Chapter X
10 Overall Conclusive Remarks
The present PhD thesis comprises the implementation of different experimental activities performed at the Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOLab) of the Department of Chemistry and Industrial Chemistry of the University of Pisa and those developed during a six month period spent at the Tissue Regeneration group of the Mira Institute of the University of Twente (Enschede, The Netherlands) within the framework of a Tuscany Region funded project on “Development of novel micro-manufactured biological and biohybrid prostheses for middle ear surgery”.
10.1 Development of Advanced Technologies for Manufacturing of Tissue Engineering Scaffolds
A significant part of this PhD research activity was dedicated to the development of advanced technologies for the manufacturing of scaffolds for tissue engineering. A computer-controlled melt extrusion-based additive manufacturing (AM) equipment was developed and combined with electrospinning technique for the fabrication of dual-scale micro/nanostructured scaffolds. Further modifications to the equipment were performed in order to produce melt-electrospun three- dimensional (3D) scaffolds by AM and to study the feasibility for the automated production of wet- spinning scaffolds with a layer-by-layer approach. Following this preliminary study, a new computer-controlled wet-spinning apparatus was specifically developed for the production of scaffolds by solution extrusion-based AM.
10.2 Dual-Scale Polymeric Constructs as Scaffolds for Tissue Engineering
During this research activity the development of dual-scale scaffold structures was investigated in order to couple the mechanical strength and structural reproducibility of 3D poly(ε-caprolactone) (PCL) microfilament constructs, fabricated by a melt extrusion-based AM technique, with the advantage of electrospun poly(lactic-co-glycolic acid) (PLGA) ultrafine fibres in enhancing cell/scaffold interactions. Dual-scale scaffolds composed of layers of parallel microsized filaments (0/90° lay-down pattern) of PCL, with a diameter of around 365 µm and interfilament distance of
around 191 µm, and PLGA electrospun fibres with a diameter of around 1 µm collected on top of the PCL constructs were developed. Cell culture experiments employing the MC3T3-E1 murine preosteoblast cell line showed good cell viability and adhesion on the dual-scale scaffolds. In particular, the influence of electrospun fibres on cell morphology and behaviour was evident, as well as in creating a structural bridging for cell colonization in the interfilament gap.
10.3 Development of 3D Wet-spun Polymeric Scaffolds Loaded with Antimicrobial Agents for Bone Engineering
Polymer scaffolds that are able to release antibiotics in a controlled manner represent an innovative therapeutic strategy for the regeneration of bone defects in the presence of infections, such as osteomyelitis. During this experimental work, 3D microfibrous non-woven scaffolds made of a star poly(ε-caprolactone) (*PCL) and fabricated by wet-spinning were developed.. Wet-spinning processing conditions were investigated in order to optimize scaffold microstructure and to endow the scaffold with antimicrobial activity by loading the microfibres with either levofloxacin or enrofloxacin. In vitro drug release kinetics from the meshes evaluated under physiological conditions, showed an initial burst release of the loaded antibiotic followed by a controlled release up to forty days. In vitro cell culture characterization with the MC3T3-E1 murine preosteoblast cell line showed good cell viability and adhesion on the wet-spun *PCL fibre scaffolds. These promising results indicate a potential application of the developed meshes as engineered bone scaffolds with antimicrobial activity.
10.4 Additive Manufacturing of Wet-spun Polymeric Scaffolds for Bone Tissue Engineering
Techniques based on a layered manufacturing strategy represent an effective approach for the control at the microscale over the internal architecture, external shape and size of TE scaffolds. In this research activity, PCL and composite PCL/hydroxyapatite (HA) scaffolds were prepared by means of a computer-controlled wet-spinning technique exploiting AM principles. The processing conditions for the production of 3D polymeric scaffolds were optimised by studying their influence on fibres morphology and alignment. Two different scaffold architectures were designed and fabricated by tuning inter-fibre distance and fibres staggering. A good reproducibility of the internal microarchitecture and external shape was achieved. Mechanical characterization of the scaffolds showed that the compressive modulus and strength were significantly influenced by
scaffold architecture and HA loading. Cell culture experiments employing the MC3T3-E1 preosteoblast cell line showed good adhesion, proliferation, alkaline phosphatase activity and bone mineralization of the cells throughout the whole scaffolds. The present study opens new possibilities for the fabrication of 3D structures with a layered manufacturing approach by employing other biodegradable polyesters that are well suited for wet-spinning processing.
10.5 Additive Manufacturing of Star Poly(ε-caprolactone) Wet- spun Scaffolds for Tissue Engineering Applications
Star polymers consist of linear polymer chains attached to a smaller central moiety and have different properties compared to the linear polymers with equivalent molecular weight. A three-arm
*PCL has been investigated by our group for TE purposes in the form of electrospun or wet-spun non-woven meshes showing promising results when tested in cell culture experiments using preosteoblast cells. The present study was aimed at the development of 3D *PCL and composite
*PCL/HA wet-spun scaffolds with a controlled and predefined internal structure and external shape by means of a novel computer-controlled wet-spinning apparatus. The influence of different processing parameters, such as polymer concentration, solution feed rate and deposition velocity, on manufacturing process and scaffold architecture was investigated. Scaffolds with different porosity, pore size and fibre diameter were achieved. Moreover, the fibre constituting the scaffold showed a porous morphology both in the outer surface and in the cross-section. Cell culture experiments performed using a murine preosteoblasts cell line showed good cell response in terms of proliferation. The obtained results suggest the suitability of this new apparatus for the production of customised scaffolds for bone tissue engineering.
10.6 Additive Manufacturing of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) Scaffolds for Engineered Bone
Development
Polyhydroxyalkanoates (PHAs), a class of biodegradable polyesters produced by many bacteria grown under unbalanced conditions, have been proposed in the last years for the fabrication of Tissue Engineering scaffolds due to their biocompatibility. In this study, the production of 3D scaffolds made of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), an elastomeric polyester belonging to the class of PHAs, by means of a novel computer-controlled wet-spinning
apparatus was investigated. Processing parameters such as, solution feed rate, deposition velocity and inter-layer needle translation, were investigated and, by applying the optimised parameters, 3D scaffolds with different architecture were produced following a layer-by-layer approach.
Morphological characterization by scanning electron microscopy showed a good control over fibre alignment and a fully interconnected network of pores. Scaffolds compressive modulus, yield compression stress and strain varied according to the produced lay-down pattern and interfibre distance. Cell culture experiments employing the MC3T3-E1 murine preosteoblast cell line showed good cell adhesion, proliferation and alkaline phosphatase activity. The results achieved indicate the suitability of the developed PHBHHx constructs as scaffolds for soft and non-load bearing TE applications.
10.7 Additive Manufacturing of 3D Melt-electrospun Star Poly(ε- caprolactone) Scaffolds
Melt-electrospinning (melt-ES) technique has gained attention for the production of highly porous microfibrous scaffolds for TE applications because of the possibility to avoid the use of organic solvents. In this study, the processing of linear PCL and *PCL using a computer-aided melt- electrospinning (melt-ES) system equipped with a screw-extruder head was investigated. Different processing parameters, such as processing temperature, extrusion flow rate and applied voltage, were studied to fabricate non-woven meshes with uniform fibre morphology. By applying the optimised parameters, 3D scaffolds with a controlled external shape were produced with a layer- by-layer approach. *PCL scaffolds were characteed for their cytocompatibility using mouse embryo fibroblasts. Results showed that the produced scaffolds were able to support cell adhesion, proliferation and migration, preserving and increasing the ability of cells to produce collagen. The main result achieved during this study was the development of an AM technique for the fabrication of melt-electrospun scaffolds with a good control over fibre collection.
10.8 Development of Novel Scaffolds for Otologic Surgery Applications
Conductive hearing loss generally induces reduction or impairment of the auditory function at middle ear level representing one of the major hearing diseases and can be caused because of the TM perforations or the loss of function of the ossicular chain. In this research activity, AM and ES techniques were used to produce partial ossicular replacement prosthesis (PORP), posterior canal
wall and tympanic membrane scaffolds, made of poly[poly(ethylene oxide) terephthalate-co- butylene terephthalate], a biocompatible and biodegradable block copolymer. Scaffolds morphology was analysed by means of scanning electron microscopy and the PORP surface topography was examined by means of atomic force microscopy. A TE approach, combining PORP scaffolds and human mesenchymal stem cells (hMSCs), was investigated performing in vitro cell culture experiments. Qualitative analyses were performed to evaluate cell distribution on the scaffolds. Moreover, quantitative analyses showed a good cellularity level along the cell culture experiment and a good osteo-differentiation level of hMSCs. These preliminary results showed the potentialities of AM and ES techniques to produce middle ear scaffolds. Moreover, biological assessment showed the capability of the PORP scaffolds to support adhesion, proliferation and differentiation of hMSCs towards osteoblastic phenotype. The developed replacements can become promising alternatives to the prostheses currently used in otologic surgery.
