Cian O'LEary

Effective tracheal tissue engineering benefits from scaffolds that mimic the native structure of the tissue, provide mechanical stability, and support spatially controlled cell seeding to encourage tissue regeneration. This study presents a novel approach for fabricating tubular scaffolds for tracheal regeneration that integrates a 3D-printed polycaprolactone (PCL) backbone with a freeze-dried collagen-hyaluronic acid (CHyA) layer. Two scaffold geometries (tubular and c-shaped) were produced and mechanically characterized, and it was demonstrated that PCL reinforcement significantly enhanced scaffold structural robustness and durability. To achieve spatially selective cell seeding, custom-designed PLA accessories facilitated the precise deposition of respiratory epithelial cells (Calu-3) onto the inner layer and lung-derived fibroblasts (Wi38) onto the outer layer of the scaffolds. Monoculture experiments showed successful cell localization, while sequential seeding established an effective coculture system with enhanced epithelial coverage and sustained fibroblast viability. This study validates a scalable and customizable method for manufacturing mechanically robust tubular scaffolds with precise spatial cell organization, providing a promising platform for tracheal tissue engineering and potentially other tubular applications such as vascular or gastrointestinal regeneration. Future work will focus on validating this method with primary human cells, incorporating air-liquid interface cultures to enhance epithelial differentiation, and scaling up the constructs to anatomically relevant sizes to advance clinical translation.

Introduction: Attempted tracheal replacement efforts thus far have had very little success. Major limiting factors have been the inability to efficiently re-vascularise and mimic the mechanical properties of native tissue. The major objective of this study was to optimise a previously developed collagen-hyaluronic acid scaffold (CHyA-B), which has shown to facilitate the growth of respiratory cells in distinct regions, as a potential tracheal replacement device.Methods: A biodegradable thermoplastic polymer was 3D-printed into different designs and underwent multi-modal mechanical assessment. The 3D-printed constructs were incorporated into the CHyA-B scaffolds and subjected to in vitro and ex vivo vascularisation.Results: The polymeric backbone provided sufficient strength to the CHyA-B scaffold, with yield loads of 1.31–5.17 N/mm and flexural moduli of 0.13–0.26 MPa. Angiogenic growth factor release (VEGF and bFGF) and angiogenic gene upregulation (KDR, TEK-2 and ANG-1) was detected in composite scaffolds and remained sustainable up to 14 days. Confocal microscopy and histological sectioning confirmed the presence of infiltrating blood vessel throughout composite scaffolds both in vitro and ex vivo.Discussion: By addressing both the mechanical and physiological requirements of tracheal scaffolds, this work has begun to pave the way for a new therapeutic option for large tracheal defects.

Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell^® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell^® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell^® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.

Although a large panel of natural antioxidants demonstrate a protective effect in preventing cellular oxidative stress, their low bioavailability limits therapeutic activity at the targeted injury site. The importance to deliver drug or cells into oxidative microenvironments can be realized with the development of biocompatible redox-modulating materials. The incorporation of antioxidant compounds within implanted biomaterials should be able to retain the antioxidant activity, while also allowing graft survival and tissue recovery. This review summarizes the recent literature reporting the combined role of natural antioxidants with biomaterials. Our review highlights how such functionalization is a promising strategy in tissue engineering to improve the engraftment and promote tissue healing or regeneration.

Although relatively rare, major trauma to the tracheal region of the airways poses a significant clinical challenge with few effective treatments. Bioengineering and regenerative medicine strategies have the potential to create biocompatible, implantable biomaterial scaffolds, with the capacity to restore lost tissue with functional neo-trachea. The main goal of this study was to develop a nanofibrous polycaprolactone-chitosan (PCL-Chitosan) scaffold loaded with a signaling molecule, all-trans retinoic acid (atRA), as a novel biomaterial approach for tracheal tissue engineering. Using the Spraybase^® electrospinning platform, polymer concentration, solvent selection, and instrument parameters were optimized to yield a co-polymer with nanofibers of 181–197 nm in diameter that mimicked tracheobronchial tissue architecture. Thereafter, scaffolds were assessed for their biocompatibility and capacity to induce mucociliary functionalization using the Calu-3 cell line. PCL-Chitosan scaffolds were found to be biocompatible in nature and support Calu-3 cell viability over a 14 day time period. Additionally, the inclusion of atRA did not compromise Calu-3 cell viability, while still achieving an efficient encapsulation of the signaling molecule over a range of atRA concentrations. atRA release from scaffolds led to an increase in mucociliary gene expression at high scaffold loading doses, with augmented MUC5AC and FOXJ1 detected by RT-PCR. Overall, this scaffold integrates a synthetic polymer that has been used in human tracheal stents, a natural polymer generally regarded as safe (GRAS), and a drug with decades of use in patients. Coupled with the scalable nature of electrospinning as a fabrication method, all of these characteristics make the biomaterial outlined in this study amenable as an implantable device for an unmet clinical need in tracheal replacement.

Within the past 25 years, tissue engineering (TE) has grown enormously as a science and as an industry. Although classically concerned with the recapitulation of tissue and organ formation in our body for regenerative medicine, the evolution of TE research is intertwined with progress in other fields through the examination of cell function and behaviour in isolated biomimetic microenvironments. As such, TE applications now extend beyond the field of tissue regeneration research, operating as a platform for modifiable, physiologically-representative in vitro models with the potential to improve the translation of novel therapeutics into the clinic through a more informed understanding of the relevant molecular biology, structural biology, anatomy, and physiology. By virtue of their biomimicry, TE constructs incorporate features of extracellular macrostructure, molecular adhesive moieties, and biomechanical properties, converging with computational and structural biotechnology advances. Accordingly, this mini-review serves to contextualise TE for the computational and structural biotechnology reader and provides an outlook on how the disciplines overlap with respect to relevant advanced analytical applications.

In a recent report, the World Health Organisation (WHO) classified antibiotic resistance as one of the greatest threats to global health, food security, and development. Methicillin-resistant Staphylococcus aureus (MRSA) remains at the core of this threat, with persistent and resilient strains detectable in up to 90% of S. aureus infections. Unfortunately, there is a lack of novel antibiotics reaching the clinic to address the significant morbidity and mortality that MRSA is responsible for. Recently, nanomedicine strategies have emerged as a promising therapy to combat the rise of MRSA. However, these approaches have been wide-ranging in design, with few attempts to compare studies across scientific and clinical disciplines. This review seeks to reconcile this discrepancy in the literature, with specific focus on the mechanisms of MRSA infection and how they can be exploited by bioactive molecules that are delivered by nanomedicines, in addition to utilisation of the nanomaterials themselves as antibacterial agents. Finally, we discuss targeting MRSA biofilms using nano-patterning technologies and comment on future opportunities and challenges for MRSA treatment using nanomedicine.

Clinical interventions for extensive tissue injury to the larger airways remain limited. Recently, respiratory tissue engineering strategies have emerged with a variety of biomimetic materials and tissue constructs to address these limitations, though rapid epithelialization of the construct with mucociliary function is still largely unresolved. The overall objective of this study was to manufacture an all-trans retinoic acid (atRA)-loaded bilayered collagen-hyaluronate (atRA-B) scaffold as a platform technology for tracheal tissue regeneration. atRA-loaded scaffolds were fabricated using a customized lyophilization process and characterized for drug loading and release properties using HPLC, followed by validation of their bioactivity using human primary tracheobronchial epithelial cells. atRA-loaded materials were reproducibly manufactured and exhibited the release of atRA following their hydration over 8–28 h that was significantly affected by collagen cross-linking. An optimal formulation consisting of 10 μg/mL atRA in a collagen-hyaluronate suspension to manufacture the scaffold film layer was identified and used to develop the atRA-B scaffold. Immunofluorescence studies and RT-PCR revealed that the atRA-loaded biomaterials increased the expression of two epithelial markers of mucociliary differentiation, MUC5AC and β-tubulin IV, via upregulation of MUC5AC and FOXJ1 genes, both in epithelial monoculture and in a 3D scaffold coculture system with lung fibroblasts. Overall, this study has demonstrated that the atRA-B scaffold can enhance functional epithelialization in primary tracheobronchial cells and can potentially pioneer the development of a novel and biocompatible device to address a currently unmet clinical need in tracheal replacement.

Currently, lung disease and major airway trauma constitute a major global healthcare burden with limited treatment options. Airway diseases such as chronic obstructive pulmonary disease and cystic fibrosis have been identified as the fifth highest cause of mortality worldwide and are estimated to rise to fourth place by 2030. Alternate approaches and therapeutic modalities are urgently needed to improve clinical outcomes for chronic lung disease. This can be achieved through tissue engineering of the respiratory tract. Interest is growing in the use of airway tissue-engineered constructs as both a research tool, to further our understanding of airway pathology, validate new drugs, and pave the way for novel drug therapies, and also as regenerative medical devices or as an alternative to transplant tissue. This review provides a concise summary of the field of respiratory tissue engineering to date. An initial overview of airway anatomy and physiology is given, followed by a description of the stem cell populations and signaling processes involved in parenchymal healing and tissue repair. We then focus on the different biomaterials and tissue-engineered systems employed in upper and lower respiratory tract engineering and give a final perspective of the opportunities and challenges facing the field of respiratory tissue engineering.