Replication of natural surface topographies to generate advanced cell culture substrates.

Surface topographies of cell culture substrates can be used to generate in vitro cell culture environments similar to the in vivo cell niches. In vivo, the physical properties of the extracellular matrix (ECM), such as its topography, provide physical cues that play an important role in modulating cell function. Mimicking these properties remains a challenge to provide in vitro realistic environments for cells. Artificially generated substrates' topographies were used extensively to explore this important surface cue. More recently, the replication of natural surface topographies has been enabling to exploration of characteristics such as hierarchy and size scales relevant for cells as advanced biomimetic substrates. These substrates offer more realistic and mimetic environments regarding the topographies found in vivo. This review will highlight the use of natural surface topographies as a template to generate substrates for in-vitro cell culture. This review starts with an analysis of the main cell functions that can be regulated by the substrate's surface topography through cell-substrate interactions. Then, we will discuss research works wherein substrates for cell biology decorated with natural surface topographies were used and investigated regarding their influence on cellular performance. At the end of this review, we will highlight the advantages and challenges of the use of natural surface topographies as a template for the generation of advanced substrates for cell culture.

The biomimetic surface topography ofRubus fruticosusleaves stimulate the induction of osteogenic differentiation of rBMSCs.

The interaction between cells and biomaterials is essential for the success of biomedical applications in which the implantation of biomaterials in the human body is necessary. It has been demonstrated that material's chemical, mechanical, and structural properties can influence cell behaviour. The surface topography of biomaterials is a physical property that can have a major role in mediating cell-material interactions. This interaction can lead to different cell responses regarding cell motility, proliferation, migration, and even differentiation. The combination of biomaterials with mesenchymal stem cells (MSCs) for bone regeneration is a promising strategy to avoid the need for autologous transplant of bone. Surface topography was also associated with the capacity to control MSCs differentiation. Most of the topographies studied so far involve machine-generated surface topographies. Herein, our strategy differentiates from the above mentioned since we selected natural surface topographies that can modulate cell functions for regenerative medicine strategies.Rubus fruticosusleaf was the selected topography to be replicated in polycaprolactone (PCL) membranes through polydimethylsiloxane moulding and using soft lithography. Afterwards, rat bone marrow stem cells (rBMSCs) were seeded at the surface of the imprinted PCL membranes to characterize the bioactive potential of our biomimetic surface topography to drive rBMSCs differentiation into the osteogenic lineage. The selected surface topography in combination with the osteogenic inductive medium reveals having a synergistic effect promoting osteogenic differentiation.

Block copolymer nanopatterns affect cell spreading: Stem versus cancer bone cells.

Bone healing after a tumor removal can be promoted by biomaterials that enhance the bone regeneration and prevent the tumor relapse. Herein, we obtained several nanopatterns by self-assembly of polystyrene-block-poly-(2-vinylpyridine) (PS-b-P2VP) with different molecular weights and investigated the adhesion and morphology of human bone marrow mesenchymal stem cells (BMMSC) and osteosarcoma cell line (SaOS-2) on these patterns aiming to identify topography and chemistry that promote bone healing. We analyzed > 2000 cells per experimental condition using imaging software and different morphometric descriptors, namely area, perimeter, aspect ratio, circularity, surface/area, and fractal dimension of cellular contour (FDC). The obtained data were used as inputs for principal component analysis, which showed distinct response of BMMSC and SaOS-2 to the surface topography and chemistry. Among the studied substrates, micellar nanopatterns assembled from the copolymer with high molecular weight promote the adhesion and spreading of BMMSC and have an opposite effect on SaOS-2. This nanopattern is thus beneficial for bone regeneration after injury or pathology, e.g. bone fracture or tumor removal.

Biomimetic surface topography as a potential modulator of macrophages inflammatory response to biomaterials.

The implantation of biomaterial devices can negatively impact the local microenvironment through several processes including the injury incurred during the implantation process and the associated host inflammatory response. Immune cell responses to implantable biomaterial devices mediate host-material interactions. Indeed, the immune system plays a central role in several biological processes required for the integration of biomaterials such as wound healing, tissue integration, inflammation, and foreign body reactions. The implant physicochemical properties such as size, shape, surface area, topography, and chemistry have been shown to provide cues to the immune system. Its induced immune-modulatory responses towards inflammatory or wound healing phenotypes can determine the success of the implant. In this work, we aim to evaluate the impact of some biomimetic surface topographies on macrophages' acute inflammatory response. For that, we selected 4 different biological surfaces to replicate through soft lithography on spin casting PCL membranes. Those topographies were: the surface of E. coli, S.eppidermidis and L929 cells cultured in polystyrene tissue culture disks, and an Eggshell membrane. We selected a model based on THP-1-derived macrophages to study the analysis of the expression of both pro-inflammatory and anti-inflammatory markers. Our results revealed that depending on the surface where these cells are seeded, they present different phenotypes. Macrophages present a M1-like phenotype when they are cultured on top of PCL membranes with the surface topography of E. coli and S. epidermidis. When cultured on membranes with L929 monolayers or Eggshell membrane surface topography, the macrophages present a M2-like phenotype. These results can be a significant advance in the development of new implantable biomaterial devices since they can help to modulate the inflammatory responses to implanted biomaterials by controlling their surface topography.

The Tumor Microenvironment: An Introduction to the Development of Microfluidic Devices.

The tumor microenvironment (TME) is like the Referee of a soccer match who has constant eyes on the activity of all players, such as cells, acellular stroma components, and signaling molecules for the successful completion of the game, that is, tumorigenesis. The cooperation among all the "team members" determines the characteristics of tumor, such as the hypoxic and acidic niche, stiffer mechanical properties, or dilated vasculature. Like in soccer, each TME is different. This heterogeneity makes it challenging to fully understand the intratumor dynamics, particularly among different tumor subpopulations and their role in therapeutic response or resistance. Further, during metastasis, tumor cells can disseminate to a secondary organ, a critical event responsible for approximately 90% of the deaths in cancer patients. The recapitulation of the rapidly changing TME in the laboratory is crucial to improve patients' prognosis for unraveling key mechanisms of tumorigenesis and developing better drugs. Hence, in this chapter, we provide an overview of the characteristic features of the TME and how to model them, followed by a brief description of the limitations of existing in vitro platforms. Finally, various attempts at simulating the TME using microfluidic platforms are highlighted. The chapter ends with the concerns that need to be addressed for designing more realistic and predictive tumor-on-a-chip platforms.

Erythrocyte-derived liposomes for the treatment of inflammatory diseases.

Effective and safe therapies to counteract persistent inflammation are necessary. We developed erythrocyte-derived liposomes (EDLs) with intrinsic anti-inflammatory activity. The EDLs were prepared using lipids extracted from erythrocyte membranes, which are rich in omega-3 fatty acids with several health benefits. Diclofenac, a widely used anti-inflammatory drug, was incorporated into EDLs in relevant therapeutic concentrations. The EDLs were also functionalised with folic acid to allow their active targeting of M1 macrophages, which are key players in inflammatory processes. In the presence of lipopolysaccharide (LPS)-stimulated macrophages, empty EDLs and EDLs incorporating diclofenac were able to reduce the levels of important pro-inflammatory cytokines, namely interleukin-6 (IL-6; ≈85% and 77%, respectively) and tumour necrosis factor-alpha (TNF-α; ≈64% and 72%, respectively). Strikingly, cytocompatible concentrations of EDLs presented similar effects to dexamethasone, a potent anti-inflammatory drug, in reducing IL-6 and TNF-α concentrations, demonstrating the EDLs potential to be used as bioactive carriers in the treatment of inflammatory diseases.

Osteogenic lithium-doped brushite cements for bone regeneration.

This study investigated the osteogenic performance of new brushite cements obtained from Li+-doped ß-tricalcium phosphate as a promising strategy for bone regeneration. Lithium (Li+) is a promising trace element to encourage the migration and proliferation of adipose-derived stem cells (hASCs) and the osteogenic differentiation-related gene expression, essential for osteogenesis. In-situ X-ray diffraction (XRD) and in-situ 1H nuclear magnetic resonance (1H NMR) measurements proved the precipitation of brushite, as main phase, and monetite, indicating that Li+ favored the formation of monetite under certain conditions. Li+ was detected in the remaining pore solution in significant amounts after the completion of hydration. Isothermal calorimetry results showed an accelerating effect of Li+, especially for low concentration of the setting retarder (phytic acid). A decrease of initial and final setting times with increasing amount of Li+ was detected and setting times could be well adjusted by varying the setting retarder concentration. The cements presented compressive mechanical strength within the ranges reported for cancellous bone. In vitro assays using hASCs showed normal metabolic and proliferative levels. The immunodetection and gene expression profile of osteogenic-related markers highlight the incorporation of Li+ for increasing the in vivo bone density. The osteogenic potential of Li-doped brushite cements may be recommended for further research on bone defect repair strategies.

Study of the immunologic response of marine-derived collagen and gelatin extracts for tissue engineering applications.

The host immunologic response to a specific material is a critical aspect when considering it for clinical implementation. Collagen and gelatin extracted from marine sources have been proposed as biomaterials for tissue engineering applications, but there is a lack of information in the literature about their immunogenicity. In this work, we evaluated the immune response to collagen and/or gelatin from blue shark and codfish, previously extracted and characterized. After endotoxin evaluation, bone marrow-derived macrophages were exposed to the materials and a panel of pro- and anti-inflammatory cytokines were evaluated both for protein quantification and gene expression. Then, the impact of those materials in the host was evaluated through peritoneal injection in C57BL/6 mice. The results suggested shark collagen as the less immunogenic material, inducing low expression of pro-inflammatory cytokines as well as inducible nitric oxide synthase (encoded by Nos2) and high expression of Arginase 1 (encoded by Arg1). Although shark gelatin appeared to be the material with higher pro-inflammatory expression, it also presents a high expression of IL-10 (anti-inflammatory cytokine) and Arginase (both markers for M2-like macrophages). When injected in the peritoneal cavity of mice, our materials demonstrated a transient recruitment of neutrophil, being almost non-existent after 24 hours of injection. Based on these findings, the studied collagenous materials can be considered interesting biomaterial candidates for regenerative medicine as they may induce an activation of the M2-like macrophage population, which is involved in suppressing the inflammatory processes promoting tissue remodeling. STATEMENT OF SIGNIFICANCE: Marine-origin biomaterials are emerging in the biomedical arena, namely the ones based in marine-derived collagen/gelatin proposed as cell templates for tissue regeneration. Nevertheless, although the major cause of implant rejection in clinical practice is the host's negative immune response, there is a lack of information in the literature about the immunological impact of these marine collagenous materials. This work aims to contribute with knowledge about the immunologic response to collagen/gelatin extracted from blue shark and codfish skins. The results demonstrated that despite some differences observed, all the materials can induce a macrophage phenotype related with anti-inflammation resolution and then act as immuno-modulators and anti-inflammatory inducible materials.

Antithrombotic and hemocompatible properties of nanostructured coatings assembled from block copolymers.

We describe the antithrombotic properties of nanopatterned coatings created by self-assembly of poly(styrene-block-2-vinylpyridine) (PS-b-P2VP) with different molecular weights. By changing the assembly conditions, we obtained nanopatterns that differ by their morphology (size and shape of the nanopattern) and chemistry. The surface exposition of P2VP block allowed quaternization, i.e. introduction of positive surface charge and following electrostatic deposition of heparin. Proteins (albumin and fibrinogen) adsorption, platelet adhesion and activation, cytocompatibility, and reendothelization capacity of the coatings were assessed and discussed in a function of the nanopattern morphology and chemistry. We found that quaternization results in excellent antithrombotic and hemocompatible properties comparable to heparinization by hampering the fibrinogen adhesion and platelet activation. In the case of quaternization, this effect depends on the size of the polymer blocks, while all heparinized patterns had similar performance showing that heparin surface coverage of 40 % is enough to improve substantially the hemocompatibility.

Porous aligned ZnSr-doped ß-TCP/silk fibroin scaffolds using ice-templating method for bone tissue engineering applications.

The bone is a complex and dynamic structure subjected to constant stress and remodeling. Due to the worldwide incidence of bone disorders, tissue scaffolds and engineered bone tissues have emerged as solutions for bone grafting, which require sophisticated scaffolding architectures while keeping high mechanical performance. However, the conjugation of a bone-like scaffold architecture with efficient mechanical properties is still a critical challenge for biomedical applications. In this sense, the present study focused on the modulating the architecture of silk fibroin (SF) scaffolds crosslinked with horseradish peroxidase and mixed with zinc (Zn) and strontium (Sr)-doped ß-tricalcium phosphate (ZnSr.TCP) to mimic bone structures. The ZnSr.TCP-SF hydrogels were tuned by programmable ice-templating parameters, and further freeze-dried, in order to obtain 3D scaffolds with controlled pore orientation. The results showed interconnected channels in the ZnSr.TCP-SF scaffolds that mimic the porous network of the native subchondral bone matrix. The architecture of the scaffolds was characterized by microCT, showing tunable pore size according to freezing temperatures (-196 °C: ∼80.2 ± 20.5 µm; -80 °C: ∼73.1 ± 20.5 µm; -20 °C: ∼104.7 ± 33.7 µm). The swelling ratio, weight loss, and rheological properties were also assessed, revealing efficient scaffold integrity and morphology after aqueous immersion. Thus, the ZnSr.TCP-SF scaffolds made of aligned porous structure were developed as affordable candidates for future applications in clinical osteoregeneration and in vitro bone tissue modelling.

Modulation of inflammation by anti-TNF α mAb-dendrimer nanoparticles loaded in tyramine-modified gellan gum hydrogels in a cartilage-on-a-chip model.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by joint inflammation. Since the inflammatory condition plays an important role in the disease process, it is important to develop and test new therapeutic approaches that specifically target and treat joint inflammation. In this study, a human 3D inflammatory cartilage-on-a-chip model was established to test the therapeutic efficacy of anti-TNFα mAb-CS/PAMAM dendrimer NPs loaded-Tyramine-Gellan Gum in the treatment of inflammation. The results showed that the proposed therapeutic approach applied to the human monocyte cell line (THP-1) and human chondrogenic primary cells (hCH) cell-based inflammation system revealed an anti-inflammatory capacity that increased over 14 days. It was also possible to observe that Coll type II was highly expressed by inflamed hCH upon the culture with anti-TNF α mAb-CS/PAMAM dendrimer NPs, indicating that the hCH cells were able maintain their biological function. The developed preclinical model allowed us to provide more robust data on the potential therapeutic effect of anti-TNF α mAb-CS/PAMAM dendrimer NPs loaded-Ty-GG hydrogel in a physiologically relevant model.

Ion-doped Brushite Cements for Bone Regeneration.

Decades of research in orthopaedics has culminated in the quest for formidable yet resorbable biomaterials using bioactive materials. Brushite cements most salient features embrace high biocompatibility, bioresorbability, osteoconductivity, self-setting characteristics, handling, and injectability properties. Such type of materials is also effectively applied as drug delivery systems. However, brushite cements possess limited mechanical strength and fast setting times. By means of incorporating bioactive ions, which are incredibly promising in directing cell fate when incorporated within biomaterials, it can yield biomaterials with superior mechanical properties. Therefore, it is a key to develop fine-tuned regenerative medicine therapeutics. A comprehensive overview of the current accomplishments of ion-doped brushite cements for bone tissue repair and regeneration is provided herein. The role of ionic substitution on the cements physicochemical properties, such as structural, setting time, hydration products, injectability, mechanical behaviour and ion release is discussed. Cell-material interactions, osteogenesis, angiogenesis, and antibacterial activity of the ion-doped cements, as well as its potential use as drug delivery carriers are also presented. STATEMENT OF SIGNIFICANCE: Ion-doped brushite cements have unbolted a new era in orthopaedics with high clinical interest to restore bone defects and facilitate the healing process, owing its outstanding bioresorbability and osteoconductive/osteoinductive features. Ion incorporation expands their application by increasing the osteogenic and neovascularization potential of the materials, as well as their mechanical performance. Recent accomplishments of brushite cements incorporating bioactive ions are overviewed. Focus was placed on the role of ions on the physicochemical and biological properties of the biomaterials, namely their structure, setting time, injectability and handling, mechanical behaviour, ion release and in vivo osteogenesis, angiogenesis and vascularization. Antibacterial activity of the cements and their potential use for delivery of drugs are also highlighted herein.

Magnetic responsive materials modulate the inflammatory profile of IL-1ß conditioned tendon cells.

Tendinopathies represent half of all musculoskeletal injuries worldwide. Inflammatory events contribute to both tendon healing and to tendinopathy conditions but the cellular triggers leading to one or the other are unknown. In previous studies, we showed that magnetic field actuation modulates human tendon cells (hTDCs) behavior in pro-inflammatory environments, and that magnetic responsive membranes could positively influence inflammation responses in a rat ectopic model. Herein, we propose to investigate the potential synergistic action of the magnetic responsive membranes, made of a polymer blend of starch with polycaprolactone incorporating magnetic nanoparticles (magSPCL), and the actuation of pulsed electromagnetic field (PEMF): 5 Hz, 4mT of intensity and 50% of duty cycle, in IL-1ß-treated-hTDCs, and in the immunomodulatory response of macrophages. It was found that the expression of pro-inflammatory (TNFα, IL-6, IL-8, COX-2) and ECM remodeling (MMP-1,-2,-3) markers tend to decrease in cells cultured onto magSPCL membranes under PEMF, while the expression of TIMP-1 and anti-inflammatory genes (IL-4, IL-10) increases. Also, CD16++ and CD206+ macrophages were only found on magSPCL membranes with PEMF application. Magnetic responsive membranes show a modulatory effect on the inflammatory profile of hTDCs favoring anti-inflammatory cues which is also supported by the anti-inflammatory/repair markers expressed in macrophages. These results suggest that magnetic responsive magSPCL membranes can contribute for inflammation resolution acting on both resident cell populations and inflammatory cells, and thus significantly contribute to tendon regenerative strategies. Statement of significance Magnetically-assisted strategies have received great attention in recent years to remotely trigger and guide cell responses. Inflammation plays a key role in tendon healing but persistent pro-inflammatory molecules can contribute to tendon disorders, and therefore provide a therapeutic target for advanced treatments. We have previously reported that magnetic fields modulate the response of human tendon cells (hTDCs) conditioned to pro-inflammatory environments (IL-1ß-treated-hTDCs), and that magnetic responsive membranes positively influence immune responses. In the present work, we combined pulsed electromagnetic field (PEMF) and magnetic responsive membranes to guide the inflammatory profile of IL-1ß-treated-hTDCs and of macrophages. The results showed that the synergistic action of PEMF and magnetic membranes supports the applicability of magnetically actuated systems to regulate inflammatory events and stimulate tendon regeneration.

Physicochemical properties and cytocompatibility assessment of non-degradable scaffolds for bone tissue engineering applications.

Bone is a dynamic tissue with an amazing but yet limited capacity of self-healing. Bone is the second most transplanted tissue in the world and there is a huge need for bone grafts and substitutes which lead to a decrease in bone banks donors. In this study, we developed three-dimensional scaffolds based on Ti6Al4V, ZrO2 and PEEK targeting bone tissue engineering applications. Experimental mechanical compressive tests and finite element analyses were carried out to study the mechanical performance of the scaffolds. Overall, the scaffolds presented different hydrophilicity properties and a reduced elastic modulus when compared with the corresponding solid materials which can in some extension minimize the phenomenon of stress shielding. The ability as a scaffold material for bone tissue regeneration applications was evaluated in vitro by seeding human osteosarcoma (SaOS-2) cells onto the scaffolds. Then, the successful culture of SaOS-2 cells on developed scaffolds was monitored by assessment of cell's viability, proliferation and alkaline phosphatase (ALP) activity up to 14 days of culturing. The in vitro results revealed that Ti6Al4V, ZrO2 and PEEK scaffolds were cytocompatible allowing the successful culture of an osteoblastic cell line, suggesting their potential application in bone tissue engineering. Statement of Significance. The work presented is timely and relevant since it gathers both the mechanical and cellular study of non-degradable cellular structures with the potential to be used as bone scaffolds. This work allow to investigate three possible bone scaffolds solutions which exhibit a significantly reduced elastic modulus when compared with conventional solid materials. While it is generally accepted that the Ti6Al4V, ZrO2 and PEEK are candidates for such applications a further study of their features and their comparison is extremely important for a better understanding of their potential.

Bactericidal nanopatterns generated by block copolymer self-assembly.

We describe the bactericidal capacity of nanopatterned surfaces created by self-assembly of block copolymers. Distinct nanotopographies were generated by spin-coating with polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP) followed by solvent vapor annealing. We demonstrate that the bactericidal efficiency of the developed coatings depends on the morphology and the chemistry of the surface: cylindrical nanotopographies presenting both blocks at the surface have stronger bactericidal effect on Escherichia coli than micellar patterns with only PS exposed at the surface. The identified mechanism of bacterial death is a mechanical stress exerted by the nanostructures on the cell-wall. Moreover, the developed nanopatterns are not cytotoxic, which makes them an excellent option for coating of implantable materials and devices. The proposed approach represents an efficient tool in the fight against bacteria, which acts via compromising the bacterial wall integrity. STATEMENT OF SIGNIFICANCE: Bacterial infections represent an important risk during biomaterial implantation in surgeries due to the increase of antibiotic resistance. Bactericidal surfaces are a promising solution to avoid the use of antibiotics, but most of those systems do not allow mammalian cell survival. Nanopatterned silicon surfaces have demonstrated to be simultaneously bactericidal and allow mammalian cell culture but are made by physical methods (e.g. plasma etching) applicable to few materials and small surfaces. In this article we show that block copolymer self-assembly can be used to develop surfaces that kill bacteria (E. coli) but do not harm mammalian cells. Block copolymer self-assembly has the advantage of being applicable to many different types of substrates and large surface areas.

Dendrimer nanoparticles for colorectal cancer applications.

Cancer nanotechnology is a prolific field of research, where nanotools are employed to diagnose and treat cancer with unprecedented precision. Targeted drug delivery is fundamental for more efficient cancer treatments. For this, nanoparticles have been extensively used during the past few years in order to improve the specificity, selectivity and controlled release of drug delivery. It holds potential in minimizing systemic toxicity through the development of functionalized particles for targeted treatment. Among all the type of nanoparticles, dendrimers display several advantages, which make them ideal candidates for improved and targeted drug delivery in cancer research. Dendrimers can transport large amounts of drug into specific areas. In addition, they can be employed for monitoring the progress of the treatment process, with an unprecedented theranostic capability. Special emphasis is given to colorectal cancer and to the preferred employed strategies for producing drug-loaded/functionalized NPs for cancer therapy in the past few years.

Micro-CT based finite element modelling and experimental characterization of the compressive mechanical properties of 3-D zirconia scaffolds for bone tissue engineering.

The present study aims at developing a computational framework with experimental validation to determine the mechanical properties of zirconia foams for bone tissue engineering. A micro-CT based finite element model that allows characterizing the mechanical property of such cellular structures is developed. Micro-CT images are filtered to vanish noises and smooth boundaries before constructing 3D zirconia foams using an adaptive Body-Centered Cubic background lattice. In addition to micro-CT images, the local material property at the scaffold struts is measured using a micro-indentation test, which shows a considerable difference with that of common zirconia owing to the manufacturing process. The computational model also takes the plastic deformation of material into account employing the Voce law, a nonlinear isotropic hardening law, as well as Von-mises yield criterion. Zirconia foams with different pore sizes are manufactured using the replica method and their mechanical properties determined experimentally. Such experimental outcomes are to validate and demonstrate the capability of the developed model, which can be used for pre-operational evaluations and preclinical tests of zirconia scaffolds. The stress magnitude and distribution within the scaffold as well as plastic strains and flow stress of the zirconia scaffold are computed and analysed. Using the proposed approach, a deep insight into the association of macroscopic behaviour of the scaffold to microscopic features, e.g. strut waviness, Plateau border, thickness variation of cells, irregularity, microstructural variability, imperfections and strut's material property associated with to the manufacturing procedure, can be gained.

Curcumin ameliorates the targeted delivery of methotrexate intercalated montmorillonite clay to cancer cells.

Montmorillonite Clay (MMT) is aimed to develop as an orally administrable drug delivery vehicle with enhanced efficacy. Aiming to enhance the therapeutic index of methotrexate, curcumin is concomitantly used with methotrexate in the present study. Being folate antagonist in nature, methotrexate is internalized into cells by folate receptor (FR); which is over-expressed in certain human cancer cells such as cervical carcinoma cells (HeLa). Firstly, montmorillonite Clay (MMT) is organically modified (OMMT) with cetyl trimethyl ammonium bromide (CTAB) and used to intercalate curcumin and methotrexate separately, designated as OMMT-Cur and OMMT-MTX, respectively. XRD pattern demonstrated successful intercalation of therapeutics and an increase in clay interlayer distance facilitated by CTAB. The dissolution kinetics of methotrexate follows Higuchi model for both Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), while the release kinetics for curcumin fitted into Higuchi model for SGF and Hixson-Crowell model for SIF, respectively. OMMT-MTX are able to discriminate FR-positive HeLa cells from FR-negative breast cancer cells (MCF7); irrespective of alike cellular phenotypes. Further, the pre-treatment of HeLa cells with curcumin improves its sensitivity towards methotrexate causing a greater killing of the Hela cells. Together, the results propose the concomitant use of curcumin and methotrexate for successfully targeting highly invasive FR-positive carcinomas by means of folate receptor using MMTs.

Colorectal tumor-on-a-chip system: A 3D tool for precision onco-nanomedicine.

Awareness that traditional two-dimensional (2D) in vitro and nonrepresentative animal models may not completely emulate the 3D hierarchical complexity of tissues and organs is on the rise. Therefore, posterior translation into successful clinical application is compromised. To address this dearth, on-chip biomimetic microenvironments powered by microfluidic technologies are being developed to better capture the complexity of in vivo pathophysiology. Here, we describe a "tumor-on-a-chip" model for assessment of precision nanomedicine delivery on which we validate the efficacy of drug-loaded nanoparticles in a gradient fashion. The model validation was performed by viability studies integrated with live imaging to confirm the dose-response effect of cells exposed to the CMCht/PAMAM nanoparticle gradient. This platform also enables the analysis at the gene expression level, where a down-regulation of all the studied genes (MMP-1, Caspase-3, and Ki-67) was observed. This tumor-on-chip model represents an important development in the use of precision nanomedicine toward personalized treatment.

Mechanical Property of Hydrogels and the Presence of Adipose Stem Cells in Tumor Stroma Affect Spheroid Formation in the 3D Osteosarcoma Model.

Osteosarcoma is one of the most common metastatic bone cancers, which results in significant morbidity and mortality. Unfolding of effectual therapeutic strategies against osteosarcoma is impeded because of the absence of adequate animal models, which can truly recapitulate disease biology of humans. Tissue engineering provides an opportunity to develop physiologically relevant, reproducible, and tunable in vitro platforms to investigate the interactions of osteosarcoma cells with its microenvironment. Adipose-derived stem cells (ASCs) are detected adjacent to osteosarcoma masses and are considered to have protumor effects. Hence, the present study focuses on investigating the role of reactive ASCs in formation of spheroids of osteosarcoma cells (Saos 2) within a three-dimensional (3D) niche, which is created using gellan gum (GG)-silk fibroin. By modifying the blending ratio of GG-silk, the optimum stiffness of the resultant hydrogels such as GG and GG75: S25 is obtained for cancer spheroid formation. This work indicates that the co-existence of cancer and stem cells can form a spheroid, the hallmark of cancer, only in particular microenvironment stiffness. The incorporation of fibrillar silk fibroin within the hydrophilic network of GG in GG75: S25 spongy-like hydrogels closely mimics the stiffness of commercially established cancer biomaterials (e.g., Matrigel, HyStem). The GG75: S25 hydrogel maintains the metabolically active construct for a longer time with elevated expression of osteopontin, osteocalcin, RUNX 2, and bone sialoprotein genes, the biomarkers of osteosarcoma, compared to GG. The GG75: S25 construct also exhibits intense alkaline phosphatase expression in immunohistochemistry compared to GG, indicating itspotentiality to serve as biomimetic niche to model osteosarcoma. Taken together, the GG-silk fibroin-blended spongy-like hydrogel is envisioned as an alternative low-cost platform for 3D cancer modeling.