3d printing in alginic acid bath of in-situ crosslinked collagen composite scaffolds

Bone-tissue regeneration is a growing field, where nanostructured-bioactive materials are designed to replicate the natural properties of the target tissue, and then are processed with technolo-gies such as 3D printing, into constructs that mimic its natural architecture. Type I bovine collagen formulations, containing functional nanoparticles (enriched with therapeutic ions or biomolecules) or nanohydroxyapatite, are considered highly promising, and can be printed using support baths. These baths ensure an accurate deposition of the material, nonetheless their full removal post-printing can be difficult, in addition to undesired reactions with the crosslinking agents often used to improve the final structural integrity of the scaffolds. Such issues lead to partial collapse of the printed constructs and loss of geometrical definition. To overcome these limitations, this work presents a new alternative approach, which consists of adding a suitable concentration of crosslinking agent to the printing formulations to promote the in-situ crosslinking of the constructs prior to the removal of the support bath. To this aim, genipin, chosen as crosslinking agent, was added (0.1 wt.%) to collagen-based biomaterial inks (containing either 38 wt.% mesoporous bioactive glasses or 65 wt.% nanohydroxyapatite), to trigger the crosslinking of collagen and improve the stability of the 3D printed scaffolds in the post-processing step. Moreover, to support the material deposition, a 15 wt.% alginic acid solution was used as a bath, which proved to sustain the printed structures and was also easily removable, allowing for the stable processing of high-resolution geometries

Long-term impact of chemical and alternative fungicides applied to grapevine CV nebbiolo on berry transcriptome

Viticulture is one of the horticultural systems in which antifungal treatments can be extremely frequent, with substantial economic and environmental costs. New products, such as biofungicides, resistance inducers and biostimulants, may represent alternative crop protection strategies respectful of the environmental sustainability and food safety. Here, the main purpose was to evaluate the systemic molecular modifications induced by biocontrol products as laminarin, resistance inducers (i.e., fosetyl-Al and potassium phosphonate), electrolyzed water and a standard chemical fungicide (i.e., metiram), on the transcriptomic profile of ‘Nebbiolo’ grape berries at harvest. In addition to a validation of the sequencing data through real-time polymerase chain reaction (PCR), for the first-time the expression of some candidate genes in different cell-types of berry skin (i.e., epidermal and hypodermal layers) was evaluated using the laser microdissection approach. Results showed that several considered antifungal treatments do not strongly affect the berry transcriptome profile at the end of season. Although some treatments do not activate long lasting molecular defense priming features in berry, some compounds appear to be more active in long-term responses. In addition, genes differentially expressed in the two-cell type populations forming the berry skin were found, suggesting a different function for the two-cell type populations

PEG-coated large mesoporous silicas as smart platform for protein delivery and their use in a collagen-based formulation for 3d printing

Silica-based mesoporous systems have gained great interest in drug delivery applications due to their excellent biocompatibility and high loading capability. However, these materials face challenges in terms of pore-size limitations since they are characterized by nanopores ranging between 6–8 nm and thus unsuitable to host large molecular weight molecules such as proteins, enzymes and growth factors (GFs). In this work, for an application in the field of bone regeneration, large-pore mesoporous silicas (LPMSs) were developed to vehicle large biomolecules and release them under a pH stimulus. Considering bone remodeling, the proposed pH-triggered mechanism aims to mimic the release of GFs encased in the bone matrix due to bone resorption by osteoclasts (OCs) and the associated pH drop. To this aim, LPMSs were prepared by using 1,3,5-trimethyl benzene (TMB) as a swelling agent and the synthesis solution was hydrothermally treated and the influence of different process temperatures and durations on the resulting mesostructure was investigated. The synthesized particles exhibited a cage-like mesoporous structure with accessible pores of diameter up to 23 nm. LPMSs produced at 140◦C for 24 h showed the best compromise in terms of specific surface area, pores size and shape and hence, were selected for further experiments. Horseradish peroxidase (HRP) was used as model protein to evaluate the ability of the LPMSs to adsorb and release large biomolecules. After HRP-loading, LPMSs were coated with a pH-responsive polymer, poly(ethylene glycol) (PEG), allowing the release of the incorporated biomolecules in response to a pH decrease, in an attempt to mimic GFs release in bone under the acidic pH generated by the resorption activity of OCs. The reported results proved that PEG-coated carriers released HRP more quickly in an acidic environment, due to the protonation of PEG at low pH that catalyzes polymer hydrolysis reaction. Our findings indicate that LPMSs could be used as carriers to deliver large biomolecules and prove the effectiveness of PEG as pH-responsive coating. Finally, as proof of concept, a collagen-based suspension was obtained by incorporating PEG-coated LPMS carriers into a type I collagen matrix with the aim of designing a hybrid formulation for 3D-printing of bone scaffolds

Electrophoretic deposition of Sr-containing mesoporous bioactive glass particles produced by spray-drying

Introduction Mesoporous bioactive glasses (MBGs) are gaining increasing interest in the biomedical field thanks to their exceptional textural characteristics (high surface area, high pore volume and highly ordered mesoporosity). These properties lead to an improved apatite kinetics formation, which allow these glasses to be successfully applied in bone tissue regeneration [1]. In this work we adopted an aerosol-based spray drying process in order to have high control and reproducibility over the morphology of particles. In order to increase their regenerative potential, the particles have been doped with strontium, element known for its osteogenic and bone antiresorptive properties [2]. Later the particles have been deposed by electrophoretic deposition (EPD) on glass-ceramic scaffolds fabricated by the polymer sponge replication method. EPD is a versatile technique which allows an easy control of the thickness of the deposited film through simple adjustment of the applied voltage and the deposition time. The scaffolds, based on a quaternary silicate glass (SCNA, SiO2–CaO–Na2O–Al2O3 oxide system), have good mechanical properties but low bioactivity [3]. Thanks to MBG particle deposition, they acquire a pronounced bioactive behaviour, thus becoming an excellent solution for bone tissue regeneration. Results and Discussion MBGs synthesized with the aerosol-based spray-drying process have a basic composition on the SiO2-CaO system and have been doped with the 1% molar of strontium (SD_Sr1). FESEM image of particles shows micro-sized spherical particles, with size mostly ranging between 500 nm and 5 µm. N2 adsorption analysis gives back a high specific surface area value, 160 m2/g, and a pore size distribution between 5 and 9 nm, which confirms the mesoporosity of the sample. Strontium incorporation inside the binary composition does not modify the bioactive behaviour of the glass: after 14 days in SBF nanoparticles are completely covered by a layer of hydroxyapatite.The EDS quantitative analysis shows that the amount of strontium effectively incorporated in the microparticles was 70% of the theoretical one, probably because of the high dimension of the ion which hinders its entrance into the glass network. Nevertheless, most of the Sr incorporated has been released after 14 days of immersion in SBF, as the coupled plasma-atomic emission spectrometry (ICP-AES) reveals. On the basis of literature data, the released concentrations are suitable for inducing osteogenesis [4]. EPD has been performed in ethanol, applying a voltage of 120 V for 5 minutes. The scaffolds, being not conductive, have been suspended between two stainless steel electrodes through a clamp. A dispersant (TEA, triethanolamine) has been used to keep the particles in suspension during the whole deposition time. The deposited layer was abundant but not uniform on the scaffold surface. After immersion for 7 days in SBF, hydroxyapatite formation has been observed on the surface of the microparticles deposited on the scaffold struts. This demonstrates that MBGs not only maintain their bioactivity after deposition but also transfer this property to scaffolds. Conclusions MBGs synthetized with aerosol-based spray-drying process and doped with strontium have excellent textural properties and a bioactive behaviour. After electrophoretic deposition, they maintain these properties and consequently they improve the bioactivity of SCNA scaffolds, which initially are almost biologically inert. In this way we demonstrate that it is possible to obtain a successful construct for bone tissue engineering with both excellent regenerative and mechanical properties

Phosphate glass fibrous scaffolds: tailoring of the properties and improvement of the bioactivity through the incorporation of mesoporous glasses

Introduction. Synthetic bone scaffolds are proposed as an alternative to the use of bone grafting technique for bone regeneration. Porous scaffold obtained from glass fibres randomly arranged into a mould shows an interconnected porosity generated by the free space between fibres and they do not need of any further material or processing step before sintering. In this work, a resorbable phosphate glass was selected for the fibre drawing and bioactive mesoporous glasses with different morphology and size were incorporated into the fibrous scaffold to combine the resorption property of the fibres with the bioactivity of the mesoporous powders. Materials and methods. Fibres of a TiO2-containing phosphate glass (TiPS2.5) were fabricated following the preform drawing approach as described elsewhere [1]. A dense silica-based bioactive glass (CEL2) [2] was produced by melt quenching as reference sample. Spherical micro-sized mesoporous glass based on SiO2-CaO system (SD_MBG) was produced by an aerosol-assisted spray-drying technique [3]. Cu-containing (85SiO2-13CaO-2CuO, % mol, referred as Cu_BGn2%) mesoporous glass nanoparticles were synthetized by an ultra-sound assisted sol-gel method to impart antibacterial properties. To fabricate the fibrous scaffolds, the selected powder and phosphate glass fibres, cut at precise length, were placed in a beaker containing 2 ml of ethanol. After ethanol evaporation, the powder/fibre mixture was randomly placed inside a zirconia cylindrical mould [4]. After the thermal treatment, the scaffolds were analyzed through micro-CT in order to investigate their inner structure. Furthermore, their ability to form hydroxyapatite was studied by soaking them in a simulated body fluid (SBF). The scaffold morphology before and after immersion in SBF was studied by FESEM. Results and discussion. FESEM micrographs show that CEL2 are not well incorporated into the fibre surface. On the contrary, SD-MBG (Figure 1.a, Figure 1.b and Figure 1.d) and Cu_BGn2% particles homogeneously cover the whole surface. Micro-CT analysis did not reveal the presence of powder agglomerates for all the observed scaffolds and showed a homogeneous porosity of 58 vol.% for CEL2/fibre scaffold, 53 vol.% for SD_MBG/scaffold (Figure 1.c) and 33% for Cu_BGn2%/scaffold. In CEL2/fibre scaffolds, glass particles were removed during soaking in SBF, leaving some pits on the fibre surface: FESEM analysis revealed few particles still anchored to the scaffold surface after 7 days. On the contrary, after 7 days in SBF, SD-MBG and Cu_BGn2% particles were clearly visible on the surface of the scaffolds and after 1 day of soaking in SBF, they appeared (Figure 2) fully covered with a HA layer, showing the typical "cauliflower-like" morphology. Conclusion. The incorporation of mesoporous bioactive glass powder in the phosphate glass fibrous scaffold resulted to be a very interesting strategy to impart multifunctional properties to the scaffold. These promising results encourage further investigation in order to fully exploit the ability of mesoporous particles to act as a system for smart release of therapeutic ions and drugs

Theta Phase Entrainment of Single-Cell Spiking in Rat Somatosensory Barrel Cortex and Secondary Visual Cortex Is Enhanced during Multisensory Discrimination Behavior

Phase entrainment of cells by theta oscillations is thought to globally coordinate the activity of cell assemblies across different structures, such as the hippocampus and neocortex. This coordination is likely required for optimal processing of sensory input during recognition and decision-making processes. In quadruple-area ensemble recordings from male rats engaged in a multisensory discrimination task, we investigated phase entrainment of cells by theta oscillations in areas along the corticohippocampal hierarchy: somatosensory barrel cortex (S1BF), secondary visual cortex (V2L), perirhinal cortex (PER), and dorsal hippocampus (dHC). Rats discriminated between two 3D objects presented in tactile-only, visual-only, or both tactile and visual modalities. During task engagement, S1BF, V2L, PER, and dHC LFP signals showed coherent theta-band activity. We found phase entrainment of single-cell spiking activity to locally recorded as well as hippocampal theta activity in S1BF, V2L, PER, and dHC. While phase entrainment of hippocampal spikes to local theta oscillations occurred during sustained epochs of task trials and was nonselective for behavior and modality, somatosensory and visual cortical cells were only phase entrained during stimulus presentation, mainly in their preferred modality (S1BF, tactile; V2L, visual), with subsets of cells selectively phase-entrained during cross-modal stimulus presentation (S1BF: visual; V2L: tactile). This effect could not be explained by modulations of firing rate or theta amplitude. Thus, hippocampal cells are phase entrained during prolonged epochs, while sensory and perirhinal neurons are selectively entrained during sensory stimulus presentation, providing a brief time window for coordination of activity

New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development