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)

Solution-based processing for scaffold fabrication in tissue engineering applications: A brief review

The fabrication of 3D scaffolds is under wide investigation in tissue engineering (TE) because of its incessant development of new advanced technologies and the improvement of traditional processes. Currently, scientific and clinical research focuses on scaffold characterization to restore the function of missing or damaged tissues. A key for suitable scaffold production is the guarantee of an interconnected porous structure that allows the cells to grow as in native tissue. The fabrication techniques should meet the appropriate requirements, including feasible reproducibility and time-and cost-effective assets. This is necessary for easy processability, which is associated with the large range of biomaterials supporting the use of fabrication technologies. This paper presents a review of scaffold fabrication methods starting from polymer solutions that provide highly porous structures under controlled process parameters. In this review, general information of solution-based technologies, including freeze-drying, thermally or diffusion induced phase separation (TIPS or DIPS), and electrospinning, are presented, along with an overview of their technological strategies and applications. Furthermore, the differences in the fabricated constructs in terms of pore size and distribution, porosity, morphology, and mechanical and biological properties, are clarified and critically reviewed. Then, the combination of these techniques for obtaining scaffolds is described, offering the advantages of mimicking the unique architecture of tissues and organs that are intrinsically difficult to design

A Biodegradable, Bio-Based Polymer for the Production of Tools for Aquaculture: Processing, Properties and Biodegradation in Sea Water

Bio-based, biodegradable polymers can dramatically reduce the carbon dioxide released into the environment by substituting fossil-derived polymers in some applications. In this work, prototypes of trays for aquaculture applications were produced via injection molding by using a biodegradable polymer, Mater-Bi-(R). A characterization carried out via calorimetric, rheological and mechanical tests revealed that the polymer employed shows properties suitable for the production of tools to be used in aquaculture applications. Moreover, the samples were subjected to a biodegradation test in conditions that simulate the marine environment. The as-treated samples were characterized from gravimetrical, morphological and calorimetric point of views. The obtained data showed a relatively low biodegradation rate of the thick molded samples. This behavior is of crucial importance since it implies a long life in marine water for these manufacts before their disappearing

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

Neurophysiological Findings in Neuronal Ceroid Lipofuscinoses

Neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of neurodegenerative diseases, characterized by progressive cerebral atrophy due to lysosomal storage disorder. Common clinical features include epileptic seizures, progressive cognitive and motor decline, and visual failure, which occur over different time courses according to subtypes. During the latest years, many advances have been done in the field of targeted treatments, and in the next future, gene therapies and enzyme replacement treatments may be available for several NCL variants. Considering that there is rapid disease progression in NCLs, an early diagnosis is crucial, and neurophysiological features might have a key role for this purpose. Across the different subtypes of NCLs, electroencephalogram (EEG) is characterized by a progressive deterioration of cerebral activity with slowing of background activity and disappearance of spindles during sleep. Some types of heterogeneous abnormalities, diffuse or focal, prevalent over temporal and occipital regions, are described in many NCL variants. Photoparoxysmal response to low-frequency intermittent photic stimulation (IPS) is a typical EEG finding, mostly described in CLN2, CLN5, and CLN6 diseases. Visual evoked potentials (VEPs) allow to monitor the visual functions, and the lack of response at electroretinogram (ERG) reflects retinal neurodegeneration. Taken together, EEG, VEPs, and ERG may represent essential tools toward an early diagnosis of NCLs

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

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

Tubular composite scaffolds produced via Diffusion Induced Phase Separation (DIPS) as a shaping strategy for anterior cruciate ligaments reconstruction

Injuries of tendons and ligaments are common, especially among the young population. Anterior cruciate ligament (ACL) injuries do not heal due to its limited vascularization and hence, surgical intervention is usually required. The ideal scaffold for ligament tissue engineering (TE) should be biocompatible and possess mechanical and functional characteristics comparable to the native ACL. The Diffusion Induced Phase Separation (DIPS) technique allows the preparation of homogenous porous tubular scaffold with micro-pores using a rather simple procedure. Composites based on biodegradable polymers and bioglass have attracted much attention in tissue reconstruction and repair because of their biological and physicochemical advantages. In this work a new approach in ACL TE will be proposed focussing on the development of a suitable technique for in vitro seeding of lapine ACL fibroblasts into tubular-shaped instructive Poly-lactic-acid (PLLA) scaffolds, supplemented or not with bioglass (BG) 1393, produced via DIPS. Tubular composite scaffold (diameters: 1.2 and 2 mm, +/- BG) were obtained through a dip coating around a cylindrical support followed by a DIPS. An 8%wt PLLA/dioxane solution was prepared with 5%wt of BG-1393 as filler. Preliminary in vitro cell culture trials were carried out by seeding lapine ACL fibroblasts inside the scaffolds (2 cm as length) employing different seeding strategies in order to find the best way that allows to obtain a homogeneous fibroblast distribution inside the tubes. (1) First trials consisted in the inoculating of the cell suspension inside the tubes and maintaining them in dynamical culture. (2) The second one was done by suspending the cells in a fibrin gel polymerized within the tubes by using of thrombin. (3) The third approach was carried out by using cell spheroids (three-dimensional self-assembled cell agglomerates). Cell attachment, viability and morphology were examined by live-death and Hematoxylin/Eosin stainings after 1, 7, 14 d and vimentin immunolabelings (7 d). Scanning electron microscopical analysis revealed that the internal surface of the tubes was homogeneously structured with micropores sized around 5 µm and a mean thickness of the wall of 60 µm. The results showed cell adhesion to the wall of the tubes with all seeding techniques applied even though with fibrin gel it was more homogenous. Furthermore, colonized areas expanded with culture time and the majority of cell survived irrespectively of seeding techniques. (1) In inoculation phase, many cells left the scaffold and attached on the plate. Even after the dynamic culture (rotating device) most cells covered only half the tube inner surface. (2) In the second trial, a fibrin gel was used to achieve a homogenous cell distribution during seeding. In the early stage (48 h) cells remained captured inside the fibrin, but after 7 d they become elongated and migrated from the fibrin to the inner tube surface forming a compact cell layer. So, the fibrin appears helpful to achieve an immediate high cell seeding efficiency and an almost homogeneous cell distribution inside the tubes. (3) Although using the spheroid technique the scaffold internal surface was not homogeneously colonized with cells, after 7 d cell migration to the inner scaffold surface from the attaching spheroids could be observed. In longitudinal sections cells were elongated like typical ligament fibroblasts parallel to the longitudinal tube axis. Therefore, it can be affirmed that employment of tubular scaffolds produced by DIPS could be a promising approach of ligament TE. In the future, it would be interesting to evaluate the effectiveness of seeding by combining the spheroids and the fibrin gel

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