Cooperativity and oscillations: Regulatory mechanisms of K-Ras nanoclusters.

K-Ras nanoclusters (NCs) concentrate all required molecules belonging to the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway in a small area where signaling events take place, increasing efficiency and specificity of signaling. Such nanostructures are characterized by controlled sizes and lifetimes distributions, but there is a poor understanding of the mechanisms involved in their dynamics of growth/decay. Here, a minimum computational model is presented to analyze the behavior of K-Ras NCs as cooperative dynamic structures that self-regulate their growth and decay according to their size. Indeed, the proposed model reveals that the growth and the local production of a K-Ras nanocluster depend positively on its actual size, whilst its lifetime is inversely proportional to the root of its size. The cooperative binding between the structural constituents of the NC (K-Ras proteins) induces oscillations in the size distributions of K-Ras NCs allowing them to range within controlled values, regulating the growth/decay dynamics of these NCs. Thereby, the size of a K-Ras NC is proposed as a key factor to regulate cell signaling, opening a range of possibilities to develop strategies for use in chronic diseases and cancer.

Modelling of chemotactic sprouting endothelial cells through an extracellular matrix.

Sprouting angiogenesis is a core biological process critical to vascular development. Its accurate simulation, relevant to multiple facets of human health, is of broad, interdisciplinary appeal. This study presents an in-silico model replicating a microfluidic assay where endothelial cells sprout into a biomimetic extracellular matrix, specifically, a large-pore, low-concentration fibrin-based porous hydrogel, influenced by chemotactic factors. We introduce a novel approach by incorporating the extracellular matrix and chemotactic factor effects into a unified term using a single parameter, primarily focusing on modelling sprouting dynamics and morphology. This continuous model naturally describes chemotactic-induced sprouting with no need for additional rules. In addition, we extended our base model to account for matrix sensing and degradation, crucial aspects of angiogenesis. We validate our model via a hybrid in-silico experimental method, comparing the model predictions with experimental results derived from the microfluidic setup. Our results underscore the intricate relationship between the extracellular matrix structure and angiogenic sprouting, proposing a promising method for predicting the influence of the extracellular matrix on angiogenesis.

Chlorine resistance property improvement of polyamide reverse osmosis membranes through cross-linking degree increment.

Highly permeable polyamide reverse osmosis (RO) membranes are desirable for reducing the energy burden and ensuring future water resources in arid and semiarid regions. One notable drawback of thin film composite (TFC) polyamide RO/NF membranes is the polyamide's sensitivity to degradation by free chlorine, the most used biocide in water purification trains. This investigation demonstrated a significant increase in the crosslinking-degree parameter by the m-phenylenediamine (MPD) chemical structure extending in the thin film nanocomposite (TFN) membrane without adding extra MPD monomers to enhance the chlorine resistance and performance. Membrane modification was carried out according to monomer ratio changes and Nanoparticle embedding into the PA layer approaches. A new class of TFN-RO membranes incorporating novel aromatic amine functionalized (AAF)-MWCNTs embedded into the polyamide (PA) layer was introduced. A purposeful strategy was carried out to use cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) as an intermediate functional group in the AAF-MWCNTs. Thus, amidic nitrogen, connected to benzene rings and carbonyl groups, assembles a structure similar to the standard PA, consisting of MPD and trimesoyl chloride. The resulting AAF-MWCNTs were mixed in the aqueous phase during the interfacial polymerization to increase the susceptible positions to chlorine attack and improve the crosslinking degree in the PA network. The characterization and performance results of the membrane demonstrated an increase in ion selectivity and water flux, impressive stability of salt rejection after chlorine exposure, and improved antifouling performance. This purposeful modification resulted in overthrowing two tradeoffs; i) high crosslink density-water flux and ii) salt rejection-permeability. The modified membrane demonstrated ameliorative chlorine resistance relative to the pristine one, with twice the increase in crosslinking degree, more than four times the enhancement of the oxidation resistance, negligible reduction in the salt rejection (0.83 %), and only 5 L/m2.h flux loss following a rigorous static chlorine exposure of 500 ppm.h under acidic conditions. The excellent performance of new chlorine resistant TNF RO membranes fabricated via AAF-MWCNTs together with the facile membrane manufacturing process offered the possibility of postulating them in the desalination field, which could eventually help the current freshwater supply challenge.

BBB-on-a-chip with integrated micro-TEER for permeability evaluation of multi-functionalized gold nanorods against Alzheimer's disease.

BACKGROUND: The lack of predictive models that mimic the blood-brain barrier (BBB) hinders the development of effective drugs for neurodegenerative diseases. Animal models behave differently from humans, are expensive and have ethical constraints. Organ-on-a-chip (OoC) platforms offer several advantages to resembling physiological and pathological conditions in a versatile, reproducible, and animal-free manner. In addition, OoC give us the possibility to incorporate sensors to determine cell culture features such as trans-endothelial electrical resistance (TEER). Here, we developed a BBB-on-a-chip (BBB-oC) platform with a TEER measurement system in close distance to the barrier used for the first time for the evaluation of the permeability performance of targeted gold nanorods for theranostics of Alzheimer's disease. GNR-PEG-Ang2/D1 is a therapeutic nanosystem previously developed by us consisting of gold nanorods (GNR) functionalized with polyethylene glycol (PEG), angiopep-2 peptide (Ang2) to overcome the BBB and the D1 peptide as beta amyloid fibrillation inhibitor, finally obtaining GNR-PEG-Ang2/D1 which showed to be useful for disaggregation of the amyloid in in vitro and in vivo models. In this work, we evaluated its cytotoxicity, permeability, and some indications of its impact on the brain endothelium by employing an animal-free device based on neurovascular human cells. RESULTS: In this work, we fabricated a BBB-oC with human astrocytes, pericytes and endothelial cells and a TEER measuring system (TEER-BBB-oC) integrated at a micrometric distance of the endothelial barrier. The characterization displayed a neurovascular network and the expression of tight junctions in the endothelium. We produced GNR-PEG-Ang2/D1 and determined its non-cytotoxic range (0.05-0.4 nM) for plated cells included in the BBB-oC and confirmed its harmless effect at the highest concentration (0.4 nM) in the microfluidic device. The permeability assays revealed that GNR-PEG-Ang2/D1 cross the BBB and this entry is facilitated by Ang2 peptide. Parallel to the permeability analysis of GNR-PEG-Ang2/D1, an interesting behavior of the TJs expression was observed after its administration probably related to the ligands on the nanoparticle surface. CONCLUSIONS: BBB-oC with a novel TEER integrated setup which allow a correct read-out and cell imaging monitoring was proven as a functional and throughput platform to evaluate the brain permeability performance of nanotherapeutics in a physiological environment with human cells, putting forward a viable alternative to animal experimentation.

Catheter design primer for neurointerventionalists.

Neurovascular catheter technology has rapidly evolved over the past decade. While performance characteristics are well known to the practitioner, the design features of these new-generation catheters and their implications on performance metrics remain a mystery to most clinicians due to the limited number of available resources. This knowledge gap hampers informed device choices and also limits collaboration between clinicians and engineers. To aid fellow neurointerventionalists, in this primer we have summarized the basic concepts of catheter design and construction.

Double stent-retriever as the first-line approach in mechanical thrombectomy: a randomized in vitro evaluation.

BACKGROUND: A repeated number of passes during mechanical thrombectomy leads to worse clinical outcomes in acute ischemic stroke. Initial experiences with the simultaneous double stent-retriever (double-SR) technique as the first-line treatment showed promising safety and efficacy results. OBJECTIVE: To characterize the potential benefits of using the double-SR as first-line technique as compared with the traditional single-SR approach. METHODS: Three types of clot analogs (soft, moderately stiff, and stiff) were used to create terminal internal carotid artery (T-ICA=44) and middle cerebral artery (MCA=88) occlusions in an in vitro neurovascular model. Sixty-six cases were randomized into each treatment arm: single-SR or double-SR, in combination with a 0.071" distal aspiration catheter. A total of 132 in vitro thrombectomies were performed. Primary endpoints were the rate of first-pass recanalization (%FPR) and procedural-related distal emboli. RESULTS: FPR was achieved in 42% of the cases. Overall, double-SR achieved a significantly higher %FPR than single-SR (52% vs 33%, P=0.035). Both techniques showed similar %FPR in T-ICA occlusions (single vs double: 23% vs 27%, P=0.728). Double-SR significantly outperformed single-SR in MCA occlusions (63% vs 38%, P=0.019), most notably in saddle occlusions (64% vs 14%, P=0.011), although no significant differences were found in single-branch occlusions (64% vs 50%, P=0.275). Double-SR reduced the maximal size of the clot fragments migrating distally (Feret diameter=1.08±0.65 mm vs 2.05±1.14 mm, P=0.038). CONCLUSIONS: This randomized in vitro evaluation demonstrates that the front-line double-SR technique is more effective than single-SR in achieving FPR when treating MCA bifurcation occlusions that present saddle thrombus.

Trackability of distal access catheters: an in vitro quantitative evaluation of navigation strategies.

BACKGROUND: In mechanical thrombectomy (MT), distal access catheters (DACs) are tracked through the vascular anatomy to reach the occlusion site. The inability of DACs to reach the occlusion site has been reported as a predictor of unsuccessful recanalization. This study aims to provide insight into how to navigate devices through the vascular anatomy with minimal track forces, since higher forces may imply more risk of vascular injuries. METHODS: We designed an experimental setup to monitor DAC track forces when navigating through an in vitro anatomical model. Experiments were recorded to study mechanical behaviors such as tension buildup against vessel walls, DAC buckling, and abrupt advancements. A multiple regression analysis was performed to predict track forces from the catheters' design specifications. RESULTS: DACs were successfully delivered to the target M1 in 60 of 63 in vitro experiments (95.2%). Compared to navigation with unsupported DAC, the concomitant coaxial use of a microcatheter/microguidewire and microcatheter/stent retriever anchoring significantly reduced the track forces by about 63% and 77%, respectively (p<0.01). The presence of the braid pattern in the reinforcement significantly reduced the track forces regardless of the technique used (p<0.05). Combined coil and braid reinforcement configuration, as compared with coil alone, and a thinner distal wall were predictors of lower track force when navigating with unsupported DAC. CONCLUSIONS: The use of microcatheter and stent retriever facilitate smooth navigation of DACs through the vascular tortuosity to reach the occlusion site, which in turn improves the reliability of tracking when positioning the DAC closer to the thrombus interface.

Microfluidic 3D platform to evaluate endothelial progenitor cell recruitment by bioactive materials.

Most of the conventional in vitro models to test biomaterial-driven vascularization are too simplistic to recapitulate the complex interactions taking place in the actual cell microenvironment, which results in a poor prediction of the in vivo performance of the material. However, during the last decade, cell culture models based on microfluidic technology have allowed attaining unprecedented levels of tissue biomimicry. In this work, we propose a microfluidic-based 3D model to evaluate the effect of bioactive biomaterials capable of releasing signaling cues (such as ions or proteins) in the recruitment of endogenous endothelial progenitor cells, a key step in the vascularization process. The usability of the platform is demonstrated using experimentally-validated finite element models and migration and proliferation studies with rat endothelial progenitor cells (rEPCs) and bone marrow-derived rat mesenchymal stromal cells (BM-rMSCs). As a proof of concept of biomaterial evaluation, the response of rEPCs to an electrospun composite made of polylactic acid with calcium phosphates nanoparticles (PLA+CaP) was compared in a co-culture microenvironment with BM-rMSC to a regular PLA control. Our results show a significantly higher rEPCs migration and the upregulation of several pro-inflammatory and proangiogenic proteins in the case of the PLA+CaP. The effects of osteopontin (OPN) on the rEPCs migratory response were also studied using this platform, suggesting its important role in mediating their recruitment to a calcium-rich microenvironment. This new tool could be applied to screen the capacity of a variety of bioactive scaffolds to induce vascularization and accelerate the preclinical testing of biomaterials. STATEMENT OF SIGNIFICANCE: For many years researchers have used neovascularization models to evaluate bioactive biomaterials both in vitro, with low predictive results due to their poor biomimicry and minimal control over cell cues such as spatiotemporal biomolecule signaling, and in vivo models, presenting drawbacks such as being highly costly, time-consuming, poor human extrapolation, and ethically controversial. We describe a compact microphysiological platform designed for the evaluation of proangiogenesis in biomaterials through the quantification of the level of sprouting in a mimicked endothelium able to react to gradients of biomaterial-released signals in a fibrin-based extracellular matrix. This model is a useful tool to perform preclinical trustworthy studies in tissue regeneration and to better understand the different elements involved in the complex process of vascularization.

Catheter tip distensibility substantially influences the aspiration force of thrombectomy devices.

BACKGROUND: A direct aspiration first pass thrombectomy (ADAPT) is a fast-growing technique for which a broad catalog of catheters that provide a wide range of aspiration forces can be used. We aimed to characterize different catheters' aspiration performance on stiff clots in an in vitro vascular model. We hypothesized that labeled catheter inner diameter (labeled-ID) is not the only parameter that affects the aspiration force (asp-F) and that thrombus-catheter tip interaction and distensibility also play a major role. METHODS: We designed an experimental setup consisting of a 3D-printed carotid artery immersed in a water deposit. We measured asp-F and distensibility of catheter tips when performing ADAPT on a stiff clot analog larger than catheter labeled-ID. Correlations between asp-F, catheter ID, and tip distensibility were statistically assessed. RESULTS: Experimental asp-F and catheter labeled-ID were correlated (r=0.9601; P<0.01). The relative difference between experimental and theoretical asp-F (obtained by the product of the tip's section area by the vacuum pressure) correlated with tip's distensibility (r=0.9050; P<0.01), evidencing that ADAPT performance is highly influenced by catheter tip shape-adaptability to the clot and that the effective ID (eff-ID) may differ from the labeled-ID specified by manufacturers. Eff-ID showed the highest correlation with experimental asp-F (r=0.9944; P<0.01), confirming that eff-ID rather than labeled-ID should be considered to better estimate the device efficiency. CONCLUSIONS: Catheter tip distensibility can induce a significant impact on ADAPT performance when retrieving a stiff clot larger than the device ID. Our findings might contribute to optimizing thrombectomy strategies and the design of novel aspiration catheters.

A microphysiological system combining electrospun fibers and electrical stimulation for the maturation of highly anisotropic cardiac tissue.

The creation of cardiac tissue models for preclinical testing is still a non-solved problem in drug discovery, due to the limitations related to thein vitroreplication of cardiac tissue complexity. Among these limitations, the difficulty of mimicking the functional properties of the myocardium due to the immaturity of the used cells hampers the obtention of reliable results that could be translated into human patients.In vivomodels are the current gold standard to test new treatments, although it is widely acknowledged that the used animals are unable to fully recapitulate human physiology, which often leads to failures during clinical trials. In the present work, we present a microfluidic platform that aims to provide a range of signaling cues to immature cardiac cells to drive them towards an adult phenotype. The device combines topographical electrospun nanofibers with electrical stimulation in a microfabricated system. We validated our platform using a co-culture of neonatal mouse cardiomyocytes and cardiac fibroblasts, showing that it allows us to control the degree of anisotropy of the cardiac tissue inside the microdevice in a cost-effective way. Moreover, a 3D computational model of the electrical field was created and validated to demonstrate that our platform is able to closely match the distribution obtained with the gold standard (planar electrode technology) using inexpensive rod-shaped biocompatible stainless-steel electrodes. The functionality of the electrical stimulation was shown to induce a higher expression of the tight junction protein Cx-43, as well as the upregulation of several key genes involved in conductive and structural cardiac properties. These results validate our platform as a powerful tool for the tissue engineering community due to its low cost, high imaging compatibility, versatility, and high-throughput configuration capabilities.

Stochastic modulation evidences a transitory EGF-Ras-ERK MAPK activity induced by PRMT5.

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway involves a three-step cascade of kinases that transduce signals and promote processes such as cell growth, development, and apoptosis. An aberrant response of this pathway is related to the proliferation of cell diseases and tumors. By using simulation modeling, we document that the protein arginine methyltransferase 5 (PRMT5) modulates the MAPK pathway and thus avoids an aberrant behavior. PRMT5 methylates the Raf kinase, reducing its catalytic activity and thereby, reducing the activation of ERK in time and amplitude. Two minimal computational models of the epidermal growth factor (EGF)-Ras-ERK MAPK pathway influenced by PRMT5 were proposed: a first model in which PRMT5 is activated by EGF and a second one in which PRMT5 is stimulated by the cascade response. The reported results show that PRMT5 reduces the time duration and the expression of the activated ERK in both cases, but only in the first model PRMT5 limits the EGF range that generates an ERK activation. Based on our data, we propose the protein PRMT5 as a regulatory factor to develop strategies to fight against an excessive activity of the MAPK pathway, which could be of use in chronic diseases and cancer.

Chemotactic TEG3 Cells' Guiding Platforms Based on PLA Fibers Functionalized With the SDF-1α/CXCL12 Chemokine for Neural Regeneration Therapy.

(Following spinal cord injury, olfactory ensheathing cell (OEC) transplantation is a promising therapeutic approach in promoting functional improvement. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical concentration differences. Here we compare the attachment, morphology, and directionality of an OEC-derived cell line, TEG3 cells, seeded on functionalized nanoscale meshes of Poly(l/dl-lactic acid; PLA) nanofibers. The size of the nanofibers has a strong effect on TEG3 cell adhesion and migration, with the PLA nanofibers having a 950 nm diameter being the ones that show the best results. TEG3 cells are capable of adopting a bipolar morphology on 950 nm fiber surfaces, as well as a highly dynamic behavior in migratory terms. Finally, we observe that functionalized nanofibers, with a chemical concentration increment of SDF-1α/CXCL12, strongly enhance the migratory characteristics of TEG3 cells over inhibitory substrates.

Polymeric Composite Dressings Containing Calcium-Releasing Nanoparticles Accelerate Wound Healing in Diabetic Mice.

Objective: Wound healing is a complex process that involves the interaction between different cell types and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of these interactions, resulting in nonhealing chronic wounds. Chronic wounds are a socioeconomic burden, one of the most prominent clinical manifestations of diabetes, however, they lack satisfactory treatment options. The objective of this study was to develop polymeric composites that deliver ions having wound healing properties and evaluate its performance using a pressure ulcer model in diabetic mice. Approach: To develop a polymeric composite wound dressing containing ion-releasing nanoparticles for chronic wound healing. This composite was chemically and physically characterized and evaluated using a pressure ulcer wound model in diabetic (db/db) mice to explore their potential as novel wound dressing. Results: This dressing exhibits a controlled ion release and a good in vitro bioactivity. The polymeric composite dressing treatment stimulates angiogenesis, collagen synthesis, granulation tissue formation, and accelerates wound closure of ischemic wounds created in diabetic mice. In addition, the performance of the newly designed composite is remarkably better than a commercially available dressing frequently used for the treatment of low-exuding chronic wounds. Innovation: The developed nanoplatforms are cell- and growth factor free and control the host microenvironment resulting in enhanced wound healing. These nanoplatforms are available by cost-effective synthesis with a defined composition, offering an additional advantage in potential clinical application. Conclusion: Based on the obtained results, these polymeric composites offer an optimum approach for chronic wound healing without adding cells or external biological factors.

Nanotechnology Approaches in Chronic Wound Healing.

Significance: The incidence of chronic wounds is increasing due to our aging population and the augment of people afflicted with diabetes. With the extended knowledge on the biological mechanisms underlying these diseases, there is a novel influx of medical technologies into the conventional wound care market. Recent Advances: Several nanotechnologies have been developed demonstrating unique characteristics that address specific problems related to wound repair mechanisms. In this review, we focus on the most recently developed nanotechnology-based therapeutic agents and evaluate the efficacy of each treatment in in vivo diabetic models of chronic wound healing. Critical Issues: Despite the development of potential biomaterials and nanotechnology-based applications for wound healing, this scientific knowledge is not translated into an increase of commercially available wound healing products containing nanomaterials. Future Directions: Further studies are critical to provide insights into how scientific evidences from nanotechnology-based therapies can be applied in the clinical setting.

Layer-by-layer modification effects on a nanopore's inner surface of polycarbonate track-etched membranes.

The control of the morphology, as well as the physical and chemical properties, of nanopores is a key issue for many applications. Reducing pore size is important in nanopore-based sensing applications as it helps to increase sensitivity. Changes of other physical properties such as surface net charge can also modify transport selectivity of the pores. We have studied how polyelectrolyte layer-by-layer (LBL) surface modification can be used to change the characteristics of nanoporous membranes. Studies were performed with a custom made three-dimensional multilayer microfluidic device able to fit membrane samples. The device allowed us to efficiently control LBL film deposition over blank low-cost commercially available polycarbonate track-etched (PCTE) membranes. We have demonstrated pore diameter reduction and deposition of the layers inside the pores through confocal and SEM images. Posterior impedance measurement studies served to evaluate experimentally the effect of the LBL deposition on the net inner nanopore surface charge and diameter. Measurements using direct current (DC) and alternative current (AC) voltages have demonstrated contrasted behaviors depending on the number and parity of deposited opposite charge layers. PCTE membranes are originally negatively charged and results evidenced higher impedance increases for paired charge LBL depositions. Impedance decreased when an unpaired positive layer was added. These results showed a different influence on the overall ion motility due to the effect of different surface charges. Results have been fit into a model that suggested a strong dependence of nanopores' impedance module to surface charge on conductive buffers, such as Phosphate Buffer Saline (PBS), even on relatively large nanopores. In AC significant differences between paired and unpaired charged LBL depositions tended to disappear as the total number of layers increased.

Hydrogel co-networks of gelatine methacrylate and poly(ethylene glycol) diacrylate sustain 3D functional in vitro models of intestinal mucosa.

Mounting evidence supports the importance of the intestinal epithelial barrier and its permeability both in physiological and pathological conditions. Conventional in vitro models to evaluate intestinal permeability rely on the formation of tightly packed epithelial monolayers grown on hard substrates. These two-dimensional models lack the cellular and mechanical components of the non-epithelial compartment of the intestinal barrier, the stroma, which are key contributors to the barrier permeability in vivo. Thus, advanced in vitro models approaching the in vivo tissue composition are fundamental to improve precision in drug absorption predictions, to provide a better understanding of the intestinal biology, and to faithfully represent related diseases. Here, we generate photo-crosslinked gelatine methacrylate (GelMA)-poly(ethylene glycol) diacrylate (PEGDA) hydrogel co-networks that provide the required mechanical and biochemical features to mimic both the epithelial and stromal compartments of the intestinal mucosa, i.e. they are soft, cell adhesive and cell-loading friendly, and suitable for long-term culturing. We show that fibroblasts can be embedded in the GelMA-PEGDA hydrogels while epithelial cells can grow on top to form a mature epithelial monolayer that exhibits barrier properties which closely mimic those of the intestinal barrier in vivo, as shown by the physiologically relevant transepithelial electrical resistance (TEER) and permeability values. The presence of fibroblasts in the artificial stroma compartment accelerates the formation of the epithelial monolayer and boosts the recovery of the epithelial integrity upon temporary barrier disruption, demonstrating that our system is capable of successfully reproducing the interaction between different cellular compartments. As such, our hydrogel co-networks offer a technologically simple yet sophisticated approach to produce functional three-dimensional (3D) in vitro models of epithelial barriers with epithelial and stromal cells arranged in a spatially relevant manner and near-physiological functionality.

Development of a novel automatable fabrication method based on electrospinning co electrospraying for rotator cuff augmentation patches.

Rotator cuff tear is one of the most common shoulder diseases. Rotator cuff augmentation (RCA) is trying to solve the high retear failure percentage after the surgery procedures (20-90%). The ideal augmentation patch must provide a temporal mechanical support during the healing process. In this work, we proposed a simple method for the fabrication of synthetic RCA patches. This method combines the use of electrospraying to produce poly-L-lactic-co-ε-caprolactone (PLC) films in an organogel form and electrospinning to produce poly(lactic) acid (PLA) nanofibers. The device consists in a combination of layers, creating a multilayered construct, enabling the possibility of tuning its mechanical properties and thickness. Besides, both techniques are simple to escalate for industrial production. A complete characterization has been performed to optimize the involved number of layers and production time of PLC films and PLA nanofibers fabrication, obtaining a final optimal configuration for RCA devices. Structural, mechanical and suture properties were evaluated. Also, the possibility of surface functionalization to improve the bioactivity of the scaffold was studied, adding aligned electrospun PLA nanofibers on the surface of the device to mimic the natural tendon topography. Surface modification was characterized by culturing adult normal human dermal fibroblasts. Lack of toxicity was detected for material presented, and cell alignment shape orientation guided by aligned fibers, mimicking tendon structure, was obtained. Cell proliferation and protein production were also evaluated.

Feasible and pure P2O5-CaO nanoglasses: An in-depth NMR study of synthesis for the modulation of the bioactive ion release.

The use of bioactive glasses (e.g. silicates, phosphates, borates) has demonstrated to be an effective therapy for the restoration of bone fractures, wound healing and vascularization. Their partial dissolution towards the surrounding tissue has shown to trigger positive bioactive responses, without the necessity of using growth factors or cell therapy, which reduces money-costs, side effects and increases their translation to the clinics. However, bioactive glasses often need from stabilizers (e.g. SiO44-, Ti4+, Co2+, etc.) that are not highly abundant in the body and which metabolization is not fully understood. In this study, we were focused on synthesizing pure calcium phosphate glasses without the presence of such stabilizers. We combined a mixture of ethylphosphate and calcium 2-methoxyethoxide to synthesize nanoparticles with different compositions and degradability. Synthesis was followed by an in-depth nuclear magnetic resonance characterization, complemented with other techniques that helped us to correlate the chemical structure of the glasses with their physiochemical properties and reaction mechanism. After synthesis, the organically modified xerogel (i.e. calcium monoethylphosphate) was treated at 200 or 350 °C and its solubility was maintained and controlled due to the elimination of organics, increase of phosphate-calcium interactions and phosphate polycondensation. To the best of our knowledge, we are reporting the first sol-gel synthesis of binary (P2O5-CaO) calcium phosphate glass nanoparticles in terms of continuous polycondensated phosphate chains structure without the addition of extra ions. The main goal is to straightforward the synthesis, to get a safer metabolization and to modulate the bioactive ion release. Additionally, we shed light on the chemical structure, reaction mechanism and properties of calcium phosphate glasses with high calcium contents, which nowadays are poorly understood. STATEMENT OF SIGNIFICANCE: The use of bioactive inorganic materials (i.e. bioactive ceramics, glass-ceramics and glasses) for biomedical applications is attractive due to their good integration with the host tissue without the necessity of adding exogenous cells or growth factors. In particular, degradable calcium phosphate glasses are completely resorbable, avoiding the retention in the body of the highly stable silica network of silicate glasses, and inducing a more controllable degradability than bioactive ceramics. However, most calcium phosphate glasses include the presence of stabilizers (e.g. Ti4+, Na+, Co2+), which metabolization is not fully understood and complicates their synthesis. The development of binary calcium phosphate glasses with controlled degradability reduces these limitations, offering a simple and completely metabolizable material with higher transfer to the clinics.

Wound healing-promoting effects stimulated by extracellular calcium and calcium-releasing nanoparticles on dermal fibroblasts.

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium-phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing.