Upscaling Osteoclast Generation by Enhancing Macrophage Aggregation Using Hollow Microgels.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.

Proteomic analysis of calcium-enriched sol-gel biomaterials.

Calcium is an element widely used in the development of biomaterials for bone tissue engineering as it plays important roles in bone metabolism and blood coagulation. The Ca ions can condition the microenvironment at the tissue-material interface, affecting the protein deposition process and cell responses. The aim of this study was to analyze the changes in the patterns of protein adsorption on the silica hybrid biomaterials supplemented with different amounts of CaCl2, which can function as release vehicles. This characterization was carried out by incubating the Ca-biomaterials with human serum. LC-MS/MS analysis was used to characterize the adsorbed protein layers and compile a list of proteins whose affinity for the surfaces might depend on the CaCl2 content. The attachment of pro- and anti-clotting proteins, such as THRB, ANT3, and PROC, increased significantly on the Ca-materials. Similarly, VTNC and APOE, proteins directly involved on osteogenic processes, attached preferentially to these surfaces. To assess correlations with the proteomic data, these formulations were tested in vitro regarding their osteogenic and inflammatory potential, employing MC3T3-E1 and RAW 264.7 cell lines, respectively. The results confirmed a Ca dose-dependent osteogenic and inflammatory behavior of the materials employed, in accordance with the protein attachment patterns.

Scalable fabrication, compartmentalization and applications of living microtissues.

Living microtissues are used in a multitude of applications as they more closely resemble native tissue physiology, as compared to 2D cultures. Microtissues are typically composed of a combination of cells and materials in varying combinations, which are dictated by the applications' design requirements. Their applications range wide, from fundamental biological research such as differentiation studies to industrial applications such as cruelty-free meat production. However, their translation to industrial and clinical settings has been hindered due to the lack of scalability of microtissue production techniques. Continuous microfluidic processes provide an opportunity to overcome this limitation as they offer higher throughput production rates as compared to traditional batch techniques, while maintaining reproducible control over microtissue composition and size. In this review, we provide a comprehensive overview of the current approaches to engineer microtissues with a focus on the advantages of, and need for, the use of continuous processes to produce microtissues in large quantities. Finally, an outlook is provided that outlines the required developments to enable large-scale microtissue fabrication using continuous processes.

Bioluminescence imaging on-chip platforms for non-invasive high-content bioimaging.

Incorporating non-invasive biosensing features in organ-on-chip models is of paramount importance for a wider implementation of these advanced in vitro microfluidic platforms. Optical biosensors, based on Bioluminescence Imaging (BLI), enable continuous, non-invasive, and in-situ imaging of cells, tissues or miniaturized organs without the drawbacks of conventional fluorescence imaging. Here, we report the first-of-its-kind integration and optimization of BLI in microfluidic chips, for non-invasive imaging of multiple biological readouts. The cell line HEK293T-GFP was engineered to express NanoLuc® luciferase under the control of a constitutive promoter and were cultured on-chip in 3D, in standard ECM-like hydrogels, to assess optimal cell detection conditions. Using real-time in-vitro dual-color microscopy, Bioluminescence (BL) and fluorescence (FL) were detectable using distinct imaging setups. Detection of the bioluminescent signals were observed at single cell resolution on-chip 20 min post-addition of Furimazine substrate and under perfusion. All hydrogels enabled BLI with higher signal-to-noise ratios as compared to fluorescence. For instance, agarose gels showed a ∼5-fold greater BL signal over background after injection of the substrate as compared to the FL signal. The use of BLI with microfluidic chip technologies opens up the potential for simultaneous in situ detection with continuous monitoring of multicolor cell reporters. Moreover, this can be achieved in a non-invasive manner. BL has great promise as a highly desirable biosensor for studying organ-on-chip platforms.

Microfluidic Generation of Thin-Shelled Polyethylene Glycol-Tyramine Microgels for Non-Invasive Delivery of Immunoprotected ß-Cells.

Transplantation of microencapsulated pancreatic cells is emerging as a promising therapy to replenish ß-cell mass lost from auto-immune nature of type I diabetes mellitus (T1DM). This strategy intends to use micrometer-sized microgels to provide immunoprotection to transplanted cells to avoid chronic application of immunosuppression. Clinical application of encapsulation has remained elusive due to often limited production throughputs and body's immunological reactions to implanted materials. This article presents a high-throughput fabrication of monodisperse, non-immunogenic, non-degradable, immunoprotective, semi-permeable, enzymatically-crosslinkable polyethylene glycol-tyramine (PEG-TA) microgels for ß-cell microencapsulation. Monodisperse ß-cell laden microgels of ≈120 µm, with a shell thickness of 20 µm are produced using an outside-in crosslinking strategy. Microencapsulated ß-cells rapidly self-assemble into islet-sized spheroids. Immunoprotection of the microencapsulated is demonstrated by inability of FITC-IgG antibodies to diffuse into cell-laden microgels and NK-cell inability to kill microencapsulated ß-cells. Multiplexed ELISA analysis on live blood immune reactivity confirms limited immunogenicity. Microencapsulated MIN6ß1 spheroids remain glucose responsive for 28 days in vitro, and able to restore normoglycemia 5 days post-implantation in diabetic mice without notable amounts of cell death. In short, PEG-TA microgels effectively protect implanted cells from the host's immune system while being viable and functional, validating this strategy for the treatment of T1DM.

Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules.

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.

The transcription factor Ndt80 is a repressor of Candida parapsilosis virulence attributes.

Candida parapsilosis is an emergent opportunistic yeast among hospital settings that affects mainly neonates and immunocompromised patients. Its most remarkable virulence traits are the ability to adhere to prosthetic materials, as well as the formation of biofilm on abiotic surfaces. The Ndt80 transcription factor was identified as one of the regulators of biofilm formation by C. parapsilosis; however, its function in this process was not yet clarified. By knocking out NDT80 (CPAR2-213640) gene, or even just one single copy of the gene, we observed substantial alterations of virulence attributes, including morphogenetic changes, adhesion and biofilm growth profiles. Both ndt80Δ and ndt80ΔΔ mutants changed colony and cell morphologies from smooth, yeast-shaped to crepe and pseudohyphal elongated forms, exhibiting promoted adherence to polystyrene microspheres and notably, forming a higher amount of biofilm compared to wild-type strain. Interestingly, we identified transcription factors Ume6, Cph2, Cwh41, Ace2, Bcr1, protein kinase Mkc1 and adhesin Als7 to be under Ndt80 negative regulation, partially explaining the phenotypes displayed by the ndt80ΔΔ mutant. Furthermore, ndt80ΔΔ pseudohyphae adhered more rapidly and were more resistant to murine macrophage attack, becoming deleterious to such cells after phagocytosis. Unexpectedly, our findings provide the first evidence for a direct role of Ndt80 as a repressor of C. parapsilosis virulence attributes. This finding shows that C. parapsilosis Ndt80 functionally diverges from its homolog in the close related fungal pathogen C. albicans.

Evaluation of the inflammatory responses to sol-gel coatings with distinct biocompatibility levels.

The immune system plays a crucial role in determining the implantation outcome, and macrophages are in the frontline of the inflammatory processes. Further, cellular oxidative stress resulting from the material recognition can influence how cell responses develop. Considering this, the aim of this study was to study oxidative stress and macrophages phenotypes in response to sol-gel materials with distinct in vivo outcomes. Four materials were selected (70M30T and 35M35G30T, with high biocompatibility, and 50M50G and 50V50G, with low biocompatibility). Gene expression, immunocytochemistry and cytokine secretion profiles for M1 and M2 markers were determined. Moreover, oxidative stress markers were studied. Immunocytochemistry and ELISA showed that 50M50G and 50V50G lead to a higher differentiation to M1 phenotype, while 70M30T and 35M35G30T promoted M2 differentiation. In oxidative stress, no differences were found. These results show that the balance between M1 and M2, more than individual quantification of each phenotype, determines a biomaterial outcome.

A possible use of melatonin in the dental field: Protein adsorption and in vitro cell response on coated titanium.

Melatonin (MLT) is widely known for regulating the circadian cycles and has been studied for its role in bone regeneration and inflammation. Its application as a coating for dental implants can condition the local microenvironment, affecting protein deposition on its surface and the cellular and tissue response. Using sol-gel coatings as a release vehicle for MLT, the aim of this work was to assess the potential of this molecule in improving the osseointegration and inflammatory responses of a titanium substrate. The materials obtained were physicochemically characterized (scanning electron microscopy, contact angle, roughness, Fourier-transform infrared spectroscopy, nuclear magnetic resonance, Si release, MLT liberation, and degradation) and studied in vitro with MC3T3-E1 osteoblastic cells and RAW264.7 macrophage cells. Although MLT application led to an increased gene expression of RUNX2 and BMP2 in 10MTL, it did not improve ALP activity. On the other hand, MLT-enriched sol-gel materials presented potential effects in the adsorption of proteins related to inflammation, coagulation and angiogenesis pathways depending on the dosage used. Using LC-MS/MS, protein adsorption patterns were studied after incubation with human serum. Proteins related to the complement systems (CO7, IC1, CO5, CO8A, and CO9) were less adsorbed in materials with MLT; on the other hand, proteins with functions in the coagulation and angiogenesis pathways, such as A2GL and PLMN, showed a significant adsorption pattern.

Characterization of serum proteins attached to distinct sol-gel hybrid surfaces.

The success of a dental implant depends on its osseointegration, an important feature of the implant biocompatibility. In this study, two distinct sol-gel hybrid coating formulations [50% methyltrimethoxysilane: 50% 3-glycidoxypropyl-trimethoxysilane (50M50G) and 70% methyltrimethoxysilane with 30% tetraethyl orthosilicate (70M30T)] were applied onto titanium implants. To evaluate their osseointegration, in vitro and in vivo assays were performed. Cell proliferation and differentiation in vitro did not show any differences between the coatings. However, four and eight weeks after in vivo implantation, the fibrous capsule area surrounding 50M50G-implant was 10 and 4 times, respectively, bigger than the area of connective tissue surrounding the 70M30T treated implant. Thus, the in vitro results gave no prediction or explanation for the 50M50G-implant failure in vivo. We hypothesized that the first protein layer adhered to the surface may have direct implication in implant osseointegration, and perhaps correlate with the in vivo outcome. Human serum was used for adsorption analysis on the biomaterials, the first layer of serum proteins adhered to the implant surface was analyzed by proteomic analysis, using mass spectrometry (LC-MS/MS). From the 171 proteins identified; 30 proteins were significantly enriched on the 50M50G implant surface. This group comprised numerous proteins of the immune complement system, including several subcomponents of the C1 complement, complement factor H, C4b-binding protein alpha chain, complement C5 and C-reactive protein. This result suggests that these proteins enriched in 50M50G surface might trigger the cascade leading to the formation of the fibrous capsule observed. The implications of these results could open up future possibilities to predict the biocompatibility problems in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1477-1485, 2018.

Silica-gelatin hybrid sol-gel coatings: A proteomic study with biocompatibility implications.

Osseointegration, including the foreign body reaction to biomaterials, is an immune-modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation-driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic Collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol-gel formulations and their combination with gelatin were applied as coatings on sandblasted, acid-etched titanium substrates, and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin-containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings.