Engineering a microparticle-loaded rough membrane for guided bone regeneration modulating osteoblast response without inducing inflammation.

Guided bone regeneration (GBR) is a widely used procedure that prevents the fast in-growth of soft tissues into bone defect. Among the different types of membranes, the use of collagen membranes is the gold standard. However, these membranes are implanted in tissue location where a severe acute inflammation will occur and can be negatively affected. The aim of this study was to develop a collagen-based membrane for GBR that incorporated alginate-hydroxyapatite microparticles. Membranes were manufactured using collagen type I and gelatin and alginate-hydroxyapatite microparticles. Membranes were assessed in terms of topography by scanning electron microscopy and confocal microscopy; stability by swelling after an overnight incubation in saline and enzymatic degradation against collagenase and mechanical properties by tensile tests. Furthermore, the biological response was assessed with SaOs-2 cells and THP-1 macrophages to determine alkaline phosphatase activity and inflammatory cytokine release. Our results showed that the incorporation of different percentages of these microparticles could induce changes in the surface topography. When the biological response was analyzed, either membranes were not cytotoxic to THP-1 macrophages or to SaOs-2 cells and they did not induce the release of pro-inflammatory cytokines. However, the different surface topographies did not induce changes in the macrophage morphology and the release of pro- and anti-inflammatory cytokines, suggesting that the effect of surface roughness on macrophage behavior could be dependent on other factors such as substrate stiffness and composition. Collagen-gelatin membranes with embedded alginate-hydroxyapatite microparticles increased ALP activity, suggesting a positive effect of them on bone regeneration, remaining unaffected the release of pro- and anti-inflammatory cytokines.

Novel strategies enhancing endodontic disinfection: Antibacterial biodegradable calcium hydroxide nanoparticles in an ex vivo model.

Due to the high failure rates associated to endodontic disinfection, this study aimed to investigate the antibacterial properties of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with Ca(OH)2 for endodontic disinfection procedures. Ca(OH)2 NPs production and physicochemical characterization were carried out as well as multiple antibacterial tests using three bacterial strains and an ex vivo model of endodontic infection with extracted human teeth. Agar diffusion test and broth dilution determined the inhibition growth zones (n = 5) and the minimal inhibitory concentration (MIC, n = 5), respectively. Cell viability was assessed using Live/Dead staining with confocal microscopy (n = 5). Data was analysed using ANOVA followed by post-hoc analysis. After 24 h of incubation, Ca(OH)2 NPs demonstrated a MIC of 10 µg/mL for Porphyromonas gingivalis (p < 0.001) and Enterococcus faecalis and 5 µg/mL for Fusobacterium nucleatum (p < 0.001). Although the agar diffusion test did not exhibit any inhibition area for Ca(OH)2 nor for Ca(OH)2 NPs, this was probably due to the buffering effect of the agar medium. However, the antibacterial capacity was confirmed in an ex vivo model, where instrumentalized teeth were infected with Enterococcus Faecalis and treated after 28 days of culture. A significant reduction in bacterial metabolic activity was confirmed for Ca(OH)2 NPs (40 % reduction with a single dose) and confirmed by Live/Dead staining. In conclusion, Ca(OH)2-loaded PLGA NPs present promising antibacterial efficacy for endodontic disinfection procedures.

Fucoidan-loaded electrospun Polyvinyl-alcohol/Chitosan nanofibers with enhanced antibacterial activity for skin tissue engineering.

The polymeric nanofiber may interact and control certain regeneration processes at the molecular level to repair damaged tissues. This research focuses on the development of characterization and antibacterial capabilities of polyvinyl alcohol (PVA)/chitosan (CS) nanofibres containing fucoidan (FUC) for tissue engineering as a skin tissue substitute. A control group consisting of 13% PVA/(0.1)% CS nanofiber was prepared. To confer antibacterial properties to the nanofiber, 10, 20, and 30 mg of FUC were incorporated into this control group. The scanning electron microscope (SEM) proved the homogeneous and beadless structures of the nanofibers. The antibacterial activity of the 13% PVA/(0.1)% CS/(10, 20, 30) FUC was tested against the S.aureus and E.coli and the results showed that with FUC addition, the antibacterial activities of the nanofibers increased. The biocompatibility test was performed with a fibroblast cell line for 1, 3, and 7 days of incubation and the results demonstrated that FUC addition enhanced the bioactivity of the 13% PVA/(0.1)% CS nanofibers. In addition, the biocompatibility results showed that 13% PVA/(0.1)% CS/10 FUC had the highest viability value for all incubation periods compared to the others. In addition, the tensile test results showed that; the maximum tensile strength value was observed for 13% PVA/(0.1)% CS/10 FUC nanofibers.

A systematic approach to improve printability and cell viability of methylcellulose-based bioinks.

Printability in 3D extrusion bioprinting encompasses extrudability, filament formation, and shape fidelity. Rheological properties can predict the shape fidelity of printed hydrogels. In particular, tan(δ), the ratio between loss (G'') and storage (G') modulus (G''/G'), is a powerful indicator of printability. This study explores the effect of different salt, sucrose, and MC concentrations on tan(δ), and therefore the printability of methylcellulose (MC) hydrogels. Salt and sucrose increased G', lowering tan(δ) and enabling printing of scaffolds with high shape fidelity. Conversely, MC concentration increased G'' and G', having a lesser effect on tan(δ). Shape fidelity of three formulations with similar G' but varying tan(δ) values were compared. Higher tan(δ) led to reduced height, while lower tan(δ) improved shape fidelity. Cell viability increased when reducing MC content, extrusion rate, and nozzle gauge. Higher MC concentration (G' > 1.5 kPa) increased the influence of needle size and extrusion rate on cell viability. Hydrogels with G' < 1 kPa could be extruded at high rates with small nozzles, minimally affecting cell viability. This work shows a direct relationship between tan(δ) and printability of MC-based hydrogels. Lowering the complex modulus of hydrogels, mitigates extrusion stress, thus improving cell survival.

Angiogenic and immunomodulation role of ions for initial stages of bone tissue regeneration.

It is widely known that bone has intrinsic capacity to self-regenerate after injury. However, the physiological regeneration process can be impaired when there is an extensive damage. One of the main reasons is due to the inability to establish a new vascular network that ensures oxygen and nutrient diffusion, leading to a necrotic core and non-junction of bone. Initially, bone tissue engineering (BTE) emerged to use inert biomaterials to just fill bone defects, but it eventually evolved to mimic bone extracellular matrix and even stimulate bone physiological regeneration process. In this regard, the stimulation of osteogenesis has gained a lot of attention especially in the proper stimulation of angiogenesis, being critical to achieve a successful osteogenesis for bone regeneration. Besides, the immunomodulation of a pro-inflammatory environment towards an anti-inflammatory one upon scaffold implantation has been considered another key process for a proper tissue restoration. To stimulate these phases, growth factors and cytokines have been extensively used. Nonetheless, they present some drawbacks such as low stability and safety concerns. Alternatively, the use of inorganic ions has attracted higher attention due to their higher stability and therapeutic effects with low side effects. This review will first focus in giving fundamental aspects of initial bone regeneration phases, focusing mainly on inflammatory and angiogenic ones. Then, it will describe the role of different inorganic ions in modulating the immune response upon biomaterial implantation towards a restorative environment and their ability to stimulate angiogenic response for a proper scaffold vascularization and successful bone tissue restoration. STATEMENT OF SIGNIFICANCE: The impairment of bone tissue regeneration when there is excessive damage has led to different tissue engineered strategies to promote bone healing. Significant importance has been given in the immunomodulation towards an anti-inflammatory environment together with proper angiogenesis stimulation in order to achieve successful bone regeneration rather than stimulating only the osteogenic differentiation. Ions have been considered potential candidates to stimulate these events due to their high stability and therapeutic effects with low side effects compared to growth factors. However, up to now, no review has been published assembling all this information together, describing individual effects of ions on immunomodulation and angiogenic stimulation, as well as their multifunctionality or synergistic effects when combined together.

Advanced Binary Guanosine and Guanosine 5'-Monophosphate Cell-Laden Hydrogels for Soft Tissue Reconstruction by 3D Bioprinting.

Soft tissue defects or pathologies frequently necessitate the use of biomaterials that provide the volume required for subsequent vascularization and tissue formation as autrografts are not always a feasible alternative. Supramolecular hydrogels represent promising candidates because of their 3D structure, which resembles the native extracellular matrix, and their capacity to entrap and sustain living cells. Guanosine-based hydrogels have emerged as prime candidates in recent years since the nucleoside self-assembles into well-ordered structures like G-quadruplexes by coordinating K+ ions and π-π stacking, ultimately forming an extensive nanofibrillar network. However, such compositions were frequently inappropriate for 3D printing due to material spreading and low shape stability over time. Thus, the present work aimed to develop a binary cell-laden hydrogel capable of ensuring cell survival while providing enough stability to ensure scaffold biointegration during soft tissue reconstruction. For that purpose, a binary hydrogel made of guanosine and guanosine 5'-monophosphate was optimized, rat mesenchymal stem cells were entrapped, and the composition was bioprinted. To further increase stability, the printed structure was coated with hyperbranched polyethylenimine. Scanning electron microscopic studies demonstrated an extensive nanofibrillar network, indicating excellent G-quadruplex formation, and rheological analysis confirmed good printing and thixotropic qualities. Additionally, diffusion tests using fluorescein isothiocyanate labeled-dextran (70, 500, and 2000 kDa) showed that nutrients of various molecular weights may diffuse through the hydrogel scaffold. Finally, cells were evenly distributed throughout the printed scaffold, cell survival was 85% after 21 days, and lipid droplet formation was observed after 7 days under adipogenic conditions, indicating successful differentiation and proper cell functioning. To conclude, such hydrogels may enable the 3D bioprinting of customized scaffolds perfectly matching the respective soft tissue defect, thereby potentially improving the outcome of the tissue reconstruction intervention.

Relevant Aspects of Titanium Topography for Osteoblastic Adhesion and Inhibition of Bacterial Colonization.

The influence of the surface topography of dental implants has been studied to optimize titanium surfaces in order to improve osseointegration. Different techniques can be used to obtain rough titanium, however, their effect on wettability, surface energy, as well as bacterial and cell adhesion and differentiation has not been studied deeply. Two-hundred disks made of grade 4 titanium were subjected to different treatments: machined titanium (MACH), acid-attacked titanium (AE), titanium sprayed with abrasive alumina particles under pressure (GBLAST), and titanium that has been treated with GBLAST and then subjected to AE (GBLAST + AE). The roughness of the different treatments was determined by confocal microscopy, and the wettability was determined by the sessile drop technique; then, the surface energy of each treatment was calculated. Osteoblast-like cells (SaOs-2) were cultured, and alkaline phosphatase was determined using a colorimetric test. Likewise, bacterial strains S. gordonii, S. oralis, A. viscosus, and E. faecalis were cultured, and proliferation on the different surfaces was determined. It could be observed that the roughness of the GBLAST and GBLAS + AE was higher, at 1.99 and 2.13 µm of Ra, with respect to the AE and MACH samples, which were 0.35 and 0.20 µm, respectively. The abrasive treated surfaces showed lower hydrophilicity but lower surface energy. Significant differences could be seen at 21 days between SaOS-2 osteoblastic cell adhesion for the blasted ones and higher osteocalcin levels. However, no significant differences in terms of bacterial proliferation were observed between the four surfaces studied, demonstrating the insensitivity of bacteria to topography. These results may help in the search for the best topographies for osteoblast behavior and for the inhibition of bacterial colonization.

Effects of Hypochlorous Acid and Hydrogen Peroxide Treatment on Bacterial Disinfection Treatments in Implantoplasty Procedures.

One of the main problems in oral implantology today is peri-implantitis, which affects almost 20% of dental implants placed in patients. One of the most commonly used techniques to eliminate bacterial biofilm is the implantoplasty, that consists of the mechanical modification of the implant surface topography followed by treatments with chemical reagents for decontamination. In this study, the main aim is to evaluate the use of two different chemical treatments based on hypochlorous acid (HClO) and hydrogen peroxide (H2O2). For this purpose, 75 titanium grade 3 discs were treated with implantoplasty according to established protocols. Twenty-five discs were used as controls, 25 were treated with concentrated HClO and 25 were treated with concentrated HClO followed by treatment with 6% H2O2. The roughness of the discs was determined using the interferometric process. Cytotoxicity with SaOs-2 osteoblastic cells was quantified at 24 and 72 h, whereas bacteria proliferation using S. gordonii and S. oralis bacteria was quantified at 5 s and 1 min of treatment. The results showed an increase in the roughness values, the control discs had an Ra of 0.33 µm and those treated with HClO and H2O2 reached 0.68 µm. Cytotoxicity was present at 72 h, together with a significant proliferation of bacteria. These biological and microbiological results can be attributed to the roughness produced by the chemical agents that triggered bacterial adsorption while inhibiting osteoblast adhesion. The results indicate that even if this treatment can decontaminate the titanium surface after implantation, the produced topography will generate an environment that will not favor long-term performance.

Optimization of guanosine-based hydrogels with boric acid derivatives for enhanced long-term stability and cell survival.

Tissue defects can lead to serious health problems and often require grafts or transplants to repair damaged soft tissues. However, these procedures can be complex and may not always be feasible due to a lack of available tissue. Hydrogels have shown potential as a replacement for tissue grafts due to their ability to support cell survival and encapsulate biomolecules such as growth factors. In particular, guanosine-based hydrogels have been explored as a potential solution, but they often exhibit limited stability which hampers their use in the biofabrication of complex grafts. To address this issue, we explored the use of borate ester chemistry and more complex boric acid derivatives to improve the stability and properties of guanosine-based hydrogels. We hypothesized that the aromatic rings in these derivatives would enhance the stability and printability of the hydrogels through added π-π stack interactions. After optimization, 13 compositions containing either 2-naphthylboronic acid or boric acid were selected. Morphology studies shows a well-defined nanofibrilar structure with good printable properties (thixotropic behaviour, print fidelity and printability). Moreover, the pH of all tested hydrogels was within the range suitable for cell viability (7.4-8.3). Nevertheless, only the boric acid-based formulations were stable for at least 7 days. Thus, our results clearly demonstrated that the presence of additional aromatic rings did actually impair the hydrogel properties. We speculate that this is due to steric hindrance caused by adjacent groups, which disrupt the correct orientation of the aromatic groups required for effective π-π stack interactions of the guanosine building block. Despite this drawback, the developed guanosine-boric acid hydrogel exhibited good thixotropic properties and was able to support cell survival, proliferation, and migration. For instance, SaOS-2 cells planted on these printed structures readily migrated into the hydrogel and showed nearly 100% cell viability after 7 days. In conclusion, our findings highlight the potential of guanosine-boric acid hydrogels as tissue engineering scaffolds that can be readily enhanced with living cells and bioactive molecules. Thus, our work represents a significant advancement towards the development of functionalized guanosine-based hydrogels.

A Novel Device for Assessment and Treatment of Upper Cervical Spine: Test-Retest Reliability Study.

INTRODUCTION: Neck pain is one of the most frequent reasons for consultation in primary care. Clinicians evaluate different variables, including movement and cervical strength, to determine the prognosis of patients. Usually, the tools employed for this purpose are expensive and bulky, or more than one is needed. This study aims to describe a novel device designed to assess the cervical spine and describe its test-retest reliability. METHODS: The Spinetrack device was designed to measure the strength of deep cervical flexor muscles and the chin-in and chin-out movement of the upper cervical spine. A test-retest reliability study was developed. The flexion, extension and strength needed to move the Spinetrack device were registered. Two measurements were developed, with one week between each assessment. RESULTS: Twenty healthy subjects were evaluated. The strength of the deep cervical flexor muscles in the first measurement was 21.18 ± 3.15 Newtons, the displacement movement during chin-in movement was 12.79 mm ± 3.46 and the displacement during chin-out movement was 35.99 mm ± 4.44. The test-retest reliability of strength was ICC 0.97 (95% CI (0.91-0.99)). CONCLUSION: The Spinetrack device has shown excellent test-retest reliability values for the measurement of the strength of the cervical flexor muscles and for the chin-in and chin-out movements.

Polydopamine Incorporation Enhances Cell Differentiation and Antibacterial Properties of 3D-Printed Guanosine-Borate Hydrogels for Functional Tissue Regeneration.

Tissue engineering focuses on the development of materials as biosubstitutes that can be used to regenerate, repair, or replace damaged tissues. Alongside this, 3D printing has emerged as a promising technique for producing implants tailored to specific defects, which in turn increased the demand for new inks and bioinks. Especially supramolecular hydrogels based on nucleosides such as guanosine have gained increasing attention due to their biocompatibility, good mechanical characteristics, tunable and reversible properties, and intrinsic self-healing capabilities. However, most existing formulations exhibit insufficient stability, biological activity, or printability. To address these limitations, we incorporated polydopamine (PDA) into guanosine-borate (GB) hydrogels and developed a PGB hydrogel with maximal PDA incorporation and good thixotropic and printability qualities. The resulting PGB hydrogels exhibited a well-defined nanofibrillar network, and we found that PDA incorporation increased the hydrogel's osteogenic activity while having no negative effect on mammalian cell survival or migration. In contrast, antimicrobial activity was observed against the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis. Thus, our findings suggest that our PGB hydrogel represents a significantly improved candidate as a 3D-printed scaffold capable of sustaining living cells, which may be further functionalized by incorporating other bioactive molecules for enhanced tissue integration.

A Novel Chitosan Composite Biomaterial with Drug Eluting Capacity for Maxillary Bone Regeneration.

Bone grafting is one of the most commonly performed treatments for bone healing or repair. Autografts, grafts from the same patient, are the most frequently used bone grafts because they can provide osteogenic cells and growth factors at the site of the implant with reduced risk of rejection or transfer of diseases. Nevertheless, this type of graft presents some drawbacks, such as pain, risk of infection, and limited availability. For this reason, synthetic bone grafts are among the main proposals in regenerative medicine. This branch of medicine is based on the development of new biomaterials with the goal of increasing bone healing capacity and, more specifically in dentistry, they aim at simultaneously preventing or eliminating bacterial infections. The use of fibers made of chitosan (CS) and hydroxyapatite (HA) loaded with an antibiotic (doxycycline, DX) and fabricated with the help of an injection pump is presented as a new strategy for improving maxillary bone regeneration. In vitro characterization of the DX controlled released from the fibers was quantified after mixing different amounts of HA (10-75%). The 1% CS concentration was stable, easy to manipulate and exhibited adequate cuttability and pH parameters. The hydroxyapatite concentration dictated the combined fast and controlled release profile of CSHA50DX. Our findings demonstrate that the CS-HA-DX complex may be a promising candidate graft material for enhancing bone tissue regeneration in dental clinical practice.

Nucleoside-Based Supramolecular Hydrogels: From Synthesis and Structural Properties to Biomedical and Tissue Engineering Applications.

Supramolecular hydrogels are of great interest in tissue scaffolding, diagnostics, and drug delivery due to their biocompatibility and stimuli-responsive properties. In particular, nucleosides are promising candidates as building blocks due to their manifold noncovalent interactions and ease of chemical modification. Significant progress in the field has been made over recent years to allow the use of nucleoside-based supramolecular hydrogels in the biomedical field, namely drug delivery and 3D bioprinting. For example, their long-term stability, printability, functionality, and bioactivity have been greatly improved by employing more than one gelator, incorporating different cations, including silver for antibacterial activity, or using additives such as boric acid or even biomolecules. This now permits their use as bioinks for 3D printing to produce cell-laden scaffolds with specified geometries and pore sizes as well as a homogeneous distribution of living cells and bioactive molecules. We have summarized the latest advances in nucleoside-based supramolecular hydrogels. Additionally, we discuss their synthesis, structural properties, and potential applications in tissue engineering and provide an outlook and future perspective on ongoing developments in the field.

Design of functional biomaterials as substrates for corneal endothelium tissue engineering.

Corneal endothelium defects are one of the leading causes of blindness worldwide. The actual treatment is transplantation, which requires the use of human cadaveric donors, but it faces several problems, such as global shortage of donors. Therefore, new alternatives are being developed and, among them, cell therapy has gained interest in the last years due to its promising results in tissue regeneration. Nevertheless, the direct administration of cells may sometimes have limited success due to the immune response, hence requiring the combination with extracellular mimicking materials. In this review, we present different methods to obtain corneal endothelial cells from diverse cell sources such as pluripotent or multipotent stem cells. Moreover, we discuss different substrates in order to allow a correct implantation as a cell sheet and to promote an enhanced cell behavior. For this reason, natural or synthetic matrixes that mimic the native environment have been developed. These matrixes have been optimized in terms of their physicochemical properties, such as stiffness, topography, composition and transparency. To further enhance the matrixes properties, these can be tuned by incorporating certain molecules that can be delivered in a sustained manner in order to enhance biological behavior. Finally, we elucidate future directions for corneal endothelial regeneration, such as 3D printing, in order to obtain patient-specific substrates.

Effect of the Size of Titanium Particles Released from Dental Implants on Immunological Response.

The techniques used in oral implantology to remove bacterial biofilm from the surface of implants by machining the titanium surface (implantoplasty) or by placing rough dental implants through friction with the cortical bone generate a large release of particles. In this work, we performed a simulation of particle generation following clinical protocols. The particles were characterized for commercially pure titanium with particle sizes of 5, 10, 15, and 30 µm. The aim was to determine the effect of particle size and chemical composition of the implant on the immune response. For this purpose, their morphology and possible contamination were characterized by scanning electron microscopy and X-ray microanalysis. In addition, the granulometry, specific surface area, release of metal ions into the medium, and studies of cytocompatibility, gene expression, and cytokine release linked to the inflammatory process were studied. The release of ions for titanium particles showed levels below 800 ppb for all sizes. Smaller particle sizes showed less cytotoxicity, although particles of 15 µm presented higher levels of cytocompatibility. In addition, inflammatory markers (TNFα and Il-1ß) were higher compared to larger titanium. Specifically, particles of 15 µm presented a lower proinflammatory and higher anti-inflammatory response as characterized by gene expression and cytokine release, compared to control or smaller particles. Therefore, in general, there is a greater tendency for smaller particles to produce greater toxicity and a greater proinflammatory response.

Biofabrication of Collagen Tissue-Engineered Blood Vessels with Direct Co-Axial Extrusion.

Cardiovascular diseases are considered one of the worldwide causes of death, with atherosclerosis being the most predominant. Nowadays, the gold standard treatment is blood vessel replacement by bypass surgery; however, autologous source is not always possible. Thereby, tissue-engineered blood vessels (TEBVs) are emerging as a potential alternative source. In terms of composition, collagen has been selected in many occasions to develop TEBVs as it is one of the main extracellular matrix components of arteries. However, it requires specific support or additional processing to maintain the tubular structure and appropriate mechanical properties. Here, we present a method to develop support-free collagen TEBVs with co-axial extrusion in a one-step procedure with high concentrated collagen. The highest concentration of collagen of 20 mg/mL presented a burst pressure of 619.55 ± 48.77 mmHg, being able to withstand perfusion of 10 dynes/cm2. Viability results showed a high percentage of viability (86.1 and 85.8% with 10 and 20 mg/mL, respectively) of human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVEC) after 24 h extrusion. Additionally, HUVEC and HASMCs were mainly localized in their respective layers, mimicking the native distribution. All in all, this approach allows the direct extrusion of collagen TEBVs in a one-step procedure with enough mechanical properties to be perfused.

Modulation of Macrophage Response by Copper and Magnesium Ions in Combination with Low Concentrations of Dexamethasone.

Macrophages have been deemed crucial for correct tissue regeneration, which is a complex process with multiple overlapping phases, including inflammation. Previous studies have suggested that divalent ions are promising cues that can induce an anti-inflammatory response, since they are stable cues that can be released from biomaterials. However, their immunomodulatory potential is limited in a pro-inflammatory environment. Therefore, we investigated whether copper and magnesium ions combined with low concentrations of the anti-inflammatory drug, dexamethasone (dex), could have a synergistic effect in macrophage, with or without pro-inflammatory stimulus, in terms of morphology, metabolic activity and gene expression. Our results showed that the combination of copper and dex strongly decreased the expression of pro-inflammatory markers, while the combination with magnesium upregulated the expression of IL-10. Moreover, in the presence of a pro-inflammatory stimulus, the combination of copper and dex induced a strong TNF-α response, suggesting an impairment of the anti-inflammatory actions of dex. The combination of magnesium and dex in the presence of a pro-inflammatory stimulus did not promote any improvement in comparison to dex alone. The results obtained in this study could be relevant for tissue engineering applications and in the design of platforms with a dual release of divalent ions and small molecules.

Multiple Ion Scaffold-Based Delivery Platform for Potential Application in Early Stages of Bone Regeneration.

Bone has the intrinsic capacity to regenerate itself, as long as the damage is small, through the sequential stimulation of specific phases, such as angiogenesis followed by osteogenesis. However, when the damage is extensive it is unable to regenerate and bone tissue engineering is used as an alternative. In this study, we developed a platform to allow the triple ion delivery with sequential delivery capacity to potentially stimulate antibacterial, angiogenic and osteogenic processes. The scaffold-based platform consisted of alginate/hydroxyapatite (HA) microparticles embedded in alginate fibers. Firstly, microparticles were developed using different ratios of alginate:HA using the spraying method, resulting in a high reproducibility of the technique. Microparticle size between 100-300 µm and ratio 1:40 resulted in a more spherical morphology and were selected for their incorporation into alginate fiber. Different amounts of copper and cobalt were added with the microparticles and alginate fiber, respectively, were used as model ions which could eventually modulate and mimic antimicrobial and angiogenic processes. Moreover, calcium ion was also incorporated in both, in order to provide the system with potential osteogenic properties together with HA. The multiple delivery of copper, cobalt and calcium released were in the therapeutic range as measured by induced coupled plasma (ICP), providing a promising delivery strategy for tissue engineering.

Assessing the potential role of copper and cobalt in stimulating angiogenesis for tissue regeneration.

The use of copper (Cu2+) and cobalt (Co2+) has been described to stimulate blood vessel formation, a key process for the success of tissue regeneration. However, understanding how different concentrations of these ions affect cellular response is important to design scaffolds for their delivery to better fine tune the angiogenic response. On the one hand, gene expression analysis and the assessment of tubular formation structures with human umbilical vein endothelial cells (HUVEC) revealed that high concentrations (10µM) of Cu2+ in early times and lower concentrations (0.1 and 1µM) at later times (day 7) enhanced angiogenic response. On the other hand, higher concentrations (25µM) of Co2+ during all time course increased the angiogenic gene expression and 0.5, 5 and 25µM enhanced the ability to form tubular structures. To further explore synergistic effects combining both ions, the non-toxic concentrations were used simultaneously, although results showed an increased cell toxicity and no improvement of angiogenic response. These results provide useful information for the design of Cu2+ or Co2+ delivery scaffolds in order to release the appropriate concentration during time course for blood vessel stimulation.

Evaluation of the immunomodulatory effects of cobalt, copper and magnesium ions in a pro inflammatory environment.

Biomaterials and scaffolds for Tissue Engineering are widely used for an effective healing and regeneration. However, the implantation of these scaffolds causes an innate immune response in which the macrophage polarization from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype is crucial to avoid chronic inflammation. Recent studies have showed that the use of bioactive ions such as cobalt (Co2+), copper (Cu2+) and magnesium (Mg2+) could improve tissue regeneration, although there is limited evidence on their effect on the macrophage response. Therefore, we investigated the immunomodulatory potential of Co2+, Cu2+ and Mg2+ in macrophage polarization. Our results indicate that Mg2+ and concentrations of Cu2+ lower than 10 µM promoted the expression of M2 related genes. However, higher concentrations of Cu2+ and Co2+ (100 µM) stimulated pro-inflammatory marker expression, indicating a concentration dependent effect of these ions. Furthermore, Mg2+ were able to decrease M1 marker expression in presence of a mild pro-inflammatory stimulus, showing that Mg2+ can be used to modulate the inflammatory response, even though their application can be limited in a strong pro-inflammatory environment.