Alfredo Ronca

Preparation of designed poly(D,L-lactide)/nanosized hydroxyapatite composite structures by stereolithography

The preparation of scaffolds to facilitate the replacement of damaged tissues and organs by means of tissue engineering has been much investigated. The key properties of the biomaterials used to prepare such scaffolds include biodegradability, biocompatibility and a well-defined three-dimensional 3-Dpore network structure. In this study a poly(D,L-lactide)/nanosized hydroxyapatite (PDLLA/nano-Hap) composite resin was prepared and used to fabricate composite films and computer designed porous scaffolds by micro-stereolithography, mixing varying quantities of nano-Hap powder and a liquid photoinitiator into a photo-crosslinkable PDLLA-diacrylate resin. The influence of nano-Hap on the rheological and photochemical properties of the resins was investigated, the materials being characterized with respect to their mechanical, thermal and morphological properties after post-preparation curing. In the cured composites stiffness was observed to increase with increasing concentration of nanoparticles. A computer designed construct with a pore network based on the Schwarz architecture was fabricated by stereolithography using PDLLA/nano-Hap composite resins.

Design of porous three-dimensional PDLLA/nano-hap composite scaffolds using stereolithography

Purpose The stereolithography process is based on the photopolymerization through a dynamic mask generator of successive layers of photocurable resin, allowing the manufactory of accurate micro objects with high aspect ratio and curved surfaces. In the present work the stereolithography technique is applied to produce nanocomposite bioactive scaffolds from Computer Assisted Design (CAD) files. Methods Porous scaffolds are designed with computer software and built with a composite poly(D,L-lactide)/nano hydroxyapatite based resin. Triply-periodic minimal surfaces are shown to be a more versatile source of biomorphic scaffold designs and scaffolds with Double Gyroid architecture are realized and characterized from morphological, mechanical and biological point of view. Results The structures show excellent reproduction of the design and good mechanical properties. Human marrow mesenchimal cells (hMSC) are seeded onto porous PDLLA composites for 3 weeks and cultured in osteogenic medium. Presence of nano-hap seems to increase the mechanical properties without affecting the morphology of the structures. The composite Double Gyroid scaffolds exhibit good biocompatibility and confirm that nano-hap enhances the scaffold bioactive and os-teoconductive potential. Conclusion The presented technology and materials enable an accurate preparation of tissue engineering composite scaffolds with a large freedom of design, and really complex internal architectures. Results indicate that the scaffolds fulfill the basic requirements of bone tissue engineering scaffold, and have the potential to be applied in orthopedic surgery.

Large defect-tailored composite scaffolds for in vivo bone regeneration.

The discovery of new strategies to repair large segmental bone defects is currently an open challenge for worldwide clinicians. In the treatment of critical-sized bone defects, an alternative strategy to traditional bone grafting is always more frequently the use of tailor-made scaffolds modelled on the final size and shape of the implant site. Here, poly-ε-caprolactone-based composite scaffolds including poly-l-lactic acid continuous fibres and hyaluronan derivates (i.e. HYAFF11®) have been investigated for the peculiar 3D architecture characterized by interconnected macroporous networks and tunable mechanical properties. Composite scaffolds were immersed in simulated body fluid solution in order to support in vivo tissue in-growth. Scaffolds loaded with autologous cells (bone marrow stromal cells) plus platelet-rich plasma and osteoconductive protein such bone morphogenetic protein-7 were also tested to evaluate eventual enhancement in bone regeneration. The morphological and mechanical properties of poly-l-lactic acid-reinforced composite scaffolds have been studied to identify the optimal scaffold design to match the implant-site requirements of sheep metatarsal defects. Dynamic mechanical tests allowed to underline the viscoelastic response of the scaffold – resulting in elastic moduli from 2.5 to 1.3 MPa, suitable to temporarily support the structural function of damaged bone tissue. In vivo preliminary investigations in a sheep model of metatarsus shaft defect also showed the attitude of the scaffold to promote osteogenesis, preferentially in association with bone marrow stromal cell and platelet-rich plasma, even if the highest amount of mature bone was reached in the case of scaffold loaded with human bone morphogenetic protein-7 released via hydrolytic degradation of HYAFF11® phases in the implant site.

Needle-like ion-doped hydroxyapatite crystals influence osteogenic properties of PCL composite scaffolds

Surface topography and chemistry both play a crucial role on influencing cell response in 3D porous scaffolds in terms of osteogenesis. Inorganic materials with peculiar morphology and chemical functionalities may be proficiently used to improve scaffold properties-in the bulk and along pore surface-promoting in vitro and in vivo osseous tissue in-growth. The present study is aimed at investigating how bone regenerative properties of composite scaffolds made of poly(Ɛ-caprolactone) (PCL) can be augmented by the peculiar properties of Mg(2+) ion doped hydroxyapatite (dHA) crystals, mainly emphasizing the role of crystal shape on cell activities mediated by microstructural properties. At the first stage, the study of mechanical response by crossing experimental compression tests and theoretical simulation via empirical models, allow recognizing a significant contribution of dHA shape factor on scaffold elastic moduli variation as a function of the relative volume fraction. Secondly, the peculiar needle-like shape of dHA crystals also influences microscopic (i.e. crystallinity, adhesion forces) and macroscopic (i.e. roughness) properties with relevant effects on biological response of the composite scaffold: differential scanning calorimetry (DSC) analyses clearly indicate a reduction of crystallization heat-from 66.75 to 43.05 J g(-1)-while atomic force microscopy (AFM) ones show a significant increase of roughness-from (78.15  ±  32.71) to (136.13  ±  63.21) nm-and of pull-off forces-from 33.7% to 48.7%. Accordingly, experimental studies with MG-63 osteoblast-like cells show a more efficient in vitro secretion of alkaline phosphatase (ALP) and collagen I and a more copious in vivo formation of new bone trabeculae, thus suggesting a relevant role of dHA to support the main mechanisms involved in bone regeneration.

Surface functionalization of acrylic based photocrosslinkable resin for 3D printing applications

The limited number of resins, available for stereolithography applications, is one of the key drivers in research applied to rapid prototyping. In this work an acrylic photocrosslinkable resin based on methyl methacrylate (MMA), butyl methacrylate (BMA) and poly(ethylene glycol) dimethacrylate (PEGDA) was developed with different composition and characterized in terms of mechanical, thermal and biological behaviour. Two different systems have been developed using different amount of reagent. The influence of every components have been evaluated on the final characteristic of the resin in order to optimize the final composition for applications in bone tissue engineering. The crosslinked materials showed good mechanical properties and thermal stabilities and moreover cytotoxicity test confirms good biocompatibility with no cytotoxic effect on cells metabolism. Moreover two different treatments have been proposed, using fetal bovine serum (FBS) and methanol (MeOH), in order to improve cell recognition of the surfaces. Samples threatened with MeOH allow cell adhesion and survival, promoting spreading, elongation and fusion of C2C12 muscle myoblast cells.

Synthesis and characterization of divinyl-fumarate Poly-ε-caprolactone for scaffolds with controlled architectures

A vinyl‐terminated polycaprolactone has been developed for tissue engineering applications using a one‐step synthesis and functionalization method based on ring opening polymerization (ROP) of ε‐Caprolactone, with hydroxyl ethyl vinyl ether (HEVE) acting both as the initiator of ROP and as photo‐curable functional group. The proposed method employs a catalyst based on aluminium, instead of the most popular Tin(II) 2‐ethylhexanoate, to reduce the cytotoxicity. Following the synthesis of the vinyl‐terminated polycaprolactone, its reaction with fumaryl chloride (FuCl) results in a divinyl‐fumarate polycaprolactone (VPCLF). The polymers obtained were thoroughly characterized using Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC) techniques. The polymer has been successfully employed, in combination with N‐vinyl pyrrolidone (NVP), to fabricate films and computer‐designed porous scaffolds by micro‐stereolithography (μ‐SL) with gyroid and diamond architectures. Characterization of the networks indicated the influence of NVP content on the network properties. Human mesenchymal stem cells adhered and spread onto VPCLF/NVP networks showing good biological properties and no cytotoxic effect. Copyright

Design of Functional Polymer and Composite Scaffolds for the Regeneration of Bone, Menisci, Osteochondral and Peripheral Nervous Tissues

In order to mimic the behaviors of natural tissue, the optimal approach for designing novel biomaterials has to be inspired to nature guidelines. One of the major challenge consists in the development of well-organized structures or scaffolds with controlled porosity in terms of pore size, pore shape and interconnection degree able to guide new tissue formation during the in vivo degradation following the scaffold implantation. Scaffolds endowed with molecular cues together to a controlled degradation profile should contribute to cell proliferation and differentiation, controlled vascularization, promoting the remodeling of neo tissue through a gradual transmission of bio-chemicals and biophysical signals as performed by the extracellular matrix (ECM). Here, different polymers and composites have been investigated to design scaffolds with peculiar micro and/or nanometric morphological features in order to satisfy all these requirements: a) bioactive scaffolds, with tailored porosity and high pores interconnectivity were developed by integrating PLA fibres, Calcium Phosphates particles or Hyaff11 phases into a Poly(ε-caprolactone) (PCL) matrix by the combination of filament winding technology and phase inversion/salt leaching technique as mineralised ECM analogue for bone regeneration; b) custom made PCL/hydroxyapatite scaffolds were designed by imaging and rapid prototyping technologies for the osteochondral defect. c) Ester of Hyaluronic Acid reinforced with degradable fibres were processed by composite technology, phase inversion and salt leaching technique, to obtain scaffolds for meniscus regeneration. d) PCL and gelatin nanofibres were obtained by highly customized fibre deposition via electrospinning to guide the nerve outgrowth in nerve regeneration. All the proposed approaches offer the chance of realizing tailor-made platforms with micro/nanoscale architecture and chemical composition suitable for the regeneration of the extracellular matrix of a large variety of natural tissues (i.e, bone, menisci, osteochondral and peripheral nervous tissues).

Bioactive composites based on double network approach with tailored mechanical, physico-chemical, and biological features: Bioactive composites based on double network approach

Hyaluronic acid (HA)-based hydrogels are one of the most promising naturally derived biomaterials for tissue engineering applications, as they can play an important role in many key cellular processes. In this study, HA was chemically functionalized with photo-cross-linkable motifs by reacting with methacrylic anhydride (MA) to obtain methacrylated hyaluronic acid (MeHA). A range of MA/HA molar ratios was used to obtain different degrees of substitution (DS) ranging from 3.5% to 74.5%, as showed by nuclear magnetic resonance and attenuated total reflection spectroscopy. By fine tuning the DS, the chemical reaction parameters, and the polymer concentration, it was demonstrated the possibility to tailor their mechanical features. Double network (DN) hydrogels were prepared through the synergic use of MeHA and polyethylene glycole diacrylate (PEGDA). To improve the biological properties of DN hydrogels, bioactive solid signals such as hydroxyapatite nanoparticles (HAp) prepared by sol-gel approach were used in combination with DN hydrogels to obtain an advanced composite material with dual function in terms of mechanical and biological support for soft/hard tissue formation. The results highlighted that composite-DN hydrogels showed a 10-time increase of the storage modulus, if compared to neat MeHA, and an early alkaline phosphatase expression from human mesenchymal stem cells in basal medium. This work can be considered a first systematic approach for the designing of photo-cross-linkable hydrogels, based on a combination of natural/synthetic polymers and HAp, that could be applied in three-dimensional additive manufacturing techniques such as stereolithography.

Multifunctional scaffolds for bone regeneration

Abstract: Biodegradable scaffolds are generally considered as indispensable elements for engineering living tissues as they are used as temporary templates with specific mechanical and biological properties similar to native extracellular matrix (ECM). They allow modulating cell adhesion, invasion, proliferation and differentiation, prior to the regeneration of biologically functional tissue or natural ECM. In the case of bone regeneration, a current challenge is to conceive new process strategies to fabricate composite or hybrid scaffolds able to provide three-dimensional templates and synthetic ECM environments. Here, we will describe current processing technologies used to achieve structural features mimicking the ECM on various levels and successfully emulate cell–ECM interactions in order to promote the regeneration of mineralized tissues such as bone.