Vincenzo Guarino

Bioactive scaffolds for bone and ligament tissue

Bone and ligament injuries present the greatest challenges in connective tissue regeneration. The design of materials for these applications lies at the forefront of material science and is the epitome of its current ambition. Indeed, its goal is to design and fabricate reproducible, bioactive and bioresorbable 3D scaffolds with tailored properties that are able to maintain their structure and integrity for predictable times, even under load-bearing conditions. Unfortunately, the mechanical properties of today’s available porous scaffolds fall short of those exhibited by complex human tissues, such as bone and ligament. The manipulation of structural parameters in the design of scaffolds and their bioactivation, through the incorporation of soluble and insoluble signals capable of promoting cell activities, are discussed as possible strategies to improve the formation of new tissues both in vitro and in vivo. This review focuses on the different approaches adopted to develop bioactive composite systems for use as temporary scaffolds for bone and anterior ligament regeneration.

Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering.

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(epsilon-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 microm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell-material interaction are simultaneously requested.

Influence of Gelatin Cues in PCL Electrospun Membranes on Nerve Outgrowth

The design of functionalized polymers that can elicit specific biological responses and the development of methods to fabricate new devices that incorporate biological cues are of great interest to the biomedical community. The realization of nanostructured matrices that exhibit biological properties and that comprise fibers with diameters of similar scale to those of the natural extracellular matrix (ECM) would enable the provision of tailored materials for tissue engineering. Accordingly, the goal of this work is to create a biologically active functionalized electrospun matrix capable of guiding neurite growth for the regeneration of nerve tissue. In this study, nanoscale electrospun membranes made of poly ε-caprolactone enhanced with gelatin from calf skin were investigated to validate their biological response under in vitro culture of PC-12 nerve cells. Preliminary observations from SEM studies supported by image analysis highlighted the nanoscale texture of the scaffold with fiber diameters equal to 0.548 ± 0.140 μm. In addition, contact angle measurements confirmed the hydrophilic behavior of the membranes, ascribable to the gelatin content. We demonstrate that the balance of morphological and biochemical properties improves all the fundamental biological events of nerve regeneration, enhancing cell adhesion, proliferation, and differentiation in comparison with PCL nanofibrous scaffolds, as well as supporting the neurite outgrowth.

Conductive PANi/PEGDA Macroporous Hydrogels For Nerve Regeneration

Only recently polymers with intrinsic conductive properties have been studied in relation to their incorporation into bioactive scaffolds for use in tissue engineering. The reason for this interest is that such scaffolds could electrically stimulate cells and thus regulate specific cellular activities, and by this means influence the process of regeneration of those tissues that respond to electrical impulses. In our work, macroporous hydrogels are developed with controlled pore morphology and conductive properties to enable sufficient cell signaling to supply events inherent to nerve regeneration. A hybrid material has been prepared by in situ precipitation of polyaniline (PANi) in polyethyleneglycol diacrylate (PEGDA) solution, followed by crosslinking via UV irradiation. A porous architecture, characterized by macropores from 136 μm to 158 μm in size, has been achieved by sodium chloride particle leaching. In this work, we demonstrate that PANi synthesis and hydrogel crosslinking combine to enable the design of materials with suitable conductive behaviour. The presence of PANi evidently increased the electrical conductivity of the hybrid material from (1.1 ± 0.5) × 10(-3) mS/cm with a PANi content of 3wt%. The hydrophilic nature of PANi also enhanced water retention/proton conductivity by more than one order of magnitude. In vitro studies confirmed that 3 wt% PANi also improve the biological response of PC12 and hMSC cells. Hybrid PANi/PEGDA macroporous hydrogels supplement new functionalities in terms of morphological and conductive properties, both of which are essential prerequisites to drive nerve cells in regenerative processes.

The role of hydroxyapatite as solid signal on performance of PCL porous scaffolds for bone tissue regeneration.

Highly porous composites made up of biodegradable poly-epsilon-caprolactone (PCL) and stoichiometric hydroxyapatite (HA) particles have been developed as substrate for bone-tissue regeneration. The processing technique consists of phase inversion and particulate (salt crystals) leaching. Three different HA contents (13, 20 and 26 vol %) in PCL-based composite were considered in this study. Pore microstructure with fully interconnected network and pore sizes ranging around a few hundred of mum (macroporosity) was obtained as a result of salt particles removal by leaching process. Several microns (microporosity) porosity was also created through phase inversion of polymer solution. Total porosity up to 95% was achieved. Human marrow stromal cells (MSC) were seeded onto porous PCL-based composites for 1-5 weeks and cultured in osteogenic medium. MSC were able to adhere and grow on PCL-based substrates with a plateau at 3-4 weeks. However, the small effect of bioactive signals on the biological response evaluated in MSC cell culture suggests a prior role of topography on the biological response. Importantly, the presence of HA as a bioactive solid signal determines an increase of mechanical properties. On the overall, the results indicated that porous PCL-based composites are potential candidate for bone substitution with beneficial influence on structural characteristics by solid signal addition.

The synergic effect of polylactide fiber and calcium phosphate particle reinforcement in poly ε-caprolactone-based composite scaffolds

In this work, three-dimensional porous composite scaffolds, based on poly(epsilon-caprolactone) (PCL), were fabricated through the combination of a filament winding technique and a phase inversion/salt leaching process. Sodium chloride crystals were used as the porogen agent, and poly(lactic acid) (PLA) fibers and calcium phosphates as reinforcement. The aim of the current work is to assess the effective synergistic role of bioactive particles (i.e. alpha-tricalcium phosphates (alpha-TCP)) and PLA fibers on the morphology and mechanical response of the final scaffold. Morphological investigations performed on fiber-reinforced composite scaffolds with different PCL/alpha-TCP volume ratios (0%, 13%, 20% and 26%) show a high porosity degree (ca. 80%), pore interconnection and a homogeneous distribution of pores within the scaffold. More specifically, a bimodal pore size distribution was observed. This comprised microporosity (pores with radii ranging from 0.1 to 10 microm, which were strictly related to solvent extraction) and macroporosity (pores with radii from 10 to 300 microm, which were ascribable to the leaching of porogen elements). Static compressive tests showed that the effect of alpha-TCP on the mechanical response was to increase the elastic modulus up to a maximum value of 2.21+/-0.24 MPa, depending on the concentration of alpha-TCP added. This effect may be explained through the interaction of calcium-deficient hydroxyapatite crystals, formed as a consequence of a hydrolysis reaction of alpha-TCP, and the fiber-reinforced polymer matrix. The correct balance between chemical composition and spatial organization of reinforcement systems allows the attainment of an ideal compromise between mechanical response and bioactive potential, facilitating the development of composite scaffolds for bone tissue engineering applications.

hMSC interaction with PCL and PCL/gelatin platforms: A comparative study on films and electrospun membranes:

Polycaprolactone (PCL) and PCL/gelatin membranes and films were fabricated by electrospinning and solvent casting. A systematic analysis of the morphology evolution, as degradation occurred, was made to separate the contribution of fiber nanotexture and gelatin biochemical signal on cell adhesion and proliferation. Field emission scanning electron microscope was used to assess the contribution of platform architecture on the gelatin degradation by the morphological changes that occurred at different times. The evaluation of human mesenchymal stem cells’ biocompatibility confirmed the role of architecture and chemical composition on cell response. The nanostructured surfaces positively affected the cell recognition by increasing the surface area. The gelatin embedded in the PCL matrix of the nanofibers improved the cell/material interaction and provided support to the proliferation. The PCL/gelatin electrospun membranes showed an increase in mineralization when conditioned in osteogenic medium; this system has promise for long-term in vitro investigations of bone regeneration.

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration.

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.

Bio-inspired composite and cell instructive platforms for bone regeneration

Abstract Biological studies indicate that numerous materials present in living tissues owe their success to an optimal combination of properties and adaptive structures, rather than to extreme properties per se. Through studying natural tissues and by biomimesis, new polymer and composite materials may be designed to emulate the structural and functional responses of bone. These materials must ensure biochemical affinity with host tissue through judicious mixing of specific chemical cues. Also, they must mimic the response under load exhibited by natural bone through complex organisation of material phases, i.e. embedding of collagen fibres in the extracellular substance. Fibre and particulate reinforced polymers are increasingly significant in the development of new biomedical materials, since they can be engineered more accurately than monolithic structures. Meanwhile, design of nanocomposites with specific morphological and chemical signals is emerging as a powerful approach to the mimesis of extracellular matrix of natural bone. In both cases, the manipulation of the main materials features at the micro- and nano-metric scale offers an intriguing strategy for improvement of biological and mechanical response. Several biodegradable and bioresorbable materials, as well as technologies and scaffold designs, will be critically reviewed, illustrating the potential of bio-inspired composites and multicomponent platforms for bone tissue engineering.

Tuning Size Scale and Crystallinity of PCL Electrospun Fibres via Solvent Permittivity to Address hMSC Response

The effect of solvent permittivity on the fibre morphology of PCL electrospun membranes for tissue engineering applications is studied. Morphological results indicate that polar solvents with higher permittivity are able to promote the formation of sub-micrometric fibres, while apolar solvents yield microfibres with an average fibre diameter of 2.86 ± 0.31 µm. Polymer/solvent interactions and electrospinning process parameters influence the mechanism of fibre and bead formation. It is shown that the dielectric properties of solvents influence the fibre size scale and crystallinity and directly contribute to the biological response of stem cells. Solvent permittivity is a key factor in controlling the morphological and physical properties of electrospun fibre meshes.