Tubular structure tissue engineering: design and validation of a novel multifunctional rotating bioreactor

Over the last 60 years, transplantation of tissues and organs, re-constructive surgical techniques, and replacement with artificial devices have significantly enhanced patient life expectance and life-style. Unfortunately, these solutions suffer from many limitations, such as donor shortage and lifelong immunosuppressive assumption, increased risk of infections, unwanted side effects, and, in some cases, finite durability1,2. This scenario let to an increasing interest in the field of tissue engineering, which merges engineering and life sciences knowledge with the final goal to develop in vitro cellularized functional substitutes able to restore or improve tissue and organ activities3,4. The three most important ingredients of tissue engineering are biomaterials, cells collected from a patient and proper environmental culture conditions (i.e., a bioreactor). In summary, a porous delivery system is needed that confines the cells to the desired location, after in vitro mechanical stimulation5. Significant progress has already been made in the field and examples of successful clinical implants of tissue-engineered products include skin substitutes6,7, nasal cartilage8, functioning bladder9 and trachea10. Whilst these are promising results, much effort is still required in vitro, to elucidate basic mechanisms regulating cell response, and the behaviour of the engineered construct during maturation4,11, and in preclinical models, to investigate the host response (e.g., neovascularization, re-modelling)12, and the behaviour of the produced substitute once grafted. The presented research project, aimed to design, fabricate and characterize an innovative multifunctional bioreactor for the regeneration of hollow organs, able to overcome the limits of the current available devices. A prototype was produced able to perform rotation of a tubular scaffold along its longitudinal axis, to allow scaffold tensioning and to connect with different hydraulic circuit in a fast and trustworthy way. Bench test results demonstrated that a reliable and easily assembled device was developed. The main characteristics of the system were: 1) ease of handling, reducing risks of contamination; 2) versatilely connectable to different perfusion system.3) compatibility with the best standard of good laboratory practice. For the testing the bioreactor potential to produce a tissue engineered tubular grafts, an innovative PCL/PLA-TMC based electrospun tubular scaffold was realized, and chemically and mechanically characterized. The three-dimensional matrix demonstrated mechanical properties comparable with native blood vessel tissue, presenting a promising candidate for vascular tissue regeneration. PCL/PLA-TMC matrix and the developed bioreactor recreated a suitable 3D environment for mesenchymal stem cells growth and differentiation. The results confirmed that 3D dynamic culture allowed for a better control over the cell fate and behaviour by facilitating mass transfer phenomena, by facilitating the medium to flow through the scaffold wall. Moreover, the transmural flow favoured cell migration trough the thickness of the tubular matrix, permitting extracellular matrix formation and deposition along all the structure. Whilst the scaffold showed both favourable mechanical properties as well as a structure, which facilitated cell colonisation for the eventual formation of a tunica media, the ability of the inner layer to support growth of cells that would form a tunica intima was not tested. With further refinement of scaffold production, it is likely that endothelial colonisation of the inner layer will be possible. This would then permit more comprehensive testing within the bioreactor, and a demonstration of the full versatility of the developed system. With the dual chamber organisation of the bioreactor, growth conditions for the development of smooth muscle and endothelial tissue layers can be independently optimised. We can state that this research led to the production of both an innovative device for tubular organs regeneration, and the characterisation of a novel scaffold for potential use in vascular grafts. With the modularity of the bioreactor along with a relative ease of use, the device holds great potential for future production of tissue engineered tubular grafts.