Natural Polymer-Polyphenol Bioadhesive Coacervate with Stable Wet Adhesion, Antibacterial Activity, and On-Demand Detachment.

Medical adhesives are emerging as an important clinical tool as adjuvants for sutures and staples in wound closure and healing and in the achievement of hemostasis. However, clinical adhesives combining cytocompatibility, as well as strong and stable adhesion in physiological conditions, are still in demand. Herein, a mussel-inspired strategy is explored to produce adhesive coacervates using tannic acid (TA) and methacrylate pullulan (PUL-MA). TA|PUL-MA coacervates mainly comprise van der Waals forces and hydrophobic interactions. The methacrylic groups in the PUL backbone increase the number of interactions in the adhesives matrix, resulting in enhanced cohesion and adhesion strength (72.7 Jm-2), compared to the non-methacrylated coacervate. The adhesive properties are kept in physiologic-mimetic solutions (72.8 Jm-2) for 72 h. The photopolymerization of TA|PUL-MA enables the on-demand detachment of the adhesive. The poor cytocompatibility associated with the use of phenolic groups is here circumvented by mixing reactive oxygen species-degrading enzyme in the adhesive coacervate. This addition does not hamper the adhesive character of the materials, nor their anti-microbial or hemostatic properties. This affordable and straightforward methodology, together with the tailorable adhesivity even in wet environments, high cytocompatibility, and anti-bacterial activity, enables foresee TA|PUL-MA as a promising ready-to-use bioadhesive for biomedical applications.

An Intracellular Metabolic Signature as a Potential Donor-Independent Marker of the Osteogenic Differentiation of Adipose Tissue Mesenchymal Stem Cells.

This paper describes an untargeted NMR metabolomics study to identify potential intracellular donor-dependent and donor-independent metabolic markers of proliferation and osteogenic differentiation of human adipose mesenchymal stem cells (hAMSCs). The hAMSCs of two donors with distinct proliferating/osteogenic characteristics were fully characterized regarding their polar endometabolome during proliferation and osteogenesis. An 18-metabolites signature (including changes in alanine, aspartate, proline, tyrosine, ATP, and ADP, among others) was suggested to be potentially descriptive of cell proliferation, independently of the donor. In addition, a set of 11 metabolites was proposed to compose a possible donor-independent signature of osteogenesis, mostly involving changes in taurine, glutathione, methylguanidine, adenosine, inosine, uridine, and creatine/phosphocreatine, choline/phosphocholine and ethanolamine/phosphocholine ratios. The proposed signatures were validated for a third donor, although they require further validation in a larger donor cohort. We believe that this proof of concept paves the way to exploit metabolic markers to monitor (and potentially predict) cell proliferation and the osteogenic ability of different donors.

Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation.

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.

NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells.

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD+); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

Metabolomic Applications in Stem Cell Research: a Review.

This review describes the use of metabolomics to study stem cell (SC) characteristics and function, excluding SCs in cancer research, suited to a fully dedicated text. The interest in employing metabolomics in SC research has consistently grown and emphasis is, here, given to developments reported in the past five years. This text informs on the existing methodologies and their complementarity regarding the information provided, comprising untargeted/targeted approaches, which couple mass spectrometry or nuclear magnetic resonance spectroscopy with multivariate analysis (and, in some cases, pathway analysis and integration with other omics), and more specific analytical approaches, namely isotope tracing to highlight particular metabolic pathways, or in tandem microscopic strategies to pinpoint characteristics within a single cell. The bulk of this review covers the existing applications in various aspects of mesenchymal SC behavior, followed by pluripotent and neural SCs, with a few reports addressing other SC types. Some of the central ideas investigated comprise the metabolic/biological impacts of different tissue/donor sources and differentiation conditions, including the importance of considering 3D culture environments, mechanical cues and/or media enrichment to guide differentiation into specific lineages. Metabolomic analysis has considered cell endometabolomes and exometabolomes (fingerprinting and footprinting, respectively), having measured both lipid species and polar metabolites involved in a variety of metabolic pathways. This review clearly demonstrates the current enticing promise of metabolomics in significantly contributing towards a deeper knowledge on SC behavior, and the discovery of new biomarkers of SC function with potential translation to in vivo clinical practice.

Engineering Strategies for Allogeneic Solid Tissue Acceptance.

Advances in allogeneic transplantation of solid organs and tissues depend on our understanding of mechanisms that mediate the prevention of graft rejection. For the past decades, clinical practice has established guidelines to prevent allograft rejection, which mostly rely on the intake of nontargeted immunosuppressants as the gold standard. However, such lifelong regimens have been reported to trigger severe morbidities and commonly fail in preventing late allograft loss. In this review, the biology of allogeneic rejection and self-tolerance is analyzed, as well as the mechanisms of cellular-based therapeutics driving suppression and/or tolerance. Bioinspired engineering strategies that take advantage of cells, biomaterials, or combinations thereof to prevent allograft rejection are addressed, as well as biological mechanisms that drive their efficacy.

One-Step All-Aqueous Interfacial Assembly of Robust Membranes for Long-Term Encapsulation and Culture of Adherent Stem/Stromal Cells.

The therapeutic effectiveness and biological relevance of technologies based on adherent cells depend on platforms that enable long-term culture in controlled environments. Liquid-core capsules have been suggested as semipermeable moieties with spatial homogeneity due to the high mobility of all components in their core. The lack of cell-adhesive sites in liquid-core structures often hampers their use as platforms for stem cell-based technologies for long-term survival and cell-directed self-organization. Here, the one-step fast formation of robust polymeric capsules formed by interfacial complexation of oppositely charged polyelectrolytes in an all-aqueous environment, compatible with the simultaneous encapsulation of mesenchymal stem/stromal cells (MSCs) and microcarriers, is described. The adhesion of umbilical cord MSCs to polymeric microcarriers enables their aggregation and culture for more than 21 days in capsules prepared either manually by dropwise addition, or by scalable electrohydrodynamic atomization, generating robust and stable capsules. Cell aggregation and secretion overtime can be tailored by providing cells with static or dynamic (bioreactor) environments.

Efficient Single-Dose Induction of Osteogenic Differentiation of Stem Cells Using Multi-Bioactive Hybrid Nanocarriers.

Bone regeneration requires the presence of specific factors to induce the differentiation of stem cells into osteoblasts. These factors induce osteogenesis by stimulating the expression of bone-related proteins, bone cell proliferation and differentiation. Herein, bioactive mesoporous silica nanoparticles are doped with calcium and phosphate ions while the porous network is loaded with dexamethasone (MSN-CaPDex). The bioactive MSN-CaPDex nanocarriers are prepared without affecting the narrow size distribution, pore structure, and morphology of the MSNs, while incorporating multi-stimuli, complementary ionic/biochemical bioactive mediators. The bioactive nanocarriers induce osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) after a single-dose administration, and without the need for further soluble osteogenic factors, in contrast to the standard continuous stimulation provided by osteogenic medium. The hBM-MSCs exhibit several biomarkers of osteogenic differentiation, including alkaline phosphatase peaking at early time points, secretion of osteopontin and osteocalcin, and deposition of a calcium-rich matrix. Overall, by inducing the osteogenic differentiation of stem cells with a single-dose administration and without requiring repeated osteogenic supplementation, the newly synthesized multi-bioactive hybrid nanocarrier shows great potential for bone tissue engineering applications.

One-Step Rapid Fabrication of Cell-Only Living Fibers.

Cellular aggregates are used as relevant regenerative building blocks, tissue models, and cell delivery platforms. Biomaterial-free structures are often assembled either as 2D cell sheets or spherical microaggregates, both incompatible with free-form deposition, and dependent on challenging processes for macroscale 3D upscaling. The continuous and elongated nature of fiber-shaped materials enables their deposition in unrestricted multiple directions. Cellular fiber fabrication has often required exogenously provided support proteins and/or the use of biomaterial-based sacrificial templates. Here, the rapid (<24 h) assembly of fiberoids is reported: living centimeter-long scaffold-free fibers of cells produced in the absence of exogenous materials or supplements. Adipose-derived mesenchymal stem cell fiberoids can be easily modulated into complex multidimensional geometries and show tissue-invasive properties while keeping the secretion of trophic factors. Proangiogenic properties studied on a chick chorioallantoic membrane in an ovo model are observed for heterotypic fiberoids containing endothelial cells. These micro-to-macrotissues may find application as morphogenic therapeutic and tissue-mimetic building blocks, with the ability to integrate 3D and 4D full biological materials.

Bioactive silica nanoparticles with calcium and phosphate for single dose osteogenic differentiation.

The differentiation of adult stem cells is usually performed in vitro, by exposing them to specific factors. Alternatively, one can use nanocarriers containing such factors, to be internalized by the cells. In this work we have reduce the size of those carriers to the nanoscale, developing bioactive silica nanoparticles with diameters under 100 nm, containing calcium and phosphate ions (SiNPs-CaP). These ions, once released inside adult stem cells, induce bone cell proliferation and differentiation, and stimulate the expression of bone-related proteins in a single dose administration. The SiNPs-CaP nanomaterials were prepared through a sol-gel approach, and the ions added with a post-synthesis functionalization method. The synthesized SiNPs-CaP have narrow size distribution, good colloidal stability, and show high levels of ion incorporation. Furthermore, the SiNPs-CaP have good cytocompatibility and promote the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC), with alkaline phosphatase, osteopontin and osteocalcin production levels comparable to the ones obtained in standard osteogenic medium. The novel bioactive SiNPs-CaP are synthesized in a simple and fast manner and show the ability to promote osteogenic differentiation after a single dose administration, independently from external osteogenic inducers, showing great potential as carriers in bone tissue engineering applications.

Sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in multiple myeloma cell lines.

INTRODUCTION: Multiple myeloma (MM) remains incurable due to high rates of relapse after various treatment regimens. WEE1 is a cell cycle related gene that regulates the G2/M checkpoint and promotes cell cycle suspension for consequent DNA repair. To date, there are clinical studies for the evaluation of WEE1 inhibitors in the treatment of solid tumors and studies on cell lines of non-MM hematological tumors. OBJECTIVES: To perform in vitro functional studies to verify the effect of the inhibition of WEE1 on MM cell lines viability and its potential as therapeutic target. MATERIAL AND METHODS: WEE1 expression was evaluated in 22 newly diagnosed MM patients and in four MM cell lines, RPMI-8226, U266 and SKO-007 and SK-MM2, by quantitative real-time PCR (qPCR). After treatment with the WEE1 inhibitor (MK-1775), with or without proteasome inhibitor (bortezomib) pretreatment, we assessed cell viability through Prestoblue functional test, microspheres formation in soft agar, and induction of apoptosis and cell cycle alterations by flow cytometry. RESULTS: All MM cell lines showed WEE1 expression by qPCR. RPMI-8226 and U266 showed a 50% reduction in cell viability after 24 h of incubation with MK-1775, at concentrations of 5 µM and 20 µM, respectively. SKO-007 showed dose and time dependence to this drug. Combination therapy with bortezomib and MK-1775 abolished the formation of soft agar microspheres in the RPMI-8226 cell line (also responsive to the use of both drugs) and U266, but SKO-007 was resistant to all drugs, isolated and combined. However, treatment of bortezomib followed by MK-1775 (sequential treatment) versus bortezomib alone showed statistically significant impact on cell lines total apoptosis: 88.8% vs 74.1% in RPMI-8222 (confirmed by cell cycle experiments); 92.5% vs 86.6% in U266; and 60.2% 30.9% on SKO-007 (p < 0.05). CONCLUSION: The sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in RPMI-8226, U266, and especially SKO-007 cell lines, more efficiently than the use of the same isolated drugs, highlighting its effect in inhibition of proliferation of tumor cells in MM cell lines. Our data suggest that WEE1 can figure as a MM target and that the sequential combination of bortezomib and MK-1775 may be explored in future clinical trials.

Screening of perfused combinatorial 3D microenvironments for cell culture.

Biomaterials combining biochemical and biophysical cues to establish close-to-extracellular matrix (ECM) models have been explored for cell expansion and differentiation purposes. Multivariate arrays are used as material-saving and rapid-to-analyze platforms, which enable selecting hit-spotted formulations targeting specific cellular responses. However, these systems often lack the ability to emulate dynamic mechanical aspects that occur in specific biological milieus and affect physiological phenomena including stem cells differentiation, tumor progression, or matrix modulation. We report a tailor-made strategy to address the combined effect of flow and biochemical composition of three-dimensional (3D) biomaterials on cellular response. We suggest a simple-to-implement device comprising (i) a perforated platform accommodating miniaturized 3D biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable perfusion chamber. The system was upscaled to parallelizable setups, increasing the number of analyzed platforms per independent experiment. As a proof-of-concept, porous chitosan scaffolds with 1 mm diameter were functionalized with combinations of 5 ECM and cell-cell contact-mediating proteins, relevant for bone and dental regeneration, corresponding to 32 protein combinatorial formulations. Mesenchymal stem cells adhesion and production of an early osteogenic marker were assessed on-chip on static and under-flow dynamic perfusion conditions. Different hit-spotted biomaterial formulations were detected for the different flow regimes using direct image analysis. Cell-binding proteins still poorly explored as biomaterials components - amelogenin and E-cadherin - were here shown as relevant cell response modulators. Their combination with ECM cell-binding proteins - fibronectin, vitronectin, and type 1 collagen - rendered specific biomaterial combinations with high cell adhesion and ALP production under flow. The developed versatile system may be targeted at widespread tissue regeneration applications, and as a disease model/drug screening platform. STATEMENT OF SIGNIFICANCE: A perfusion system that enables cell culture in arrays of three-dimensional biomaterials under dynamic flow is reported. The effect of 31 cell-binding protein combinations in the adhesion and alkaline phosphatase (ALP) production of mesenchymal stem cells was assessed using a single bioreactor chamber. Flow perfusion was not only assessed as a classical enhancer/accelerator of cell growth and early osteogenic differentiation. We hypothesized that flow may affect cell-protein interactions, and that key components driving cell response may differ under static or dynamic regimes. Indeed, hit-spotted formulations that elicited highest cell attachment and ALP production on static cell culture differed from the ones detected for dynamic flow assays. The impacting role of poorly studied proteins as E-cadherin and amelogenin as biomaterial components was highlighted.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines.

INTRODUCTION: Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR). METHODS: We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors. RESULTS: For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload. CONCLUSION: Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

Targeting the polarization of tumor-associated macrophages and modulating mir-155 expression might be a new approach to treat diffuse large B-cell lymphoma of the elderly.

Aging immune deterioration and Epstein-Barr (EBV) intrinsic mechanisms play an essential role in EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly (EBV + DLBCLe) pathogenesis, through the expression of viral proteins, interaction with host molecules and epigenetic regulation, such as miR-155, required for induction of M1 phenotype of macrophages. This study aims to evaluate the relationship between macrophage polarization pattern in the tumor microenvironment and relative expression of miR-155 in EBV + DLBCLe and EBV-negative DLBCL patients. We studied 28 EBV + DLBCLe and 65 EBV-negative DLBCL patients. Tumor-associated macrophages (TAM) were evaluated by expression of CD68, CD163 and CD163/CD68 ratio (degree of M2 polarization), using tissue microarray. RNA was extracted from paraffin-embedded tumor samples for miR-155 relative expression study. We found a significantly higher CD163/CD68 ratio in EBV + DLBCLe compared to EBV-negative DLBCL. In EBV-negative DLBCL, CD163/CD68 ratio was higher among advanced-staged/high-tumor burden disease and overexpression of miR-155 was associated with decreased polarization to the M2 phenotype of macrophages. The opposite was observed in EBV + DLBCLe patients: we found a positive association between miR-155 relative expression and CD163/CD68 ratio, which was not significant after outlier exclusion. We believe that the higher CD163/CD68 ratio in this group is probably due to the presence of the EBV since it directly affects macrophage polarization towards M2 phenotype through cytokine secretion in the tumor microenvironment. Therapeutic strategies modulating miR-155 expression or preventing immuno-regulatory and pro-tumor macrophage polarization could be adjuvants in EBV + DLBCLe therapy since this entity has a rich infiltration of M2 macrophages in its tumor microenvironment.

Association between IL1A and IL1B polymorphisms and primary open angle glaucoma in a Brazilian population.

Abstract: The aim of this study was to investigate the association of five polymorphisms in the IL1A and IL1B genes in Brazilian patients with primary open angle glaucoma (POAG). A case­control study, including 214 unrelated POAG patients and 187 healthy individuals, was conducted to evaluate the frequency of polymorphisms in the IL1A and IL1B genes. Ophthalmic evaluation was performed and genomic DNA was obtained from all participants. Five single nucleotide polymorphisms (SNPs): IL1A (­889C/T: rs1800587:C > T, +4845G/T:rs17561G>T) and IL1B (­31C/T:rs1143627:T > C, ­511C/T:rs16944C>T and +3954C/T:rs1143634:C > T) were genotyped through direct sequencing. The association of individual SNPs was tested using logistic regression. There was an association between the ­31C/T and ­511 C/T polymorphisms in the IL1B gene with POAG (p = 0.002 and p = 0.009, respectively). High linkage disequilibrium was observed between the ­31C/T and ­511C/T polymorphisms. The statistical analysis showed that the T/C haplotype (­31/­511) in the IL1B gene is more frequent in controls (p = 0.011) and the C/T haplotype (­31/­511) is more common in POAG patients (p = 0.018). Among POAG cases, the genotypic distribution of the ­31C/T and ­511 C/T SNPs was significantly different in patients who underwent anti-glaucomatous surgery compared to patients without surgery (p = 0.016 and 0.023, respectively). There was no statistically significant difference for the remaining SNPs between POAG patients and controls. In conclusion, the C allele of the ­31C/T and the T allele of the ­511C/T polymorphisms in the IL1B gene may represent a "risk haplotype" for the development of POAG in Brazilian individuals. Further studies with larger cohorts of patients are necessary to substantiate these findings.

Anti-myeloma effects of ruxolitinib combined with bortezomib and lenalidomide: A rationale for JAK/STAT pathway inhibition in myeloma patients.

JAK proteins have been linked with survival and proliferation of multiple myeloma (MM) cells; therefore, JAK inhibition could be a therapeutic strategy for MM. We evaluated JAK1 and JAK2 expression in MM patients and the effects of JAK/STAT pathway inhibition on apoptosis, cell cycle, gene and protein expression in RPMI-8226 and U266 MM cell lines. 57% of patients presented overexpression of JAK2 and 27%, of JAK1. After treatment with ruxolitinib and bortezomib, RPMI-8226 and U266 presented 50% of cells in late apoptosis, reduction of anti-apoptotic genes expression and higher number of cells in SubG0 phase. Co-culture with stromal cells protected RPMI-8226 cells from apoptosis, which was reversed by lenalidomide addition. Combination of ruxolitinib, bortezomib and lenalidomide induced 72% of cell death, equivalent to bortezomib, lenalidomide and dexamethasone, combination used in clinical practice. Many JAK/STAT pathway genes, after treatment, had their expression reduced, mainly in RPMI-8226, with insignificant changes in U266. In this scenario, JAK/STAT pathway could pose as a new therapeutic target to be exploited, since it is constitutively active and contributes to survival of MM tumor cells.

Increased risk of developing schizophrenia in animals exposed to cigarette smoke during the gestational period.

Cigarette smoking during the prenatal period has been investigated as a causative factor of obstetric abnormalities, which lead to cognitive and behavioural changes associated with schizophrenia. The aim of this study was to investigate behaviour and AChE activity in brain structures in adult rats exposed to cigarette smoke during the prenatal period. Pregnant rats were divided into non-PCSE (non-prenatal cigarette smoke exposure) and PCSE (prenatal cigarette smoke exposure) groups. On post-natal day 60, the rats received saline or ketamine for 7days and were subjected to behavioural tasks. In the locomotor activity task, the non-PCSE+ketamine and PCSE+ketamine groups exhibited increased locomotor activity compared with the saline group. In the social interaction task, the non-PCSE+ketamine and PCSE+ketamine groups exhibited an increased latency compared with the control groups. However, the PCSE+ketamine group exhibited a decreased latency compared with the non-PCSE+ketamine group, which indicates that the cigarette exposure appeared to decrease, the social deficits generated by ketamine. In the inhibitory avoidance task, the non-PCSE+ketamine, PCSE, and PCSE+ketamine groups exhibited impairments in working memory, short-term memory, and long-term memory. In the pre-pulse inhibition (PPI) test, cigarette smoke associated with ketamine resulted in impaired PPI in 3 pre-pulse (PP) intensity groups compared with the control groups. In the biochemical analysis, the AChE activity in brain structures increased in the ketamine groups; however, the PCSE+ketamine group exhibited an exacerbated effect in all brain structures. The present study indicates that exposure to cigarette smoke during the prenatal period may affect behaviour and cerebral cholinergic structures during adulthood.

The influence of surface modified poly(l-lactic acid) films on the differentiation of human monocytes into macrophages.

Macrophages play a crucial role in the biological performance of biomaterials, as key factors in defining the optimal inflammation-healing balance towards tissue regeneration and implant integration. Here, we investigate how different surface modifications performed on poly(l-lactic acid) (PLLA) films would influence the differentiation of human monocytes into macrophages. We tested PLLA films without modification, surface-modified by plasma treatment (pPLLA) or by combining plasma treatment with different coating materials, namely poly(l-lysine) and a series of proteins from the extracellular matrix: collagen I, fibronectin, vitronectin, laminin and albumin. While all the tested films are non-cytotoxic, differences in cell adhesion and morphology are observed. Monocyte-derived macrophages (MDM) present a more rounded shape in non-modified films, while a more elongated phenotype is observed containing filopodia-like and podosome-like structures in all modified films. No major differences are found for the expression of HLA-DR+/CD80+ and CD206+/CD163+ surface markers, as well as for the ability of MDM to phagocytize. Interestingly, MDM differentiated on pPLLA present the highest expression of MMP9. Upon differentiation, MDM in all surface modified films present lower amounts of IL-6 and IL-10 compared to non-modified films. After stimulating MDM with the potent pro-inflammatory agent LPS, pPLLA and poly(l-lysine) and fibronectin-modified films reveal a significant reduction in IL-6 secretion, while the opposite effect is observed with IL-10. Of note, in comparison to non-modified films, all surface modified films induce a significant reduction of the IL-6/IL-10 ratio, a valuable prognosticator of the pro- versus anti-inflammatory balance. These findings provide important insights into MDM-biomaterial interactions, while strengthening the need for designing immune-informed biomaterials.

Injectable PEGylated fibrinogen cell-laden microparticles made with a continuous solvent- and oil-free preparation method.

A new methodology is reported for the continuous, solvent- and oil-free production of photopolymerizable microparticles containing encapsulated human dermal fibroblasts. A precursor solution of cells in photoreactive poly(ethylene glycol) (PEG)-fibrinogen (PF) polymer was transported through a transparent injector exposed to light irradiation before being atomized in a jet-in-air nozzle. Shear rheometry data revealed the crosslinking kinetics of the PF/cell solution, which was then used to determine the amount of irradiation required to partially polymerize the mixture just prior to atomization. The partially polymerized drops of PF/cells fell into a gelation bath for further crosslinking until fully polymerized hydrogel microparticles were formed. As the drops of solution exited the air-in-jet nozzle, their viscosity was designed to be sufficiently high so as to prevent rapid mixing and/or dilution in the gelation bath, but without undergoing complete gelation in the nozzle. Several parameters of this system were varied to control the size and polydispersity of the microparticles, including the cell density, the flow rate and the air pressure in the nozzle. The system was capable of producing cell-laden microparticles with an average diameter of between 88.1 to 347.1 µm, and a dispersity of between 1.1 and 2.4, depending on the parameters chosen. Varying the precursor flow rate and/or cell density was beneficial in controlling the size and polydispersity of the microparticles; all microparticles exhibited very high cell viability, which was not affected by these parameters. In conclusion, this dropwise photopolymerization methodology for preparing cell-laden microparticles is an attractive alternative to existing techniques that use harsh solvents/oils and offer limited control over particle size and polydispersity.

Superhydrophobic chips for cell spheroids high-throughput generation and drug screening.

We suggest the use of biomimetic superhydrophobic patterned chips produced by a benchtop methodology as low-cost and waste-free platforms for the production of arrays of cell spheroids/microtissues by the hanging drop methodology. Cell spheroids have a wide range of applications in biotechnology fields. For drug screening, they allow studying 3D models in structures resembling real living tissues/tumors. In tissue engineering, they are suggested as building blocks of bottom-up fabricated tissues. We used the wettability contrast of the chips to fix cell suspension droplets in the wettable regions and evaluated on-chip drug screening in 3D environment. Cell suspensions were patterned in the wettable spots by three distinct methods: (1) by pipetting the cell suspension directly in each individual spot, (2) by the continuous dragging of a cell suspension on the chip, and (3) by dipping the whole chip in a cell suspension. These methods allowed working with distinct throughputs and degrees of precision. The platforms were robust, and we were able to have static or dynamic environments in each droplet. The access to cell culture media for exchange or addition/removal of components was versatile and opened the possibility of using each spot of the chip as a mini-bioreactor. The platforms' design allowed for samples visualization and high-content image-based analysis on-chip. The combinatorial analysis capability of this technology was validated by following the effect of doxorubicin at different concentrations on spheroids formed using L929 and SaOs-2 cells.