Francesco Carfì Pavia

Synthesis, characterization and foaming of PHEA–PLLA, a new graft copolymer for biomedical engineering

In this study a chemical grafting procedure was set up in order to link high molecular weight poly L-lactic acid (PLLA) chains to the hydrophilic α,β-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) backbone. A graft copolymer named PHEA-g-PLLA (or simply PHEA-PLLA) was obtained bearing a degree of derivatization of 1.0 mol.% of PLLA as grafted chain. This new hybrid derivative offers both the opportune crystallinity necessary for the production of scaffolds trough a thermally induced phase separation (TIPS) technique and the proper chemical reactivity to perform further functionalizations with bio-effectors and drugs. PHEA-PLLA porous scaffolds for tissue engineering applications were successfully obtained via TIPS and characterized. Structures with an open porosity and a good level of interconnection were detected. As the applicability of the scaffold is mainly dependent on its pore size, preliminary studies about the mechanisms governing scaffolds pore diameter were carried out.

Morphology and thermal properties of foams prepared via thermally induced phase separation based on polylactic acid blends

Blends of poly-l-lactic acid with two different types of polylactic acid with different average molecular weights (50,000 and 175,000 g/mol, respectively) in different proportions (90/10, 80/20 and 70/30) were utilized in order to produce biodegradable and biocompatible scaffolds for soft tissue engineering applications. The scaffolds were produced via thermally induced phase separation starting from ternary systems where dioxane was the solvent and water the non-solvent. Morphology (average pore size and interconnection) was evaluated by scanning electron microscopy. Foams apparent density was also evaluated (porosity ranges from 87% to 92%). Moreover, a differential scanning calorimetry analysis was carried out on the as-obtained scaffold, so as to obtain information about their thermal properties (enthalpy of melting and crystallization). The results showed that is possible to prepare scaffolds of poly-l-lactic acid/polylactic acid via thermally induced phase separation with both polylactic acids and to tune their average pore size (from 40 to 70 µm) by changing some experimental parameters (e.g. demixing temperature). Moreover, the average molecular weight of the polylactic acid in the blend seems to influence the thermally induced phase separation process in terms of demixing temperatures, which resulted higher than pure poly-l-lactic acid for the blends containing the high molecular weight polylactic acid, and lower for the blends containing the low molecular weight polylactic acid. Finally, a decrease in the crystallinity of the foams when increasing polylactic acid content in poly-l-lactic acid/polylactic acid blends was observed, as witnessed by a drop in the enthalpy of melting and crystallization. The results confirm that the morphology and the mechanical properties of the scaffold can be tuned up, starting from poly-l-lactic acid and blending it in different proportions with polylactic acid with different molecular weights.

PLLA scaffolds produced by thermally induced phase separation (TIPS) allow human chondrocyte growth and extracellular matrix formation dependent on pore size

Damage of hyaline cartilage species such as nasoseptal or joint cartilage requires proper reconstruction, which remains challenging due to the low intrinsic repair capacity of this tissue. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair. The aim of this work was to assess the viability, attachment, morphology, extracellular matrix (ECM) production of human articular and nasoseptal chondrocytes cultured in vitro in porous poly(l-lactic) (PLLA) scaffolds of two selected pore sizes (100 and 200μm). The PLLA scaffolds with 100 and 200μm pore sizes were prepared via ternary thermally induced phase separation (TIPS) technique and analyzed using scanning electron microscopy (SEM). Articular and nasoseptal chondrocytes were seeded on the scaffold and cultures maintained for 7 and 14days. Live/dead staining, (immuno-)histology and gene expression analysis of type II, type I collagen, aggrecan and SOX9 were performed to assess scaffold cytocompatibility and chondrocyte phenotype. The majority of both chondrocyte types survived on both scaffolds for the whole culture period. Hematoxylin-eosin (HE), alcian blue (visualizing glycosaminoglycans) stainings, immunoreactivity and gene expression of ECM proteins and cartilage marker (type II, I collagen, aggrecan, SOX9) of the chondrocyte scaffold constructs indicated that the smaller pore dimensions promoted the differentiation of the chondrocytes compared with the larger pore size. The present work revealed that the scaffold pore size is an important factor influencing chondrocyte differentiation and indicated that the scaffolds with 100μm pores serve as a cytocompatible basis for further future modifications.

Proteolytic enzymes clustered in specialized plasma-membrane domains drive endothelial cells' migration

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

Galvanic deposition and characterization of brushite/hydroxyapatite coatings on 316L stainless steel

In this work, brushite and brushite/hydroxyapatite (BS, CaHPO4·H2O; HA, Ca10(PO4)6(OH)2) coatings were deposited on 316L stainless steel (316LSS) from a solution containing Ca(NO3)2·4H2O and NH4H2PO4 by a displacement reaction based on a galvanic contact, where zinc acts as sacrificial anode. Driving force for the cementation reaction arises from the difference in the electrochemical standard potentials of two different metallic materials (316LSS and Zn) immersed in an electrolyte, so forming a galvanic contact leading to the deposition of BS/HA on nobler metal. We found that temperature and deposition time affect coating features (morphology, structure, and composition). Deposits were characterized by means of several techniques. The morphology was investigated by scanning electron microscopy, the elemental composition was obtained by X-ray energy dispersive spectroscopy, whilst the structure was identified by Raman spectroscopy and X-ray diffraction. BS was deposited at all investigated temperatures covering the 316LSS surface. At low and moderate temperature, BS coatings were compact, uniform and with good crystalline degree. On BS layers, HA crystals were obtained at 50°C for all deposition times, while at 25°C, its presence was revealed only after long deposition time. Electrochemical studies show remarkable improvement in corrosion resistance.

Modulation of physical and biological properties of a composite PLLA and polyaspartamide derivative obtained via thermally induced phase separation (TIPS) technique

In the present study, blend of poly l-lactic acid (PLLA) with a graft copolymer based on α,β-poly(N-hydroxyethyl)-dl-aspartamide and PLA named PHEA-PLA, has been used to design porous scaffold by using Thermally Induced Phase Separation (TIPS) technique. Starting from a homogeneous ternary solution of polymers, dioxane and deionised water, PLLA/PHEA-PLA porous foams have been produced by varying the polymers concentration and de-mixing temperature in metastable region. Results have shown that scaffolds prepared with a polymer concentration of 4% and de-mixing temperature of 22.5°C are the best among those assessed, due to their optimal pore size and interconnection. SEM and DSC analysis have been carried out respectively to study scaffold morphology and the influence of PHEA-PLA on PLLA crystallization, while DMF extraction has been carried out in order to quantify PHEA-PLA into the final scaffolds. To evaluate scaffold biodegradability, a hydrolysis study has been performed until 56days by incubating systems in a media mimicking physiological environment (pH7.4). Results obtained have highlighted a progressive increase in weight loss with time in PLLA/PHEA-PLA scaffolds, conceivably due to the presence of PHEA-PLA and polymers interpenetration. Viability and adhesion of bovine chondrocytes seeded on the scaffolds have been studied by MTS test and SEM analysis. From results achieved it appears that the presence of PHEA-PLA increases cells affinity, allowing a faster adhesion and proliferation inside the scaffold.

In vitro degradation and bioactivity of composite poly-l-lactic (PLLA)/bioactive glass (BG) scaffolds: comparison of 45S5 and 1393BG compositions

AbstractThe objective of this study was to compare the effect of two bioglass (BG) compositions 45S5 and 1393 in poly-l-lactic composite scaffolds in terms of morphology, mechanical properties, biodegradation, water uptake and bioactivity. The scaffolds were produced via thermally induced phase separation starting from a ternary polymer solution (polymer/solvent/non-solvent). Furthermore, different BG to polymer ratios have been selected (1, 2.5, 5% wt/wt) to evaluate the effect of the amount of filler on the composite structure. Results show that the addition of 1393BG does not affect the scaffold morphology, whereas the 45S5BG at the highest amount tends to appreciably modify the scaffold architecture interacting with the phase separation process. Bioactivity tests confirmed the formation of a hydroxycarbonateapatite-layer in both types of BGs (detected via scanning electron microscopy, X-ray diffractometry and Fourier Transform Infrared Spectroscopy). Overall, the results showed that 1393BG composition affects the experimental preparation protocol to a minimal extent thus allowing a better control of the scaffold’s morphology compared to 45S5BG.

PLLA Scaffold via TIPS for Bone Tissue Engineering

Tissue engineering offers a promising new approach to repair bone fractures, fractures that do not heal, and fractures due to bone tumors. In this work, two different approaches were tested in order to obtain Poly-LLactic Acid (PLLA) porous scaffolds via Thermally Induced Phase Separation (TIPS) for bone tissue engineering application. First, the possibility to produce a composite material, by incorporating Hydroxyapatite (HA) particles in a Poly-L-lactic acid (PLLA) matrix was investigated. Two PLLA/HA weight ratios (70/30 and 50/50) were tested. The results showed that the presence of HA does not influence the phase separation process, i.e. the composite scaffolds microstructure is similar to pure PLLA scaffolds. WAXD analysis confirmed the full incorporation of HA particles into the polymer matrix. Moreover, compression tests showed a fourfold increase of Young module with respect to pure PLLA scaffold. Since the production of scaffolds for bone tissue regeneration is a challenging target, scaffolds must mimic the bone morphology, thus requiring a gradient of pore dimension and morphology along one dimension. To attain this goal, the second part of the work describes the design, set up and test of an experimental apparatus able to set different thermal histories on the two sides of a sample. Scaffolds were produced by following various thermal protocols on both sample surfaces. The results showed that through this technique it is possible to produce scaffolds with a pore size that increases along sample thickness. As a matter of fact, the obtained average pore dimension on one side of the sample was about 70 μm, whereas it was around 240 μm on the opposite surface. By moving along the sample thickness, the pore dimension increased steadily. All things considered, a reliable route for the production of composite PLLA/HA scaffolds with a controlled pore size distribution was assessed, thus offering a valid support to bone tissue engineering.

PHEA‐PLLA: A New Polymer Blend For Tissue Engineering Applications

One of most important features that a material should have in order to be utilized for tissue engineering applications is its biocompatibility and its chemical surface. These properties are required for a high degree of cell adhesion on the scaffold. Poly‐L‐lactid acid (PLLA) is a biocompatible synthetic polymer approved by the Food and Drug Administration for human clinical applications. It has been largely employed, in the last years, as a constituent of surgical and implantable devices. PHEA is a biocompatible water‐soluble synthetic polymer, with a protein‐like structure, whose use as a drug carrier and as starting material for many other biomedical and pharmaceutical applications has been reported in the literature. In this work a copolymer (PHEA‐PLLA) of the aforementioned polymers was synthesized and characterized the possibility to produce porous scaffolds with it was assessed. The results have shown that is possible to prepare scaffolds of PHEA‐PLLA via Thermally Induced Phase Separation (TIPS). ...

Preparation, characterization and in vitro test of composites poly-lactic acid/hydroxyapatite scaffolds for bone tissue engineering

In this work, the possibility to produce composite Poly-L-lactic acid (PLLA)/Hydroxyapatite (HA) porous scaffolds via Thermally Induced Phase Separation (TIPS) for bone tissue engineering applications was investigated. Several PLLA/HA wt/wt ratios (95/5, 90/10, 70/30, 50/50, 34/66) were tested and the as-obtained scaffolds were characterized via Scanning Electron Microscopy, Wide Angle X-Ray Diffraction, Thermogravimetric analysis, Gas Pycnometry, Differential Scanning Calorimetry and mechanical compression test. Morphological analysis revealed an open structure with interconnected pores and HA particles embedded in the polymer matrix. Finally, cell cultures were carried out into the composite scaffolds in order to evaluate the effect of HA on the proliferation and differentiation of osteoblastic cells, showing a higher alkaline phosphatase activity on composite scaffolds compared to neat PLLA ones.