Levofloxacin-loaded star poly(ε-caprolactone) scaffolds by additive manufacturing

The employment of a tissue engineering scaffold able to release an antimicrobial agent with a controlled kinetics represents an effective tool for the treatment of infected tissue defects as well as for the prevention of scaffolds implantation-related infectious complications. This research activity was aimed at the development of additively manufactured star poly(ε-caprolactone) (*PCL) scaffolds loaded with levofloxacin, investigated as antimicrobial fluoroquinolone model. For this purpose a computer-aided wet-spinning technique allowing functionalizing the scaffold during the fabrication process was explored. Scaffolds with customized composition, microstructure and anatomical external shape were developed by optimizing the processing parameters. Morphological, thermal and mechanical characterization showed that drug loading did not compromise the fabrication process and the final performance of the scaffolds. The developed *PCL scaffolds showed a sustained in vitro release of the loaded antibiotic for 5 weeks. The proposed computer-aided wet-spinning technique appears well suited for the fabrication of anatomical scaffolds endowed with levofloxacin-releasing properties to be tested in vivo for the regeneration of long bone critical size defects in a rabbit model

Tailored star poly (ε-caprolactone) wet-spun scaffolds for in vivo regeneration of long bone critical size defects

One of the most challenging requirements of a successful bone tissue engineering approach is the development of scaffolds specifically tailored to individual tissue defects. Besides materials chemistry, well-defined scaffold’s structural features at the micro- and macro-levels are needed for optimal bone in-growth. In this study, polymeric fibrous scaffolds with a controlled internal network of pores and modelled on the anatomical shape and dimensions of a critical size bone defect in a rabbit’s radius model were developed by employing a computer-aided wet-spinning technique. The tailored scaffolds made of star poly(ε caprolactone) or star poly(ε-caprolactone)– hydroxyapatite composite material were implanted into 20-mm segmental defects created in radial diaphysis of New Zealand white rabbits. Bone regeneration and tissue response were assessed by X-rays and histological analysis at 4, 8 and 12 weeks after surgery. No signs of macroscopic and microscopic inflammatory reactions were detected, and the developed scaffolds showed a good ability to support and promote the bone regeneration process. However, no significant differences in osteoconductivity were observed between star poly(ε-caprolactone) and star poly(ε-caprolactone)–hydroxyapatite scaffolds. Long-term study on implanted star poly(ε-caprolactone) scaffolds confirmed the presence of signs of bone regeneration and remodelling, particularly evident at 24 weeks

PROGETTAZIONE, PREPARAZIONE E CARATTERIZZAZIONE STRUTTURALE E BIOLOGICA DI SUPPORTI POLIMERICI TRIDIMENSIONALI PER APPLICAZIONI DI INGEGNERIA TISSUTALE

L’ingegneria tissutale ha suscitato grande interesse negli ultimi anni per il suo potenziale utilizzo nel campo della medicina rigenerativa. Uno dei concetti fondamentali alla base di questa disciplina prevede la combinazione di una matrice biodegradabile, comunemente denominata scaffold, con cellule e/o molecole biologicamente attive, al fine di sviluppare un supporto in grado di promuovere la rigenerazione di tessuti danneggiati. Il presente lavoro di tesi è incentrato sullo sviluppo di scaffold con attività antimicrobica, forma e dimensioni anatomiche, e microstruttura porosa adatta al loro impiego nell’ingegnerizzazione del tessuto osseo. Per tale scopo, è stata sviluppata una metodologia basata sulla tecnica di wet-spinning automatizzata per la preparazione di scaffold a base di poli(caprolattone) con struttura molecolare a stella e caricati con un antibiotico fluorochinolonico (levofloxacina). Gli scaffold sviluppati sono stati caratterizzati da un punto di vista strutturale, chimico-fisico, meccanico e biologico. L’analisi mediante microscopia elettronica a scansione ha mostrato che la tecnica impiegata permette di ottenere, con buona riproducibilità della microstruttura e della geometria esterna, scaffold costituiti da strati sovrapposti di fibre porose con orientazione controllata, diametro medio di circa 200 μm e dimensioni dei pori interfibra dell’ordine di qualche centinaia di micrometri. L’efficienza di caricamento, il loading e la cinetica di rilascio in vitro dell’antibiotico contenuto negli scaffold sono stati analizzati tramite cromatografia liquida ad alta prestazione. In base alla concentrazione di antibiotico nella soluzione di partenza, l’efficienza di caricamento è risultata variare nell’intervallo 5% - 16% e il loading nell’intervallo 0.15% - 2%. Lo studio della cinetica di rilascio ha mostrato un rilascio rapido di antibiotico nei primi 4 giorni seguito da un rilascio che si mantiene a velocità circa costante fino alla quinta settimana del test. Studi di analisi termogravimetrica e calorimetria differenziale a scansione non hanno evidenziato variazioni significative delle proprietà termiche del polimero dovute al processo di produzione degli scaffold o al caricamento dell’antibiotico. Lo studio delle proprietà meccaniche ha evidenziato che gli scaffold sono caratterizzati da valori di modulo e di resistenza di pochi megapascal sia in compressione che in trazione. La caratterizzazione biologica in vitro, condotta utilizzando una linea cellulare di preosteoblasti murini, ha mostrato una buona risposta cellulare in termini di vitalità, proliferazione e differenziamento in senso osteoblastico. Infine, i risultati ottenuti da sperimentazione in vivo, effettuata su conigli di razza New Zealand White, suggeriscono l’effettiva applicabilità degli scaffold prodotti nell’ingegneria tissutale dell’osso

Preparation and characterization of micro/nanostructured three-dimensional scaffolds based on renewable polymers for tissue engineering applications

Tissue Engineering (TE) applications range from the treatment of hard tissues like bone, cartilage, teeth, and soft tissues such as muscles and epithelia, thus leading to an increasing need to develop new materials with tailorable properties and new processing technologies to customize the support’s characteristics based on the specific application. At present, there is also an upsurge of interest in polymeric materials for biomedical applications obtained from natural and sustainable resources, to limit the depletion of fossil resources. Aim of the present PhD work was the design and the development of micro/nanostructured threedimensional supports, based on renewable polymers and bioactive molecules, to use as scaffolds in TE applications and Regenerative Medicine. For this objective, biodegradable polymers from renewable sources, including polysaccharides, proteins and microbial polyesters, were investigated to assess their potential use as matrices for the production of three-dimensional scaffolds. The research activity was performed under a multidisciplinary approach and it was structured in three main topics: • Additive manufacturing of wet-spun poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyexanoate] (PHBHHx) scaffolds tailored on a critical size long bone defect: Predefined three-dimensional PHBHHx scaffolds were produced layer upon layer by a computer controlled wet-spinning technique. The scaffolds were designed with anatomical geometry and dimensions modeled on a critical size defect in the radius of New Zealand White rabbits. Scaffolds with a longitudinal central channel to facilitate cell penetration in the inner parts of the constructs were also designed and produced. Optical Microscopy analysis showed a good reproducibility of the internal architecture and high pores interconnection. FTIR spectroscopy and thermal analysis showed that the employed technique did not affect the material’s chemical-physical properties. The compressive and tensile mechanical properties of the structure were shown to be correlated to the direction of the stress relative to the fibers and scaffold axis. • Development of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyexanoate] (PHBHHx) and poly (ε-caprolactone) (PCL) biodegradable stents by computer-aided wet-spinning: PHBHHx and PCL biodegradable stents were developed for the treatment of injured small caliber (< 3 mm) blood vessels. A novel computer-aided wet-spinning apparatus for the production of threedimensional microstructured polymeric constructs with a tubular geometry was designed and assembled. The produced stents exhibited a well defined tubular microfibrous structure. By tuning the fabrication parameters, it was possible to produce stents with different morphological characteristics (length, porosity and wall thickness), underlining the versatility of the developed technique in customizing stent structural and dimensional features. By axial and radial mechanical compression tests, PHBHHx stents demonstrated great elasticity, in particular a full elastic recovery up to radial deformation of 70 % of the diameter, thus showing potential compliance with the treated artery. PCL stents showed mechanical strength comparable with PCL stents produced by different techniques and the ability to expand through a coronary stent system balloon, maintaining the expanded state after deployment. • Wet-spun poly(ε-caprolactone) and poly(ε-caprolactone)/hydroxyapatite scaffolds for the development of an osteochondral interface tissue and vascularized bone construct: A model of an osteochondral tissue interface and a vascularized bone construct were developed using PCL and PCL/HA scaffolds in combination with methacrylated gelatin. Both scaffold components were seeded with human bone marrow derived mesenchymal stem cells (hMSCs) and supplied with chondrogenic and osteogenic media through a dual chamber bioreactor that allows the simultaneous, separate flow of each medium, for the simultaneous differentiation of each compartment towards a cartilaginous or osseous lineage to recreate the osteochondral complex. Human umbilical veins endothelial cells (HUVECs) have been used to create a capillary-like network in an engineered bone construct, produced by the combination of PCL-PCL/HA-gelMA construct seeded with hMSCs

Design, fabrication and characterization of tailored poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyexanoate] scaffolds by computer-aided wet-spinning

Purpose – The purpose of this paper is to describe the fabrication and characterization of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyexanoate] (PHBHHx) tissue engineering scaffolds with anatomical shape and customized porous structure. Design/methodology/approach – Scaffolds with external shape and size modeled on a critical size segment of a rabbit’s radius model and an internal macrochanneled porous structure were designed and fabricated by means of a computer-aided wet-spinning (CAWS) technique. Morphological, thermal and mechanical characterization were carried out to assess the effect of the fabrication process on material properties and the potential of the PHBHHx scaffolds in comparison with anatomical star poly(e-caprolactone) (*PCL) scaffolds previously validated in vivo. Findings – The CAWS technique is well suited for the layered manufacturing of anatomical PHBHHx scaffolds with a tailored porous architecture characterized by a longitudinal macrochannel. Morphological analysis showed that the scaffolds were composed by overlapping layers of microfibers with a spongy morphology, forming a 3D interconnected network of pores. Physical-chemical characterization indicated that the used technique did not affect the molecular structure of the processed polymer. Analysis of the compressive and tensile mechanical properties of the scaffolds highlighted the anisotropic behavior of the porous structure and the effect of the macrochannel in enhancing scaffold compressive stiffness. In comparison to the *PCL scaffolds, PHBHHx scaffolds showed higher compressive stiffness and tensile deformability. Originality/value – This study shows the possibility of using renewable microbial polyester for the fabrication of scaffolds with anatomical shape and internal architecture tailored for in vivo bone regeneration studies

Engineering in-vitro stem cell-based vascularized bone models for drug screening and predictive toxicology

Abstract The production of veritable in-vitro models of bone tissue is essential to understand the biology of bone and its surrounding environment, to analyze the pathogenesis of bone diseases (e.g., osteoporosis, osteoarthritis, osteomyelitis, etc.), to develop effective therapeutic drug screening, and to test potential therapeutic strategies. Dysregulated interactions between vasculature and bone cells are often related to the aforementioned pathologies, underscoring the need for a bone model that contains engineered vasculature. Due to ethical restraints and limited prediction power of animal models, human stem cell-based tissue engineering has gained increasing relevance as a candidate approach to overcome the limitations of animals and to serve as preclinical models for drug testing. Since bone is a highly vascularized tissue, the concomitant development of vasculature and mineralized matrix requires a synergistic interaction between osteogenic and endothelial precursors. A number of experimental approaches have been used to achieve this goal, such as the combination of angiogenic factors and three-dimensional scaffolds, prevascularization strategies, and coculture systems. In this review, we present an overview of the current models and approaches to generate in-vitro stem cell-based vascularized bone, with emphasis on the main challenges of vasculature engineering. These challenges are related to the choice of biomaterials, scaffold fabrication techniques, and cells, as well as the type of culturing conditions required, and specifically the application of dynamic culture systems using bioreactors

Integrated three-dimensional fiber/hydrogel biphasic scaffolds for periodontal bone tissue engineering

Combining a tissue engineering scaffold made of a load-bearing polymer with a hydrogel represents a powerful approach to enhancing the functionalities of the resulting biphasic construct, such as its mechanical properties or ability to support cellular colonization. This research activity was aimed at the development of biphasic scaffolds through the combination of an additively manufactured poly(εlunate-caprolactone) (PCL) fiber construct and a chitosan/poly(γ-glutamic acid) polyelectrolyte complex hydrogel. By investigating a set of layered structures made of PCL or PCL/hydroxyapatite composite, biphasic scaffold prototypes with good integration of the two phases at the macroscale and microscale were developed. The biphasic constructs were able to absorb cell culture medium up to 10-fold of their weight, and the combination of the two phases had a significant influence on compressive mechanical properties compared with hydrogel or PCL scaffold alone. In addition, due to the presence of chitosan in the hydrogel phase, biphasic scaffolds exerted a broad-spectrum antibacterial activity. The developed biphasic systems appear well suited for application in periodontal bone regenerative approaches in which a biodegradable porous structure providing mechanical stability and a hydrogel phase functioning as absorbing depot of endogenous proteins are simultaneously required. © 2016 Society of Chemical Industr

Modeling In Vitro Osteoarthritis Phenotypes in a Vascularized Bone Model Based on a Bone-Marrow Derived Mesenchymal Cell Line and Endothelial Cells

The subchondral bone and its associated vasculature play an important role in the onset of osteoarthritis (OA). Integration of different aspects of the OA environment into multi-cellular and complex human, in vitro models is therefore needed to properly represent the pathology. In this study, we exploited a mesenchymal stromal cell line/endothelial cell co-culture to produce an in vitro human model of vascularized osteogenic tissue. A cocktail of inflammatory cytokines, or conditioned medium from mechanically-induced OA engineered microcartilage, was administered to this vascularized bone model to mimic the inflamed OA environment, hypothesizing that these treatments could induce the onset of specific pathological traits. Exposure to the inflammatory factors led to increased network formation by endothelial cells, reminiscent of the abnormal angiogenesis found in OA subchondral bone, demineralization of the constructs, and increased collagen production, signs of OA related bone sclerosis. Furthermore, inflammation led to augmented expression of osteogenic (alkaline phosphatase (ALP) and osteocalcin (OCN)) and angiogenic (vascular endothelial growth factor (VEGF)) genes. The treatment, with a conditioned medium from the mechanically-induced OA engineered microcartilage, also caused increased demineralization and expression of ALP, OCN, ADAMTS5, and VEGF; however, changes in network formation by endothelial cells were not observed in this second case, suggesting a possible different mechanism of action in inducing OA-like phenotypes. We propose that this vascularized bone model could represent a first step for the in vitro study of bone changes under OA mimicking conditions and possibly serve as a tool in testing anti-OA drugs