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Biomaterials for Tracheal Replacement in Age-related Cancer via a Humanly Engineered Airway

Periodic Report Summary 2 - BIOTRACHEA (Biomaterials for Tracheal Replacement in Age-related Cancer via a Humanly Engineered Airway)

Project Context and Objectives:
Primary tracheal cancers are neoplastic lesions of the airways with a high mortality rate, significantly impacting upon the lives and careers of thousands of patients each year throughout Europe. In almost all cases, surgical resection with primary reconstruction is regarded as the gold standard for treatment. However, as definitive diagnosis is often reached when more than 50% of the total tracheal length is affected by primary malignant disease, it is only possible to treat patients palliatively. For these patients prognosis is poor with a 5-year survival rate of about 5%. Even for patients with operable tumours, the proportion of complete tumour resection is less than 60%, due to limitations in performing the reconstruction of the trachea. The outcome for patients with primary tracheal cancers would be radically transformed if a trachea replacement with similar anatomical, physiological and biomechanical properties to those of the native trachea, could be available. This clinical need has given rise to the concept of manufacturing a tissue engineered trachea.
Constructing a tissue engineered trachea involves utilising a donor trachea which undergoes a decellularization process to remove all donor cells and antigens. This process should ideally provide a non-immunogenic, pro-angiogenic tracheal scaffold which also maintains its biomechanical properties. Subsequently, airway epithelium cells are seeded intra-luminally and mesenchymal stem cell-derived chondrocytes extraluminally to allow recellularization of the tracheal scaffold prior to its implantation in the patient in need of trachea replacement. An alternative approach to obtaining a trachea scaffold is to construct it using biosynthetic materials, which can mimic the properties of the native trachea. While this option can possibly provide a made-to-measure scaffold for each patient and does not rely on the availability of donor organs, meeting the physiochemical, structural and mechanical properties of the native trachea is a challenge.
The BIOtrachea EU consortium, consisting of 12 EU-based, clinical, academic and commercial participants brings together their expertise in Tissue Engineering, Airway Epithelium and Stem Cell Biology, Pharmacology, Stem Cell Mobilisation, Cell Therapy, Cellular and Molecular Immunology, Pre-clinical and Translational study Design, Radiobiology, Proteomics, Membrane bioreactors and Membrane technology, GMP manufacturing and development, Bioethics and commercialisation of bioreactors, in order to optimise the production and implantation of Tissue Engineered Tracheas. Once this is achieved, it will become possible to offer a curative treatment to trachea cancer patients who are currently only offered palliative therapy. Furthermore, it will be possible to apply the knowledge gained to Tissue Engineering protocols for other organs.
The overall objective of this programme is to both improve and optimise the approach using decellularized biological scaffold and to develop synthetic biomaterial scaffolds by using innovative next generation polymeric nanocomposite materials. At the same time, new pharmacological strategies will be developed to improve tracheal regeneration. The BIOtrachea consortium will ensure that the synergistic outputs of the project will far exceed the sum of each individual component.

The main objectives of the BIOtrachea programme are:
1. Iterative improvement of the procedure and widening of our clinical experience with tracheal implantation.
2. Tissue engineering and clinical methods development for airway regeneration using synthetic scaffolds.
3. To scale-up airway engineering methods and develop Good Manufacturing Practice (GMP) procedures for commercial production.
4. To develop airway tissue engineering as a model.

Project Results:
During P1 the consortium focussed on developing a number of platform technologies, developing the synthetic scaffold for in vitro and in vivo testing, performing in vitro assays to examine the efficacy of regenerative factors on the growth of human tracheal epithelial cells (HTEpC) and human mesenchymal stem cells (HMSC) in the context of inflammatory cytokines and establishing a bioethics committee. In vitro testing of the synthetic POSS-PCU was reported. An in vitro trauma model was established showing that specific regenerative pharmacological factors can increase the proliferation of HTEpC in the presence of trauma cytokines under both normoxic and hypoxic conditions. Experiments performed with HMSCs showed that G-CSF and TGFβ were similarly effective in stimulating proliferation of MSCs in the context of trauma and ischemia. A hollow organ bath system was developed and a bioreactor was designed.
The project second period (P2, 18 months) has focussed on the developing a number of procedures to optimize the processing of biological tracheas including their decelullarization, storage, and reseeding with HMSCs cells. A bioreactor has been developed to perform these operations in an automated fashion and SOPs have been established for harvesting MSCs. The cell metabolic rates have been determined, epithelial cells have been isolated from a human trachea biopsy, and two proteomic analysis have been performed. The delivery of synthetic scaffolds for testing in large animals has not been achieved but different partners have received synthetic scaffolds for in vitro, biomechanical, biocompatibility and toxicological studies. A total of 23 deliverables have been submitted and three milestones achieved. Thus ten WPs had deliverables during P2 and the work performed is summarized here. The SOP for decellularized scaffold production has been established and computer modelling has been used to study oxygen tensions across the trachea on the re-population of the decellularized trachea. SOPs have established for quality control and manufacturing methods of POSS-PCU under GMP and synthesis and conjugation of RGD peptides into POSS-PCU polymers have been accomplished. Protocols for harvesting HMSCs and seeding both the synthetic and biological tracheas have been established. Cell metabolic reaction rates on the tracheal constructs have been determined, and the biological response of MSCs to gamma irradiation has been reported. An automated, software-controlled bioreactor has been developed. The first human biopsy has been obtained. Culture of HTEpC has been successful, the cells preserving all their proliferative and differentiation capacity. An SOP for cell seeding onto scaffolds has been established. The safety of the natural trachea implantation has been demonstrated and proteomics has revealed different protein expression patterns in native and reseeded tracheal scaffolds. An inflammatory response has been observed in healthy lung tissue after irradiation accompanied by collagen deposition. A preclinical dossier for biological and synthetic tracheas has been compiled. Two databases have been fully implemented and the first generation transport and storage tools have been developed. While other WPs did not have any specified deliverables for P2 on-going work towards future deliverables has been performed. Regenerative factors encapsulated hydrogels are able to maintain the factors activity and can be used for construction of biotrachea and the prolonged presence can lead to significant increase in angiogenesis. Proteomic analysis of epithelial cells treated with two airway toxins revealed a difference in the protein patterns. Mesenchymal stem cells used to seed the trachea scaffold provide a strong stimulus to promote angiogenesis due to their ability to recruit angiogenic monocytes

Potential Impact:
The main driver behind the whole BIOtrachea programme is to combine basic, translational and clinical research in order to generate robust protocols for the construction and use of Bioengineered Tracheal Implants in patients with primary tracheal malignancies. These implants can facilitate a major leap in treatment of these patients who currently face a very poor prognosis even in cases where the disease is not metastatic. Due to the termination of the project after approximately two and half years the consortium will not be able to accomplish the final goal of the project that is to develop an airway tissue engineered trachea for clinical studies.

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