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.

Collagen-based bioinks for hard tissue engineering applications: a comprehensive review.

In the last few years, additive manufacturing (AM) has been gaining great interest in the fabrication of complex structures for soft-to-hard tissues regeneration, with tailored porosity, and boosted structural, mechanical, and biological properties. 3D printing is one of the most known AM techniques in the field of biofabrication of tissues and organs. This technique opened up opportunities over the conventional ones, with the capability of creating replicable, customized, and functional structures that can ultimately promote effectively different tissues regeneration. The uppermost component of 3D printing is the bioink, i.e. a mixture of biomaterials that can also been laden with different cell types, and bioactive molecules. Important factors of the fabrication process include printing fidelity, stability, time, shear-thinning properties, mechanical strength and elasticity, as well as cell encapsulation and cell-compatible conditions. Collagen-based materials have been recognized as a promising choice to accomplish an ideal mimetic bioink for regeneration of several tissues with high cell-activating properties. This review presents the state-of-art of the current achievements on 3D printing using collagen-based materials for hard tissue engineering, particularly on the development of scaffolds for bone and cartilage repair/regeneration. The ultimate aim is to shed light on the requirements to successfully print collagen-based inks and the most relevant properties exhibited by the so fabricated scaffolds. In this regard, the adequate bioprinting parameters are addressed, as well as the main materials properties, namely physicochemical and mechanical properties, cell compatibility and commercial availability, covering hydrogels, microcarriers and decellularized matrix components. Furthermore, the fabrication of these bioinks with and without cells used in inkjet printing, laser-assisted printing, and direct in writing technologies are also overviewed. Finally, some future perspectives of novel bioinks are given.

Triggering the activation of Activin A type II receptor in human adipose stem cells towards tenogenic commitment using mechanomagnetic stimulation.

Stem cell therapies hold potential to stimulate tendon regeneration and homeostasis, which is maintained in response to the native mechanical environment. Activins are members of the mechano-responsive TGF-ß superfamily that participates in the regulation of several downstream biological processes. Mechanosensitive membrane receptors such as activin can be activated in different types of stem cells via magnetic nanoparticles (MNPs) through remote magnetic actuation resulting in cell differentiation. In this work, we target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), using anti-ActRIIA functionalized MNPs, externally activated through a oscillating magnetic bioreactor. Upon activation, the phosphorylation of Smad2/3 is induced allowing translocation of the complex to the nucleus, regulating tenogenic transcriptional responses. Our study demonstrates the potential remote activation of MNPs tagged hASCs to trigger the Activin receptor leading to tenogenic differentiation. These results may provide insights toward tendon regeneration therapies.

Biofunctional Ionic-Doped Calcium Phosphates: Silk Fibroin Composites for Bone Tissue Engineering Scaffolding.

The treatment and regeneration of bone defects caused by traumatism or diseases have not been completely addressed by current therapies. Lately, advanced tools and technologies have been successfully developed for bone tissue regeneration. Functional scaffolding materials such as biopolymers and bioresorbable fillers have gained particular attention, owing to their ability to promote cell adhesion, proliferation, and extracellular matrix production, which promote new bone growth. Here, we present novel biofunctional scaffolds for bone regeneration composed of silk fibroin (SF) and ß-tricalcium phosphate (ß-TCP) and incorporating Sr, Zn, and Mn, which were successfully developed using salt-leaching followed by a freeze-drying technique. The scaffolds presented a suitable pore size, porosity, and high interconnectivity, adequate for promoting cell attachment and proliferation. The degradation behavior and compressive mechanical strengths showed that SF/ionic-doped TCP scaffolds exhibit improved characteristics for bone tissue engineering when compared with SF scaffolds alone. The in vitro bioactivity assays using a simulated body fluid showed the growth of an apatite layer. Furthermore, in vitro assays using human adipose-derived stem cells presented different effects on cell proliferation/differentiation when varying the doping agents in the biofunctional scaffolds. The incorporation of Zn into the scaffolds led to improved proliferation, while the Sr- and Mn-doped scaffolds presented higher osteogenic potential as demonstrated by DNA quantification and alkaline phosphatase activity. The combination of Sr with Zn led to an influence on cell proliferation and osteogenesis when compared with single ions. Our results indicate that biofunctional ionic-doped composite scaffolds are good candidates for further in vivo studies on bone tissue regeneration.

Biomechanical and cellular segmental characterization of human meniscus: building the basis for Tissue Engineering therapies.

OBJECTIVE: To overcome current limitations of Tissue Engineering (TE) strategies, deeper comprehension on meniscus biology is required. This study aims to combine biomechanical segmental analysis of fresh human meniscus tissues and its correlation with architectural and cellular characterization. METHOD: Morphologically intact menisci, from 44 live donors were studied after division into three radial segments. Dynamic mechanical analysis (DMA) was performed at physiological-like conditions. Micro-computed tomography (CT) analysis of freeze-dried samples assessed micro-structure. Flow cytometry, histology and histomorphometry were used for cellular study and quantification. RESULTS: Anterior segments present significantly higher damping properties. Mid body fresh medial meniscus presents higher values of E' compared to lateral. Cyclic loads influence the viscoelastic behavior of menisci. By increasing the frequency leads to an increase in stiffness. Conversely, with increasing frequencies, the capacity to dissipate energy and damping properties initially decrease and then rise again. Age and gender directly correlate with higher E' and tan δ. Micro-CT analysis revealed that mean porosity was 55.5 (21.2-89.8)% and 64.7 (47.7-81.8)% for freeze-dried lateral and medial meniscus, respectively. Predominant cells are positive for CD44, CD73, CD90 and CD105, and lack CD31, CD34 and CD45 (present in smaller populations). Histomorphometry revealed that cellularity decreases from vascular zone 1 to zone 3. Anterior segments of lateral and medial meniscus have inferior cellularity as compared to mid body and posterior ones. CONCLUSION: Menisci are not uniform structures. Anterior segments have lower cellularity and higher damping. Cyclic loads influence viscoelastic characteristics. Future TE therapies should consider segmental architecture, cellularity and biomechanics of fresh tissue.

Bottom-up approach to construct microfabricated multi-layer scaffolds for bone tissue engineering.

The use of bottom-up approaches in tissue engineering applications is advantageous since they enable the combination of various layers that could be made from different materials and/or incorporate different biochemical cues. Regarding the complex structure and the vascular system of the bone tissue, the aim of this study was to develop an innovative bottom-up approach that allows the construction of 3D biodegradable scaffolds from 2D microfabricated membranes with precise shape, pore size and porosity. For that purpose, poly (caprolactone) (PCL) and starch ­ poly (caprolactone) (SPCL (30 % starch)) blended sheets were used as substrates to produce the microfabricated membranes using micro hotembossing. The use of this micro fabrication process allowed accurately imprinting micropillars and microholes in reproducible way. The assembling of the microfabricated membranes was performed using an easy, highly reproducible and inexpensive approach based on its successive stacking. Additionaly, the suitability of the microfabricated membranes to support the attachment and the cytoskeletal organization of human bone marrow stem cells (hBMSCs), macrovascular endothelial cells and osteoblasts derived from hBMSCs was demonstrated. Furthermore, hBMSCs proliferated and maintained the expression of the stromal progenitor marker STRO-1 when cultured on both PCL and SPCL microfabricated membranes. The proposed methodology constitutes a promising alternative to the traditional processing methods used to prepare tissue engineering scaffolds.

Photopatterned antibodies for selective cell attachment.

We present a phototriggerable system that allows for the spatiotemporal controlled attachment of selected cell types to a biomaterial using immobilized antibodies that specifically target individual cell phenotypes. o-Nitrobenzyl caged biotin was used to functionalize chitosan membranes and mediate site-specific coupling of streptavidin and biotinylated antibodies after light activation. The ability of this system to capture and immobilize specific cells on a surface was tested using endothelial-specific biotinylated antibodies and nonspecific ones as controls. Homogeneous patterned monolayers of human umbilical vein endothelial cells were obtained on CD31-functionalized surfaces. This is a simple and generic approach that is applicable to other ligands, materials, and cell types and shows the flexibility of caged ligands to trigger and control the interaction between cells and biomaterials.

Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds.

Among the wide range of strategies to target skin repair/regeneration, tissue engineering (TE) with stem cells at the forefront, remains as the most promising route. Cell sheet (CS) engineering is herein proposed, taking advantage of particular cell-cell and cell-extracellular matrix (ECM) interactions and subsequent cellular milieu, to create 3D TE constructs to promote full-thickness skin wound regeneration. Human adipose derived stem cells (hASCs) CS were obtained within five days using both thermoresponsive and standard cell culture surfaces. hASCs-based constructs were then built by superimposing three CS and transplanted into full-thickness excisional mice skin wounds with delayed healing. Constructs obtained using thermoresponsive surfaces were more stable than the ones from standard cell culture surfaces due to the natural adhesive character of the respective CS. Both CS-generating strategies lead to prolonged hASCs engraftment, although no transdifferentiation phenomena were observed. Moreover, our findings suggest that the transplanted hASCs might be promoting neotissue vascularization and extensively influencing epidermal morphogenesis, mainly through paracrine actions with the resident cells. The thicker epidermis, with a higher degree of maturation characterized by the presence of rete ridges-like structures, as well as a significant number of hair follicles observed after transplantation of the constructs combining the CS obtained from the thermoresponsive surfaces, reinforced the assumptions of the influence of the transplanted hASCs and the importance of the higher stability of these constructs promoted by cohesive cell-cell and cell-ECM interactions. Overall, this study confirmed the potential of hASCs CS-based constructs to treat full-thickness excisional skin wounds and that their fabrication conditions impact different aspects of skin regeneration, such as neovascularisation, but mainly epidermal morphogenesis.

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.

Peripheral mineralization of a 3D biodegradable tubular construct as a way to enhance guidance stabilization in spinal cord injury regeneration.

Spinal cord injuries (SCI) present a major challenge to therapeutic development due to its complexity. Combinatorial approaches using biodegradable polymers that can simultaneously provide a tissue scaffold, a cell vehicle, and a reservoir for sustained drug delivery have shown very promising results. In our previous studies we have developed a novel hybrid system consisting of starch/poly-e-caprolactone (SPCL) semi-rigid tubular porous structure, based on a rapid prototyping technology, filled by a gellan gum hydrogel concentric core for the regeneration within spinal-cord injury sites. In the present work we intend to promote enhanced osteointegration on these systems by pre-mineralizing specifically the external surfaces of the SPCL tubular structures, though a biomimetic strategy, using a sodium silicate gel as nucleating agent. The idea is to create two different cell environments to promote axonal regeneration in the interior of the constructs while inducing osteogenic activity on its external surface. By using a Teflon cylinder to isolate the interior of the scaffold, it was possible to observe the formation of a bone-like poorly crystalline carbonated apatite layer continuously formed only in the external side of the tubular structure. This biomimetic layer was able to support the adhesion of Bone Marrow Mesenchymal Stem Cells, which have gone under cytoskeleton reorganization in the first hours of culture when compared to cells cultured on uncoated scaffolds. This strategy can be a useful route for locally stimulate bone tissue regeneration and facilitating early bone ingrowth.

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.

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.

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.

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.

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.

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.

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.

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.