Zhilong Shi

The effect of VEGF functionalization of titanium on endothelial cells in vitro.

One of the key challenges in bone healing and regeneration is the engineering of an implant with surface properties that can enhance revascularization to meet the metabolic demands of recovery. Successful implant integration into the surrounding tissue is highly dependent on the crucial role of blood supply in driving bone repair and development. Therapeutic application of vascular endothelial growth factor (VEGF) is a promising approach to enhance blood supply and healing through revascularization around an engineered implant in a regulated manner. In this in vitro study, we investigated the effects of immobilized VEGF on titanium alloy substrates coated with thin adherent polydopamine film. X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces at various stages of surface functionalization to verify the successful deposition of polydopamine and VEGF on the metal surface. Surface topography was evaluated from the surface profile determined by atomic force microscopy (AFM). The functionalized surfaces showed a significant increase in human dermal microvascular endothelial cells (HDMECs) attachment, viability and proliferation compared to the pristine substrate. Furthermore the immobilized VEGF was able to induce the differentiation of human mesenchymal stem cells (hMSCs) into endothelial cells. Therefore utilizing the reactivity of polydopamine films to immobilize VEGF onto metal substrates may provide a promising approach for application in situations where revascularization around implants would be beneficial in improving bone healing and implant integration.

Surface Functionalization of Titanium with Carboxymethyl Chitosan and Immobilized Bone Morphogenetic Protein-2 for Enhanced Osseointegration

Orthopedic implant failure has been attributed mainly to loosening of the implant from host bone, which may be due to poor bonding of the implant material to bone tissue, as well as to bacterial infection. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that simultaneously enhances bone cell function while decreasing bacterial adhesion. In this in vitro study, the surfaces of titanium alloy substrates were functionalized by first covalently grafting carboxymethyl chitosan (CMCS), followed by the conjugation of bone morphogenetic protein-2 (BMP-2) to the CMCS-grafted surface. Bacterial adhesion on the substrates was assayed with Staphylococcus aureus and Staphylococcus epidermidis . Cell functions were investigated using osteoblasts and human bone marrow-derived mesenchymal stem cells. The results showed that bacterial adhesion on both the CMCS and CMCS-BMP-2 functionalized surfaces was significantly reduced compared to that on the pristine substrates. In addition, the CMCS-BMP-2 modified substrates significantly promoted attachment, alkaline phosphatase activity, and calcium mineral deposition of both osteoblast and human bone marrow-derived mesenchymal stem cells. The achievement of the dual functions of bacterial adhesion reduction and cell function promotion by the CMCS-BMP-2 modified titanium substrates illustrates the good potential of such surfaces for enhancement of tissue integration and implant longevity.

An Investigation on the Antibacterial and Antibiofilm Efficacy of Cationic Nanoparticulates for Root Canal Disinfection

This study aimed to investigate the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. Experiments were performed in two stages. In stage 1, experiments were conducted to examine the physical properties of three types of nanoparticulates. The antibacterial properties of nanoparticulates alone and nanoparticulates mixed with zinc oxide-eugenol-based sealer were studied. In stage 2, the ability of nanoparticulates-treated dentin to prevent bacterial adherence was examined. Zinc oxide nanoparticulates, chitosan nanoparticulates, a mixture of zinc oxide and chitosan nanoparticulates, and zinc oxide nanoparticulates with multilayered coating of chitosan were tested. This study showed that the incorporation of nanoparticulates did not alter the flow characteristics of sealer but improved the direct antibacterial property and the ability to leach out antibacterial components. There was a significant reduction in the adherence of Enterococcus faecalis to nanoparticulates-treated dentin (p < 0.05). These experiments highlighted the potential advantage of nanoparticulates in root canal disinfection.

An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion

The long-term success of orthopedic implants may be compromised by defective osseointegration and bacterial infection. An effective approach to minimize implant failure would be to modify the surface of the implant to make it habitable for bone-forming cells and anti-infective at the same time. In this in vitro study, the surfaces of titanium (Ti) substrates were functionalized by first covalently grafting either dopamine followed by carboxymethyl chitosan (CMCS) or hyaluronic acid-catechol (HAC). Vascular endothelial growth factor (VEGF) was then conjugated to the polysaccharide-grafted surface. Antibacterial assay with Staphylococcus aureus (S. aureus) showed that the polysaccharide-modified substrates significantly decrease bacterial adhesion. The CMCS-functionalized Ti demonstrated better antibacterial property than the HAC-functionalized Ti since CMCS is bactericidal while HA only inhibits the adhesion of bacteria without killing them. Osteoblast attachment, as well as alkaline phosphatase (ALP) activity and calcium deposition were enhanced by the immobilized VEGF on the polysaccharide-grafted Ti. Thus, Ti substrates modified with polysaccharides conjugated with VEGF can promote osteoblast functions and concurrently reduce bacterial adhesion. Since VEGF is also known to enhance angiogenesis, the VEGF-polysaccharide functionalized substrates will have promising applications in the orthopedic field.

Antioxidant and antibacterial activities of eugenol and carvacrol-grafted chitosan nanoparticles.

Essential oils are known to possess antimicrobial and antioxidant activity while chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. In this work, nanoparticles with both antioxidant and antibacterial properties were prepared by grafting eugenol and carvacrol (two components of essential oils) on chitosan nanoparticles. Aldehyde groups were first introduced in eugenol and carvacrol, and the grafting of these oils to chitosan nanoparticles was carried out via the Schiff base reaction. The surface concentration of the grafted essential oil components was determined by X‐ray photoelectron spectroscopy (XPS). The antioxidant activities of the carvacrol‐grafted chitosan nanoparticles (CHCA NPs) and the eugenol‐grafted chitosan nanoparticles (CHEU NPs) were assayed with diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a representative gram‐negative bacterium, Escherichia coli (E. coli) and a gram‐positive bacterium, Staphylococcus aureus (S. aureus). The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component‐grafted chitosan nanoparticles achieved an antibacterial activity equivalent to or better than that of the unmodified chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils. Biotechnol. Bioeng. 2009; 104: 30–39

Titanium with Surface-Grafted Dextran and Immobilized Bone Morphogenetic Protein-2 for Inhibition of Bacterial Adhesion and Enhancement of Osteoblast Functions

Failure of bone and joint implants has been attributed mainly to poor bonding of the implant to bone tissue, and to bacterial infection. The probability of successful osseointegration or implant infection depends on the race for the surface between tissue cells and bacteria. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that increases bone cell (osteoblast) function while decreasing bacterial adhesion. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted oxidized dextran, which is known to have activity against bacterial adhesion. Bone morphogenetic protein-2 (BMP-2) was then covalently linked to dextran-grafted surfaces through a chemical conjugation process. The composition and properties of the surface were investigated by X-ray photoelectron spectroscopy and by measuring the surface density of BMP-2 using an enzyme-linked immunosorbent assay. Bacterial adhesion was assayed with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial adhesion on both the dextran and dextran-BMP-2-functionalized surfaces was significantly decreased compared to that on the pristine substrates. Further, the dextran-BMP-2 modified substrates with a surface protein density of >50 ng/cm(2) or higher significantly promoted osteoblast spreading, alkaline phosphatase activity, and calcium mineral deposition. Thus, the results from this study suggest that surface grafting of dextran in conjunction with the bone growth factor BMP-2 on metal surfaces can enhance tissue integration of implants through the dual functions of reducing bacterial adhesion and promoting osteoblast functions.

(Carboxymethyl)chitosan-Modified Superparamagnetic Iron Oxide Nanoparticles for Magnetic Resonance Imaging of Stem Cells

Magnetic resonance imaging (MRI) is emerging as a powerful tool for in vivo noninvasive tracking of magnetically labeled stem cells. In this work, we present an efficient cell-labeling approach using (carboxymethyl)chitosan-modified superparamagnetic iron oxide nanoparticles (CMCS-SPIONs) as contrast agent in MRI. The CMCS-SPIONs were prepared by conjugating (carboxymethyl)chitosan to (3-aminopropyl)trimethoxysilane-treated SPIONs. These nanoparticles were internalized into human mesenchymal stem cells (hMSCs) via endocytosis as confirmed by Prussian Blue staining and electron microscopy investigation and quantified by inductively coupled plasma mass spectrometry. A MTT assay of the labeled cells showed that CMCS-SPIONs did not possess significant cytotoxicity. In addition, the osteogenic and adipogenic differentiations of the hMSCs were not influenced by the labeling process. The in vitro detection threshold of cells after incubation with 0.05 mg/mL of CMCS-SPIONs for 24 h was estimated to be about 40 cells. The results from this study indicate that the biocompatible CMCS-SPIONs show promise for use with MRI in visualizing hMSCs.

Inhibition of escherichia coli and proteus mirabilis adhesion and biofilm formation on medical grade silicone surface

Silicone has been utilized extensively for biomedical devices due to its excellent biocompatibility and biodurability properties. However, its surface is easily colonized by bacteria which will increase the probability of nosocomial infection. In the present work, a hydrophilic antimicrobial carboxymethyl chitosan (CMCS) layer has been grafted on medical grade silicone surface pre‐treated with polydopamine (PDA). The increase in hydrophilicity was confirmed from contact angle measurement. Bacterial adhesion tests showed that the PDA‐CMCS coating reduced the adhesion of Escherichia coli and Proteus mirabilis by ≥90%. The anti‐adhesion property was preserved even after the aging of the functionalized surfaces for 21 days in phosphate‐buffered saline (PBS), and also after autoclaving at 121°C for 20 min. Both E. coli and P. mirabilis readily form biofilms on the pristine surface under static and flow conditions but with the PDA‐CMCS layer, biofilm formation is inhibited. The flow experiments indicated that it is more difficult to inhibit biofilm formation by the highly motile P. mirabilis as compared to E. coli. No significant cytotoxicity of the modified substrates was observed with 3T3 fibroblasts. Biotechnol. Bioeng. 2012; 109:336–345.

Human bone marrow-derived mesenchymal stem cells and osteoblast differentiation on titanium with surface-grafted chitosan and immobilized bone morphogenetic protein-2

Circulating progenitor cells are known to home to various organs to repair injured tissues or to routinely replace old cells and maintain tissue integrity. Similarly, circulating progenitor bone cells can possibly home to a bone implant, differentiate, and eventually osteointegrate with the prosthesis. Osteointegration of bone cells with the prosthesis can help to reduce the risk of implant failure due to constant movement between bone tissue and implant surface. In this study, we aim to investigate if immobilized bone morphogenetic protein-2 (BMP2) on chitosan-grafted titanium substrate (Ti-CS-BMP2) will enhance bone marrow-derived mesenchymal stem cell (BMMSC) adhesion onto the substrate surface and further induce their differentiation into osteoblasts. The results show that our Ti-CS-BMP2 substrate is able to retain adsorbed BMP2, and is capable of slow release of this growth factor. Despite the lesser number of BMMSCs initially attached onto the Ti-CS-BMP2 substrates and consequently the lower level of cell proliferation, Ti-CS-BMP2 cells had the highest level of ALP activity. RT-PCR results show that Ti-CS-BMP2 cells had a relatively higher level of transcription activity of Runx2, compared with that of bone cell-derived osteoblasts (BC-OB), an indication that the BMMSCs were actively differentiating into osteoblasts. Finally, alizarin red staining reveals the presence of calcium deposits in the differentiated cells. Hence our Ti-CS-BMP2 substrates possess an osteoconductive effect and can possibly be used to fabricate bone implants that can osteointegrate with host bone tissue.

Structural stability and bioapplicability assessment of hyaluronic acid–chitosan polyelectrolyte multilayers on titanium substrates

Since bacterial infections associated with implants remain a major cause of their failure, this study investigated the use of polyelectrolyte multilayers (PEMs) comprising hyaluronic acid (HA) and chitosan (CH) to confer antibacterial properties on titanium (Ti). HA and CH were deposited on Ti using the layer-by-layer deposition method. The antibacterial efficacy of the functionalized Ti substrates was assessed using Escherichia coli and Staphylococcus aureus. The number of adherent bacteria on Ti functionalized with HA and CH PEMs was up to an order of magnitude lower than that on the pristine Ti. The effects of chemical crosslinking of the PEMs on the structural stability and antibacterial efficacy were investigated. The chemical crosslinking of the PEMs imparts greater structural stability and preserves the antibacterial properties even after the prolonged immersion in phosphate-buffered saline. The cytotoxicity of the PEMs to osteoblasts was evaluated using the MTT assay. The results showed that the biocompatible and long-lasting antibacterial nature of the functionalized Ti substrates offers great potential for reducing implant-associated infections.