Three-dimensional phase separation for the manufacture of elastomer scaffolds in soft tissue engineering

摘要

Introduction: 3D-printing holds promising potential for the manufacture of patient specific implants and tissue-regeneration scaffolds. Despite this, limitations in the choice of printable materials, and a lack of capacity of 3D-prinlers to produce structures with hierarchical porosity, often results in printed structures which lack the ability to mimic the intricate structure of extracellular matrix (ECM) In this study, we propose a novel 3D manufacturing technique, which produced digitally controlled soft scaffolds from Polyhedral Oligomeric Silsesquioxane grafted Poly(carbonate-urea)urethane (POSS-PCUU), a solution based polymer nanocomposite. Through the manipulation of temperature controlled phase-separation of this block-copolymer nanocomposite, confined within a sacrificial 3D-printed preform, we were able to produce a range of polymer constructs from the same polymer solution, each possessing distinctly different mechanical and cell-guiding properties. These constructs maintained their complex external architectures, whilst containing a completely interconnected internal porous structure, with multi-level hierarchical porosities. Additionally auricular and nasal constructs (from patient data) were fabricated successfully using our technique as model structures. Methods: PVApre-form shells were translated from 3D data using a fusion deposition modelling 3D printer. They were filled with POSS-PCUU, and subjected to phase-separation conditions at different temperatures. The structure and mechanical properties of the scaffolds were characterised. In vitro cell culture was conducted using human dermal ftbroblasts (HDF), with metabolic activity measured using alamar blue and total DNA quantification at days 1,3,5,7 and 10. Confocal microscopy was used to obtain cell-scaffold images by live/dead fluorescence assay and immunofluorescent staining respectively. Finally cell-scaffold samples were harvested at different time points and subjected to SEM and histological analysis. Results: We fabricated patient-specific POSS-PCUU soft constructs, with controllable and interconnected macro-levels of porosity. Simultaneously, through temperature controlled phase separation, the self-assembly of the POSS-PCUU nano-phase structure was also manipulated. The controlled crystallisation that was produced during the micro-phase transition of our nanocomposite, imparted the greatest biomechanical properties upon scaffold structure. Moreover, the crystalline structures produced through temperature controlled phase separation demonstrated the greatest infinity for HDF attachment and three-dimensional proliferation in vitro. Discussion: To our knowledge, the dual-level regulation of porosity demonstrated by our technique, where design is used to control macro- to micro scale changes; and phase-separation used to govern nano-scale structures, has not been described previously. It also permits immense flexibility in the architecture of fabricated constructs, tunability that is not presently achievable through phase-separation or current direct printing techniques alone. This low cost method, with short-lead time, shows capability of shaping a variety of otherwise unprintable degradable and non-degradable polymers with their nanocomposites, into such highly interconnected 3D constructs at multiscale.