Hypoxic Microenvironment Reconstruction with Synergistic Biofunctional Ions Promotes Diabetic Wound Healing.

Chronic hypoxia and ischemia make diabetic wounds non-healing. Cellular functions of diabetic chronic wounds are inhibited under a pathological environment. Therefore, this work develops a composite hydrogel system to promote diabetic wound healing. The composite hydrogel system consists of ε-poly-lysine (EPL), calcium peroxide (CP), and borosilicate glass (BG). The hydrogel supplies continuous dissolved oxygen molecules to the wound that can penetrate the skin tissue to restore normal cellular function and promote vascular regeneration. Biofunctional ions released from BGs can recruit more macrophages through neovascularization and modulate macrophage phenotypic transformation. Combining oxygen-mediated vascular regeneration and ion-mediated inflammatory regulation significantly accelerated diabetic wound healing. These findings indicate that this composite hydrogel system holds promise as a novel tissue engineering material.

Dual stimulus responsive borosilicate glass (BSG) scaffolds promote diabetic alveolar bone defectsrepair by modulating macrophage phenotype.

The regeneration of alveolar bone is still clinical challenge, particularly accompanied with diabetes, causing metabolic disorder with a protracted low-grade inflammatory phenotype. As a result, the anticipated loading of biomaterials is highly suspicious in spontaneous modulation of cells function, which is mostly disturbed by constant inflammation. In this study, we developed glucose and hydrogen peroxide dual-responsive borosilicate glass (BSG) scaffolds loaded with epigallocatechin gallate (EGCG) to synergistically modulate the abnormal inflammation of diabetic alveolar bone defects. It was found that the release of EGCG by BSG could directly regulate the shift of macrophages from M1 to the M2 phenotype by promoting autophagy and lessening the inhibition of autophagic flux. Moreover, EGCG can also indirectly regulate the polarization phenotype of macrophages by reducing the activation of NF-κb in stem cells and restoring its immunoregulatory capacity. Therefore, the addition of EGCG to BSG scaffold in diabetes allows for a more striking modulation of the macrophage phenotype in a timely manner. The altered macrophage phenotype reduces local inflammation and thus increases the ability to repair diabetic alveolar bone, showing promise for the treatment of alveolar defect in diabetic patients.

Unusual Surface Coagulation Activation Patterns of Crystalline and Amorphous Silicate-Based Biominerals.

Activation of coagulation cascades, especially FX and prothrombin, prevents blood loss and reduces mortality from hemorrhagic shock. Inorganic salts are efficient but cannot stop bleeding completely in hemorrhagic events, and rebleeding carries a significant mortality risk. The coagulation mechanism of biominerals has been oversimplified in the past two decades, limiting the creation of novel hemostats. Herein, at the interface, the affinity of proteins, the protease activity, fibrinolysis, hydration shell, and dynamic microenvironment are monitored at the protein level. Proteomic analysis reveals that fibrinogen and antithrombin III's affinity for kaolin's interface causes a weak thrombus and rebleeding during hemostasis. Inspiringly, amorphous bioactive glass (BG) with a transient-dynamic ion microenvironment breaches the hydration layer barrier and selectively and slightly captures procoagulant components of kiniogen-1, plasma kallikrein, FXII, and FXI proteins on its interface, concurrently generating a continuous biocatalytic interface to rapidly activate both intrinsic and extrinsic coagulation pathways. Thus, prothrombin complexes are successfully hydrolyzed to thrombin without platelet membrane involvement, speeding production of high-strength clots. This study investigates how the interface of inorganic salts assists in coagulation cascades from a more comprehensive micro-perspective that may help elucidate the clinical application issues of kaolin-gauze and pave the way to new materials for managing hemorrhage.

Spontaneous immunomodulation and regulation of angiogenesis and osteogenesis by Sr/Cu-borosilicate glass (BSG) bone cement to repair critical bone defects.

Injectable bone biomaterials like bone cement should be designed and fabricated with certain biological criteria, which include: 1) recruitment and polarization of the macrophages from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype, 2) enhance vascularization, and 3) activate osteogenic differentiation of bone marrow-derived stem cells to promote bone healing. So far, no injectable biomaterials could spontaneously regulate the entire bone healing process that involves inflammation, angiogenesis, and osteogenesis. Therefore, in this study, we designed bone cement comprised of strontium and copper-incorporated borosilicate glass (Sr/Cu-BSG) in the liquid phase of chitosan to modulate bone healing. In vitro studies showed that the controlled release of Sr and Cu ions up-regulated anti-inflammatory genes(IL-1Ra and TGF-ß1) while down-regulating pro-inflammatory genes(IL-1ß and IL-6) in macrophages at 3 days. Sr and Cu ions also increased the expressions of angiogenic genes (VEGF and bFGF) in HUVECs at 5 days and osteogenic genes (Runx-2, OCN, and OPN) in hBMSCs at 7, 14, and 21 days. 5Sr3Cu-BSG bone cement exhibited the best anti-inflammatory, angiogenic, and osteogenic properties among the bone cement groups with different Sr and Cu ratios. Short-term and long-term implantation of Sr/Cu-BSGs in femoral condylar bone defects of rats and rabbits confirmed the in vitro results, where the degradation rate of Sr/Cu-BSG matched the bone healing rate. Similar to in vitro, the 5Sr3Cu-BSG group also showed the highest bone formation in vivo. Excellent physical and chemical properties, along with its bone repairing ability, make the Sr/Cu-BSG bone cement a good candidate biomaterial for treating bone defects.

Biological Fixation of Bioactive Bone Cement in Vertebroplasty: The First Clinical Investigation of Borosilicate Glass (BSG) Reinforced PMMA Bone Cement.

PMMA bone cement has been clinically used for decades in vertebroplasty due to its high mechanical strength and satisfactory injectability. However, the interface between bone and PMMA is fragile and more prone to refracture in situ because PMMA lacks a proper biological response from the host bone with minimal bone integration and dense fibrous tissue formation. Here, we modified PMMA by incoporating borosilicate glass (BSG) with a dual glass network of [BO3] and [SiO4], which spontaneously modulates immunity and osteogenesis. In particular, the BSG modified PMMA bone cement (abbreviated as BSG/PMMA cement) provided an alkaline microenvironment that spontaneously balanced the activities between osteoclasts and osteoblasts. Furthermore, the trace elements released from the BSGs enhanced the osteogenesis to strengthen the interface between the host bone and the implant. This study shows the first clinical case after implantation of BSG/PMMA for three months using the dual-energy CT, which found apatite nucleation around PMMA instead of fibrous tissues, indicating the biological interface was formed. Therefore, BSG/PMMA is promising as a biomaterial in vertebroplasty, overcoming the drawback of PMMA by improving the biological response from the host bone.

A New Method of Myostatin Inhibition in Mice via Oral Administration of Lactobacillus casei Expressing Modified Myostatin Protein, BLS-M22.

Myostatin is a member of the transforming growth factor-beta superfamily and is an endogenous negative regulator of muscle growth. This study aimed to determine whether an oral administration of Lactobacillus casei expressing modified human myostatin (BLS-M22) could elicit sufficient levels of myostatin-specific antibody and improve the dystrophic features of an animal model of Duchenne muscular dystrophy (DMD; mdx mouse). BLS-M22 is a recombinant L. casei engineered to harbor the pKV vector and poly-gamma-glutamic acid gene linked to a modified human myostatin gene. Serological analysis showed that anti-myostatin IgG titers were significantly increased, and serum creatine kinase was significantly reduced in the BLS-M22-treated mdx mice compared to the control mice. In addition, treatment of BLS-M22 resulted in a significant increase in body weight and motor function (Rotarod behavior test). Histological analysis showed an improvement in the dystrophic features (fibrosis and muscle hypertrophy) of the mdx mice with the administration of BLS-M22. The circulating antibodies generated after BLS-M22 oral administration successfully lowered serum myostatin concentration. Myostatin blockade resulted in serological, histological, and functional improvements in mdx mice. Overall, the findings suggest the potential of BLS-M22 to treat DMD; however, further clinical trials are essential to ascertain its efficacy and safety in humans.

Cytotoxic effect of galvanically coupled magnesium-titanium particles on Escherichia coli.

Orthopedic device-related infections (ODRIs) are difficult to control due to microbial biofilm formation and associated with high-level resistance to conventional antibiotics. In many cases, the only treatment option for ODRI is explantation. Previous studies have shown that application of cathodic potentials at the metal surface can eradicate biofilms, and Mg and Mg-Ti particles have the same effect as cathodic potentials. This study investigated the effects of Mg and Mg-Ti particles on established biofilms and planktonic cells E. coli. Bacterial cultures with developed biofilms or planktonic cells were treated with Mg or Mg-Ti particles, and the viability were assessed using flow cytometry or visual assessment methods (i.e., observation from SEM images and opacity of the solution). It was found that viability of biofilms treated with 16.67 mg/ml of Mg was 2.8 ± 0.96% at the end of 6-hr killing compared to untreated controls. This extent of killing was more significant compared to 24-hr grown biofilms treated with ofloxacin, an antibiotic known to be effective against these bacteria. Biofilms treated with 50 and 100 µg/ml of ofloxacin had 62 ± 4.6% and 52 ± 19.3% survival, respectively, where ofloxacin at these concentrations is known to kill planktonic counterparts very effectively. Inhibition zone tests revealed that biofilms within 2 mm of Mg or Mg-Ti particle clusters were effectively killed. These results demonstrated the potential of Mg or Mg-Ti particles in killing microbial biofilms and potential for controlling ODRI.

Programming Colloidal Self-Assembled Patterns (cSAPs) into Thermo-Responsible Hybrid Surfaces for Controlling Human Stem Cells and Macrophages.

Hybrid surfaces with tunable topography, chemistry, and stiffness have potential to rebuild native extracellular matrix (ECM) and manipulate cell behavior in vitro. However, the fabrication of controllable hybrid surfaces is still challenging. In this study, colloidal self-assembly technology was used to program particles into highly ordered structures with hybrid chemistry and stiffness at biointerfaces. These colloidal self-assembled patterns (cSAPs), including unary, binary, and ternary cSAPs, composed of silicon (Si), polystyrene (PS), and/or poly(N-isopropylacrylamide) (pNIPAM) nanogels (PNGs), were fabricated using either coassembly or layer-by-layer (LBL) methods. The selected binary cSAPs (i.e., PS/PNG and PNG/PS) have a tunable surface topography and wettability between 25 and 37 °C; thus, they can be used as dynamic cell culture substrates. Human adipose-derived mesenchymal stem cells (hASCs), bone marrow-derived mesenchymal stem cells (hBMSCs), and macrophages (THP-1) were investigated on these hybrid cSAPs under a static or dynamic system. The results showed that hybrid cSAPs significantly influenced the focal adhesions, cell morphology, cell migration, and gene expressions of stem cells. In general, stem cells had more vinculin puncta, smaller spreading size, and faster migration speed than the TCPS control. Hybrid cSAPs up-regulated gene expressions of focal adhesion kinase (FAK) and chondrocytes (AGG and SOX9) under static culture, while they also up-regulated osteocytes (COL1 and RUNX2) under dynamic culture. THP-1 macrophages were at M0 state on all cSAPs under static culture. However, cells became sensitive under dynamic culture. For example, some M1 genes (i.e., IL6, CD68, and TNFα) and M2 genes (i.e., IL10 and CD206) were down-regulated, while other M1 genes (i.e., IL1ß) and M2 genes (i.e., TGF-ß and IL1ra) were up-regulated, depending on the particle combinations. In conclusion, new hybrid cSAPs with thermoresponsive surface properties are versatile materials for stem cells and macrophages manipulation.

Design, Material, and Seating Load Effects on In Vitro Fretting Corrosion Performance of Modular Head-Neck Tapers.

BACKGROUND: The short-term corrosion and micromechanical behavior of 32 unique head-neck taper design/material/assembly conditions was tested using an incremental cyclic fretting corrosion (ICFC) test method previously developed. METHODS: Seven materials, design, and simulated surgical parameters were evaluated, each being assigned 2 conditions for testing, using a 27-2 (7 factor, quarter factorial) design of experiments test matrix. The factors explored were (1) seating load, (2) head-neck offset, (3) material combination, (4) taper diameter, (5) taper roughness, (6) angular mismatch/engagement, and (7) taper length. Each sample underwent assembly, ICFC testing, pull off. RESULTS: Low seating load and high head offset correlated with increased fretting corrosion (P < .05). High head offset also contributed to a lower onset load for fretting current and higher micromotion (P < .05). Head subsidence measured over the ICFC test for samples seated at 100 N was significantly higher than samples seated at 4000 N. Micromotion for 12-mm head offsets was statistically higher than samples with a 1.5-mm head offset. A number of interactive effects were observed. For example, samples seated at 4000 N were less sensitive to head offset than samples seated at 100 N in terms of the resulting fretting current. CONCLUSION: Taper locking position, material combination, taper engagement length, taper roughness, and taper dimensions all had weak or no correlation with fretting current and taper micromotion. This test method and experimental design is a versatile means of assessing potential new taper designs in the future.

In vitro cytotoxicity of galvanically coupled magnesium-titanium particles on human osteosarcoma SAOS2 cells: A potential cancer therapy.

Osteosarcoma is a malignant bone cancer that occurs mostly in children and young adults. This study investigated the cytotoxicity of Mg and Mg-Ti microparticles to human osteosarcoma cells. Osteosarcoma cells were killed in a dosage-dependent manner when cells, with a cell seeding density of 30,000 cells/cm2 , were cultured with 0 to 2500 µg/mL of Mg or Mg-Ti in cell culture media for 24-72 h. Mg-Ti killed cells more effectively, where 1250 µg/mL of Mg-Ti killed cells completely by 24 h, while 2500 µg/mL of Mg killed nearly all cells, but not all. Killing due to particle corrosion occurred mostly during the first 24 h, and so the percent cell viability between 24 and 72 h showed not much variability. However, the measurement of live and dead cell numbers, over the timeframe of 24-72 h, showed more insight, such as cell recovery. If particle concentrations were low, the number of live cells increased after 24 h, indicating cell proliferation. If particle concentrations were high, the number of live cells either remained steady or decreased, indicating cell quiescence or continued killing, respectively. Increase in the number of dead cells also indicated killing, while plateau meant discontinued killing. In addition, repeated killing of recovered cells exhibited the same dose-dependent killing profile as the initial experiment, implying little development of cell resistance to treatment. These results, together, show that osteosarcoma cells are susceptible to killing by way of exposure to corroding particles, showing highly effective killing using the galvanic couple of Mg-Ti. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 178-189, 2019.

The effect of cell density, proximity, and time on the cytotoxicity of magnesium and galvanically coupled magnesium-titanium particles in vitro.

Magnesium (Mg) and galvanically coupled magnesium-titanium (Mg-Ti) particles in vitro have been reported previously to kill cells in a dosage-dependent manner. Mg-Ti particles kill cells more effectively than Mg alone, due to the galvanic effect of Mg and Ti. This study further investigated the in vitro cytotoxicity of Mg and Mg-Ti in terms of particle concentration, cell density, time, and proximity. Cell density has an effect on cell viability only at low particle concentrations (below 250 µg/mL), where cell viability dropped only for lower cell densities (5000-10,000 cells/cm2 ) and not for higher cell densities (20,000-30,000 cells/cm2 ), showing that the particles cannot kill if there are more cells present. Cytotoxicity of Mg and Mg-Ti particles is quick and temporary, where the particles kill cells only during particle corrosion (first 24 h). Depending on the percentage of surviving cells, particle concentrations, and ongoing corrosion activity, the remaining live cells either proliferated and recovered, or just remained viable and quiescent. The particle killing is also proximity-dependent, where cell viability was significantly higher for cells far away from the particles (greater than ∼1 mm) compared to those close to the particles (less than ∼1 mm). Although the increase of pH does affect cell viability negatively, it is not the sole killing factor since cell viability is significantly dependent on particle type and proximity but not pH. Mg and Mg-Ti particles used in this study are large enough to prevent direct cell phagocytosis so that the cell killing effect may be attributed to solely electrochemical reactions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1428-1439, 2018.

Cytotoxic effect of galvanically coupled magnesium-titanium particles.

Recent work has shown that reduction reactions at metallic biomaterial surfaces can induce significant killing of cells in proximity to the surface. To exploit this phenomenon for therapeutic purposes, for example, for cancer tumor killing or antibacterial effects (amongst other applications), magnesium metal particles, galvanically coupled to titanium by sputtering, have been evaluated for their cell-killing capability (i.e. cytotoxicity). Magnesium (Mg) particles large enough to prevent particle phagocytosis were investigated, so that only electrochemical reactions, and not particle toxicity per se, caused cytotoxic effects. Titanium (Ti) coated magnesium particles, as well as magnesium-only particles were introduced into MC3T3-E1 mouse pre-osteoblast cell cultures over a range of particle concentrations, and cells were observed to die in a dosage-dependent manner. Ti-coated magnesium particles killed more cells at lower particle concentration than magnesium alone (P<0.05), although the pH measured for magnesium and magnesium-titanium had no significant difference at similar particle concentrations. Complete cell killing occurred at 750µg/ml and 1500µg/ml for Mg-Ti and Mg, respectively. Thus, this work demonstrates that galvanically coupled Mg-Ti particles have a significant cell killing capability greater than Mg alone. In addition, when the pH associated with complete killing with particles was created using NaOH only (no particles), then the percentage of cells killed was significantly less (P<0.05). Together, these findings show that pH is not the sole factor associated with cell killing and that the electrochemical reactions, including the reduction reactions, play an important role. Reduction reactions on galvanically coupled Mg-Ti and Mg particles may generate reactive oxygen intermediates that are able to kill cells in close proximity to the particles and this approach may lead to potential therapies for infection and cancer. STATEMENT OF SIGNIFICANCE: This paper demonstrates that during active corrosion of both Mg and Mg-Ti particles cells cultured with the particles are killed in a dose-dependent particle concentration fashion. Additionally, galvanically-coupled magnesium-titanium microparticles kill cells more effectively than magnesium particles alone. The killing effect was shown to not be due to pH shifts since no differences were seen for different particle types and pH adjusted medium without particles did not exhibit the same level of killing. The significance of this work is the recognition of this killing effect with Mg particles and the potential therapeutic applications in infection control and cancer treatment that this process may provide.

Intravitreal tanibirumab, a fully human monoclonal antibody against vascular endothelial growth factor receptor 2, partially suppresses and regresses laser-induced choroidal neovascularization in a rat model.

PURPOSE: The study investigated the effect of intravitreally administered tanibirumab, a fully human monoclonal antibody against vascular endothelial growth factor receptor 2, in a rat model of laser-induced choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on day 0 in the eyes of Brown Norway rats. Intravitreal injection of tanibirumab or phosphate-buffered saline (PBS) was done on day 0 (prevention arm) or day 7 (treatment arm). Seven days after injection, the eyes were enucleated and retinal pigment epithelium-choroid-sclera flat mounts were prepared. Areas of CNV were determined in the flat mounts using tetramethylrhodamine isothiocyanate Bandeiraea simplicifolia (BS) isolectin labeling and intravenously administered fluorescein isothiocyanate-dextran and quantified using an image analysis program. RESULTS: In the prevention arm, the mean area of CNV measured by BS isolectin labeling was reduced by 28.2% and 53.9% in tanibirumab-treated eyes (20 and 60 µg, respectively) compared with PBS-treated control eyes on day 7 (P=0.038 and P<0.001, respectively). In the treatment arm, the mean area of CNV measured by BS isolectin labeling was reduced by 28.7% and 46.0% in tanibirumab-treated eyes (20 and 60 µg, respectively) compared with PBS-treated control eyes on day 14 (P=0.048 and P<0.001, respectively). CONCLUSIONS: Intravitreally administered tanibirumab partially suppressed the formation of new CNV and partially regressed preformed laser-induced CNV in the rat model. Tanibirumab may be a feasible treatment for CNV associated with age-related macular degeneration or other causes.

Stanniocalcin-1 protects retinal ganglion cells by inhibiting apoptosis and oxidative damage.

Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.

Discovery of gliotoxin as a new small molecule targeting thioredoxin redox system.

Thioredoxin redox system has been implicated as an intracellular anti-oxidant defense system leading to reduction of cellular oxidative stresses utilizing electrons from NADPH. From high content screening of small molecules targeting the system, gliotoxin, a fungal metabolite, was identified as an active compound. Gliotoxin potently accelerates NADPH oxidation and reduces H(2)O(2). The compound reduces H(2)O(2) to H(2)O by replacing the function of peroxiredoxin in vitro and decreases intracellular level of H(2)O(2) in HeLa cells. The anti-oxidant activity of gliotoxin was further validated H(2)O(2)-mediated cellular phenotype of angiogenesis. The proliferation of endothelial cells was inhibited by the compound at nanomolar range. In addition, H(2)O(2)-induced tube formation and invasion of the cells were blocked by gliotoxin. Together, these results demonstrate that gliotoxin is a new small molecule targeting thioredoxin redox system.

Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system.

2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.