Symposium F.SM08—Regenerative Engineering and Synthetic Biology

Porous membranes with well-defined pore structures find plenty of uses in research and industrial areas. Well-defined pore structures afford sharp selectivity in filtration and thus applications in biomedical uses are also expected such as cell/spheroid separation and immunoprotection. Track-etching is a well-known technology to fabricate porous membranes with relatively defined pores, but undesired overlapped spots result in loss of selectivity. Against the background, we developed a novel microfabrication methodology to prepare porous membrane with strictly defined pores. We successfully developed porous PTFE membrane with 5-10 um thickness with tunable pore sizes. We will discuss the potential applications of microfabricated PTFE membranes in biomedical uses.

An ability to control the assembly of functional proteins with designable organizations and an unpresented density in multidimensional space, provides routes to the next-generation nano-systems, biomaterials and complex biomolecular scaffolds. To achieve this, DNA nanotechnology offers exciting opportunities to modulate assembly processes with nano-scale resolution and spatial addressability. In this work, we developed a protein encapsulation strategy combined with the DNA programmable assembly for a bottom-up fabrication of biologically functional protein arrays with 2D and 3D prescribed organizations. We showed that ferritin, an iron storage protein, was integrated with DNA frames, of which each vertex and their internal space were encoded with addressable strands to program the protein position and vertex-to-vertex interconnection. Such designed arrays allow to maintain a biological activity of ferritin in the nanoscale lattices at relevant environments, and meanwhile, small molecules can access the iron core of ferritin within the 3D array and convert it into a core-less form, apoferritin. Our study, through the use of in situ small angle X-ray scattering, electron microscopy and tomographic modelling, revealed the complex arrangement of designed protein arrays and their ability to perform functions within 3D arrays, thus confirming a new strategy for creating bio-active protein-containing materials.

Defects in bone tissue have been more common in recent years, based on this a section has been dedicated in the area of biomaterials for research. Currently, knowledge about new biomaterials has led to the discovery of sets of precursors that work effectively in the recovery of damaged tissue, which must meet certain requirements to be recognized as biomaterials, that is, they must be biocompatible, biodegradable, non-toxic, have mechanical resistance, as well as the appropriate density, weight, shape and size. Once these characteristics are known, it has been decided to experiment with three basic precursors in bone regeneration. The work that was carried out aims to manufacture three-dimensional matrices using precursors such as hydroxyapatite, polylactic acid and polycaprolactone. The characterizations were carried out to verify the purity of such biomaterials. Differential Scanning Calorimetry for both tests in a nitrogen/air atmosphere (80/20) at a temperature range of 100 °C to 1000 °C with a heating of 10 °C/min; on the other hand, for polylactic acid an analysis was carried out using Fourier Transform Infrared Spectrometry. Once the results were obtained, the scaffolding was manufactured using leaching and solvent casting techniques. With this, two groups of samples were developed with concentrations of 30% HAp -10% PLA -60% PCL and 10% HAp - 10% PLA - 80% PCL, they were mixed with an organic solvent and sodium chloride. The resulting scaffolds were subjected to mechanical compression and tension tests. Obtaining promising results for application in bone tissue.

Silver nanoparticles play a significant role in public health. They have been proven to be effective substrates for high-intensity surface-enhanced Raman spectroscopy (SERS) in biomedical applications. The nanoparticles are able to detect organic molecules at nanomolar concentration and has the potential for early disease detection or virus killing. Since SERS technology has gained traction in public health uses recently, the synthesis of AgNPs which is considered as the essential and fundamental key of applications, has attracted numerous researchers. In this study, we propose a green, wet-chemical way to control the synthesis of AgNPs and explore three different reducing agents under similar conditions. Sodium citrate, L-cysteine, and oxalic acid, all used as reducing agents, are respectively added into the AgNO₃ aqueous solution where the Ag⁺ concentration is 2.5mmol/L in room temperature. L-cysteine, a specially selected amino acid, exists in every group acting as the capping agent to restrict the aggregation and control the size of nanoparticles. According to the surface-enhanced Raman spectrum and SEM images, sodium citrate demonstrates the best reducing ability under the same concentration of the carboxy group and can be used for a quick synthesis. On the other hand, the particle size of oxalic acid carpets is more controlled regardless of concentration. It is suitable for precise synthesis associated with the time when the surface-enhanced Raman spectrum indicates the size growth of silver nanoparticles as time increases. Some mechanisms during the process are revealed as well. After analyzing the spectrum, we deem that Ag⁺ ion combines with carboxy groups more easily than sulfur in this system. Consequently, these three reducing agents exhibit their unique advantages which provide us with various selections for the synthesis of silver nanoparticles under different requirements for different applications in public health.

Acknowledgment
The first author is grateful to the Academy for Advanced Research and Development (AARD, website: www.ardacademy.org; contact email: [email protected]) for the Intern Research Sponsorship through the collaboration with the Center for Nanoscale Systems at Harvard University.

The biophysical and biochemical properties of the extracellular matrix (ECM) (e.g., mechanics, multiscale structure, receptor-binding sites) are essential for the function and regeneration of connective tissues throughout the body, from the collagen-rich loose irregular connective tissue of the lung to the dense regular connective tissue of tendon and ligament. To study the complex process of connective tissue healing outside of the body toward ultimately directing it in vivo, we have designed soft biomaterials, engineered synthetic hydrogels, that mimic key aspects of the collagen-rich ECM found in the early stages of tissue healing. These materials are constructed by bottom-up assembly of multifunctional collagen mimetic peptides (mfCMPs) designed with hydrogen bonding blocks and sticky ends for the formation of nano- and micro-fibrils and decorated with reactive handles for covalent crosslinking and labeling to control mechanical properties and bioactivity. We have established robust, scalable workflows for synthesis of these materials to facilitate their use in translational applications. Wound healing human mesenchymal stem cells (hMSCs) encapsulated within these materials exhibit good viability (over 85%) and significant elongation during 3D culture, with deposition of cell-secreted collagen I. Further, in response to a chemokine gradient, hMSCs exhibit directional invasion and increased motility within these materials relative to hydrogels without mfCMPs. Our on-going investigations demonstrate the relevance of these collagen mimetic materials for fundamental studies to examine cell responses in fibrous wound-healing microenvironments and for translational studies to implement these materials as scaffolds for enhanced tissue regeneration.

Stem cells isolated from the dental pulp (dental pulp stem cells (DPSCs)) were shown the pluripotent differentiation properties, where the differentiation lineage can be regulated by both soluble and non-soluble factors. In our previous studies, we have shown that substrate modulus can trigger DPSCs to biomineralize when the modulus is higher than a critical value. However, the incomplete differentiation and amorphous particle-like mineral deposits were observed on flat hard substrates. Surface coating with TiO₂, on the other hand, was found to be able to induce osteogenic differentiation and hard tissue formation, regardless of substrate moduli. Instead of directly inducing the differentiation by substrate properties, lateral signals, cell-cell communication was also found to be important to regulate cell functions. Therefore, half-coating the polybutadiene (PB) substrates with TiO₂ by atomic layer deposition (ALD) were used to study DPSCs communication between cultures on TiO₂ coating and non-coating region of the substrate. The results showed that the cell proliferation was improved and biomineralization was induced from the cultures on the non-coating region, indicating the ability of lateral signal transduction in DPSCs. However, amorphous particle-like mineral deposits were present on the non-coating region while collagen fibers templated minerals were observed on the TiO₂ coating region. Hence, even though proliferation and biomineralization could be induced without directly contact with TiO₂-coated surface, while the ability to form fibers templated minerals, the structure required for bone or dentin tissue formation, is a function of surface properties, particularly the role of TiO₂ to nucleate banded collage fibers in enabling hard tissue formation.
This study address the importance of both integrin signals from substrate properties and transmembrane signals from cell-cell contact are important to investigate due to the dynamic in vivo environment, and was found to be able to regulate DPSCs proliferation and differentiation.

Native extracellular matrixes (ECM) acts as signaling repositories to regulate cell behavior and function. Installation of implantable medical devices disrupts these matrices and destroys spatiotemporal presentation and interaction of cells with microenvironmental cues. Regeneration of tissue niches is critical for long-term stability and integration of medical devices in patients. Here, we present a new, broadly-applicable physicochemical strategy for percutaneous devices to leverage secreted, nascent ECM for instructing epithelial cell behavior. We synthesized a set of photopolymerizable polymers with catechol and structurally-similar moieties, easily applied to percutaneous device surfaces like catheters, dental restorative materials, or orthopaedic implants, with systematically varied physicochemical properties but similar bulk mechanical properties. Tuning surface physicochemical properties, specifically charge and polarity, enabled us to tune keratinocyte hemidesmosome formation, adhesive structures that anchor cells to ECM, formation and downstream signaling. A series of inhibitor and knock-down experiments demonstrated the bioinstructive effect of our materials on elaborated ECM, laminin332 in particular, in an integrin-dependent manner. We propose our biostructure polymers direct cell responses via modulation of laminin332’s structure in the pericellular ECM. Finally, we challenged our engineered materials in a course of experiments, including ex vivo, meant to reflect the harsh environment experienced during clinical therapeutic use and showed our materials retain their mechanical and bioinstructive properties. Taken together, we present a strategy to leverage the secreted matrix, via surface physicochemical properties, for control of ECM-mediated signals toward enhancing biological responses critical for long-term stability and integration of percutaneous medical devices.

The extracellular matrix directs stem cell function through a complex choreography of biomacromolecular interactions in a tissue-dependent manner. Far from static, this hierarchical milieu of biochemical and biophysical cues presented within the native cellular niche is both spatially complex and ever changing. As these pericellular reconfigurations are vital for tissue morphogenesis, disease regulation, and healing, in vitro culture platforms that recapitulate such dynamic environmental phenomena would be invaluable for fundamental studies in stem cell biology, as well as in the eventual engineering of functional human tissue. In this talk, I will discuss some of our group’s recent success in reversibly modifying both the chemical and physical aspects of synthetic cell culture platforms with user-defined spatiotemporal control, emphasizing new material strategies that are genetically encoded. Results will highlight our ability to modulate intricate cellular behavior including stem cell differentiation, protein secretion, and cell-cell interactions in 4D.

Various functionalized polythiophenes, particularly poly(3,4-ethylenedioxythophene) (PEDOT), have been investigated as tissue interfacing materials in medical devices and implants. Polythiophenes have the ability to communicate both electronically and ionically; thus, allowing it to communicate both with implanted electronic devices and electrolytically-active organs. They possess mechanical properties intermediate to hard inorganic electronics and soft wet tissue. Additionally, they are mechanically and chemically stable. These features make PEDOTs prime candidates for neural regeneration applications. However, despite the continued interest in PEDOT, there are still many unanswered questions. A major challenge is the relative insolubility of PEDOTs in most major solvents which prevents an accurate analysis of the degree of polymerization.
PEDOT and its copolymers, including the polymer formed from the carboxyl-substituted monomer P(EDOT-acid), have received specific attention because of their chemical stability and excellent charge transport properties. We have been investigating the use of vibrational spectroscopic techniques along with Gaussian molecular simulations to obtain a better understanding of the polymerization process. This complements ongoing research in our laboratory using in-situ Transmission Electron Microscopy (TEM) and hot stage optical microscopy (OM).
We were able to collect a Gel Permeation Chromatogram of P(EDOT-acid) thus allowing us for the first time to associate the vibrational spectra of a polymer with its molecular weight. Complimenting this with Density Functional Theory calculations and experimental vibrational spectroscopy (Raman and FTIR) data for various oligomers we are able to develop a deeper understanding about the chemistry and the material properties of these materials. We are examining the influence of chain length and conformation on the experimental and simulated vibrational spectra of PEDOT and P(EDOT-acid) oligomers. We have identified characteristic peaks associated with the reactive hydrogens on the thiophene ring that allow us to monitor the process of polymerization. We have specifically studied the vibrational modes associated with the reactive protons and conjugation of the thiophene network. The 3110 cm^-1 thiophene C-H stretch peak is of particular interest, since it decreases with intensity as the monomers react. In addition, we have monitored the peak at 1422 cm^—1 associated with the C=C stretch and the peaks at 835-1184 cm^-1 associated with backbone conjugation. The peaks at 1540 cm^-1 and 1570 cm^-1 are associated with the quinoidal structure of the PEDOT. These clues provide insight into the changes occurring during the polymerization reaction. By correlating these spectroscopic results with data using microscopy, we are developing a deeper understanding of the thiophene electropolymerization reaction.

Heart disease is the leading cause of mortality across the world and myocardial infarctions (MIs) are individually linked to at least 50% of deaths. During MI, blood supply to cardiac tissue is compromised, initiating a tissue remodeling response, which includes the loss of contractile cardiomyocytes and the replacement of healthy tissue with non-contractile fibrotic tissue. One goal in the treatment of MI is the replacement of these lost cardiomyocytes to improve cardiac function; however, cell-based therapies have not been successful to do this. An alternative to cell delivery is to promote endogenous cardiomyocyte proliferation using growth factors, small molecules, and gene transfer. Specifically, microRNA-based therapies that target cardiomyocyte proliferation have great potential for the treatment of MI, including the miR-302/367 cluster.

We developed an injectable hyaluronic acid (HA) hydrogel for the local and sustained delivery of miR-302 mimics to the heart. The hydrogel is based on the assembly of HA through modification with host-guest chemical groups, namely cyclodextrin (host) and adamantane (guest). These modifications permit the fabrication of shear-thinning and self-healing hydrogels that can be injected into cardiac tissue. The miR-302 mimics released from hydrogels in vitro over a week and promoted cardiomyocyte proliferation and a single injection of the hydrogel in the mouse heart led to local and sustained cardiomyocyte proliferation for two weeks. After MI, miR-302 hydrogel delivery caused local clonal proliferation and increased cardiomyocyte numbers in the border zone of Confetti mice. Further, the hydrogel delivered miR-302 decreased cardiac end-diastolic (39%) and end-systolic (50%) volumes and improved ejection fraction (32%) and fractional shortening (64%) four weeks after MI and injection, compared to controls. This is quite promising towards the development of new therapies in cardiac regeneration and we are currently investigating other delivered therapeutics, such as extracellular vesicles.

To better understand the action of molecules on tissues, in vitro models are needed. However, current models are limited in the engineering of functional tissue models with requisite cell densities and heterogeneity to appropriately model cell and tissue behaviors, such as cardiac tissue after MI. To address this, we developed a new bioprinting approach to transfer cellular spheroids into self-healing support hydrogels at high resolution, enabling their patterning and fusion into high-cell density microtissues of prescribed spatial organization. We bioprinted induced pluripotent stem cell-derived cardiac microtissue models with spatially controlled cardiomyocyte and fibroblast cell ratios to replicate the structural and functional features of scarred cardiac tissue that arise following MI, including reduced contractility and irregular electrical activity. The bioprinted in vitro model was then combined with functional readouts to probe how various pro-regenerative microRNA treatment regimes influenced tissue regeneration and recovery of function as a result of cardiomyocyte proliferation. This method is useful for a range of biomedical applications, including the development of precision models to mimic diseases and for the screening of drugs, particularly where high cell densities and heterogeneity are important.

Cell proliferation and osteogenic differentiation of mesenchymal stem cells were evaluated in contact with biodegradable and bioactive ceramic scaffolds. These structures were generated by 3D printing with Schwarz D geometry based on periodic minimum surfaces (TPMS). For its printing, composition by weight of the ceramic paste was used was 66.5% calcium phosphate β-TCP or 66.5% calcium phosphate with Magnesium β-TCP / Mg, 28.5% Bioglass Borate (BGBS ), 3% Atapulgite and 2% of water. The osteogenic differentiation of mesenchymal stem cells (hMSC) from human adipose tissue (ADSCs) was performed. The scaffolds were characterized by X-ray diffraction (XRD) and infrared spectroscopy (FTIR), morphology, porosity and interconnectivity of the pores by scanning electron microscopy (SEM), degradability was tested by ISO 10993-14 standard, in vitro bioactivity by means of immersion tests in SBF by Kokubo test in different time intervals, cell proliferation and osteogenic differentiation by means of alkaline phosphatase activity.
The structural results showed calcium phosphate associated with its respective crystalline phases (β –TCP) and (β –TCP)/ Mg, in addition to the amorphous character of Bioglass Borate (BGBS). Calcium phosphate scaffold with Magnesium (β –TCP)/Mg showed better degradability and bioactivity compared to the (β –TCP) scaffold. Both scaffolds showed porosity and interconnectivity of pores. As for the mesenchymal stem cells from adipose tissue, they showed good adhesion and cell proliferation in contact with the scaffolds, being the scaffolds doped with Mg a better promoter of cell proliferation, in addition to this; they do not generate any cytotoxic effect in the cells, and finally the differentiation to an osteogenic environment was verified by means of alkaline phosphatase activity. As a result of this study, the 3D printed scaffolds showed good performance on cells proliferation of adipose tissue derived mesenchymal stem cells and the influence of magnesium is crucial for the development of osteogenic differentiation

Injectable materials that can conform to the shape of a desired space are used in a variety of fields including medicine. The ability to fill a tissue defect with an injectable material can be used for example to deliver drugs, augment tissue volume, or promote repair of an injury. This talk will explore the development of injectable materials that are based on assembled particle building blocks, for tissue repair. We find that using microparticle building blocks to build the scaffold generates a porous network by the space left behind between adjacent building blocks. Due to the injecability of this microporous material we have explored its wide applicability to tissue repair applicatins ranging from skin to brain wounds. In this talk, I will describe how MAP scaffolds can modulate the wound healing immune response and lead to regenerative wound healing.

Epithelial tissues contain three-dimensional (3D) microstructures that guide cell self-organization at the tissue level. In the small intestine, crypts and finger-like villi microstructures improve its absorbance function, provide specific microenvironments and compartmentalize cell types [1–3]. Despite its physiological relevance, tissue architecture and multicellular population are neglected in the standard in vitro models, thus compromising their predictive capabilities [4]. Our efforts in addressing these shortcomings by including key elements to mimic the native tissue in vitro will be discussed in this talk. First, this will include strategies to promote cell’s self-organization capabilities giving rise to crypt-villus domains on 2D monolayers [5], and strategies to engineer cell spatial positioning through micropatterning. Then, our approach to include the 3D architecture of the tissue will be addressed. In here, light-based biofabrication techniques to produce 3D villus-like structures [6,7] will be discussed. Finally, I will introduce our biofabrication proposal to produce tissue engineered models that include the epithelial and the stromal compartments [8]. Improving the prediction capabilities of cell-based assays is a growing strategy to lead to more efficient drug development processes. As 2D-based systems are showing their limits, new 3D strategies are gaining acceptance among the scientific community [9]. Our approaches aim to further accelerate this trend by providing feasible strategies to routinely incorporate 3D multicellular structures at the tissue level in cell culture systems.

[1] G.J. Tortora, B.H. Derrickson, Principles of Anatomy and Physiology, 14th Ed., Wiley Global Education (2014).
[2] M.N. Marsh, J.A. Swift, Gut, 10 940–949 (1969).
[3] P. Kelly, I. Menzies, R. Crane, I. Zulu, C. Nickols, R. Feakins, J. Mwansa, V. Mudenda, M. Katubulushi, S. Greenwald, M. Farthing, Am. J. Trop. Med. Hyg. 70, 412–419 (2004)
[4] Torras, N., García-Díaz, M., Fernández-Majada, V., Martínez, E. Front. Bioeng. Biotech. 6, 197 (2018)
[5] Altay, G.; Larrañaga, E.; Tosi, S.; Barriga, F.M.; Batlle, E.; Fernandez-Majada, V.; Martinez, E., Scientific Reports 9(1), 10140 (2019).
[6] Castaño, AG; Garcia-Diaz, M; Torras, N; Altay, G; Comelles, J; Martinez, E. Biofabrication 11(2) 025007 (2019).
[7] Altay, G.; Tosi, S.; García-Díaz, M.; Martínez, E. Front. Bioeng. and Biotech. 8, 294 (2020).
[8] Vila, A., Torras, N., Castaño, A.G., García-Díaz, M., Comelles, J., Pérez-Berezo, T., Corregidor, C., Castaño, Ó., Engel, E., Fernández-Majada, V., Martínez, E. Biofabrication 12 (2), 025008, (2020).
[9] Martínez, E.; St-Pierre JF.; Variola, F. Advances in Physics:X, 4:1, 1622451 (2019).

The morphogenetic patterning that generates three-dimensional (3D) tissues requires dynamic concerted rearrangements of individual cells with respect to each other. We have developed microfluidic approaches to investigate the mechanical forces and downstream signaling responsible for generating the airways of the lung. I will discuss how we combine these experimental techniques with computational models to uncover the physical forces that drive development of complex epithelial geometries. I will also describe efforts to uncover and actuate the different physical mechanisms used to build the airways in lungs from birds, mammals, and reptiles.

Spatially controlled arrangement of bacterial adhesion and microcolonies (clusters of cells) has the great potential to unveil the dynamics of multicellular systems and especially complex biological phenomena such as cell-cell communication¹. We present here a simple Au/SiO₂ device to spatially control bacterial cluster formation so that the basic biofilm framework can be studied in detail. Gold (Au) surfaces are biocompatible, chemically inert, and commonly used in diverse biomedical applications. On the other hand, thiol groups, which strongly interact with Au, are key functional molecules involved in adhesion mechanisms of several bacteria², including a plant pathogen Xylella fastidiosa.

In this work, Au patterned SiO₂ substrates have been prepared with direct laser writing lithography for selective bacterial adhesion of X. fastidiosa. The bacterial adhesion and aggregation were specifically tuned with well-defined geometry of Au array and different disk spacings. Ordered nucleated clusters on the Au patterned samples allowed us an extensive analysis for cell length populations with different growth time that represent unambiguous length distribution of typical cells (~3-4 µm) and filamentous cells (>9 µm) of X. fastidiosa³. Two filamentous cell populations are observed which relate well to the geometric distances between first and second neighbors in the disk array. Further, Au arrays with smaller spacings and closely organized clusters present larger fractions of filamented cells, when compared to Au arrays with larger spacings. We readily observed adjacent bacterial clusters interconnected by filamentous cells which predominantly depend on cluster sizes and disk distances. Upon continued growth, the clusters were interconnected by multiple filamentous cells that facilitate the macro-scale biofilm architecture of X. fastidiosa³. The results indirectly confirm quorum sensing based chemical signaling involved in the formation of filamentous cells associated with bacterial clusters of X. fastidiosa. The present approach based on Au patterned arrays is not only promising to understand various complex phenomenon of multicellular assembly but also offers new directions to engineering biological system.

References:
1. Chen, F, et al. Bacterial photolithography: patterning Escherichia coli biofilms with high spatial control using photocleavable adhesion molecules. Adv. Biosyst. 3, 1800269 (2019).
2. Newton, G. L. et al. Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J. Bacteriol. 178, 1990–1995 (1996).
3. Janissen, R. et al. Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation. Sci. Rep. 5, 9856 (2015).

Current paradigms for the treatment of autoimmune diseases (e.g. rheumatoid arthritis [RA]) are woefully inadequate, often missing the mark on desired physiological responses and not targeting the root cause of the disease. Predictably, novel approaches to re-establish immune homeostasis in patients afflicted by autoimmune conditions are now under intense investigation. Notably, we are developing an array of multifunctional, biomaterial-based ‘anti-vaccines’ that can be easily administered to remediate some of the prevalent autoimmune diseases. In this talk, I will focus on two particulate systems currently under development in my lab, which attempt to control critical cellular and humoral mediators that engender conditions such as RA and autoimmune autism. Previously, our group demonstrated that subcutaneous injection of a dual-sized, biodegradable PLGA MP regulatory vaccine (REGvac), comprising two ∼1 μm phagocytosable MPs (Vit D3 and collagen-II (coll-II)-loaded MPs) and two ∼30 μm non-phagocytosable MPs (TGF-β1-loaded MPs and GM-CSF-loaded MPs), resulted in remission of the characteristic symptoms of RA. In this iteration, we used a singly encapsulated formulation of the REGvac, meaning each microparticle had one agent. To increase the translatability of this system we re-engineered this REGvac and applied it to a more rigorous model of RA, the collagen-induced arthritis/citrullinated fibrinogen induced arthritis model (CIA/FIA). Further, we co-encapsulated each factor previously described in the REGvac into one small microparticle containing coll-II, citrullinated fibrinogen (cit fib) and Vit D3, and one large particle containing TGF-β1 and GM-CSF (Duo REGvac). Additionally, we tuned the release of each of the factors, fabricating the small MP from an increased molecular weight PLGA polymer and the large MP from a lower molecular weight PLGA polymer. This co-encapsulated formulation allows for the spatiotemporal control over delivery of each factor. The quick release of GM-CSF and TGF-β1 allows for rapid infiltration and recruitment of DCs to the injection site hours after the injection. The slower release of encapsulated factors from the small MP allows for a higher therapeutic payload to be delivered inside the cell. Moreover, Vit D3 co-encapsulation with antigen allows for tolerogenic cues to be delivered directly with antigen in a spatiotemporal context. We describe our efforts to improve the REGvac system and assess its ability to intervene in late stage RA in the CIA/FIA model. We are also heavily invested in developing a nanoparticle-based prophylactic for Maternal Autoantibody-related (MAR) autism. We previously demonstrated that LDH B peptide-functionalized citrate-coated dextran iron oxide nanoparticles (DIONPs) [Systems for Nanoparticle-based Autoantibody Reception and Entrapment (SNARE)] are capable of capturing LDH B autoantibodies from mothers of children with MAR autism in vitro. Further, we found that SNAREs intravenously injected in C57BL/6j pregnant dams demonstrated slow clearance from the blood (24 hours) and deposited in the liver and the lungs confirmed via histology and magnetic resonance imaging (MRI) analyses. Here, we investigated the ability of SNAREs to capture MAR autoantibodies in immunized pregnant dams. Remarkably, a single injection of 100 mg/kg SNAREs led to a 62% reduction of LDH B autoantibody maternal blood titer within 4 hours and 54% within 24 hours of injection with dampened titer levels for 5 days thereafter. The ability of SNAREs to capture MAR LDH B autoantibodies in vivo sets the basis for investigating its efficacy to prevent disease in a MAR autism mouse model.

Introduction: Therapeutic angiogenesis is a promising treatment for ischemic tissue disorders and for vascularizing biomaterials in regenerative medicine. Macrophages are important regulators of angiogenesis, playing distinct roles in different stages of angiogenesis due to their diverse phenotypes. Interleukin 4 (IL-4)-stimulated macrophages (M2) play a role in the later stage of angiogenesis, likely by stabilizing and supporting new blood vessels, although their precise functions are not known. Biomaterials that control macrophages may be advantageous compared to strategies targeting other cell types because macrophages regulate many different cell types involved in angiogenesis and tissue repair. However, macrophages are highly plastic cells that can easily change their phenotype upon encountering different stimuli at a wound site. Consequently, a localized drug delivery system is necessary to release polarizing signals to macrophages in a sustained manner to maintain and promote phenotype. As a potent stimulator of the pro-angiogenic M2 phenotype, IL-4 was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles for sustained release. To test their effects on macrophage phenotype and angiogenesis, the microparticles were co-delivered with fluorescent macrophages into a murine model of hindlimb ischemia three days post-surgery.

Materials and Methods: Using double emulsion, IL-4 was encapsulated into PLGA microparticles (30-40 µm). Green fluorescent protein-labeled (GFP+) murine bone marrow derived macrophages were gathered and cultured to an M2 phenotype via stimulation with IL-4. These macrophages and microparticles were injected intramuscularly into the calf of a hindlimb ischemia murine model 3 days post injury. Mice were sacrificed two days later, and their calves harvested for flow cytometry analysis and immunohistochemistry (IHC). Using collagenase to digest the muscle, a single cell solution of the calf was stained with multiple macrophage phenotype markers (F4/80, CD206, ARG1, CD38, CD86) and Live/ Dead (L/D) markers. Separately, the solution was also stained with F4/80 and endothelial cell marker CD31. Flow data were analyzed using tSNE dimensionality reduction and Phenograph and FlowSOM clustering algorithms. Calf muscle sections were stained with DAPI and CD31, and images were analyzed with CellProfiler for CD31+ structures. All animal studies were approved by the Drexel Institutional Animal Use and Care Committee.

Results and Discussion: The number of GFP- macrophages was very high while the number of recovered GFP+ macrophages was exceedingly low, indicating that any effects of microparticles on angiogenesis and macrophage phenotype was caused by host macrophages. IL-4 releasing microparticles caused macrophages to take on a complex phenotypic profile. IL-4 affected macrophages were highest in clusters expressing both M1 and M2 markers. Additionally, treatment with IL-4 releasing microparticles increased the amount of endothelial cells (F4/80-CD31+), suggesting angiogenesis. Analysis of CD31+ structures showed that IL-4-releasing microparticles increased the density of vessels of 5-18 µm in diameter, while small capillaries (2-5 µm diameter) were unaffected. IL-4 release from the microparticles may have cause the M2 macrophages to help support recently created blood vessels, thereby increasing the number of larger structures.

Conclusions: This study shows that IL-4 releasing microparticles can control macrophage phenotype to support the development of blood vessels in vivo.

Lower limb amputations are a major source of morbidity and mortality throughout the world. They principally result from a trifecta of atherosclerosis, limb ischemia, and the formation of non-healing chronic wounds. At the core of these afflictions is dysregulated behavior of macrophages, which are critical regulators of homeostatic tissue turnover and the healing of injured tissues. Therefore, biomaterials that can correct dysfunctional macrophage behavior hold promise for promoting wound healing in patients at risk for amputation.
In successful wound healing, macrophages transition from a population that is largely pro-inflammatory (M1) to a heterogeneous population of macrophages associated with the resolution of inflammation and wound healing (M2), although the extent of the diversity of this M2 population is not known. Because M1 macrophages normally switch to M2, and because chronic wounds are characterized by deficiencies in M1 as well as M2 activation, we investigated the consequences of prior M1 polarization on subsequent M2 polarization. M2 macrophages that were derived from M1 macrophages exhibited a distinct gene, protein, and functional signature compared to M2 macrophages derived directly from unactivated macrophages, suggesting that M1 polarization is an important regulator of M2 activation. These findings, in combination with our previous work describing important roles for M1 macrophages in initiating angiogenesis, indicate that immunomodulatory biomaterials should allow for a robust M1 phase prior to mediating a transition to M2. To investigate this concept, we developed PLGA microparticles that released IL4, a main cytokine responsible for promoting a key M2 subtype. When administered locally to the site of injury at 3 days following injury in a murine hindlimb ischemia model, these microparticles increased the number of larger blood vessels without affecting the number of small blood vessels. These findings support a potential role for M2 macrophages in the maturation stage of angiogenesis. Then, because patients at risk for amputation may suffer from impaired immune cell trafficking, we designed a biomaterial-mediated macrophage cell therapy strategy to control the phenotype of macrophages following their administration, a long-standing problem in macrophage cell therapy due to the inherent plasticity of these cells. In a proof-of-concept study, the anti-inflammatory drug dexamethasone was incorporated into PLGA microparticles and loaded into human peripheral blood-derived monocytes via phagocytosis. The microparticles were retained intracellularly for several weeks in vitro and they did not interfere with monocyte-to-macrophage differentiation nor subsequent phagocytosis of tissue debris or bacteria. After 7 days in pro-inflammatory media, macrophages containing dexamethasone-loaded microparticles decreased expression of pro-inflammatory genes, increased expression of M2 genes associated with dexamethasone treatment, and decreased secretion of the potent pro-inflammatory cytokine tumor necrosis factor-alpha (TNFa), even in the presence of pro-inflammatory stimuli.
Collectively, these studies highlight the importance of temporal control over macrophage phenotype, and suggest that immunomodulatory biomaterials that target macrophages may be effective in treatments designed to rescue angiogenesis and wound repair.

Cancer is referred to as a group of diseases in which abnormal growth of cells interferes with the function of vital organs and finally stops the body from functioning. Cancer patients have higher chances of survival if their cancer diseases are diagnosed in the early stage of development. Detection of cancer via cancer biomarkers is one of the well-established ways for early diagnosis of cancer diseases. Cancer biomarker is referred to as any substance that can indicate the presence of cancer in a body. Thus far, many cancer biomarkers have been discovered, which can be collected from tissue, blood, and urine. Among the cancer biomarkers detectable from blood, platelet-derived growth factor-BB (PDGF-BB) has been associated with development and metastasis of various malignant tumors in vital organs including breast, brain, ovarian, pancreas, and liver. A feasible means for early detection of cancers is developing point-of-care biosensors based on DNA aptamers, which are referred to as aptasensors. Various advanced technologies have been investigated for developing cancer biomarker aptasensors, including optical, piezoelectric, and electrochemical based techniques. Among the developed technologies, label-free electrochemical aptasensors are highly compelling since they can reduce the cost and complications of sample preparation, and the electrochemical cells can be efficiently miniaturized and integrated with available lab-on-chips and MEMS technologies. Nevertheless, the sensing performance of the label-free electrochemical cancer aptasensors, including selectivity, limit of detection, and dynamic range requires further enhancements to be at similar levels to clinical testing. In the light of the importance of PDGF-BB detection and demanded enhancements for label-free electrochemical aptasensors, we have developed label-free electrochemical aptasensors based on photoresist derived carbon microelectrodes. The active electrode of this aptasensor was synthesized via well-established C-MEMS (carbon microelectromechanical systems) fabrication technology. C-MEMS platforms have unique features such as high resolution, low background capacitance, high stability when they exposed to different physical/chemical treatments, biocompatibility, and good electrical conductivity. The applied C-MEMS fabrication in this study includes photopatterning of SU-8 25 negative photoresist-based microelectrodes and carbonization of the developed microelectrodes at high temperatures and oxygen-free tube furnaces. The carbonized microelectrodes were functionalized utilizing oxygen plasma oxidation pretreatment to introduce carboxyl groups to the surfaces of the carbon microelectrodes. The PDGF-BB affinity aptamers were covalently immobilized via amid binding of amino-tag terminated aptamers and carboxyl groups covered carbon surfaces. The Fourier-transform infrared spectroscopy (FTIR) confirmed the successful carboxyl group functionalization of the C-MEMS microelectrodes and covalent immobilization of affinity aptamers via amide biding. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for characterizing the C-MEMS based aptasensors in different stages of development and sensing performances. The turn-on sensing strategy was implemented via measuring the charge transfer resistance (R_CT) from EIS Nyquist plots, which yielded to wide sensing linear range of 0.005 – 50 nM with a high sensitivity of 14.82 × 10³ Ω.(Log (M))^-1 and low limit of detection of 1.9 pM (S/N=3). The turn-off sensing strategy was applied via measuring capacitance from CV curves, which conceded to a wide linear response range of 0.01 – 50 nM with a high sensitivity of 29.97 mF.cm^-2.(Log (M))^-1 and low limit of detection of 7 pM (S/N=3) toward the PDGF-BB. The developed label-free electrochemical aptasensor exhibited good selectivity, stability, and repeatability, which is highly promising for point-of-care cancer diagnosis technologies.

In situ gelation of hydrogels via near-infrared (NIR) light can implant drug reservoirs at deep tumor tissues with minimal invasion. Ultraviolet or visible light is often used for photocrosslinking but has limited application for deep tissue therapy due to the shallow penetration depths. NIR is of particular interest because of the therapeutic window where light has its maximum depth of penetration in tissue. However, NIR-triggered formation of hydrogels is rarely reported due to limited tools of NIR photochemistry. In this study, we demonstrate in situ gelation by combining with a NIR-responsive photothermal agent, molybdenum disulfide (MoS₂), with a temperature-responsive phase transition polymer, poly(N-isopropyl acrylamide) (PNIPAm). Molybdenum is thiophilic, and sulfur has a strong affinity toward forming disulfide bonds, together they offer facile binding centers for thiolated molecules. Therefore, thiolated-PNIPAm is synthesized to form crosslinks with defect-rich MoS₂ nanosheets. Moreover, dynamic polymer-nanoparticle interactions can be leveraged to control the formation of hydrogels spatiotemporally. With the increase in temperature by the photothermal effect of MoS₂, the hydrophobic interactions of MoS₂ nanosheets and PNIPAm chains are strengthened, and thus facilitate the gelation. Further, the combinatorial chemo-photothermal therapy of the MoS₂ hydrogels showed significant inhibition of cancer cells, indicating its effective role in chemotherapy. Overall, the hazardous-free gelling behavior of the MoS₂ hydrogels has strong potential for a range of biomedical applications, including phototherapy, 3D printing, and therapeutic delivery.

Neurological disorders such as Epilepsy and Parkinson’s (PD) are associated with the alteration of neural signals. Current techniques to modulate neural activity such as implantable microelectronic devices, pharmacological agents or optical stimulation, are nonspecific to cell-type and damaging to biological tissues ^1–3. Recently, magnetic hyperthermia has been proposed in neuromodulation as a wireless technology that could reach deep into brain due to the weak magnetic properties and low electrical conductivity of brain tissues ⁴. This technology is highly dependent of the hysteresis power loss of magnetic nanoparticles (MNPs) under an applied alternating magnetic field (AMF). Thus, maximizing magnetic heating of MNPs will allow a more efficient energy conversion with lower cytotoxic effects.
Here, we investigate the magnetic heating properties of core and core-shell cubic MNPs for neuromodulation applications. Core MNPs are composed of Fe and Zn (Fe₂O₄Zn) at different molar compositions were synthesized to obtain nanoparticles (NPs) with different sizes (28 to 89 nm). It was observed that Fe₂O₄Zn NPs coercivity (H_c) increases with NPs size up to a saturation point due to a change in the multi-magnetic domains. On the other hand, it was found that the hybridization of Fe₂O₄Zn NPs with a Fe-Co shell (CoFe₂O₄) allowed us to control their H_c. Hybridization with Fe-Co shell can enhance Fe₂O₄Zn NPs H_c from 13 to 170 times depending on the shell molar composition. After optimization, Fe₂O₄Zn NPs (28, 35 and 55 nm) and Fe₂O₄Zn-CoFe₂O₄ NPs (with different Co concentration) were functionalized with polyacrylic acid (PAA) to stabilize them in biological fluids. Calorimetry studies were done to evaluate the specific power loss (SLP) of these MNPs when exposed to an AMF. SLP measurements were consistent with H_c values (dependent on size and Co concentration). Electrokinetic potential and polydispersity of MNPs in different biological fluids was assed using dynamic light scattering, and reveling high colloidal stability in phosphate buffer and media with high concentration of proteins. The cytotoxicity of these MNPs with and without AMFs was evaluated with a MTT Assay, where it was observed that MNPs functionalized with PAA (MNPs/PAA) with or without AMF cause no cytotoxic effects.
Finally, we tested our MNPs/PAA with the highest SLP and H_c to evoke neural activity in neurons expressing the heat-sensitive ion channel TRPV1. We isolated primary hippocampal neurons from new-born rats. After 1 week of culturing, we label the neurons with the Calcium indicator Fluo-4 in order to monitor neural activity through Calcium influx. Hippocampal neurons were exposed to cubic MNPs (with and without shell) and AMFs. Neural activity was recorded in real time using a calcium fluorescence imaging. We were able to evoke neural activity with all the MNPs tested. The heat dissipated by our MNPs was enough to trigger the response of the heat-sensitive transmembrane ion channels expressed on hippocampal neurons. The most remarkable achievement from this preliminary stimulation results was our ability to excite neural activity using 50X and 100X less the amount of MNPs in comparison with previously reported work ⁴. This finding corroborates our hypothesis for maximizing hysteretic losses by magnetic anisotropy. Moreover, the optimized MNPs phase transfer procedure allowed us to reduce the possibility of agglomeration and bulk heating.
References

1. Polikov, V. S., et al., J. Neurosci. Methods 148, 1–18 (2005).
2. Strazielle, N, et al., Mol. Pharm. 10, 1473–1491 (2013).
3. Roet, M. et al., Prog. Neurobiol. 177, 1–14 (2019).
4. Chen, R., et al., Science 347, 1477–1480 (2015).

Cellular grafts for the regeneration of cartilage and bone have been engineered using a variety of cell sources, scaffolds and manufacturing systems. Clinical implementation of some of these approaches by the own group has led to promising outcome results (Fulco+, Lancet 2014; Mumme+, Lancet 2016; Saxer+, Stem Cells 2016), but is still associated with manufacturing and robustness challenges. Alternative strategies have been conceived to gain repeatability of processes, by delivering signals capable to recapitulate developmental events, with proofs of principle in the context of bone and cartilage regeneration (Scotti+, PNAS 2013; Occhetta+, PNAS 2018). Along this line, it was identified that regeneration-inductive signals may not require living cells to be efficiently delivered, but could be encoded in cell-laid and subsequently devitalized extracellular matrices (Bourgine+, PNAS 2014; Bourgine+, Adv Funct Mater 2017). The combination of multiple cytokines and morphogens, physiologically presented by a set of extracellular matrix molecules, would synergistically potentiate their effects. This new class of off-the-shelf biomaterials, which would function by offering the primordial template for the development of the target tissue, may offer an alternative strategy to synthetic matrices. These cell-free extracellular matrices, as germs for de novo tissue development, could be generated based on highly standardized processes, thanks to the use of cell lines and bioreactor-based systems, and at the same time customized to address specific disease stages and patient profiles, in a perspective of personalized medicine (Haumer+, Adv Drug Del Rev 2017).

Introduction: Damage to the pleural tissue lining the lungs can occur in a multitude of ways, resulting in fluid leak into the pleural cavity, which, left untreated, can result in mortality. Historically such cases were treated via pleurodesis. Recent developments of lung tissue sealants serve as alternatives, but few options are available. While popular sealant tests can deliver qualitative pass/fail data, there is lack of quantitative in vivo methods to characterize sealing capabilities of these materials, particularly for pleural applications. In addition, commercial products have failed due to poor design and administration. Herein, we describe our development of a powder sealant capable of adhering to tissue and crosslinking to form an elastic hydrogel, circumventing limitations of current tissue sealants.

Experimental methods: Methacrylated alginate (AMA) was synthesized and characterized via ¹H-NMR spectroscopy. To enable gelation, visible green light crosslinkers were incorporated into the polymer solution. A sodium ion exchange was performed, the solution was lyophilized, and the product was processed into a powder. To investigate physical material properties, gelation kinetics and the viscosities of reconstituted solutions were collected at 37C, via an oscillatory time sweep at 10% radial strain and 1 Hz during exposure to green light. Quantitative in vitro experiments: dynamic burst pressure experiments were conducted using a custom-built burst pressure testing device, using a modified ASTM F2392 standard. Quantitative in vivo experiments: a murine model was developed in which we controlled respiratory dynamics to test sealant mechanics. Mice were anesthetized with pentobarbital sodium, tracheostomized, and paralized using pancuronium. Body temperature was maintained by keeping the animal under a lamp. Mice were connected to a computer-controlled small animal mechanical ventilator (flexiVent, Scireq) and ventilated. A series of deep inflation maneuvers (Total Lung Capacity and Trapezoidal deep inflation waveform) and a composite signal comprising of mutually prime frequencies ranging from 0-20 Hz were performed to establish baseline for the animal prior to injury. The thoracic cavity of the animal was opened and lungs were punctured, followed by another series of baseline measurements to calibrate testing mechanics and verify injury. The injury was treated by the application of the AMA powder and tests were repeated to compare lung mechanics pre-injury and post-repair, allowing for real-time pressure data collection of lung mechanics during sealant testing. After testing, animals underwent euthanasia.

Results: After acute traumatic injury using a murine model, there was a loss in pressure, confirming the injury model. Successful post-repair (i.e., application of the AMA powder, hydration and crosslinking) tests indicated that the sealant maintained physiologically-relevant lung pressure and returned lung capacity to pre-injury values, and that the sealant did not fail. A failure in the patch would result in a failure to hold pressure. Initial results show physiologically relevant burst pressure values (i.e. 30 cmH2O) for the powder sealant collected during in vitro testing.

Conclusions: The data indicates efficacy of a novel powder-based elastic sealant for recapitulating lung mechanics post injury. During an in vitro burst pressure experiment, the powder formed an crosslinked sealant capable of maintaining physiological pressure before failure. The powder-based sealant has the advantages of adhering to tissue upon application in situ and subsequent hydration, remaining in place while crosslinking via light exposure took place. Future work will explore long term in vivo studies to investigate lung tissue healing after repair with the novel powder sealant material.

Acknowledgements: This work was funded in part by NIH R01EB020964 (Oldinski) and the University of Vermont.
Disclosure of Interest: None Declared.

To enable the understanding and repair of complex biological systems such as the brain, we are creating novel optical tools that enable molecular-resolution maps of large scale systems, as well as technologies for observing and controlling high-speed physiological dynamics in such systems. First, we have developed a method for imaging large 3-D specimens with nanoscale precision, by embedding them in a swellable polymer, homogenizing their mechanical properties, and exposing them to water – which causes them to expand isotropically manyfold. This method, which we call expansion microscopy (ExM), enables scalable, inexpensive diffraction-limited microscopes to do large-volume nanoscopy, in a multiplexed fashion – important, for example, for brain mapping. Second, we have developed a set of genetically-encoded reagents, known as optogenetic tools, that when expressed in specific neurons, enable their electrical activities to be precisely driven or silenced in response to millisecond timescale pulses of light. Finally, we are developing novel reagents, such as fluorescent voltage indicators, and systems, such as novel microscope architectures, to enable the imaging of fast physiological processes in 3-D with millisecond precision. In this way we aim to enable the systematic mapping, control, and dynamical observation of complex biological systems like the brain.

Antimicrobial resistance to antibiotics (AMR) and drug-resistant cancer are two of the main concerns that the healthcare system is facing nowadays [1,2]. Current chemotherapy treatments and antibiotic-based therapies are becoming ineffective or have plenty of drawbacks that are increasing the prevalence of resistance in both hospital and community settings. Therefore, new alternatives are needed, and nanotechnology might offer a suitable answer.

Although different nanomaterials are being used as a method to fight pathogens and cancer, traditionally synthesized nanoparticles present drawbacks, such as the production of toxic by-products, increased cytotoxicity in the cell and lack of selectivity. Consequently, green nanotechnology appears as an alternative approach, which employs living organisms and biomolecules as raw materials in a cost-effective and environmentally-friendly approach [3]. Among all these methods, microbially-produced nanomaterials have presented a significant potential, rendering nanomaterials with a powerful antimicrobial activity that can be tailored to the infection.

With the objective to demonstrate the antimicrobial and biocompatibility efficacy of bacterial-produced nanoparticles in skin bacterial infections, top pathogenic strains found in cutaneous wounds were employed for the synthesis of metalloid selenium nanoparticles (SeNPs), including Methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. Once generated, the SeNPs were analyzed in terms of their physicochemical features and the composition was extensively studied. After purification, the SeNPs were used as antimicrobial and anticancer agents through colony counting unit and MTS assays, respectively, as well as characterized for the mechanisms of death associated to their activities. At the same time, low cytotoxicity was demonstrated over the same range of antimicrobial efficiency.

Consequently, microbiological agents were successfully used as a biofactories for the generation of SeNPs of different compositions with biomedical properties. Therefore, bacteria were presented as a suitable approach for the synthesis of nanomaterials in a green fashion, overcoming the limitations of traditional nanotechnology.
References
[1] Ventola, C. Lee. Pharmacy and Therapeutics 40.4 (2015): 277–283.
[2] Katz, Steven J. Journal of Oncology Practice 9.3 (2013): 114–115.
[3] De la Guardia, Miguel. BioImpacts: BI 4.1 (2014): 1–2.

A major barrier in tissue engineering is a lack of understanding of how to integrate vascular networks into 3D tissue constructs. Without perfusable vascular networks, large constructs containing living cells develop necrotic cores due to nutrient, oxygen, and waste diffusion limitations. To address this challenge, we have developed approaches to generate perfusable vascular networks that support 3D culture of assembled constructs, provide a means to study how to regulate vascular remodeling and angiogenesis, and improve the integration of such vasculature to host circulation in implanted constructs. We demonstrate a critical role for material properties such as stiffness or degradability, and also an important role for the organization of materials and cells to provide functional perfusable vasculature. Ultimately, these approaches will inform how we establish solutions to generate functional vascular beds for engineered tissues both in vitro and in vivo.

Endogenous endothelial progenitor cell (EPC) recruitment to sites of vascular injury is a multi-step process mediated by selectins, chemokines, and extracellular matrix (ECM) proteins. New vascular implants with surface-immobilized capture antibodies targeting circulating EPCs have shown clinical promise in accelerating the formation of neo-endothelium to cover the implant. The absence of subsequent adhesion-based signaling needed for EPC proliferation and differentiation could explain the lack of long-term improvement in vascular re-stenosis and overall vascular recovery. Antibodies can mimic the cell tethering effect of selectins but lack necessary EPC signaling effects introduced by cytokines and ECM proteins. We have previously shown that ECM-derived peptides, such as RGD, can promote the adhesion and enhance the clonal expansion of EPC-derived endothelial colony forming cells (ECFCs) via integrin based signaling. In this study, we developed a novel bifunctional surface modification strategy to produce surfaces that combine immobilized capture-antibodies and surface grafted ECM-derived peptides with controlled surface concentrations and ligand orientation. We hypothesize that this bifunctional strategy would further accelerate the re-endothelialization process while maximizing downstream endothelial function and vascular recovery via an integrin-dependent process.
Peripheral blood from 22 adult human donors was used to obtain donor-specific ECFCs. Aminated culture surfaces were modified by either the covalent conjugation of a customized RGD peptide, immobilization of vascular endothelial growth factor receptor-2 (VEGFR-2) antibody, or combining both biomolecules at different ratios using an optimized process. Unmodified surfaces were used as a negative control. Surface concentration of the peptide and antibody were analyzed using fluorescence microscopy and an enzyme-linked immunosorbent assay. First, the expanded ECFCs were seeded under static conditions and cell adhesion was studied using immunocytochemistry. ECFC interactions with the bifunctional surfaces were then interrogated under more physiologically relevant dynamic flow conditions in a microchannel perfusion loop at 1 dyn/cm² wall shear stress and analyzed through live cell imaging followed by immunocytochemistry.
Surfaces with immobilized anti-VEGFR-2 antibodies displayed significantly enhanced ECFC capture potential compared to RGD and unmodified controls as quantified by the number of adhered cells under both static and dynamic conditions. Average ECFC spreading and focal adhesion formation were significantly increased on RGD surfaces compared to the anti-VEGFR-2 and unmodified controls under both static and dynamic conditions. Total endothelial coverage and the total number of focal adhesions were maximized on the bifunctional surfaces combining the anti-VEGFR-2 and RGD due to a combined increase in both cell number and cell spreading. By increasing the peptide concentration on the bifunctional surfaces, ECFC spreading was increased without significantly affecting the number of adhered cells. These results suggest an additive (i.e. independent) effect of capture antibodies on ECFC tethering vs. RGD peptides on subsequent ECFC spreading and integrin-dependent cell signaling.
To our knowledge, this is the first study showcasing the ability of a bifunctional antibody/peptide surface modification strategy in promoting ECFC capture and adhesion. Our results also display a clear distinction between the mechanisms by which human-derived ECFCs react to immobilized capture antibodies and conjugated ECM-peptides. This strategy creates sequential steps of EPC trafficking to mimic endogenous processes of vascular healing which can potentially create an effective regenerative capacity for vascular implants.

Monitoring vascular permeability represents a significant challenge in materials science. The study of vascular permeabilization is of great interest to the scientific community in its relevance to extravasation during tumor growth, transport across the blood-brain barrier, etc. However, studying vascular permeability in vitro requires development of materials systems that not only support cell growth but also produce real-time data on cell behavior. Native vasculature consists of a tube with an interior lining of barrier-forming cells called endothelial cells. These cells selectively allow transport of different factors, of which small molecules and ions primarily pass through intercellular junctions. In this study, we developed a bioelectronic platform for vascular tissue engineering to actively monitor the integrity of the vascular membrane during culture.
Previously, we developed bioelectronic monitoring systems for gauging various cellular phenomena. These systems have frequently used conductive polymers, such as poly(ethylene dioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), which are biocompatible and ion-sensitive. These polymers can be designed for use as electrodes or transistors to monitor local ion flux. As cells deposit extracellular matrix (ECM) and form intercellular junctions, these structures inhibit ion flow, which can be sensed using conductive polymeric devices. These polymers can also be fabricated as porous scaffolds through ice templating to support cell growth.
To produce this vascular monitoring system, we constructed a bioelectronic device, which can simultaneously promote and monitor the development of a vascular membrane. This device was constructed using a porous, electroactive PEDOT:PSS scaffold to support cell culture with surrounding electrical architecture to monitor ion flux into the scaffolds. We produced tissue engineered vasculature by seeding fibroblasts onto the top of the scaffold. These cells were allowed to proliferate, producing a bed of ECM on top of and within the pores of the scaffold. Subsequently, vascular endothelial cells were deposited on top of this culture and allowed to proliferate. After a period of static culture, the devices were placed on an orbital shaker to mimic blood flow. During the following culture period, the devices were monitored using impedance spectroscopy to gauge the formation of a vascular membrane.
Over the course of the culture period, we observed a continuous increase in impedance, indicative of the deposition of ECM and the formation of vascular junctions. At the termination of culture, we found the scaffolds showed complete coverage and junctional formation across the entirety of the 6 mm scaffolds. We will now seek to apply biological factors that affect vascular permeability, such as TNF-α, to promote extravasation in the tissue engineered cultures allowing for determination of the efficacy of using this system to monitor vascular permeabilization. This system provides a platform for studying various physiological phenomena associated with transport through the vascular wall and highlights the capabilities of in vitro bioelectronic tissue engineering.

Introduction: Platelets play a role in hemostasis and are the first responders to arrest bleeding during an injury [1]. Therefore, any aberration in their function can cause bleeding or thrombus formation. Nanoparticles (NPs) when present in the bloodstream interact with the different components of blood and have been shown to modulate platelet functions [2,3]. However, these interactions are not well understood. In this study, we are using six formulations of PVA (polyvinyl alcohol) coated-SPIONs based on differences in molecular weight (M.W.) and charge of the polymer coating to study the effect caused by their presence on platelet function of activation and aggregation, and additionally, we have explored possible mechanisms to explain the observed effect.
Materials and Methods: Human platelet-rich plasma was obtained from healthy donor blood. Platelet activation and aggregation were assessed in the presence of PVA-SPIONs with 12 kDa and 31 kDa PVA, as well as positive, neutral, or negative surface charges, by using flow cytometry and light transmission aggregometry. The agonist adenosine diphosphate was used to induce platelet activation and aggregation. Platelets were incubated with up to 500 ug/mL PVA-SPIONs. ELISAs were used to quantify the presence of plasma proteins fibrinogen and von-Willebrand factor (vWF) in platelet-poor plasma incubated with SPIONs. Fibrinogen and vWF binding to platelet GPIIb-IIIa was also studied using flow cytometry, while their structural integrity with SPION incubation was assessed by UV-vis.
Results and Discussion: Platelets were activated in the presence of positively charged SPIONs at 500 ug/mL, irrespective of M.W. of PVA. A dose-dependent inhibitory effect was also observed on platelet aggregation when platelets were incubated with SPIONs. This antiplatelet effect was independent of the M.W. and surface charge of the PVA-SPIONs. Therefore, we tested three hypotheses to explain this antiplatelet effect. Based on the important role of plasma proteins fibrinogen and vWF in platelet aggregation and the formation of protein corona due to the adsorption of proteins on the surface of NPs [4], we tested if the proteins were significantly adsorbed on the surface of the NP, thus depleting them from the plasma for potential platelet aggregation. Although adsorption of proteins was observed, there was no significant decrease in the concentration of available proteins for platelet aggregation. Next, we tested if the NPs interfere with the normal process of fibrinogen and/or vWF binding to platelet GPIIb-IIIa. In agreement with our platelet activation study, there was an increase in binding of fibrinogen to the receptor GPIIb-IIIa when the PRP was incubated with positively charged SPIONs. In addition, there was no change in the binding efficiency for both fibrinogen and vWF with other SPION formulations. Lastly, we investigated whether the PVA-SPIONs would interfere with the protein structure, and thus with their role in platelet aggregation.
Conclusions: PVA-SPIONs induced an inhibitory effect on platelet aggregation regardless of their size and charge. Our results suggest that the inhibitory effect could be ascribed to a change in fibrinogen conformation, induced by the presence of these nanoparticles, thus impairing the platelet-platelet bridging ability of the protein.
Acknowledgments: This work has been supported by the George Washington University startup funds.
References:
[1] M. A. Packham, “Role of platelets in thrombosis and hemostasis,” Can. J. Physiol. Pharmacol., 72, 3, 278–284, 1994.
[2] S. Shrivastava, T. Bera, S. K. Singh, G. Singh, P. Ramachandrarao, D. Dash, “Characterization of antiplatelet properties of silver nanoparticles.,” ACS Nano, 3, 6, 1357–1364, 2009.
[3] U. Sakulkhu, M. Mahmoudi, L. Maurizi, J. Salaklang, H. Hofmann, “Protein corona composition of superparamagnetic iron oxide nanoparticles with various physico-chemical properties and coatings,” Sci. Rep., 4, 5020, 2014.

Native extracellular matrixes (ECM) acts as signaling repositories to regulate cell behavior and function. Installation of implantable medical devices disrupts these matrices and destroys spatiotemporal presentation and interaction of cells with microenvironmental cues. Regeneration of tissue niches is critical for long-term stability and integration of medical devices in patients. Here, we present a new, broadly-applicable physicochemical strategy for percutaneous devices to leverage secreted, nascent ECM for instructing epithelial cell behavior. We synthesized a set of photopolymerizable polymers with catechol and structurally-similar moieties, easily applied to percutaneous device surfaces like catheters, dental restorative materials, or orthopaedic implants, with systematically varied physicochemical properties but similar bulk mechanical properties. Tuning surface physicochemical properties, specifically charge and polarity, enabled us to tune keratinocyte hemidesmosome formation, adhesive structures that anchor cells to ECM, formation and downstream signaling. A series of inhibitor and knock-down experiments demonstrated the bioinstructive effect of our materials on elaborated ECM, laminin332 in particular, in an integrin-dependent manner. We propose our biostructure polymers direct cell responses via modulation of laminin332’s structure in the pericellular ECM. Finally, we challenged our engineered materials in a course of experiments, including ex vivo, meant to reflect the harsh environment experienced during clinical therapeutic use and showed our materials retain their mechanical and bioinstructive properties. Taken together, we present a strategy to leverage the secreted matrix, via surface physicochemical properties, for control of ECM-mediated signals toward enhancing biological responses critical for long-term stability and integration of percutaneous medical devices.

Introduction: Therapeutic angiogenesis is a promising treatment for ischemic tissue disorders and for vascularizing biomaterials in regenerative medicine. Macrophages are important regulators of angiogenesis, playing distinct roles in different stages of angiogenesis due to their diverse phenotypes. Interleukin 4 (IL-4)-stimulated macrophages (M2) play a role in the later stage of angiogenesis, likely by stabilizing and supporting new blood vessels, although their precise functions are not known. Biomaterials that control macrophages may be advantageous compared to strategies targeting other cell types because macrophages regulate many different cell types involved in angiogenesis and tissue repair. However, macrophages are highly plastic cells that can easily change their phenotype upon encountering different stimuli at a wound site. Consequently, a localized drug delivery system is necessary to release polarizing signals to macrophages in a sustained manner to maintain and promote phenotype. As a potent stimulator of the pro-angiogenic M2 phenotype, IL-4 was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles for sustained release. To test their effects on macrophage phenotype and angiogenesis, the microparticles were co-delivered with fluorescent macrophages into a murine model of hindlimb ischemia three days post-surgery.

Materials and Methods: Using double emulsion, IL-4 was encapsulated into PLGA microparticles (30-40 µm). Green fluorescent protein-labeled (GFP+) murine bone marrow derived macrophages were gathered and cultured to an M2 phenotype via stimulation with IL-4. These macrophages and microparticles were injected intramuscularly into the calf of a hindlimb ischemia murine model 3 days post injury. Mice were sacrificed two days later, and their calves harvested for flow cytometry analysis and immunohistochemistry (IHC). Using collagenase to digest the muscle, a single cell solution of the calf was stained with multiple macrophage phenotype markers (F4/80, CD206, ARG1, CD38, CD86) and Live/ Dead (L/D) markers. Separately, the solution was also stained with F4/80 and endothelial cell marker CD31. Flow data were analyzed using tSNE dimensionality reduction and Phenograph and FlowSOM clustering algorithms. Calf muscle sections were stained with DAPI and CD31, and images were analyzed with CellProfiler for CD31+ structures. All animal studies were approved by the Drexel Institutional Animal Use and Care Committee.

Results and Discussion: The number of GFP- macrophages was very high while the number of recovered GFP+ macrophages was exceedingly low, indicating that any effects of microparticles on angiogenesis and macrophage phenotype was caused by host macrophages. IL-4 releasing microparticles caused macrophages to take on a complex phenotypic profile. IL-4 affected macrophages were highest in clusters expressing both M1 and M2 markers. Additionally, treatment with IL-4 releasing microparticles increased the amount of endothelial cells (F4/80-CD31+), suggesting angiogenesis. Analysis of CD31+ structures showed that IL-4-releasing microparticles increased the density of vessels of 5-18 µm in diameter, while small capillaries (2-5 µm diameter) were unaffected. IL-4 release from the microparticles may have cause the M2 macrophages to help support recently created blood vessels, thereby increasing the number of larger structures.

Conclusions: This study shows that IL-4 releasing microparticles can control macrophage phenotype to support the development of blood vessels in vivo.

Introduction: Damage to the pleural tissue lining the lungs can occur in a multitude of ways, resulting in fluid leak into the pleural cavity, which, left untreated, can result in mortality. Historically such cases were treated via pleurodesis. Recent developments of lung tissue sealants serve as alternatives, but few options are available. While popular sealant tests can deliver qualitative pass/fail data, there is lack of quantitative in vivo methods to characterize sealing capabilities of these materials, particularly for pleural applications. In addition, commercial products have failed due to poor design and administration. Herein, we describe our development of a powder sealant capable of adhering to tissue and crosslinking to form an elastic hydrogel, circumventing limitations of current tissue sealants.

Experimental methods: Methacrylated alginate (AMA) was synthesized and characterized via ¹H-NMR spectroscopy. To enable gelation, visible green light crosslinkers were incorporated into the polymer solution. A sodium ion exchange was performed, the solution was lyophilized, and the product was processed into a powder. To investigate physical material properties, gelation kinetics and the viscosities of reconstituted solutions were collected at 37C, via an oscillatory time sweep at 10% radial strain and 1 Hz during exposure to green light. Quantitative in vitro experiments: dynamic burst pressure experiments were conducted using a custom-built burst pressure testing device, using a modified ASTM F2392 standard. Quantitative in vivo experiments: a murine model was developed in which we controlled respiratory dynamics to test sealant mechanics. Mice were anesthetized with pentobarbital sodium, tracheostomized, and paralized using pancuronium. Body temperature was maintained by keeping the animal under a lamp. Mice were connected to a computer-controlled small animal mechanical ventilator (flexiVent, Scireq) and ventilated. A series of deep inflation maneuvers (Total Lung Capacity and Trapezoidal deep inflation waveform) and a composite signal comprising of mutually prime frequencies ranging from 0-20 Hz were performed to establish baseline for the animal prior to injury. The thoracic cavity of the animal was opened and lungs were punctured, followed by another series of baseline measurements to calibrate testing mechanics and verify injury. The injury was treated by the application of the AMA powder and tests were repeated to compare lung mechanics pre-injury and post-repair, allowing for real-time pressure data collection of lung mechanics during sealant testing. After testing, animals underwent euthanasia.

Results: After acute traumatic injury using a murine model, there was a loss in pressure, confirming the injury model. Successful post-repair (i.e., application of the AMA powder, hydration and crosslinking) tests indicated that the sealant maintained physiologically-relevant lung pressure and returned lung capacity to pre-injury values, and that the sealant did not fail. A failure in the patch would result in a failure to hold pressure. Initial results show physiologically relevant burst pressure values (i.e. 30 cmH2O) for the powder sealant collected during in vitro testing.

Conclusions: The data indicates efficacy of a novel powder-based elastic sealant for recapitulating lung mechanics post injury. During an in vitro burst pressure experiment, the powder formed an crosslinked sealant capable of maintaining physiological pressure before failure. The powder-based sealant has the advantages of adhering to tissue upon application in situ and subsequent hydration, remaining in place while crosslinking via light exposure took place. Future work will explore long term in vivo studies to investigate lung tissue healing after repair with the novel powder sealant material.

Acknowledgements: This work was funded in part by NIH R01EB020964 (Oldinski) and the University of Vermont.
Disclosure of Interest: None Declared.

Antimicrobial resistance to antibiotics (AMR) and drug-resistant cancer are two of the main concerns that the healthcare system is facing nowadays [1,2]. Current chemotherapy treatments and antibiotic-based therapies are becoming ineffective or have plenty of drawbacks that are increasing the prevalence of resistance in both hospital and community settings. Therefore, new alternatives are needed, and nanotechnology might offer a suitable answer.

Although different nanomaterials are being used as a method to fight pathogens and cancer, traditionally synthesized nanoparticles present drawbacks, such as the production of toxic by-products, increased cytotoxicity in the cell and lack of selectivity. Consequently, green nanotechnology appears as an alternative approach, which employs living organisms and biomolecules as raw materials in a cost-effective and environmentally-friendly approach [3]. Among all these methods, microbially-produced nanomaterials have presented a significant potential, rendering nanomaterials with a powerful antimicrobial activity that can be tailored to the infection.

With the objective to demonstrate the antimicrobial and biocompatibility efficacy of bacterial-produced nanoparticles in skin bacterial infections, top pathogenic strains found in cutaneous wounds were employed for the synthesis of metalloid selenium nanoparticles (SeNPs), including Methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. Once generated, the SeNPs were analyzed in terms of their physicochemical features and the composition was extensively studied. After purification, the SeNPs were used as antimicrobial and anticancer agents through colony counting unit and MTS assays, respectively, as well as characterized for the mechanisms of death associated to their activities. At the same time, low cytotoxicity was demonstrated over the same range of antimicrobial efficiency.

Consequently, microbiological agents were successfully used as a biofactories for the generation of SeNPs of different compositions with biomedical properties. Therefore, bacteria were presented as a suitable approach for the synthesis of nanomaterials in a green fashion, overcoming the limitations of traditional nanotechnology.
References
[1] Ventola, C. Lee. Pharmacy and Therapeutics 40.4 (2015): 277–283.
[2] Katz, Steven J. Journal of Oncology Practice 9.3 (2013): 114–115.
[3] De la Guardia, Miguel. BioImpacts: BI 4.1 (2014): 1–2.

Endogenous endothelial progenitor cell (EPC) recruitment to sites of vascular injury is a multi-step process mediated by selectins, chemokines, and extracellular matrix (ECM) proteins. New vascular implants with surface-immobilized capture antibodies targeting circulating EPCs have shown clinical promise in accelerating the formation of neo-endothelium to cover the implant. The absence of subsequent adhesion-based signaling needed for EPC proliferation and differentiation could explain the lack of long-term improvement in vascular re-stenosis and overall vascular recovery. Antibodies can mimic the cell tethering effect of selectins but lack necessary EPC signaling effects introduced by cytokines and ECM proteins. We have previously shown that ECM-derived peptides, such as RGD, can promote the adhesion and enhance the clonal expansion of EPC-derived endothelial colony forming cells (ECFCs) via integrin based signaling. In this study, we developed a novel bifunctional surface modification strategy to produce surfaces that combine immobilized capture-antibodies and surface grafted ECM-derived peptides with controlled surface concentrations and ligand orientation. We hypothesize that this bifunctional strategy would further accelerate the re-endothelialization process while maximizing downstream endothelial function and vascular recovery via an integrin-dependent process.
Peripheral blood from 22 adult human donors was used to obtain donor-specific ECFCs. Aminated culture surfaces were modified by either the covalent conjugation of a customized RGD peptide, immobilization of vascular endothelial growth factor receptor-2 (VEGFR-2) antibody, or combining both biomolecules at different ratios using an optimized process. Unmodified surfaces were used as a negative control. Surface concentration of the peptide and antibody were analyzed using fluorescence microscopy and an enzyme-linked immunosorbent assay. First, the expanded ECFCs were seeded under static conditions and cell adhesion was studied using immunocytochemistry. ECFC interactions with the bifunctional surfaces were then interrogated under more physiologically relevant dynamic flow conditions in a microchannel perfusion loop at 1 dyn/cm² wall shear stress and analyzed through live cell imaging followed by immunocytochemistry.
Surfaces with immobilized anti-VEGFR-2 antibodies displayed significantly enhanced ECFC capture potential compared to RGD and unmodified controls as quantified by the number of adhered cells under both static and dynamic conditions. Average ECFC spreading and focal adhesion formation were significantly increased on RGD surfaces compared to the anti-VEGFR-2 and unmodified controls under both static and dynamic conditions. Total endothelial coverage and the total number of focal adhesions were maximized on the bifunctional surfaces combining the anti-VEGFR-2 and RGD due to a combined increase in both cell number and cell spreading. By increasing the peptide concentration on the bifunctional surfaces, ECFC spreading was increased without significantly affecting the number of adhered cells. These results suggest an additive (i.e. independent) effect of capture antibodies on ECFC tethering vs. RGD peptides on subsequent ECFC spreading and integrin-dependent cell signaling.
To our knowledge, this is the first study showcasing the ability of a bifunctional antibody/peptide surface modification strategy in promoting ECFC capture and adhesion. Our results also display a clear distinction between the mechanisms by which human-derived ECFCs react to immobilized capture antibodies and conjugated ECM-peptides. This strategy creates sequential steps of EPC trafficking to mimic endogenous processes of vascular healing which can potentially create an effective regenerative capacity for vascular implants.