Cellular Affinity Research Articles

Preparation and cytocompatibility of a novel bismuth aluminate/calcium phosphate cement with high radiopacity.

In a minimally invasive surgery, using a bone cement being radiologically detectable is vital to the success of the procedure and avoiding cement leakage in the early stage. The radiopacity of calcium phosphate cement (CPC) is inadequate, thus limiting its clinic application in this area. In this work, bismuth aluminate (BiA) was employed as a radiopaque agent for CPC. The influences of BiA on physicochemical, radiopaque and in vitro biocompatible properties of CPC were investigated. With the increasing content of BiA, the setting time and the compressive strength of CPC were augmented, while the injectability of the cement pastes was reduced. The radiopacity of CPC was significantly improved by adding more than 6 wt.% BiA. CPC specimens with less than 12 wt.% BiA showed good cellular affinity. Moreover, the CPC containing 6 and 9 wt.% BiA promoted the cell growth and ALP activity of mouse bone marrow mesenchymal stem cells when compared with the control. On the basis of its improved radiopacity and cytocompatibility, the radiopaque CPC with 6 ~ 9 wt.% BiA is expected to be a potential substitute for bone defect restoration via minimally invasive surgery. CPC with bismuth aluminate reveals better radiopacity and cell affinity along with proper physicochemical properties.

Preclinical evaluation of 99mTc direct labeling ZHER2:V2 for HER2 positive tumors imaging.

The present study aimed to label ZHER2:V2 with technetium-99m (99mTc) using a simple method and to evaluate its clinical potential as a diagnostic probe for human epidermal growth factor receptor type 2 (HER2)-positive tumors. The ZHER2:V2 (Affibody molecule of ZHER2:2395-C, which is based on the ZHER2:342 binding sequence with C-terminal engineered cysteine) with C-terminal chelating sequence GGGC was designed and labeled with 99mTc. The 99mTc-ZHER2:V2 labeling efficiency was analyzed. The cellular uptake, retention and binding affinity, and the stability of the probe were examined in vitro. 99mTc-ZHER2:V2 biodistribution analysis and imaging were performed in BALB/c nude mice bearing SKOV3 (HER2-overexpression) xenografts. Furthermore, imaging of the probe was performed in MCF-7 (HER2 low-expression) xenografts. The 99mTc-ZHER2:V2 labeling efficiency was identified as 98.99±0.99% (n=6), and was stable in physiological saline and fresh human serum at 37°C in vitro. The cellular uptake peak of SKOV3 cells at 24 h was 6.15±0.18%, the cellular retention ratio of the probe was 48.58±4.52% at 6 h following interrupted incubation, and ~70% of 99mTc-ZHER2:V2 was membrane bound following 24 h. 99mTc-ZHER2:V2 was blocked by excess amounts of unlabeled ZHER2:V2 in SKOV3 cells. 99mTc-ZHER2:V2 exhibited high distribution (10.07% ID/g) in SKOV3 ×enografts at 6 h following injection. The single photon emission computed tomography (SPECT) imaging revealed clear localization of 99mTc-ZHER2:V2 in the SKOV3 ×enografts at 4 h. However, there was low uptake in MCF-7 tumors on the SPECT images. The SKOV3 ×enograft imaging could be blocked by excess amounts unlabelled ZHER2:V2. 99mTc-ZHER2:V2 is an easy and quick labeling method, with high labeling yields, and radiochemical purity. 99mTc-ZHER2:V2 is a promising probe for the diagnosis of HER2-overexpression tumors and the monitoring of therapy response.

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( Kd = 6-9 nM).

Evaluating the efficacy of the anticancer drug cetuximab by atomic force microscopy.

Cetuximab is a monoclonal antibody that binds to the epidermal growth factor receptor, which is important in the growth of many cancers. However, the biophysical characteristics of cetuximab as an anti-cancer drug remain elusive. In this study, we adopted atomic force microscopy to measure the mechanical properties of cancer cells following cetuximab treatment and the biomechanical properties of cetuximab and epidermal growth factor receptor interactions. Atomic force microscopy can be implemented as a platform for further investigations that target the cellular stiffness and affinity of ligand–receptor as a therapeutic choice.

Structure Reconstruction of LyP-1: Lc(LyP-1) Coupling by Amide Bond Inspires the Brain Metastatic Tumor Targeted Drug Delivery.

The stability and binding affinity of targeting ligands are very important in active targeting drug delivery. Herein we used LyP-1 peptide as a model peptide to investigate chemical-biology-based strategies in the design of peptide ligands for active targeting. LyP-1 is a short peptide cyclized with a disulfide bond. It can specifically bind to tumor cells and tumor lymphatics through the interaction with cell-surface protein p32/gC1qR. Lc(LyP-1), with a same sequence of LyP-1, is coupled by amide bond. It showed better cellular uptake and stability in blood in our previous research. Further, usually d-peptide demonstrates higher stability than l-peptide, and it may contribute to better active targeting ability in vivo. Herein, we designed a retro-inverso isomer of Lc(LyP-1), termed Dc(LyP-1), expecting to inspire brain metastatic tumor targeted drug delivery. However, although Lc(LyP-1) showed lower stability than Dc(LyP-1) in fresh rat bold serum, both the 4T1 cellular uptake capacity (89.20%) and p32 protein binding affinity (7.39 × 10-6) were significantly higher than those (33.41%, 1.37 × 10-5) of Dc(LyP-1). Further, Lc(LyP-1) modified PEG-PLA micelles displayed much higher in vivo distribution in brain metastatic tumor than Dc(LyP-1). All results suggested that Lc(LyP-1) had a better performance than Dc(LyP-1) in brain metastatic tumor-targeted drug delivery.

Designing a multifunctional Ti-2Cu-4Ca porous biomaterial with favorable mechanical properties and high bioactivity

The appropriate combination between bones and implants is an important issue for the long-term stability of biomaterials. Hence, a Ti implant alloy with stable osteointegration and simultaneously low modulus is extremely desirable. Ca possesses superb bioactivity and biodegradability in orthopedic applications. Cu is not only favorable for angiogenesis but also exhibits prolonged antibacterial effects. In our present work, Cu and Ca are thus selected as alloying elements to develop a multifunctional Ti-2Cu-4Ca biomaterial with controllable pore structure through spark plasma sintering (SPS). The developed porous Ti-2Cu-4Ca alloy with interconnected pores, a porosity of 43% ± 5% and an average pore size of 460 μm, exhibits bone-like mechanical properties and enhanced cellular affinity (ROS1728 cells). The favorable cell viability and growth inside the pores of the alloy arise from the rough porous surface and the release of bioactive Ca and Cu ions into the biological environment. Our work provides a strategy to design multifunctional biomaterials and, specifically, the porous Ti-2Cu-4Ca alloy is expected to be a promising bone-implant biomaterial.

Regulating the physicochemical and biological properties in vitro of octacalcium phosphate by substitution with strontium in a large doping range

Octacalcium phosphate (OCP) is a key precursor of biological apatite in bone and teeth with excellent osteoconductive and biodegradable properties for bone regeneration. In this work, strontium was employed for improving the poor bioactivity and osteogenesis of OCP by ionic substitution using a homogeneous precipitation method. Sr was successfully incorporated in the structure within a fairly large doping range, even up to 23.25 mol.%, leading to lattice expansion and preferred growth. Sr-OCP samples with better cellular affinity had significantly enhanced the viability, proliferation and osteogenic differentiation of bone mesenchymal stem cells, compared with control. Dynamically released biological ions were involved in the biological apatite deposition for biomineralization/cellular calcification, and remarkably promoted the osteogenic ability. Results indicated the effective enhancements of Sr substitution on the biocompatibility, bioactivity and osteogenesis of OCP biomaterials, providing a potential strategy for the accounts of ionic substitution in biomaterials and the following bone repair applications.

Abstract 465: Downregulation of miR-193a and its correlation with clinical and pathological behavior of colorectal cancer

Abstract Aim: Deregulation of miR-193a expression was reported to have significant roles in cancer development and progression. This study aimed to investigate the expression pattern and clinicopathological implication of miR-193a-3p in patients with colorectal cancer. Also, the bio-physiological effects of miR-193a in colon cancer cells were examined. Methodology: Fresh frozen tissues from 70 matched colorectal cancers (adenocarcinomas) and adjacent noncancer tissue were prospectively collected with no selection bias. Expression level of miR193a-3p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to detect the expression level of K-ras protein as a predicted downstream target for miR-193a. Restoration of the miR-193a level in the cell lines was achieved by permanent transfection. Multiple functional and immunological assays were performed to analyze miR-193’s cellular implications and target affinity in colon cancer cell lines. Results: Downregulation of miR-193a-3p was noted in the majority of the colorectal cancer tissues when compared to nontumor colorectal tissues (70%, n= 49/70). In addition, high prevalence of miR-193a downregulation was noted in early stage colon cancer tissues and cell lines (SW480) when compared to advanced staged colorectal carcinomas. Immunohistochemical analysis revealed inverse correlation between miR-193a and K-ras expression in which high K-ras protein expression was reported in 28/49 cases with miR-193a downregulation. Analogues findings were also observed in cell lines using Western blot (P value&amp;lt; 0.05). Overexpression of miR-193a in colon cancer cell lines resulted in reduced cell proliferation, increased apoptosis and a less accumulation of cells in the G0-G1 phase cell cycle. Conclusion: The current study confirms the downregulation of miR-193a-3P in colorectal cancer and its tumour suppressor effects in vitro plays a key role in the modulation of K-ras regulated molecular signalling pathways in colon cancers Note: This abstract was not presented at the meeting. Citation Format: Afraa M. Mamoori, Riajul Wahab, Farhadul Islam, Katherine Lee, Robert A. Smith, Vinod Gopalan, Alfred Ky Lam. Downregulation of miR-193a and its correlation with clinical and pathological behavior of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2017-465

The LFA1 ligand, CD54, is not required for the differentiation of TFH cells or germinal center formation

Abstract In germinal centers (GC), B cells undergo cellular proliferation and affinity maturation, and ultimately differentiate into either high-affinity antibody-secreting cells (ASCs) or memory B cells. GC B cells present antigen to T follicular helper (TFH) cells in order to elicit survival and proliferation signals. The integrin, Lymphocyte Function-Associated Antigen 1 (LFA1), is required for this process, as mice lacking LFA1 or treated with LFA1 blocking antibodies fail to form GCs or TFH cells. We hypothesized that the counter-receptor for LFA1, ICAM-1 or CD54, would be similarly required for GC formation and TFH differentiation. To test this hypothesis, we infected CD54-deficient (KO) and wild-type (WT) mice with influenza virus (PR8) and enumerated ASCs and GC B cells on days 7, 9, and 14. We found that KO mice had more GC B cells than WT mice on days 9 and 14, but observed no difference in the number of ASCs or TFH cells. To determine if this result was intrinsic to the B cell lineage, we generated 50:50 (KO:WT) bone marrow chimeras. Following reconstitution, we infected the mice with PR8 and assayed donor-specific GC B cells and ASCs on days 9 and 14. On day 9, we found that GC B cells were highly enriched for KO cells, whereas the ASCs were highly enriched for WT cells. In contrast, TFH cells were represented equally in WT and KO populations. On day 14, we found that GC B cells were still highly enriched for KO cells, whereas there was no significant difference between WT and KO ASCs at this time. These results lead us to the surprising conclusion that, although the loss of LFA1 leads to the failure of TFH and GC responses, the loss of CD54 does not affect the TFH compartment and actually improves GC B cell responses.

Rapid fabrication of function-structure-integrated NiTi alloys: Towards a combination of excellent superelasticity and favorable bioactivity

Porous NiTi has brought new expectations to the field of orthopaedic implants due to its excellent mechanical properties such as high strength and superelasticity together with good biocompatibility. In order to facilitate the surrounding bone tissue ingrowth into the implanted porous alloy, reasonably large sized pores and a high amount of porosity are required. There is, however, a major challenge for clinical applications: the higher the porosity, the worse are the mechanical properties and the superelasticity. In this work, therefore, function-structure-integrated NiTi alloys consisting of a central solid and an outer porous layer were fabricated by spark plasma sintering (SPS). When sintered at 750 °C, the NiTi alloy with 14% porosity in the inner part and 49% porosity as well as 350 μm average pore size in the outer layer exhibits an exceptionally high compressive strength (∼1375 MPa), together with an excellent superelastic recovery strain (>4%) and favorable cellular affinity (ROS1728 osteoblasts). Altogether, this work provides a strategy to design materials with function-structure integration and suggests that properly designed function-structure integrated NiTi alloys may be promising as advanced bone implants.

Cellular characteristics and growth behavior of iron‐limited Microcystis aeruginosa in nutrient‐depleted and nutrient‐replete chemostat systems

Cellular responses of Fe-limited Microcystis aeruginosa were investigated under nutrient-depleted and -replete conditions. Cellular growth, Fe quota and Fe uptake kinetics were examined in chemostat systems using nutrient-replete Fraquil* (where all nutrients except for Fe are present at sufficient level to achieve optimal growth) and nutrient-deplete Fraquil* (where some nutrients in addition to Fe are potentially growth-limiting factors). For both nutrient conditions, cellular Fe quota increased with increasing dilution rate in a manner consistent with Droop theory. However, the Fe quota in nutrient-deplete Fraquil* was determined to be lower, indicating lower cellular Fe requirement in the nutrient-depleted condition. Short-term Fe uptake assays indicated that cells acclimated in nutrient-replete conditions adjust to various degrees of Fe stress by solely increasing maximum Fe uptake rate, consistent with expected negative feedback regulation. In contrast, the maximum Fe uptake rate decreased with increasing degree of Fe limitation in the nutrient-depleted chemostat (particularly nitrate and molybdenum in this study). This non-negative feedback regulation is likely associated with lower Fe requirement for specific functions (e.g., intracellular nitrate reduction). Cellular affinity for Fe uptake and cellular size were independent of degree of Fe stress for both nutrient conditions.

Protease-Resistant and Cell-Permeable Double-Stapled Peptides Targeting the Rab8a GTPase.

Small GTPases comprise a family of highly relevant targets in chemical biology and medicinal chemistry research and have been considered "undruggable" due to the persisting lack of effective synthetic modulators and suitable binding pockets. As molecular switches, small GTPases control a multitude of pivotal cellular functions, and their dysregulation is associated with many human diseases such as various forms of cancer. Rab-GTPases represent the largest subfamily of small GTPases and are master regulators of vesicular transport interacting with various proteins via flat and extensive protein-protein interactions (PPIs). The only reported synthetic inhibitor of a PPI involving an activated Rab GTPase is the hydrocarbon stapled peptide StRIP3. However, this macrocyclic peptide shows low proteolytic stability and cell permeability. Here, we report the design of a bioavailable StRIP3 analogue that harbors two hydrophobic cross-links and exhibits increased binding affinity, combined with robust cellular uptake and extremely high proteolytic stability. Localization experiments reveal that this double-stapled peptide and its target protein Rab8a accumulate in the same cellular compartments. The reported approach offers a strategy for the implementation of biostability into conformationally constrained peptides while supporting cellular uptake and target affinity, thereby conveying drug-like properties.

Positive feedback during sulfide oxidation fine-tunes cellular affinity for oxygen

Sulfide (H2S in the gas form) is the third gaseous transmitter found in mammals. However, in contrast to nitric oxide (NO) or carbon monoxide (CO), sulfide is oxidized by a sulfide quinone reductase and generates electrons that enter the mitochondrial respiratory chain arriving ultimately at cytochrome oxidase, where they combine with oxygen to generate water. In addition, sulfide is also a strong inhibitor of cytochrome oxidase, similar to NO, CO and cyanide. The balance between the electron donor and the inhibitory role of sulfide is likely controlled by sulfide and oxygen availability. The present study aimed to evaluate if and how sulfide release and oxidation impacts on the cellular affinity for oxygen. Results: i) when sulfide delivery approaches the maximal sulfide oxidation rate cells become exquisitely dependent on oxygen; ii) a positive feedback makes the balance between sulfide-releasing and -oxidizing rates the relevant parameter rather than the absolute values of these rates, and; iii) this altered dependence on oxygen is detected with sulfide concentrations that remain in the low micromolar range. Conclusions: i) within the context of continuous release of sulfide stemming from cellular metabolism, alterations in the activity of the sulfide oxidation pathway fine-tunes the cell's affinity for oxygen, and; ii) a decrease in the expression of the sulfide oxidation pathway greatly enhances the cell's dependence on oxygen concentration.

High‐Performance Multifunctional Graphene‐PLGA Fibers: Toward Biomimetic and Conducting 3D Scaffolds

The development of electrically conducting fibers based on known cytocompatible materials is of interest to those engaged in tissue regeneration using electrical stimulation. Herein, it is demonstrated that with the aid of rheological insights, optimized formulations of graphene containing spinnable poly(lactic‐co‐glycolic acid) (PLGA) dopes can be made possible. This helps extend the general understanding of the mechanics involved in order to deliberately translate the intrinsic superior electrical and mechanical properties of solution‐processed graphene into the design process and practical fiber architectural engineering. The as‐produced fibers are found to exhibit excellent electrical conductivity and electrochemical performance, good mechanical properties, and cellular affinity. At the highest loading of graphene (24.3 wt%), the conductivity of as‐prepared fibers is as high as 150 S m−1 (more than two orders of magnitude higher than the highest conductivity achieved for any type of nanocarbon‐PLGA composite fibers) reported previously. Moreover, the Young's modulus and tensile strength of the base fiber are enhanced 647‐ and 59‐folds, respectively, through addition of graphene.

Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts

The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs.

Enhanced cellular affinity for poly(lactic acid) surfaces modified with titanium oxide

Poly(lactic acid) (PLA) with titanium oxide (TiOx) composite films were successfully prepared using a simple sol–gel method. Once TiOx was incorporated into the PLA surface, initial cell adhesion and proliferation on the scaffolds were enhanced due to the high biocompatibility of TiOx derivatives.

Detection and isolation of anti-hapten antibody-secreting cells by cellular affinity matrix technology

We developed a method to detect and isolate plasma cells that produce antigen-specific antibodies. An affinity matrix of hapten was constructed on a cell surface, and subsequent incubation allowed cells to secrete antibodies. Anti-hapten antibodies preferentially bound to the affinity matrix on the cells from which they were secreted. We showed that the combination of surface biotinylation and streptavidin which was conjugated with a high valence of hapten was suitable for sensitive detection of antibody binding. Using this protocol, anti-hapten plasma cells from immunized mouse spleen were detected and enriched by flow cytometry. This method allows for isolation of intact plasma cells according to the antibody specificity and may be useful for highly efficient and precise analysis of an antibody repertoire.

Bone marrow-derived mesenchymal cells feature selective migration behavior on submicro- and nano-dimensional multi-patterned substrates

This study investigated whether cells have an intrinsic ability to recognize nanopatterns, which could lead to their accumulation or diminution on a biomaterial. A multi-patterned “biochip” was made, containing 36 differently designed surfaces, including squares and grooves varying in feature sizes between 10 and 1000nm. The grooved patterns could additionally be subdivided into three groups having ridge to groove ratios of 1:1, 1:3 and 3:1. These substrates were used for culture of rat bone marrow derived mesenchymal cells. In time cells should accumulate on patterns of preference, while migrating away from patterns of disfavor. A regression analysis model was designed for the analysis of the obtained data.Results showed that strong differences existed between the tested patterns regarding the cellular affinity. All sizes of squares showed strong cell-repelling capacity, with the biggest sized squares displaying up to 40% less cells compared to the smooth surface. Among the nano-grooved patterns cell repelling was seen for the grooves with the ridge to groove ratio of 1:3, while grooves with the ridge to groove ratio of 3:1 partially showed cell attraction. Such effects were shown to be based on selective migration rather than proliferation.In conclusion, the use of a multi-patterned biochip setup allows for enhanced evaluation of cell behavior, as compared to uniformly patterned setups. Cells exhibit the ability to actively avoid or migrate to surfaces featuring certain topographies on nanometric scale. Such phenomena may be utilized for the development of biomaterials in regenerative medicine.

Hydroxyapatite whisker reinforced 63s glass scaffolds for bone tissue engineering.

Bioactive glass (BG) is widely used for bone tissue engineering. However, poor mechanical properties are the major shortcomings. In the study, hydroxyapatite nanowhisker (HANw) was used as a reinforcement to improve the mechanical properties. 63s glass/HANw scaffolds were successfully fabricated by selective laser sintering (SLS). It was found that the optimal compressive strength and fracture toughness were achieved when 10 wt.% HANw was added. This led to 36% increase in compressive strength and 83% increase in fracture toughness, respectively, compared with pure 63s glass scaffolds. Different reinforcement mechanisms were analyzed based on the microstructure investigation. Whisker bridging and whisker pulling-out were efficient in absorbing crack propagating energy, resulting in the improvement of the mechanical properties. Moreover, bioactivity and biocompatibility of the scaffolds were evaluated in vitro. The results showed that composite scaffolds with 10 wt.% HANw exhibited good apatite-forming ability and cellular affinity.

Long-term antibiotic delivery by chitosan-based composite coatings with bone regenerative potential

Composite coatings with bone-bioactivity and drug-eluting capacity are considered as promising materials for titanium bone implants. In this work, drug-eluting chitosan-bioactive glass coatings were fabricated by a single-step electrophoretic deposition technique. Drug-loading and -releasing capacity of the composite coatings were carried out using the vancomycin antibiotic. Uniform coatings with a thickness of ∼55μm containing 23.7wt% bioactive glass particles and various amounts of the antibiotic (380–630μg/cm2) were produced. The coatings were bioactive in terms of apatite-forming ability in simulated body fluid and showed favorable cell adhesion and growth. In vitro biological tests also indicated that the composite coatings had better cellular affinity than pristine chitosan coatings. The in vitro elution kinetics of the composite coating revealed an initial burst release of around 40% of the drug within the first elution step of 1h and following by a continuous eluting over 4 weeks, revealing long-term drug-delivering potential. Antibacterial tests using survival assay against Gram-positive Staphylococcus aureus bacteria determined the effect of vancomycin release on reduction of infection risk. Almost no bacteria were survived on the coatings prepared from the EPD suspension containing ≥0.5g/l vancomycin. The developed chitosan-based composite coatings with bone bioactivity and long-term drug-delivery ability may be potentially useful for metallic implants to reduce infection risk.