Electrospinning of poly (ester amide) fibres for mesenchymal progenitor cell differentiation

The in vitro vascular tissue engineering paradigm seeks to produce biologically responsive vascular substitutes using cells, biodegradable scaffolds, and bioreactors to mature the tissue for the potential treatment of vascular occlusions and to create 3D tissue models for pre-clinical testing. In this work, a poly (ester amide) (PEA) derived from from L-phenylalanine, sebacoyl chloride and 1,4 butanediol was synthesized and electrospun to form both 3D fibrous mats and tubular constructs. Both the polymer solution concentration and mandrel rotation speed were optimized to fabricate bead-free fibres. Cytocompatibility and proliferation studies using mesenchymal progenitor 10T1/2 cells showed PEA fibres were not cytotoxic and were able to support proliferation for 14 days. 10T1/2 cells demonstrated increased attachment and spreading for up to 7 days on fibrous mats but perfusion bioreactor studies on tubular scaffolds did not demonstrate sufficient cell infiltration. 10T1/2 cell differentiation studies using qPCR and Western blot showed a TGFβ1 induced upregulation in both the gene and protein expression of vascular smooth muscle cell (VSMC) specific markers smooth muscle alpha-actin (SM- a-actin) and smooth muscle myosin heavy chain (SM-MHC) on PEA fibres, with the differentiation further confirmed using immunofluorescence staining. Overall, this in vitro model of 10T1/2 cell differentiation may serve as a potential platform to fabricate small-diameter tissue engineered vascular grafts

Stiffness memory of 3D-TIPS elastomer nanohybrid scaffolds for biologically responsive bespoke tracheal implants

Advancements in materials science and 3D printing have inspired the development of bespoke stimuli responsive scaffolds as an attempt to handle challenging issues in tracheal tissue engineering, especially epithelialization and re-vascularization. Poly(urea-urethane)-polyhedral oligomeric silsequioxane (PUU-POSS) elastomers were selected for their appealing mechanical properties and in vitro responses with several cell lines. However, manufacturing PUU-POSS into 3D tracheal structures using conventional printing techniques remains challenging. In this thesis, a reverse 3D printing technique, based on controlled thermally-induced phase separation (TIPS) (3D-TIPS) of a PUU-POSS nanohybrid polymer solution and microphase separation of soft and hard segments of PUU-POSS, was developed to manufacture a wide range of soft elastomer scaffolds with hierarchically porous structure and tuneable stiffness. The dynamic changes of structure, mechanical properties and cellular responses to those scaffolds in vitro and in vivo were systematically characterized. The thermoresponsive stiffness softening of the scaffold was observed at body temperature, which is near the crystal-to-rubber phase transition of the soft segments of PUU-POSS. A potential application of a synthetic trachea based on the 3D-TIPS scaffolds was demonstrated. A successful submucosal tissue analogue of the trachea has been developed based on the multi-layered co-culture of human bronchial epithelial cells (hBEpiCs), human bronchial fibroblast cell (hBFs) or human bone-marrow derived mesenchymal stem cells (hBM-MSCs) supported by collagen hydrogel impregnated the scaffolds as matrix, reminiscent of the native tracheobronchial epithelium architecture. Furthermore, cellular responses of using human dermal fibroblasts (HDFs) and hBM-MSCs on the scaffolds and rat animal model proved the different roles of the hierarchical porous structure, initial stiffness and stiffness softening in modulating cell growth and differentiation, tissue ingrowth and vascularization. Overall, thermoresponsive biomimetic scaffolds by 3D-TIPS hold promise for personalized and biologically responsive soft tissue implants and implantable device with better mechanical matches, angiogenesis and tissue integration