Poly-Left-Lactic Acid tubular scaffolds via Diffusion Induced Phase Separation (DIPS): control of morphology

n this work, tubular poly-left-lactic acid scaffolds for vascular tissue engineering applications were produced by an innovative two-step method. The scaffolds were obtained by performing a dip-coating around a nylon fiber, followed by a diffusion induced phase separation process. Morphological analysis revealed that the internal lumen of the as-obtained scaffold is equal to the diameter of the fiber utilized; the internal surface is homogeneous with micropores 1–2 μm large. Moreover, a porous open structure was detected across the thickness of the walls of the scaffold. An accurate analysis of the preparation process revealed that it is possible to tune up the morphology of the scaffold (wall thickness, porosity, and average pore dimension), simply by varying some experimental parameters. Preliminary in vitro cell culture tests were carried out inside the scaffold. The results showed that cells are able to grow within the internal surface of the scaffolds and after 3 weeks they begin to form a “primordial” vessel-like structure. Modeling predictions of the dip-coating process display always an underestimate of experimental data (dependence of wall thickness upon extraction rate).In this work, tubular poly-left-lactic acid scaffolds for vascular tissue engineering applications were produced by an innovative two-step method. The scaffolds were obtained by performing a dip-coating around a nylon fiber, followed by a diffusion induced phase separation process. Morphological analysis revealed that the internal lumen of the as-obtained scaffold is equal to the diameter of the fiber utilized; the internal surface is homogeneous with micropores 1–2 lm large. Moreover, a porous open structure was detected across the thickness of the walls of the scaffold. An accurate analysis of the preparation process revealed that it is possible to tune up the morphology of the scaffold (wall thickness, porosity, and average pore dimension), simply by varying some experimental parameters. Preliminary in vitro cell culture tests were carried out inside the scaffold. The results showed that cells are able to grow within the internal surface of the scaffolds and after 3 weeks they begin to form a ‘‘primordial’’ vessel-like structure. Modeling predictions of the dipcoating process display always an underestimate of experimental data (dependence of wall thickness upon extraction rate)

PLLA biodegradable scaffolds for angiogenesis via Diffusion Induced Phase Separation (DIPS)

A critical obstacle in tissue engineering is the inability to maintain large masses of living cells upon transfer from the in vitro culture conditions into the host in vivo. Capillaries, and the vascular system, are required to supply essential nutrients, including oxygen, remove waste products and provide a biochemical communication “highway”. For this reason it is mandatory to manufacture an implantable structure where the process of vessel formation – the angiogenesis – can take place. In this work PLLA scaffolds for vascular tissue engineering were produced by dip-coating via Diffusion Induced Phase Separation (DIPS) technique. The scaffolds, with a vessel-like shape, were obtained by performing a DIPS process around a nylon fibre whose diameter was 700 μm. The fibre was first immersed into a 4% PLLA dioxane solution and subsequently immersed into a second bath containing distilled water. The covered fibre was then rinsed in order to remove the excess of dioxane and dried; finally the internal nylon fibre was pulled out so as to obtain a hollow biodegradable PLLA fiber. SEM analysis revealed that the scaffolds have a lumen of ca. 700 μm. The internal surface is homogeneous with micropores 1–2 μm large. Moreover, a cross section analysis showed an open structure across the thickness of the scaffold walls. A cell culture of endothelial cells was carried out into the as-prepared scaffolds. The result showed that cells are able to grow within the scaffolds and after 3 weeks they begin to form a “primordial” vessel-like structure

Galvanic deposition of Chitosan-AgNPs as antibacterial coating

Thanks to mechanical properties similar human bones, metallic materials represent the best choice for fabrication of orthopedic implants. Although metals could be widely used in the field of biomedical implants, corrosion phenomena could occur, causing metal ions releasing around periprosthetic tissues leading, in the worst cases, to the development of infections. In these cases, patients need prolonged antibiotic therapies that may cause bacterial resistance. Preventing bacterial colonization of biomedical surfaces is the key to limiting the spread of infections. Antibacterial coatings have become a very active field of research, strongly stimulated by the increasing urgency of identifying alternatives to the traditional administration of antibiotics. Nowadays, the research was focused on coating science to deal with these issues. In particular, the development of the antibacterial composite coatings could be a viable way to provide not only a corrosion resistance but also an antibacterial action and biocompatibility. Chitosan is a great biomaterial used in medicine. It is a natural bioactive polymer and is the second most abundant in nature polysaccharide after cellulose. Chitosan comes from the deacetylation of chitin, a homopolymer of beta-(1-4)-N-acetyl-D-glucosamine, derived from exoskeleton of crustaceans. It is high biocompatible and it is also used in drug delivery. In addition, chitosan has chelating properties due to the amino groups of polysaccharide that are responsible of selective chelation with metal ions. In particular, the attention has been paid to silver nanoparticles for their high stability, low toxicity, biocompatibility and antibacterial properties. These ones are incorporated in polymeric matrix (e.g. chitosan) and they are capable to interact physically with cell walls of bacteria. In this study Chitosan-Silver nanoparticles composite coating on AISI 304L was investigated. These coatings were realized by an alternative method of deposition respect to traditional ones based on galvanic coupling. This process doesn’t request any external power supply and is very easy to carried out. The difference of the electrochemical redox potential between the substrate (cathode) and a sacrificial anode is the pivotal role of the process. Deposition rate is controlled by the ratio of cathodic and anodic area. In practice, electrons generated by anode corrosion flow towards to more noble metal thanks to a short-circuit. As soon electrons arrive to the cathode, the base electrogeneration reactions of nitrate ions and water molecules occur. Production of hydroxyl ions causes an increasing of pH at substrate/solution interface. Hence, deprotonation of amine group leads precipitation of chitosan (pKa=6.4) onto surface. At the same time, silver nanoparticles are incorporated in polymeric matrix of chitosan. Physical-chemical characterizations of the coatings were carried out in order to investigate morphology and chemical composition. In addition, corrosion tests (potentiodynamic polarization and electrochemical impedance spectroscopy) were executed in a simulated body fluid to scrutinize the corrosion resistance. Furthermore, the release of silver nanoparticles from coating in SBF were studied

Galvanic Deposition of Hydroxyapatite/Chitosan/Collagen Coatings on 304 Stainless Steel

The galvanic deposition method was used to deposit Hydroxyapatite/Chitosan/Collagen coatings on 304 stainless steel. Galvanic deposition is an alternative and valid way to fabricate bio-coatings with high biocompatibility and good anticorrosion properties. Physical-chemical characterizations were carried out to investigate chemical composition and morphology of the samples. Coatings consist of a mixture of calcium phosphate (Brushite and Hydroxyapatite) with chitosan and collagen. Corrosion tests were performed in the simulated body fluid (SBF) after different aging times. Results show that, in comparison with bare 304 stainless steel, coating shifts corrosion potential to anodic values and reduces corrosion current density. Nevertheless, the aging in SBF led to a completely conversion of brushite into hydroxyapatite. The release of metal ions, measured after 21 days of aging in SBF solution, is very low due to the presence of coating that slow-down the corrosion rate of steel

Galvanic Deposition of Calcium Phosphate/Bioglass Composite Coating on AISI 316L

Calcium phosphate/Bioglass composite coatings on AISI 316L were investigated with regard to their potential role as a beneficial coating for orthopedic implants. These coatings were realized by the galvanic co-deposition of calcium phosphate compounds and Bioglass particles. A different amount of Bioglass 45S5 was used to study its effect on the performance of the composite coatings. The morphology and chemical composition of the coatings were investigated before and after their aging in simulated body fluid. The coatings uniformly covered the AISI 316L substrate and consisted of a brushite and hydroxyapatite mixture. Both phases were detected using X-ray diffraction and Raman spectroscopy. Additionally, both analyses revealed that brushite is the primary phase. The presence of Bioglass was verified through energy-dispersive X-ray spectroscopy, which showed the presence of a silicon peak. During aging in simulated body fluid, the coating was subject to a dynamic equilibrium of dissolution/reprecipitation with total conversion in only the hydroxyapatite phase. Corrosion tests performed in simulated body fluid at different aging times revealed that the coatings made with 1 g/L of Bioglass performed best. These samples have a corrosion potential of −0.068V vs. Ag/AgCl and a corrosion current density of 8.87 × 10−7 A/cm2. These values are better than those measured for bare AISI 316L (−0.187 V vs. Ag/AgCl and 2.52 × 10−6 A/cm2, respectively) and remained superior to pure steel for all 21 days of aging. This behavior indicated the good protection of the coating against corrosion phenomena, which was further confirmed by the very low concentration of Ni ions (0.076 ppm) released in the aging solution after 21 days of immersion. Furthermore, the absence of cytotoxicity, verified through cell viability assays with MC3T3-E1 osteoblastic cells, proves the biocompatibility of the coatings

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 56 days by incubating systems in a media mimicking physiological environment (pH 7.4). 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

3D cultures of primary astrocytes on Poly-L-lactic acid scaffolds

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro tissues with morphological and functional features similar to the biological tissue of the human body. Polymeric materials can be used in contact with biological systems in replacing destroyed tissue by transplantation [1]. Several biopolymers, including poly L (lactic acid) (PLLA), have been used in biomedical applications to set scaffolds with ductile proprieties and biodegradation kinetics [2]. In particular, the PLLA scaffold topography mimics the natural extracellular matrix and makes it a good candidate for neural tissue engineering. We report about of 3D system the PLLA porous scaffolds prepared via thermally-induced phase separation (TIPS) [3], and utilized as substrate for primary rat astrocytes 3D growth. Interestingly astrocytes adapt well to these porous matrices, not only remaining on the surface, but also penetrating inside the scaffolds. They colonize the matrix acquiring a typical star-like morphology; they form cell contacts and, in addition produce EVs as in vivo [4]. These results suggest that the chosen conditions could be a good starting point for 3D brain culture systems. PLLA scaffolds could be further enriched to host two or three different brain cell types, in order to set an in vitro model of blood brain barrier. The future use of co-culture systems may be involved in drug delivery studies, and in the formulation of new therapeutic strategies for the treatment of neurological diseases. [1]Langer R, Vacanti JP. Tissue engineering. Science. 1993; 260: 920 [2]Nejati E, et al. Appl. Sci. Manuf. 2008; 39: 1589–1596 [3]Scaffaro R, et al. J. Mech. Behav. Biomed. Mater. 2016; 54:8-20 [4]Schiera G, et al. Biomed Res Int 2015: 152926, 201

Use of Modified 3D Scaffolds to Improve Cell Adhesion and Drive Desired Cell Responses.

In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement.In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement. Copyright © 2012, AIDIC Servizi S.r.l

PLA/PLLA scaffold for vascular tissue engineering applications

A critical obstacle encountered by tissue engineering is the inability to maintain large masses of living cells upon transfer from the in vitro culture conditions to host in vivo. Capillaries, and the vascular system, are required to supply essential nutrients, including oxygen, remove waste products and provide a biochemical communication “highway”. Another task in this research field is the possibility to tune the biodegradability of the scaffold. After implantation, the scaffold must be gradually populated by cells and replaced by extra cellular matrix; with this respect, it is crucial that this replacement takes place with appropriate dynamics and a well-defined timescale. A premature degradation, in fact, could lead to a collapse of the structure as the newly generated tissue could not have reached yet the suitable mechanical properties. Conversely, a long degradation time could delay or completely interrupt the development of the new tissue. In this work scaffolds for vascular tissue engineering were produced by dip-coating followed by Diffusion Induced Phase Separation (DIPS). Several PLA-PLLA blends (100/0, 75/25, 25/75, 10/90 wt/wt) were utilized to produce the scaffolds. Moreover preliminary biodegradation tests were carried out in order to evaluate the degradation rates

Preparation and characterization of PLLA-HA scaffolds for bone tissue engineering

In this work, the possibility to produce composite - Poly-L-lactic acid (PLLA) and Hydroxyapatite (HA) - porous scaffolds via Thermally Induced Phase Separation for bone tissue engineering applications was investigated. Several PLLA/HA ratios were tested (70/30, 50/50, 30/70 and 20/80 wt/wt) and the as-obtained scaffolds were characterized via Scanning Electron Microscopy (SEM), Wide Angle X-Ray Diffraction (WAXD) and compression test. The results showed that the presence of HA does not influence the phase separation process. Morphological analysis revealed an open structure with interconnected pores and HA embedded in the polymer matrix. This evidence was confirmed by WAXD analysis; where the typical peaks of PLLA and HA appear. Finally, an increase of the young modulus was observed in the composite scaffolds with respect to pure PLLA scaffolds