Dario Puppi

Additive manufacturing techniques for the production of tissue engineering constructs

‘Additive manufacturing’ (AM) refers to a class of manufacturing processes based on the building of a solid object from three‐dimensional (3D) model data by joining materials, usually layer upon layer. Among the vast array of techniques developed for the production of tissue‐engineering (TE) scaffolds, AM techniques are gaining great interest for their suitability in achieving complex shapes and microstructures with a high degree of automation, good accuracy and reproducibility. In addition, the possibility of rapidly producing tissue‐engineered constructs meeting patients specific requirements, in terms of tissue defect size and geometry as well as autologous biological features, makes them a powerful way of enhancing clinical routine procedures. This paper gives an extensive overview of different AM techniques classes (i.e. stereolithography, selective laser sintering, 3D printing, melt–extrusion‐based techniques, solution/slurry extrusion‐based techniques, and tissue and organ printing) employed for the development of tissue‐engineered constructs made of different materials (i.e. polymeric, ceramic and composite, alone or in combination with bioactive agents), by highlighting their principles and technological solutions. Copyright

Novel electrospun polyurethane/gelatin composite meshes for vascular grafts

Novel polymeric micro-nanostructure meshes as blood vessels substitute have been developed and investigated as a potential solution to the lack of functional synthetic small diameter vascular prosthesis. A commercial elastomeric polyurethane (Tecoflex® EG-80A) and a natural biopolymer (gelatin) were successfully co-electrospun from different spinnerets on a rotating mandrel to obtain composite meshes benefiting from the mechanical characteristics of the polyurethane and the natural biopolymer cytocompatibility. Morphological analysis showed a uniform integration of micrometric (Tecoflex®) and nanometric (gelatin) fibers. Exposure of the composite meshes to vapors of aqueous glutaraldehyde solution was carried out, to stabilize the gelatin fibers in an aqueous environment. Uniaxial tensile testing in wet conditions demonstrated that the analyzed Tecoflex®–Gelatin specimens possessed higher extensibility and lower elastic modulus than conventional synthetic grafts, providing a closer matching to native vessels. Biological evaluation highlighted that, as compared with meshes spun from Tecoflex® alone, the electrospun composite constructs enhanced endothelial cells adhesion and proliferation, both in terms of cell number and morphology. Results suggest that composite Tecoflex®–Gelatin meshes could be promising alternatives to conventional vascular grafts, deserving of further studies on both their mechanical behaviour and smooth muscle cell compatibility.

Development of 3D wet-spun polymeric scaffolds loaded with antimicrobial agents for bone engineering

Three-dimensional wet-spun microfibrous meshes of a star poly(∈-caprolactone) were developed as potential scaffolds endowed with antimicrobial activity. The in vitro release kinetics of the meshes, under physiological conditions, was initially fast and then a sustained release for more than one month was observed. Cell cultures of a murine pre-osteoblast cell line showed good cell viability and adhesion on the wet-spun star poly(∈-caprolactone) fiber scaffolds. These promising results indicate a potential application of the developed meshes as engineered bone scaffolds with antimicrobial activity.

Nano/microfibrous polymeric constructs loaded with bioactive agents and designed for tissue engineering applications: A review

Nano/microfibrous polymeric constructs present various inherent advantages, such as highly porous architecture and high surface to volume ratio, making them attractive for tissue engineering purposes. Electrospinning is the most preferred technique for the fabrication of polymeric nanofibrous assemblies that can mimic the physical functions of native extracellular matrix greatly favoring cells attachment and thus influencing their morphology and activities. Different approaches have been developed to apply polymeric microfiber fabrication techniques (e.g. wet-spinning) for the obtainment of scaffolds with a three-dimensional network of micropores suitable for effective cells migration. Progress in additive manufacturing technology has led to the development of complex scaffolds shapes and microfibrous structures with a high degree of automation, good accuracy and reproducibility. Various loading methods, such as direct blending, coaxial electrospinning and microparticles incorporation, are enabling to develop customized strategies for the biofunctionalization of nano/microfibrous scaffolds with a tailored kinetics of release of different bioactive agents, ranging from small molecules, such as antibiotics, to protein drugs, such as growth factors, and even cells. Recent activities on the combination of different processing techniques and loading methods for the obtainment of biofunctionalized polymeric constructs with a complex multiscale structure open new possibilities for the development of biomimetic scaffolds endowed with a hierarchical architecture and a sophisticated release kinetics of different bioactive agents. This review is aimed at summarizing current advances in technologies and methods for manufacturing nano/microfibrous polymeric constructs suitable as tissue engineering scaffolds, and for their combination with different bioactive agents to promote tissue regeneration and therapeutic effects.

Poly(vinyl alcohol)-based electrospun meshes as potential candidate scaffolds in regenerative medicine

Fibrous meshes based on three different poly(vinyl alcohol) (PVA) polymers, with 12% vinyl acetate monomeric units and molar weights of 37,000, 67,000, and 130,000 were developed as potential scaffolds for regenerative medical applications. The meshes were electrospun and characterized by molecular weight, concentration, applied voltage, and needle—collector distance. The influence of feed rate and the electrodes configuration (needle-to-tip and screen-to-screen system) was determined. Highly porous, 3D structures composed of randomly oriented ultrafine fibers, with an average fiber diameter of a few hundred nanometers were developed. Solutions of PVA and human serum albumin were successfully electrospun and the fibrous mesh was stabilized with glutaraldehyde. The influence of these operations on the mechanical properties was evaluated by uniaxial tensile testing.

Development of Electrospun Three-arm Star Poly(ε-caprolactone) Meshes for Tissue Engineering Applications

We have developed three-dimensional electrospun microfibrous meshes of a novel star branched three-arm poly(ε-caprolactone) (*PCL) as potential scaffolds for tissue engineering applications. The processing conditions required to obtain uniform fibers were optimized by studying their influence on fiber morphology and size. Polymer molecular weight and solution feed rate influenced both the mesh microstructure and the tensile properties of the developed mats. Electrospun samples were also tested for their mechanical properties in wet conditions, showing higher yield strength and strain in comparison to that observed in dry conditions. Cell culture experiments employing MC3T3-E1 osteoblast like cells showed good cell viability adhesion and collagen production on the *PCL scaffolds.

Additive manufacturing of star poly(ε-caprolactone) wet-spun scaffolds for bone tissue engineering applications

Three-dimensional fibrous scaffolds made of a three-arm star poly(ε-caprolactone) were developed by employing a novel computer-aided wet-spinning apparatus to precisely control the deposition pattern of an extruded polymeric solution as a filament into a coagulation bath. Star poly(ε-caprolactone)/hydroxyapatite composite scaffolds composed of fibres with a porous morphology both in the outer surface and in the cross section were successfully produced with a layer-by-layer approach achieving good reproducibility of the internal architecture and external shape. Changes in processing parameters were used to fabricate scaffolds with different architectural parameters in terms of average pore size in the xy-axes (from 190 to 297 µm) and in the z-axis (from 54 to 126 µm) and porosity (in the range of 20%–60%). Based on the mechanical characterization, processing variations and hydroxyapatite loading have an influence on scaffold compression properties. Cell cultures, using a murine pre-osteoblast cell line, had good cell responses in terms of proliferation and osteoblastic differentiation. Thus, this technique appears to be an effective method for producing customized polymeric scaffolds for bone tissue engineering applications.

Dual-Scale Polymeric Constructs as Scaffolds for Tissue Engineering

This research activity was aimed at the development of dual-scale scaffolds consisting of three-dimensional constructs of aligned poly(ε-caprolactone) (PCL) microfilaments and electrospun poly(lactic-co-glycolic acid) (PLGA) fibers. PCL constructs composed by layers of parallel microsized filaments (0/90° lay-down pattern), with a diameter of around 365 μm and interfilament distance of around 191 μm, were produced using a melt extrusion-based additive manufacturing technique. PLGA electrospun fibers with a diameter of around 1 μm were collected on top of the PCL constructs with different thicknesses, showing a certain degree of alignment. Cell culture experiments employing the MC3T3 murine preosteoblast cell line showed good cell viability and adhesion on the dual-scale scaffolds. In particular, the influence of electrospun fibers on cell morphology and behavior was evident, as well as in creating a structural bridging for cell colonization in the interfilament gap.

Fibrous star poly(ε-caprolactone) melt-electrospun scaffolds for wound healing applications

Polymeric fibrous scaffolds based on the biocompatible and biodegradable three-arm-branched star poly(ε-caprolactone) (Mw = 189,000 g/mol) were prepared by a melt electrospinning technique. The possibility of processing polymers without the use of organic solvents is one of the main advantages over solution electrospinning. Scaffolds were biologically tested for their ability of supporting skin tissue regeneration. For this purpose, mouse embryo fibroblast (BALB/3T3 clone A31) and human keratinocyte (HaCaT) cell lines were selected as models, and seeded onto the polymeric supports both as single and co-culture. Cell viability, proliferation, and collagen production were assessed by WST-1 assay and Direct Red 80 dye, respectively. Cell morphology and colonization of the supports were evaluated by scanning electron microscopy and confocal laser scanning microscopy. Results highlighted that the star poly(ε-caprolactone) scaffolds were able to promote collagen production by fibroblasts. In co-culture studies, scaffolds supported adhesion, proliferation, and spatial organization of both cell lines. By virtue of the observed results, the developed polymeric scaffolds appeared suitable as biodegradable and biocompatible three-dimensional supports for skin tissue regeneration in wound healing dressing.

Multiscale fabrication of biomimetic scaffolds for tympanic membrane tissue engineering

The tympanic membrane (TM) is a thin tissue able to efficiently collect and transmit sound vibrations across the middle ear thanks to the particular orientation of its collagen fibers, radiate on one side and circular on the opposite side. Through the combination of advanced scaffolds and autologous cells, tissue engineering (TE) could offer valuable alternatives to autografting in major TM lesions. In this study, a multiscale approach based on electrospinning (ES) and additive manufacturing (AM) was investigated to fabricate scaffolds, based on FDA approved copolymers, resembling the anatomic features and collagen fiber arrangement of the human TM. A single scale TM scaffold was manufactured using a custom-made collector designed to confer a radial macro-arrangement to poly(lactic-co-glycolic acid) electrospun fibers during their deposition. Dual and triple scale scaffolds were fabricated combining conventional ES with AM to produce poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer scaffolds with anatomic-like architecture. The processing parameters were optimized for each manufacturing method and copolymer. TM scaffolds were cultured in vitro with human mesenchymal stromal cells, which were viable, metabolically active and organized following the anisotropic character of the scaffolds. The highest viability, cell density and protein content were detected in dual and triple scale scaffolds. Our findings showed that these biomimetic micro-patterned substrates enabled cell disposal along architectural directions, thus appearing as promising substrates for developing functional TM replacements via TE.