Engineering a 3D Biomimetic Peptides Functionalized-Polyethylene Glycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes: Enhancing In Vitro Blood-Brain Barrier Biomimicry.

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm2) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 µg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

Assessment of the efficiency of dental excavation methods using laser speckle imaging.

This paper introduces a novel application of the laser speckle technique in dentistry, focusing on assessing the efficiency of dental excavation methods used to remove decayed tooth structure. The aim is to evaluate the efficiency of two chemo-mechanical agents and the high-speed drill using the laser speckle technique, which offers objective, non-invasive, and real-time evaluation capabilities. Extracted human primary molars with active occlusal carious lesions were sectioned into three parts, with each part allocated to one of three groups: Group 1 (Brix3000®), Group 2 (Papacarie DUO®), and Group 3 (High-speed drill mechanical caries removal). Caries removal was performed using the designated agent or method for each group. After caries excavation, speckle imaging using a 632.8 nm laser was conducted. Additionally, SEM was used to acquire micro-photographs of the surface morphology of the treated samples. The findings reveal insights into the comparative efficiency of the three dental excavation agents and methods using the laser speckle technique. The speckle parameters extracted from speckle patterns generated by treated teeth provide valuable information for evaluating the performance of the excavation methods. The scanning electron microscopy images also offer detailed visual evidence to support the analysis. This paper demonstrates the potential of the laser speckle technique for assessing the efficiency of dental excavation methods. The objective, non-invasive, and real-time evaluation provided offers advantages over subjective visual assessment and manual measurements.

Effect of heat application on the physicochemical properties of new endodontic sealers: an in vitro and SEM study.

Understanding the properties of endodontic sealers is vital for treatment planning. Calcium silicate-based sealers are important in modern endodontics. This study investigates the effect of heat on the physicochemical properties of new calcium silicate sealers, addressing concerns raised by clinicians seeking to combine their benefits with the gutta-percha obturation technique. Five endodontic sealers (AH Plus Bioceramic®, Total Fill® BC®, One-fil® Bioceramic, K-Biocer, Any-seal®) were evaluated. Each sealer (n = 16/group) was either kept at 37 °C or subjected to heat at 60 or 200 °C for 30 s. ISO 6876-2012 standards were used to measure setting time, flow, film thickness, and dimensional changes over time. SEM and EDS were utilized for surface and chemical analysis. Data analysis employed IBM SPSS Statistics version 26 with a 5% significance level for two-sided tests. The sealers' setting times were shortened by heat, except for Total Fill® BC with extended setting time. All sealers had significantly longer setting times than manufacturer specifications. Film thickness increased with temperature, while flow values decreased. K-Biocer sealer showed the highest flow (16.89 ± 0.57 mm) at 200 °C, while Total Fill® sealer had the lowest (15.32 ± 0.62 mm). Shrinkage was significant at 60 °C and 200 °C, with greater shrinkage at 200 °C. Heat caused surface deformations in all sealers. The 200 °C groups exhibited more voids in AH Plus Bioceramic®, Any-seal®, and One-fil® sealers, and higher void area in Total Fill®, One-fil®, and K-Biocer sealers (p value < 0.001). EDX analysis confirmed heat-induced chemical and elemental changes in all tested sealers. Elevated temperature affects the physicochemical properties and structure of the tested endodontic sealers. The consideration of endodontic sealer compatibility is essential when warm gutta-percha obturation techniques are used.

Comparison of Fracture Resistance between Single-cone and Warm Vertical Compaction Technique Using Bio-C Sealer® in Mandibular Incisors: An In Vitro Study.

AIM: The aim of the study was to compare the fracture resistance of the single-cone technique with the warm vertical compaction technique (WVCT) in mandibular incisors using Bio-C sealer®, by applying a compressive force using a universal testing machine (UTM) (Instron 5943; Instron, Norwood, Massachusetts, USA). MATERIALS AND METHODS: Twenty-two mandibular incisors were selected and divided into two groups after applying the same shaping protocol. To assess the influence of the wave vertical compaction technique on the fracture resistance, the first group was obturated by a single-cone obturation technique (SCOT) (n = 12), and the second group was obturated with a WVCT (n = 10). Bio-C sealer® (Angelus, Hague Netherlands) was used in the two obturation techniques. Wax-coated roots were put in an acrylic mold and loaded to compressive strength fracture in a mechanical material testing machine (UTM) (Instron 5943; Instron, Norwood, Massachusetts, USA), with Bluehill 3 software (version 3.15.1343) recording the maximum load at fracture. Fracture loads were compared statistically, and data were examined with the Mann-Whitney U test with a level of significance set at p ≤0.05. RESULTS: No statistically significant difference was registered between the SCOT (264.97 ± 83.975 N) and WVCT (313.35 ± 89.149 N) concerning the endodontically treated mandibular incisors' fracture resistance (p = 0.159). CONCLUSION: Warm vertical compaction technique (WVCT) did not affect the fracture resistance of endodontically treated mandibular incisors when compared to SCOT canal preparation. CLINICAL SIGNIFICANCE: General practitioners and endodontists face challenges during root canal treatment such as cracks and root fractures. This article aims to guide experts in choosing between the single-cone and the continuous WVCT aiming for higher long-term quality of root canal filling.

The Effectiveness of Different Irrigation Techniques on Debris and Smear Layer Removal in Primary Mandibular Second Molars: An In Vitro Study.

AIMS: The aim of the present in vitro study was to compare the effectiveness of passive ultrasonic irrigation (PUI), sonic irrigation, and mechanic dynamic activation on the removal of debris and smear layer from primary mandibular second molars during pulpectomy. MATERIALS AND METHODS: Mesial roots of 48 primary mandibular second molars were prepared with an R-motion 21 mm file (30/0.04) (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland), irrigated with 1% sodium hypochlorite (NaOCl) and 17% ethylenediaminetetraacetic acid (EDTA), and divided into four groups (n = 24 canals) according to the final irrigation activation technique: control group without activation, PUI with Ultra-X (Eighteeth, Changzhou, China), mechanical activation with XP-endo Finisher (FKG), and sonic irrigation with EQ-S (Meta Biomed, Chungcheongbuk-do, Korea). The roots were split longitudinally and analyzed using scanning electron microscopy (SEM). The presence of debris and smear layer was assessed using a 5-grade scoring scale with 200× and 1000× magnification, respectively. The Kruskal-Wallis and Friedman tests were used for data analysis. RESULTS: The activation of the irrigant significantly improved debris and smear layer removal (p < 0.001). There was no significant difference between Ultra-X, XP-endo Finisher, and EQ-S (p > 0.05). No activation technique was able to completely eliminate debris and smear layer from the root canals of primary mandibular second molars. CONCLUSIONS: During pediatric pulpectomy, the irrigation protocol must include activation of the irrigation solutions using either ultrasonic, sonic, or mechanical activation techniques to enhance the removal of debris and smear layer for a better prognosis. CLINICAL SIGNIFICANCE: During root canal treatment on primary teeth, the clinician must incorporate an activation technique in the irrigation protocol to enhance the removal of debris and smear layer and increase the success of the treatment.

Anti-Tumor Effects of Biomimetic Sulfated Glycosaminoglycans on Lung Adenocarcinoma Cells in 2D and 3D In Vitro Models.

Lung cancer development relies on cell proliferation and migration, which in turn requires interaction with extracellular matrix (ECM) components such as glycosaminoglycans (GAGs). The mechanisms through which GAGs regulate cancer cell functions are not fully understood but they are, in part, mediated by controlled interactions with cytokines and growth factors (GFs). In order to mechanistically understand the effect of the degree of sulfation (DS) of GAGs on lung adenocarcinoma (LUAD) cells, we synthesized sulfated alginate (AlgSulf) as sulfated GAG mimics with DS = 0.0, 0.8, 2.0, and 2.7. Human (H1792) and mouse (MDA-F471) LUAD cell lines were treated with AlgSulf of various DSs at two concentrations 10 and 100 µg/mL and their anti-tumor properties were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue exclusion, and wound healing assays for 2D models and sphere formation assay for the 3D model. The proliferation and number of live MDA-F471 cells at the concentration of 100 µg/mL decreased significantly with the increase in the DS of biomimetic GAGs. In addition, the increase in the DS of biomimetic GAGs decreased cell migration (p < 0.001 for DS = 2.0 and 2.7 compared to control) and decreased the diameter and number of spheres formed (p < 0.001). The increased DS of biomimetic GAGs attenuated the expression of cancer stem cell (CSC)/progenitor markers in the 3D cultures. In conclusion, GAG-mimetic AlgSulf with increased DS exhibit enhanced anti-proliferative and migratory properties while also reducing growth of KRAS-mutant LUAD spheres in vitro. We suggest that these anti-tumor effects by GAG-mimetic AlgSulf are possibly due to differential binding to GFs and consequential decreased cell stemness. AlgSulf may be suitable for applications in cancer therapy after further in vivo validation.

Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K, PKC and MAPKs.

Chondrocytes rapidly lose their phenotypic expression of collagen II and aggrecan when grown on 2D substrates. It has generally been observed that a fibroblastic morphology with strong actin-myosin contractility inhibits chondrogenesis, whereas chondrogenesis may be promoted by depolymerization of the stress fibers and/or disruption of the physical link between the actin stress fibers and the ECM, as is the case in 3D hydrogels. Here we studied the relationship between the actin-myosin cytoskeleton and expression of chondrogenic markers by culturing fibroblastic chondrocytes in the presence of cytochalasin D and staurosporine. Both drugs induced collagen II re-expression; however, renewed glycosaminoglycan synthesis could only be observed upon treatment with staurosporine. The chondrogenic effect of staurosporine was augmented when blebbistatin, an inhibitor of myosin/actin contractility, was added to the staurosporine-stimulated cultures. Furthermore, in 3D alginate cultures, the amount of staurosporine required to induce chondrogenesis was much lower compared to 2D cultures (0.625 nM vs. 2.5 nM). Using a selection of specific signaling pathway inhibitors, it was found that PI3K-, PKC- and p38-MAPK pathways positively regulated chondrogenesis while the ERK-pathway was found to be a negative regulator in staurosporine-induced re-differentiation, whereas down-regulation of ILK by siRNA indicated that ILK is not determining for chondrocyte re-differentiation. Furthermore, staurosporine analog midostaurin displayed only a limited chondrogenic effect, suggesting that activation/deactivation of a specific set of key signaling molecules can control the expression of the chondrogenic phenotype. This study demonstrates the critical importance of mechanobiological factors in chondrogenesis suggesting that the architecture of the actin cytoskeleton and its contractility control key signaling molecules that determine whether the chondrocyte phenotype will be directed along a fibroblastic or chondrogenic path.

Development of alginate and alginate sulfate/polycaprolactone nanoparticles for growth factor delivery in wound healing therapy.

Connective tissue growth factor (CTGF) holds great promise for enhancing the wound healing process; however, its clinical application is hindered by its low stability and the challenge of maintaining its effective concentration at the wound site. Herein, we developed novel double-emulsion alginate (Alg) and heparin-mimetic alginate sulfate (AlgSulf)/polycaprolactone (PCL) nanoparticles (NPs) for controlled CTGF delivery to promote accelerated wound healing. The NPs' physicochemical properties, cytocompatibility, and wound healing activity were assessed on immortalized human keratinocytes (HaCaT), primary human dermal fibroblasts (HDF), and a murine cutaneous wound model. The synthesized NPs had a minimum hydrodynamic size of 200.25 nm. Treatment of HaCaT and HDF cells with Alg and AlgSulf2.0/PCL NPs did not show any toxicity when used at concentrations <50 µg/mL for up to 72 h. Moreover, the NPs' size was not affected by elevated temperatures, acidic pH, or the presence of a protein-rich medium. The NPs have slow lysozyme-mediated degradation implying that they have an extended tissue retention time. Furthermore, we found that treatment of HaCaT and HDF cells with CTGF-loaded Alg and AlgSulf2.0/PCL NPs, respectively, induced rapid cell migration (76.12% and 79.49%, P<0.05). Finally, in vivo studies showed that CTGF-loaded Alg and AlgSulf2.0/PCL NPs result in the fastest and highest wound closure at the early and late stages of wound healing, respectively (36.49%, P<0.001 on day 1; 90.45%, P<0.05 on day 10), outperforming free CTGF. Double-emulsion NPs based on Alg or AlgSulf represent a viable strategy for delivering heparin-binding GF and other therapeutics, potentially aiding various disease treatments.

Effect of Continuous Chelation Irrigation Using DualRinse HEDP+3% NaOCl with or without High-power Sonic Activation on Debris and Smear Layer Removal.

OBJECTIVE: This study aimed to assess the effect of sodium hypochlorite (NaOCl) combined with a novel chelating agent DualRinse HEDP (Medcem GmbH, Weinfelden, Switzerland), a product consisting of 0.9 g of 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) powder, with or without high-power sonic activation on debris and smear layer removal. METHODS: Seventy-five mandibular premolars were divided into 5 groups (n=15) and treated with different irrigation protocols: group 1 (D3N), DualRinse HEDP+3% NaOCl without activation; group 2 (D3NA), DualRinse HEDP+3% NaOCl with activation (EDDY, VDW, Munich, Germany) during the final irrigation; group 3 (3NE), 3% NaOCl+17% Ethylenediaminetetracetic acid (EDTA)+3% NaOCl without activation; group 4 (3NEA), 3% NaOCl+17% EDTA+3% NaOCl with activation during the final irrigation; group 5 (NC), negative control group, 0.9% saline. Samples were analysed by scanning electron microscopy (SEM) to evaluate residual debris and smear layer at 3 levels of the root canal: coronal, middle, and apical. Statistical analysis was performed with a level of significance set at p<0.05. The normality distribution of scores within each group was assessed using Kolmogorov-Smirnov and Shapiro-Wilk tests. A Kruskal-Wallis test followed by multiple comparison tests was used to compare scores among the 5 groups on the apical, middle, and coronal levels of the root canal. A Friedman test followed by multiple comparison tests was used to compare scores within the apical, middle, and coronal levels for each treatment group. RESULTS: Debris score was significantly the lowest for D3NA, followed by D3N, 3NEA and 3NE at all root levels (p<0.05). The smear layer score was significantly the lowest for D3NA, followed by D3N, 3NEA and 3NE only at the apical level, while no significant difference was found in the middle and coronal levels between the groups (p<0.05). DualRinse HEDP resulted in less debris and smear layer compared to the classic approach of NaOCl without activation. Implementing sonic activation further improved debris and smear layer removal. CONCLUSION: DualRinse HEDP+3% NaOCl improved debris removal at all levels and smear layer elimination at the apical level of the root canal. These results were further enhanced when adding high-power sonic activation. (EEJ-2022-09-116).

Organ-on-a-chip platforms as novel advancements for studying heterogeneity, metastasis, and drug efficacy in breast cancer.

Breast cancer has the highest cancer incidence rate in women worldwide. Therapies for breast cancer have shown high success rates, yet many cases of recurrence and drug resistance are still reported. Developing innovative strategies for studying breast cancer may improve therapeutic outcomes of the disease by providing better insight into the associated molecular mechanisms. A novel advancement in breast cancer research is the utilization of organ-on-a-chip (OOAC) technology to establish in vitro physiologically relevant breast cancer biomimetic models. This emerging technology combines microfluidics and tissue culturing methods to establish organ-specific micro fabricated culture models. Here, we shed light on the advantages of OOAC platforms over conventional in vivo and in vitro models in terms of mimicking tissue heterogeneity, disease progression, and facilitating pharmacological drug testing with a focus on models of the mammary gland in both normal and breast cancer states. By highlighting the various designs and applications of the breast-on-a-chip platforms, we show that the latter propose means to facilitate breast cancer-related studies and provide an efficient approach for therapeutic drug screening in vitro.

Er,Cr:YSGG Laser Surface Modification Effect on Dentin Bonding to Zirconia: An In Vitro Study.

Objective: This study investigated the effect of dentin surface treatment with the erbium, chromium-doped yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser on the bond strength of zirconia to dentin. Background data: Although it is well-known that resin cement (RC) provides adequate bond strength of zirconia restorations to the tooth structure, many clinicians were not convinced in bonding reliability to zirconia materials. So, they preferred cementing their crowns with glass-ionomer cement (GIC). Pretreating the dentin surface is recommended to improve the adhesion of crowns cemented with GIC or RC. Recently, the Er,Cr:YSGG laser has been widely used for a cavity preparation, conditioning the tooth structure. However, there is not enough research on the bond strength of zirconia crowns cemented on a Er,Cr:YSGG laser-treated dentin. Methods: Forty-eight molars were cut horizontally at the crest of curvature. The flat dentin surface of 24 molars was left untreated, whereas the dentin surface of the other half was treated using an Er,Cr:YSGG laser 2.78 µm (4.5 W, 8.18 J/cm2, 90 mJ, 60 µs, 50 Hz, 60% air and 80% water). In addition, 48 plates of zirconia were prepared to be cemented with GIC or RC on the dentin surface. So, the molars were divided into four groups as follows: GIC+untreated dentin; GIC+treated dentin; RC+untreated dentin; and RC+treated dentin. All the specimens were subjected to shear bond strength test. The mode of failure was determined and additional samples were prepared to evaluate the cement-dentin interface using a scanning electron microscope. Data were analyzed with two-way analysis of variance accompanied by univariate analyses. Results: The bond strength of zirconia cemented with GIC or bonded with RC significantly increased on a Er,Cr:YSGG laser-treated dentin surface (p < 0.05). Conclusions: Dentin preconditioning with an Er,Cr:YSGG laser significantly improved the bond strength of zirconia plates to dentin as compared with untreated dentin.

Polymeric nanoparticles in the diagnosis and treatment of myocardial infarction: Challenges and future prospects.

Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide. Despite extensive efforts to provide early diagnosis and adequate treatment regimens, detection of MI still faces major limitations and pathological MI complications continue to threaten the recovery of survivors. Polymeric nanoparticles (NPs) represent novel noninvasive drug delivery systems for the diagnosis and treatment of MI and subsequent prevention of fatal heart failure. In this review, we cover the recent advances in polymeric NP-based diagnostic and therapeutic approaches for MI and their application as multifunctional theranostic tools. We also discuss the in vivo behavior and toxicity profile of polymeric NPs, their application in noninvasive imaging, passive, and active drug delivery, and use in cardiac regenerative therapy. We conclude with the challenges faced with polymeric nanosystems and suggest future efforts needed for clinical translation.

Layer-by-layer films made from extracellular matrix macromolecules on silicone substrates.

The layer-by-layer (LbL) technique has been widely used to produce nanofilms for biomedical applications. Naturally occurring polymers such as ECM macromolecules are attractive candidates for LbL film preparation. In this study, we assessed the build-up of type I collagen (Col1)/chondroitin sulfate (CS) or Col1/Heparin (HN) on polydimethylsiloxane (PDMS) substrates. The build-up was assessed by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Integrin-mediated cell adhesion was assessed by studying the cytoskeletal organization of mammalian primary cells (chondrocytes) seeded on different end layers and number of layers. Data generated from the QCM-D observations showed a consistent build-up of films with more adsorption in the case of Col1/HN. Col1/CS films were stable in media, whereas Col1/HN films were not. AFM analysis showed that the layers were fibrillar in structure for both systems and between 20 and 30 nm thick. The films promoted cell adhesion when compared with tissue culture plastic in serum-free media with cycloheximide. Crosslinking of the films resulted in constrained cell spreading and a ruffled morphology. Finally, beta1 integrin blocking antibodies prevented cell spreading, suggesting that cell adhesion and spreading were mediated mainly by interaction with the collagen fibrils. The ability to construct stable ECM-based films on PDMS has particular relevance in mechanobiology, microfluidics, and other biomedical applications.

Sulfated alginate/polycaprolactone double-emulsion nanoparticles for enhanced delivery of heparin-binding growth factors in wound healing applications.

Diabetic foot ulcers (DFUs) that are not effectively treated could lead to partial or complete lower limb amputations. The lack of connective tissue growth factor (CTGF) and insulin-like growth factor (IGF-I) in DFUs results in limited matrix deposition and poor tissue repair. To enhance growth factor (GF) availability in DFUs, heparin (HN)-mimetic alginate sulfate/polycaprolactone (AlgSulf/PCL) double emulsion nanoparticles (NPs) with high affinity and sustained release of CTGF and IGF-I were synthesized. The NPs size, encapsulation efficiency (EE), cytotoxicity, cellular uptake and wound healing capacity in immortalized primary human adult epidermal cells (HaCaT) were assessed. The sonication time and amplitude used for NPs synthesis enabled the production of particles with a minimum of 236 ± 25 nm diameter. Treatment of HaCaT cells with up to 50 µg mL-1 of NPs showed no cytotoxic effects after 72 h. The highest bovine serum albumin EE (94.6 %, P = 0.028) and lowest burst release were attained with AlgSulf/PCL. Moreover, cells treated with AlgSulf/CTGF (250 ng mL-1) exhibited the most rapid wound closure compared to controls while maintaining fibronectin synthesis. Double-emulsion NPs based on HN-mimetic AlgSulf represent a novel approach which can significantly enhance diabetic wound healing and can be expanded for applications requiring the delivery of other HN-binding GFs.

Biomimetic sulfated glycosaminoglycans maintain differentiation markers of breast epithelial cells and preferentially inhibit proliferation of cancer cells.

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4-2 (T4-2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue and bromodeoxyuridine proliferation (BrdU) assays, immunofluorescence staining and transwell invasion assay.  Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4-2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In the ELISA assay, a trend of EGF binding was detected for sulfated polysaccharides while QCM-D analysis showed negligible binding of insulin and EGF to sulfated substrates. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells may in part be growth factor dependent. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

Alginate Sulfate Substrates Control Growth Factor Binding and Growth of Primary Neurons: Toward Engineered 3D Neural Networks.

Sulfated glycosaminoglycans (sGAGs) are vital molecules of the extracellular matrix (ECM) of the nervous system known to regulate proliferation, migration, and differentiation of neurons mainly through binding relevant growth factors. Alginate sulfate (AlgSulf) mimics sGAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, thin films of biotinylated AlgSulf (b-AlgSulfn ) are engineered with sulfation degrees (DS = 0.0 and 2.7) and the effect of polysaccharide concentration on FGF-2 and nerve growth factor (ß-NGF) binding and subsequent primary neural viability and neurite outgrowth is assessed. An increase in b-AlgSulfn concentration results in higher FGF-2 and ß-NGF binding as demonstrated by greater frequency and dissipation shifts measured with quartz crystal microbalance with dissipation monitoring (QCM-D). Primary neurons seeded on the 2D b-AlgSulfn films maintain high viability comparable to positive controls grown on poly-d-lysine. Neurons grown in 3D AlgSulf hydrogels (DS = 0.8) exhibit a significantly higher viability, neurite numbers and mean branch length compared to neurons grown in nonsulfated controls. Finally, a first step is made toward constructing 3D neuronal networks by controllably patterning neurons encapsulated in AlgSulf into an alginate carrier. The substrates and neural networks developed in the current study can be used in basic and applied neural applications.

The sulfation of biomimetic glycosaminoglycan substrates controls binding of growth factors and subsequent neural and glial cell growth.

Sulfated glycosaminoglycans (GAGs) are key structural and functional extracellular matrix (ECM) molecules involved in numerous signaling pathways mainly through their interaction with growth factors. Alginate sulfate mimics sulfated GAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, natural biomimetic substrates were engineered by immobilizing biotinylated alginate sulfates with varying degrees of sulfation (DS, from 0 to 2.7) on gold and polystyrene substrates using biotin-streptavidin binding. The build-up of films and the effect of the DS and biotinylation method on FGF-2 binding were assessed using quartz crystal microbalance with dissipation monitoring (QCM-D) and immunohistochemistry. The role of substrate sulfation and FGF-2 loading on the growth of A172 (human glioblastoma multiforme), SH-SY5Y (human neuroblastoma), and PC-12 (rat pheochromocytoma) cell lines was evaluated in vitro using proliferation and neurite outgrowth assessment. An increase in the DS of alginates resulted in augmented FGF-2 binding as evidenced by higher frequency and dissipation shifts measured with QCM-D and confirmed with immunostaining. All sulfated alginate substrates supported the attachment and growth of neural/glial cell lines better than controls with the highest increase in cell proliferation observed for the highest DS (p < 0.05 for all the cell lines). Moreover, FGF-2 loaded substrates with the highest DS induced the most significant increase in neurite-positive PC-12 cells and average neurite length. The developed biomimetic coatings can be used to functionalize substrates for biosensing applications (e.g. gold substrates) and to induce defined cellular responses via controlled growth factor delivery for basic and applied sciences.

Cell-Free Approaches in Synthetic Biology Utilizing Microfluidics.

Synthetic biology is a rapidly growing multidisciplinary branch of science which aims to mimic complex biological systems by creating similar forms. Constructing an artificial system requires optimization at the gene and protein levels to allow the formation of entire biological pathways. Advances in cell-free synthetic biology have helped in discovering new genes, proteins, and pathways bypassing the complexity of the complex pathway interactions in living cells. Furthermore, this method is cost- and time-effective with access to the cellular protein factory without the membrane boundaries. The freedom of design, full automation, and mimicking of in vivo systems reveal advantages of synthetic biology that can improve the molecular understanding of processes, relevant for life science applications. In parallel, in vitro approaches have enhanced our understanding of the living system. This review highlights the recent evolution of cell-free gene design, proteins, and cells integrated with microfluidic platforms as a promising technology, which has allowed for the transformation of the concept of bioprocesses. Although several challenges remain, the manipulation of biological synthetic machinery in microfluidic devices as suitable 'homes' for in vitro protein synthesis has been proposed as a pioneering approach for the development of new platforms, relevant in biomedical and diagnostic contexts towards even the sensing and monitoring of environmental issues.

Development of Microplatforms to Mimic the In Vivo Architecture of CNS and PNS Physiology and Their Diseases.

Understanding the mechanisms that govern nervous tissues function remains a challenge. In vitro two-dimensional (2D) cell culture systems provide a simplistic platform to evaluate systematic investigations but often result in unreliable responses that cannot be translated to pathophysiological settings. Recently, microplatforms have emerged to provide a better approximation of the in vivo scenario with better control over the microenvironment, stimuli and structure. Advances in biomaterials enable the construction of three-dimensional (3D) scaffolds, which combined with microfabrication, allow enhanced biomimicry through precise control of the architecture, cell positioning, fluid flows and electrochemical stimuli. This manuscript reviews, compares and contrasts advances in nervous tissues-on-a-chip models and their applications in neural physiology and disease. Microplatforms used for neuro-glia interactions, neuromuscular junctions (NMJs), blood-brain barrier (BBB) and studies on brain cancer, metastasis and neurodegenerative diseases are addressed. Finally, we highlight challenges that can be addressed with interdisciplinary efforts to achieve a higher degree of biomimicry. Nervous tissue microplatforms provide a powerful tool that is destined to provide a better understanding of neural health and disease.