Luigi Ambrosio

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.

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.

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.

Bone regeneration potential of a soybean-based filler: experimental study in a rabbit cancellous bone defects.

Autologous and allogenic bone grafts are considered as materials of choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of alternative biomaterials. The present work compares the bone regeneration potential of a soybean based bone filler (SB bone filler) in comparison to a commercial 50:50 poly(d,l lactide–glycolide)-based bone graft (Fisiograft® gel) when implanted into a critical size defect (6-mm diameter, 10-mm length) in rabbit distal femurs. The histomorphometric and microhardness analyses of femoral condyles 4, 8, 16 and 24xa0weeks after surgery showed that no significant difference was found in the percentage of both bone repair and bone in-growth in the external, medium and inner defect areas. The SB filler-treated defects showed significantly higher outer bone formation and microhardness results at 24xa0weeks than Fisiograft® gel (Pxa0<xa00.05). Soybean-based biomaterials clearly promoted bone repair through a mechanism of action that is likely to involve both the scaffolding role of the biomaterial for osteoblasts and the induction of their differentiation.

Soybean-based biomaterials: preparation, properties and tissue regeneration potential

Future successes in regenerative medicine will depend on the development of new biodegradable biomaterials able to control tissue regeneration in vitro and in vivo. None of the products currently available to surgeons can combine all the essential characteristics for biodegradable biomaterials, which are tunable degradation rate, controlled inflammatory reaction, no toxicity and stimulation of tissue regeneration. These clinical features should be provided, together with ease of handling during surgery and cost-effective production. Here, an overview is presented of a novel class of soybean-based biomaterials, which can be manufactured as different hydrogel formulations, all tailored for specific clinical applications. ln vitro and in vivo studies have ascertained their activity on various biochemical and cellular components of regenerating tissues. Beyond their use, the ascertained bioactivity of some of the soybean components may open new investigations and commercial routes in regenerative medicine.

Image processing and fractal box counting: user-assisted method for multi-scale porous scaffold characterization

Image analysis has gained new effort in the scientific community due to the chance of investigating morphological properties of three dimensional structures starting from their bi-dimensional gray-scale representation. Such ability makes it particularly interesting for tissue engineering (TE) purposes. Indeed, the capability of obtaining and interpreting images of tissue scaffolds, extracting morphological and structural information, is essential to the characterization and design of engineered porous systems. In this work, the traditional image analysis approach has been coupled with a probabilistic based percolation method to outline a general procedure for analysing tissue scaffold SEM micrographs. To this aim a case study constituted by PCL multi-scaled porous scaffolds was adopted. Moreover, the resulting data were compared with the outputs of conventionally used techniques, such as mercury intrusion porosimetry. Results indicate that image processing methods well fit the porosity features of PCL scaffolds, overcoming the limits of the more invasive porosimetry techniques. Also the cut off resolution of such IP methods was discussed. Moreover, the fractal dimension of percolating clusters, within the pore populations, was addressed as a good indication of the interconnection degree of PCL bi-modal scaffolds. Such findings represent (i) the bases for a novel approach complementary to the conventional experimental procedure used for the morphological analysis of TE scaffolds, in particular offering a valid method for the analysis of soft materials (i.e., gels); also (ii) providing a new perspective for further studies integrating to the structural and morphological data, fluid-dynamics and transport properties modelling.

Self-hardening calcium deficient hydroxyapatite/gelatine foams for bone regeneration

In this work gelatine was used as multifunctional additive to obtain injectable self-setting hydroxyapatite/gelatine composite foams for bone regeneration. The foaming and colloidal stabilization properties of gelatine are well known in food and pharmaceutical applications. Solid foams were obtained by foaming liquid gelatine solutions at 50°C, followed by mixing them with a cement powder consisting of alpha tricalcium phosphate. Gelatine addition improved the cohesion and injectability of the cement paste. After setting the foamed paste transformed into a calcium deficient hydroxyapatite. The final porosity, pore interconnectivity and pore size were modulated by modifying the gelatine content in the liquid phase.

The biocompatibility of silver-containing Na2O·CaO·2SiO2 glass prepared by sol–gel method: In vitro studies

This study was performed to determine the in vitro biocompatibility of a bioactive, antibacterial silver based gel. A human osteoblast-like cell line (HOS TE85) was used to test cytocompatibility in the presence of eluants and the biological response in direct contact with Na(2)O.CaO.2SiO(2) gel (containing 0.5050 wt % Ag(2)O prepared by a sol-gel process) sterilized by two different methods; autoclave and gamma-irradiation. MTT assay was used for the indirect evaluation of eluant toxicity and alamar Blue was performed to assess the biological response, which included cell viability and proliferation, and the results showed that these materials were cytocompatible.

Chemical-physical characterization and in vitro preliminary biological assessment of hyaluronic acid benzyl ester-hydroxyapatite composite

HYAFF®11 is a biocompatible, biodegradable benzyl ester of hyaluronic acid. However, in order to use it for orthopedic application, its mechanical performance needs to be improved. In this study, a novel composite based on HYAFF®11 polymer matrix reinforced with hydroxylapatite (HA) has been developed. Its advantage is having a similar component of the mineral phase of bone resulting in favorable osteoconductive properties. The present study has examined the compressive mechanical and surface chemical-physical properties of the novel HYAFF®11-HA composite. Preliminary biological investigations, including pH and cytotoxicity studies of the material extracts, have also been performed using an in vitro primary human osteoblast-like cell model. Moreover, protein, especially fibronectin adsorption has been investigated following incubation in culture medium and human plasma. The results show a grainy surface topography composed mainly of C, P, and Ca, with a Ca/P atomic ratio indicating HA on the composite surface. Mechanical analysis shows an improvement of the compressive properties of HYAFF11 matrix, both in the dry and swollen states, with values in the range of that of spongy bone. No cytotoxic effects and no inhibition of cell proliferation have been observed in the presence of the material extracts with pH values within acceptable ranges for cell vitality. Protein studies reveal a similar pattern, but a higher amount of fibronectin following incubation in human plasma when compared with culture medium. The results show that the novel HYAFF®11-HA composite shows a great potential for application in orthopedic fields, especially as vertebral trabecular bone substitute.