Self-assembling antimicrobial peptides on nanotubular titanium surfaces coated with calcium phosphate for local therapy.

Bacterial infection is a serious medical problem leading to implant failure. The current antibiotic based therapies rise concerns due to bacterial resistance. The family of antimicrobial peptides (AMP) is one of the promising candidates as local therapy agents due to their broad-spectrum activity. Despite AMPs receive increasing attention to treat infection, their effective delivery to the implantation site has been limited. Here, we developed an engineered dual functional peptide which delivers AMP as a biomolecular therapeutic agent onto calcium phosphate (Ca-P) deposited nanotubular titanium surfaces. Dual functionality of the peptide was achieved by combining a hydroxyapatite binding peptide-1 (HABP1) with an AMP using a flexible linker. HABP functionality of the peptide provided a self-coating property onto the nano-topographies that are designed to improve osteointegration capability, while AMP offered an antimicrobial protection onto the implant surface. We successfully deposited calcium phosphate minerals on nanotubular titanium oxide surface using pulse electrochemical deposition (PECD) and characterized the minerals by XRD, FT-IR, FE-SEM. Antimicrobial activity of the engineered peptide was tested against S. mutans (gram- positive) and E. coli (gram-negative) both in solution and on the Ca-P coated nanotubular titanium surface. In solution activity of AMP and dual functional peptide have the same Minimum Inhibitory Concentration (MIC) (32 mg/mL). The peptide also resulted in the reduction of the number of bacteria both for E.coli and S. mutans compare to control groups on the surface. Antimicrobial features of dual functional peptides are strongly correlated with their structures suggesting tunability in design through linkers regions. The dual-function peptide offers single-step solution for implant surface functionalization that could be applicable to any implant surface having different topographies.

pH-Controlled Cerium Oxide Nanoparticle Inhibition of Both Gram-Positive and Gram-Negative Bacteria Growth.

Here, the antibacterial activity of dextran-coated nanoceria was examined against Pseudomonas aeruginosa and Staphylococcus epidermidis by varying the dose, the time of treatment, and the pH of the solution. Findings suggested that dextran-coated nanoceria particles were much more effective at killing P. aeruginosa and S. epidermidis at basic pH values (pH = 9) compared to acidic pH values (pH = 6) due to a smaller size and positive surface charge at pH 9. At pH 9, different particle concentrations did cause a delay in the growth of P. aeruginosa, whereas impressively S. epidermidis did not grow at all when treated with a 500 µg/mL nanoceria concentration for 24 hours. For both bacteria, a 2 log reduction and elevated amounts of reactive oxygen species (ROS) generation per colony were observed after 6 hours of treatment with nanoceria at pH 9 compared to untreated controls. After 6 hours of incubation with nanoceria at pH 9, P. aeruginosa showed drastic morphological changes as a result of cellular stress. In summary, this study provides significant evidence for the use of nanoceria (+4) for a wide range of anti-infection applications without resorting to the use of antibiotics, for which bacteria are developing a resistance towards anyway.

Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants.

Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property.

Reducing bacteria and macrophage density on nanophase hydroxyapatite coated onto titanium surfaces without releasing pharmaceutical agents.

Reducing bacterial density on titanium implant surfaces has been a major concern because of the increasing number of nosocomial infections. Controlling the inflammatory response post implantation has also been an important issue for medical devices due to the detrimental effects of chronic inflammation on device performance. It has recently been demonstrated that manipulating medical device surface properties including chemistry, roughness and wettability can control both infection and inflammation. Here, we synthesized nanophase (that is, materials with one dimension in the nanoscale) hydroxyapatite coatings on titanium to reduce bacterial adhesion and inflammatory responses (as measured by macrophage functions) and compared such results to bare titanium and plasma sprayed hydroxyapatite titanium coated surfaces used clinically today. This approach is a pharmaceutical-free approach to inhibit infection and inflammation due to the detrimental side effects of any drug released in the body. Here, nanophase hydroxyapatite was synthesized in sizes ranging from 110-170 nm and was subsequently coated onto titanium samples using electrophoretic deposition. Results indicated that smaller nanoscale hydroxyapatite features on titanium surfaces alone decreased bacterial attachment in the presence of gram negative (P. aeruginosa), gram positive (S. aureus) and ampicillin resistant gram-negative (E. coli) bacteria as well as were able to control inflammatory responses; properties which should lead to their further investigation for improved medical applications.

pH-Dependent Activity of Dextran-Coated Cerium Oxide Nanoparticles on Prohibiting Osteosarcoma Cell Proliferation.

Cerium oxide nanoparticles (or nanoceria) have demonstrated great potential as antioxidants in various cell culture models. Despite such promise for reducing reactive oxygen species and an ability for surface functionalization, nanoceria has not been extensively studied for cancer applications to date. Herein, we engineered the surface of nanoceria with dextran and observed its activity in the presence bone cancer cells (osteosarcoma cells) at different pH values resembling the cancerous and noncancerous environment. We found that dextran coated nanoceria was much more effective at killing bone cancer cells at slightly acidic (pH 6) compared to physiological and basic pH values (pH 7 and pH 9). In contrast, minimal toxicity was observed for healthy (noncancerous) bone cells when cultured with nanoceria at pH = 6 after 1 day of treatment in the concentration range of 10-1000 µg/mL. Although healthy bone cancer cell viability decreased after treatment with high ceria nanoparticle concentrations (250-1000 µg/mL) for longer time periods at pH 6 (3 days and 5 days), approximately 2-3 fold higher healthy bone cell viabilities were observed compared to osteosarcoma cell viability at similar conditions. Very low toxicity was observed for healthy osteoblasts cultured with nanoceria for any concentration at any time period at pH 7. In this manner, this study provides the first evidence that nanoceria can be a promising nanoparticle for treating bone cancer without adversely affecting healthy bone cells and thus deserves further investigation.

Controlling self-assembly of engineered peptides on graphite by rational mutation.

Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio-solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range-ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered via simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides via their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio-solid interfaces.

Biofunctionalization of materials for implants using engineered peptides.

Uncontrolled interactions between synthetic materials and human tissues are a major concern for implants and tissue engineering. The most successful approaches to circumvent this issue involve the modification of the implant or scaffold surfaces with various functional molecules, such as anti-fouling polymers or cell growth factors. To date, such techniques have relied on surface immobilization methods that are often applicable only to a limited range of materials and require the presence of specific functional groups, synthetic pathways or biologically hostile environments. In this study we have used peptide motifs that have been selected to bind to gold, platinum, glass and titanium to modify surfaces with poly(ethylene glycol) anti-fouling polymer and the integrin-binding RGD sequence. The peptides have several advantages over conventional molecular immobilization techniques; they require no biologically hostile environments to bind, are specific to their substrates and could be adapted to carry various active entities. We successfully imparted cell-resistant properties to gold and platinum surfaces using gold- and platinum-binding peptides, respectively, in conjunction with PEG. We also induced a several-fold increase in the number and spreading of fibroblast cells on glass and titanium surfaces using quartz and titanium-binding peptides in conjunction with the integrin ligand RGD. The results presented here indicate that control over the extent of cell-material interactions can be achieved by relatively simple and biocompatible surface modification procedures using inorganic binding peptides as linker molecules.

An in vitro evaluation of the Ca/P ratio for the cytocompatibility of nano-to-micron particulate calcium phosphates for bone regeneration.

Calcium phosphate based bioceramics have been widely used for orthopedic applications due to their chemical similarity to natural bone. The Ca/P stoichiometry of calcium phosphates strongly influences their performance under biological conditions, which have not yet been fully elucidated to date. For this reason, the objective of this in vitro study was to understand the relationship between the Ca/P ratio of nano-to-micron particulate calcium phosphate substrates and their biological properties, such as osteoblast (bone-forming cell) viability, collagen production, alkaline phosphatase activity and nitric oxide (NO) production. A group of calcium phosphates with Ca/P ratios between 0.5 and 2.5 were obtained by intentionally adjusting the Ca/P stoichiometry of the initial reactants necessary for calcium phosphate precipitation. For samples with 0.5 and 0.75 Ca/P ratios, tricalcium phosphate (TCP) and Ca(2)P(2)O(7) phases were observed. In contrast, for samples with 1.0 and 1.33 Ca/P ratios, the only stable phase was TCP. For samples with a 1.5 Ca/P ratio, the TCP phase was dominant; however, small amounts of the hydroxyapatite (HA) phase started to appear. For samples with a 1.6 Ca/P ratio, the HA phase was dominant. Lastly, for samples with 2.0 and 2.5 Ca/P ratios, the CaO phase started to appear in the HA phase which was the dominant phase. Moreover, the average grain size and the average pore size decreased from micron-scale (e.g. 1370nm for a 0.5 Ca/P ratio) to nano-scale (e.g. 262nm for a 2.5 Ca/P ratio) with increasing Ca/P ratios. The porosity (%) of calcium phosphate substrates also decreased with increasing Ca/P ratios. Previous in vitro results demonstrated increased osteoblast adhesion on calcium phosphates with higher Ca/P ratios (up to 2.5). The present study showed that the collagen production by osteoblasts was similar between all the calcium phosphates but slightly lower with a 1.6 Ca/P ratio. Greater alkaline phosphatase activity by osteoblasts was observed in all the cultures with various calcium phosphates (0.5-2.5 Ca/P ratios) than in the control (only cells in culture). Ca/P ratios of <2 and 1 optimized osteoblast viability and promoted alkaline phosphatase activity in osteoblasts, respectively. However, the presence of the CaO phase in Ca/P ratios 2.0 increased osteoblast NO production and decreased osteoblast viability. In summary, this study provided evidence that the Ca/P ratio of calcium phosphate is a very important factor that should be considered when selecting nano-to-micron particulate calcium phosphates for various orthopedic applications.

Mechanical properties and biocompatibility of plasma-nitrided laser-cut 316L cardiovascular stents.

The effect of surface modification of laser-cut 316L cardiovascular stents by low-T plasma nitriding was evaluated in terms of mechanical properties and biocompatibility of the stents. The plasma nitriding was performed at 400, 450 or 500 degrees C using various ratios of nitrogen-hydrogen gas mixtures. The flexibility and radial strength were measured in crimped and expanded state of the stents, respectively. The mechanical properties could be adjusted and improved by plasma nitriding conducted at temperatures lower than 450 degrees C and/or nitrogen content less than 10% in the treatment gas. An osteoblast cell culture model system was utilized to investigate the effect of plasma nitriding of the stents on the biological response towards the stents, using biological criteria such as cell viability, alkaline phosphatase and nitric oxide production. In terms of cell viability and alkaline phosphatase production, the plasma nitriding procedure did not appear to negatively affect the biocompatibility of the 316L steel stents. However, in terms of nitric oxide production that was slightly increased in the presence of the plasma-nitrided stents, an indirect improvement in the biocompatibility could possibly be expected.

BRCA1 and BRCA2 mutations in Turkish familial and non-familial ovarian cancer patients: a high incidence of mutations in non-familial cases.

Ovarian cancer is a clinically important cancer in Turkey. The contribution of BRCA1 and BRCA2 to ovarian cancer in Turkish patients has not previously been described. In this study we investigated the presence of BRCA1 and BRCA2 mutations in 102 consecutively ascertained, hospital-based, ovarian cancer cases. Four out of 15 (26.7%, 95% confidence interval (CI), 7.8%-55.1%) familial cases were found to carry mutations in BRCA1. Thirteen of the 87 (14.9%, 95% CI, 7.5%-22.4%) non-familial cases had BRCA1 and BRCA2 mutations, six in BRCA1, and seven in BRCA2. We have further studied the non-familial ovarian cancer cases to determine which subgroups have a likelihood of carrying clinically important mutations. Our study shows that those Turkish ovarian cancer patients with serous histopathology harbor a high proportion of mutations (12/58, 20.7%, 95% CI, 10.3%-31.1%) compared to all non-familial cases (14.9%) regardless of pathology. Within the serous sub-group, those that were also diagnosed below age 50 have an even greater percentage of mutations (8/28, 28.6%, 95% CI, 11.8%-45.3%). Our findings demonstrate that a substantial proportion of Turkish ovarian cancer patients, both with and without a family history, carry BRCA1 and BRCA2 mutations, demonstrating the importance of BRCA1 and BRCA2 in the development of ovarian cancer in this population.