Lymphocyte Activation Gene-3 Regulates Dendritic Cell Metabolic Programing and T Cell Priming Function.

Deficiency of lymphocyte activation gene-3 (LAG3) is significantly associated with increased cardiovascular disease risk with in vitro results demonstrating increased TNF-α and decreased IL-10 secretion from LAG3-deficient human B lymphoblasts. The hypothesis tested in this study was that Lag3 deficiency in dendritic cells (DCs) would significantly affect cytokine expression, alter cellular metabolism, and prime naive T cells to greater effector differentiation. Experimental approaches used included differentiation of murine bone marrow-derived DCs (BMDCs) to measure secreted cytokines, cellular metabolism, RNA sequencing, whole cell proteomics, adoptive OT-II CD4+Lag3 +/+ donor cells into wild-type (WT) C57BL/6 and Lag3 -/- recipient mice, and ex vivo measurements of IFN-γ from cultured splenocytes. Results showed that Lag3 -/- BMDCs secreted more TNF-α, were more glycolytic, used fewer fatty acids for mitochondrial respiration, and glycolysis was significantly reduced by exogenous IL-10 treatment. Under basal conditions, RNA sequencing revealed increased expression of CD40 and CD86 and other cytokine-signaling targets as compared with WT. Whole cell proteomics identified a significant number of proteins up- and downregulated in Lag3 -/- BMDCs, with significant differences noted in exogenous IL-10 responsiveness compared with WT cells. Ex vivo, IFN-γ expression was significantly higher in Lag3 -/- mice as compared with WT. With in vivo adoptive T cell and in vitro BMDC:T coculture experiments, Lag3 -/- BMDCs showed greater T cell effector differentiation and proliferation, respectively, compared with WT BMDCs. In conclusion, Lag3 deficiency in DCs is associated with an inflammatory phenotype that provides a plausible mechanism for increased cardiovascular disease risk in humans with LAG3 deficiency.

Bioactive organic-inorganic poly(CLMA-co-HEA)/silica nanocomposites.

A series of novel poly(CLMA-co-HEA)/silica nanocomposites is synthesized from caprolactone 2-(methacryloyloxy)ethyl ester (CLMA) and 2-hydroxyethyl acrylate (HEA) as organic comonomers and the simultaneous sol-gel polymerization of tetraethyloxysilane (TEOS) as silica precursor, in different mass ratios up to a 30 wt% of silica. The nanocomposites are characterized as to their mechanical and thermal properties, water sorption, bioactivity and biocompatibility, reflecting the effect on the organic matrix provided by the silica network formation. The nanocomposites nucleate the growth of hydroxyapatite (HAp) on their surfaces when immersed in the simulated body fluid of the composition used in this work. Proliferation of the MC3T3 osteoblast-like cells on the materials was assessed with the MTS assay showing their biocompatibility. Immunocytochemistry reveals osteocalcin and type I collagen production, indicating that osteoblast differentiation was promoted by the materials, and calcium deposition was confirmed by von Kossa staining. The results indicate that these poly(CLMA-co-HEA)/silica nanocomposites could be a promising biomaterial for bone tissue engineering.

Methacrylate-endcapped caprolactone and FM19G11 provide a proper niche for spinal cord-derived neural cells.

Spinal cord injury (SCI) is a cause of paralysis. Although some strategies have been proposed to palliate the severity of this condition, so far no effective therapies have been found to reverse it. Recently, we have shown that acute transplantation of ependymal stem/progenitor cells (epSPCs), which are spinal cord-derived neural precursors, rescue lost neurological function after SCI in rodents. However, in a chronic scenario with axon repulsive reactive scar, cell transplantation alone is not sufficient to bridge a spinal cord lesion, therefore a combinatorial approach is necessary to fill cavities in the damaged tissue with biomaterial that supports stem cells and ensures that better neural integration and survival occur. Caprolactone 2-(methacryloyloxy) ethyl ester (CLMA) is a monomer [obtained as a result of ε-caprolactone and 2-hydroxyethyl methacrylate (HEMA) ring opening/esterification reaction], which can be processed to obtain a porous non-toxic 3D scaffold that shows good biocompatibility with epSPC cultures. epSPCs adhere to the scaffolds and maintain the ability to expand the culture through the biomaterial. However, a significant reduction of cell viability of epSPCs after 6 days in vitro was detected. FM19G11, which has been shown to enhance self-renewal properties, rescues cell viability at 6 days. Moreover, addition of FM19G11 enhances the survival rates of mature neurons from the dorsal root ganglia when cultured with epSPCs on 3D CLMA scaffolds. Overall, CLMA porous scaffolds constitute a good niche to support neural cells for cell transplantation approaches that, in combination with FM19G11, offer a new framework for further trials in spinal cord regeneration.

Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds.

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.

Platelet-rich plasma favors proliferation of canine adipose-derived mesenchymal stem cells in methacrylate-endcapped caprolactone porous scaffold niches.

Osteoarticular pathologies very often require an implementation therapy to favor regeneration processes of bone, cartilage and/or tendons. Clinical approaches performed on osteoarticular complications in dogs constitute an ideal model for human clinical translational applications. The adipose-derived mesenchymal stem cells (ASCs) have already been used to accelerate and facilitate the regenerative process. ASCs can be maintained in vitro and they can be differentiated to osteocytes or chondrocytes offering a good tool for cell replacement therapies in human and veterinary medicine. Although ACSs can be easily obtained from adipose tissue, the amplification process is usually performed by a time consuming process of successive passages. In this work, we use canine ASCs obtained by using a Bioreactor device under GMP cell culture conditions that produces a minimum of 30 million cells within 2 weeks. This method provides a rapid and aseptic method for production of sufficient stem cells with potential further use in clinical applications. We show that plasma rich in growth factors (PRGF) treatment positively contributes to viability and proliferation of canine ASCs into caprolactone 2-(methacryloyloxy) ethyl ester (CLMA) scaffolds. This biomaterial does not need additional modifications for cASCs attachment and proliferation. Here we propose a framework based on a combination of approaches that may contribute to increase the therapeutical capability of stem cells by the use of PRGF and compatible biomaterials for bone and connective tissue regeneration.

Blending polysaccharides with biodegradable polymers. I. Properties of chitosan/polycaprolactone blends.

Blends of polycaprolactone (PCL) and chitosan (CHT) were prepared by casting from a solution. CHT and PCL were dissolved by using acetic acid/water mixtures. Both solutions were slowly mixed to cast blend films containing 10%, 20%, 30%, and 40% by weight of CHT. PCL and CHT form phase separated blends. The phase morphology is in large extent controlled by the casting procedure. Even if casting of the film starts from a clear solution, the solvent composition determines the form in which phase separation takes place and consequently the morphology of the resulting blend after solvent evaporation. The blend containing 20% CHT presents cocontinuous phases. The sample presents a high elastic modulus even at temperatures above melting of PCL. Blends with higher CHT contents consist of disperse PCL domains in a CHT matrix and the contrary occurs in the blend containing 10% CHT in which disperse CHT domains with a network morphology appear inside the spherulites of PCL. In all the blends, the nucleation effect of CHT accelerates the crystallization of PCL from the melt, although in the blends with high CHT contents a part of the PCL mass included in large domains might not be affected by the presence of CHT. The sample containing 20% CHT has a peculiar behavior with respect to the crystallization of PCL, only a small part of PCL crystallizes in isothermal treatments although this fraction crystallizes faster than in the rest of the blends.

Blending polysaccharides with biodegradable polymers. II. Structure and biological response of chitosan/polycaprolactone blends.

Blends of polycaprolactone (PCL) and chitosan (CHT) were prepared by casting from the mixture of solutions of both components in suitable solvents. PCL, and CHT, form phase separated blends with improved mechanical properties and increased water sorption ability with respect to pure PCL. The morphology of the system was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and confocal microscopy. Dispersed domains of CHT in the semicrystalline PCL matrix were found in samples with less than 20% CHT but cocontinuous phase morphologies are found in blends with 20% or more CHT. This feature was corroborated by the temperature dependence of the elastic modulus measured by dynamic mechanical properties as a function of temperature. It was observed that for those blends above 20 wt% CHT, the mechanical stability of the system was kept even after melting of the PCL phase. Primary human chondrocytes were cultured on the different substrates. Cell morphology was studied by SEM and the viability and proliferation was investigated by the colorimetric MTT assay. Different protein conformations were found by AFM on CHT and PCL samples which were related to the biological performance of the substrates. Hydrophilicty of the material is not directly related to the biological response and the sample with 20 wt% CHT shows better results than the other blends with respect to chondrocyte viability and proliferation. However, the results obtained in the blends are worse than in pure PCL. It seems to be correlated with the surface energy of the different blends rather than hydrophilicity.