows amid the PCL rings by pouring followed by lyophilization. The biomimetic scaffold exhibited bionic radial rigidity based on compressive tests and longitudinal flexibility based on three-point bending tests. Furthermore, the biomimetic scaffold was recolonized by chondrocytes and developed tracheal cartilage in vitro. In vivo experiments showed substantial deposition of tracheal cartilage and formation of a biomimetic trachea mimicking the native trachea both structurally and mechanically. Finally, a long-segment tracheal replacement experiment in a rabbit model showed that the engineered biomimetic trachea elicited a satisfactory repair outcome. These results highlight the advantage of a biomimetic trachea with a separated-ring structure that mimics the native trachea both structurally and mechanically and demonstrates its promise in repairing long-segment tracheal defects.

Regeneration of partially decellularized tracheal scaffolds in a mouse model of orthotopic tracheal replacement

Decellularized tracheal scaffolds offer a potential solution for the repair of long-segment tracheal defects. However, complete decellularization of trachea is complicated by tracheal collapse. We created a partially decellularized tracheal scaffold (DTS) and characterized regeneration in a mouse model of tracheal transplantation. All cell populations except chondrocytes were eliminated from DTS. DTS maintained graft integrity as well as its predominant extracellular matrix (ECM) proteins. We then assessed the performance of DTS in vivo. Grafts formed a functional epithelium by study endpoint (28 days). While initial chondrocyte viability was low, this was found to improve in vivo. We then used atomic force microscopy to quantify micromechanical properties of DTS, demonstrating that orthotopic implantation and graft regeneration lead to the restoration of native tracheal rigidity. We conclude that DTS preserves the cartilage ECM, supports neo-epithelialization, endothelialization and chondrocyte viability, and can serve as a potential solution for long-segment tracheal defects.