Contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium

Chitosan, a derivative of the bio-polysaccharide chitin, has shown promise as a bioactive material for implant, tissue engineering and drug-delivery applications. The aim of this study was to evaluate the contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium. Rough ground titanium (Ti) coupons were solution cast and bonded to 91.2% de-acetylated chitosan (1 wt% chitosan in 0.2% acetic acid) coatings via silane reactions. Non-coated Ti was used as controls. Samples were sterilized by ethylene oxide gas prior to experiments. Contact angles on all surfaces were measured using water. 5 x 10(4) cells/ml of ATCC CRL 1486 human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, were used for the cell attachment study. SEM evaluations were performed on cells attached to all surfaces. Contact angles and cell attachment on all surfaces were statistically analyzed using ANOVA. The chitosan-coated surfaces (76.4 +/- 5.1 degrees) exhibited a significantly greater contact angle compared to control Ti surfaces (32.2 +/- 6.1 degrees). Similarly, chitosan-coated surfaces exhibited significantly greater (P < 0.001) albumin adsorption, fibronectin adsorption and cell attachment, as compared to the control Ti surfaces. Coating chitosan on Ti surfaces decreased the wettability of the Ti, but increased protein adsorption and cell attachment. Increased protein absorption and cell attachment on the chitosan-coated Ti may be of benefit in enhancing osseointegration of implant devices.     

Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants

Chitosan is a biopolymer that exhibits osteoconductive, enhanced wound healing and antimicrobial properties which make it attractive for use as a bioactive coating to improve osseointegration of orthopaedic and craniofacial implant devices. Coatings made from 91.2% de-acetylated chitosan were chemically bonded to titanium coupons via silane-glutaraldehyde molecules. The bond strength of the coatings was evaluated in mechanical tensile tests, and their dissolution and cytocompatibility were evaluated in vitro using cell-culture medium and UMR 106 osteoblastic cells, respectively. The results showed that the chitosan coatings were chemically bonded to the titanium substrate and that the bond strengths (1.5-1.8 MPa) were not affected by gas sterilization. However, the chitosan bond strengths were less than those reported for calcium-phosphate coatings. The gas-sterilized coatings exhibited little dissolution over 8 weeks in cell-culture solution, and the attachment and growth of the UMR 106 osteoblast cells was greater on the chitosan-coated samples than on the uncoated titanium. These results indicated that chitosan has the potential to be used as a biocompatible, bioactive coating for orthopaedic and craniofacial implant devices.     

Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants

Chitosan is a biopolymer that exhibits osteoconductive, enhanced wound healing and antimicrobial properties which make it attractive for use as a bioactive coating to improve osseointegration of orthopaedic and craniofacial implant devices. Coatings made from 91.2% de-acetylated chitosan were chemically bonded to titanium coupons via silane-glutaraldehyde molecules. The bond strength of the coatings was evaluated in mechanical tensile tests, and their dissolution and cyto-compatibility were evaluated in vitro using cell-culture medium and UMR 106 osteoblastic cells, respectively. The results showed that the chitosan coatings were chemically bonded to the titanium substrate and that the bond strengths (1.5-1.8 MPa) were not affected by gas sterilization. However, the chitosan bond strengths were less than those reported for calcium-phosphate coatings. The gas-sterilized coatings exhibited little dissolution over 8 weeks in cell-culture solution, and the attachment and growth of the UMR 106 osteoblast cells was greater on the chitosan-coated samples than on the uncoated titanium. These results indicated that chitosan has the potential to be used as a biocompatible, bioactive coating for orthopaedic and craniofacial implant devices.     

Protein adsorption on titanium surfaces and their effect on osteoblast attachment

The objective of this study was to investigate the adsorption of albumin and fibronectin on titanium (Ti) surfaces and the effect of preadsorbed albumin and fibronectin on osteoblast attachment in vitro. Bovine serum albumin and bovine fibronectin were used in this study. Maximum adsorption of bovine serum albumin and fibronectin on Ti surfaces was observed to occur after 180-min incubation. In the presence of preadsorbed proteins, osteoblast attachment on Ti surfaces was observed to be enhanced compared to control Ti surfaces. However, cell attachment was affected by the types of protein adsorbed. Preadsorbed albumin was observed to have no significant effect on the amount of osteoblast cells attached. In comparison to control Ti surface and Ti surfaces preadsorbed with albumin, Ti surfaces preadsorbed with fibronectin for 15 min was observed to significantly increase osteoblast cell attachment, whereas Ti surfaces preadsorbed with fibronectin for 180 min did not affect cell attachment. In addition, cell morphology of the attached cells on protein preadsorbed Ti surfaces was not affected by the type of protein used in this study. It was concluded from this study that the concentration of fibronectin adsorbed on Ti surfaces was higher compared to albumin. In addition, it was also concluded that the concentration of fibronectin on Ti surfaces plays a role in governing cell attachment.     

Osteoblast response to phospholipid modified titanium surface

The objective of this study was to evaluate the effect of different phospholipid coatings on osteoblast responses in vitro. Commercially available phospholipids [phosphatidylcholine (PC), phosphatidyl-serine (PS) and phosphatidylinositol (PI)] were converted to their Ca-PL-PO(4) and were coated on commercially pure titanium (Ti) grade 2 disks. Using uncoated Ti surfaces as controls, cell responses to phospholipid-coated surfaces were evaluated using the American Type Culture Collection (Manassas, VA, USA) CRL-1486 human embryonic palatal mesenchyme cells (HEPM), an osteoblast precursor cell line, over a 14-day period. Total protein synthesis and alkaline phosphatase specific activity at 0, 7, and 14 days were measured. It was observed that Ti surfaces coated with PS exhibited enhanced protein synthesis and alkaline phosphatase specific activity compared to other phospholipids and uncoated surfaces. These results indicate the possible usefulness of PS-coated Ti surfaces for inducing enhanced bone formation and are very encouraging for bone and dental implantology.     

Use of polyelectrolyte thin films to modulate osteoblast response to microstructured titanium surfaces

The microstructure and wettability of titanium (Ti) surfaces directly impact osteoblast differentiation in vitro and in vivo. These surface properties are important variables that control initial interactions of an implant with the physiological environment, potentially affecting osseointegration. The objective of this study was to use polyelectrolyte thin films to investigate how surface chemistry modulates response of human MG63 osteoblast-like cells to surface microstructure. Three polyelectrolytes, chitosan, poly(L-glutamic acid), and poly(L-lysine), were used to coat Ti substrates with two different microtopographies (PT, Sa = 0.37 μm and SLA, Sa = 2.54 μm). The polyelectrolyte coatings significantly increased wettability of PT and SLA without altering micron-scale roughness or morphology of the surface. Enhanced wettability of all coated PT surfaces was correlated with increased cell numbers whereas cell number was reduced on coated SLA surfaces. Alkaline phosphatase specific activity was increased on coated SLA surfaces than on uncoated SLA whereas no differences in enzyme activity were seen on coated PT compared to uncoated PT. Culture on chitosan-coated SLA enhanced osteocalcin and osteoprotegerin production. Integrin expression on smooth surfaces was sensitive to surface chemistry, but microtexture was the dominant variable in modulating integrin expression on SLA. These results suggest that surface wettability achieved using different thin films has a major role in regulating osteoblast response to Ti, but this is dependent on the microtexture of the substrate.     

Effects of electrodeposited poly(ethylene glycol) on biofilm adherence to titanium

Protein-resistant coatings have been studied for inhibiting biofilm formation on implant devices. In this study, titanium (Ti) surfaces were biofunctionalized with poly(ethylene glycol) (PEG) by electrodeposition and were evaluated as biofilm substrates under an oral simulated environment. Streptococcus gordonii, an early colonizer of oral biofilms, was inoculated on Ti and PEG-electrodeposited Ti (PEG-Ti) surfaces and was analyzed quantitatively and topographically. Streptococcus mutans supplemented with sucrose, a late colonizer mainly found in dental plaque, was also used to form biofilms on the surfaces of Ti and PEG-Ti for 20 h followed by sonication as a means of detaching the biofilms. The results indicated that the attachment of S. gordonii on PEG-Ti surfaces was inhibited compared with Ti, and the S. mutans biofilm was easier to be detached from the surface of PEG-Ti than that of Ti. Moreover, the presence of PEG electrodeposited on Ti surface inhibited salivary protein adsorption. The degree of detachment of biofilms from PEG-Ti was associated with the inhibition of the salivary protein adsorption, suggesting weak basal attachment of the biofilms to the electrodeposited surfaces. Therefore, controlling protein adsorption at the initial stage of biofilm formation may be an effective strategy to protect metal surfaces from bacterial contamination not only in dental manipulations but also in orthopedic applications.     

Attachment,Spreading, and Adhesion Strength of Human Bone Marrow Cells on Chitosan

The successful integration of an orthopedic implant into bone depends on the mechanisms at the tissue–implant interface and mostly on the osteoblast attachment phenomenon. Chitosan has emerged as an attractive biomacromolecule favoring osseointegration. In this study highly deacetylated chitosan coatings, with roughness of about 1 nm, were bonded to glass surfaces via silane–glutaraldehyde molecules. Human osteoblasts were used to study the development of attachment during the first 60 min. Chitosan favored the number of the attached cells compared to the uncoated surfaces for 30 min seeding time (t _s). For t _s up to 60 min the attached cell area was almost 210% significantly higher on the chitosan surfaces, indicating an enhanced spreading process. To determine the cell attachment strength, a micropipette aspiration method was used, where the value of the term I = ∫Fdt is representative of the single cell attachment–adhesion procedure and quantitatively reflects the strength evolution during attachment: F equals the detaching force applied on the cell. The results showed higher strength values on the chitosan surfaces. The findings reinforce the favorable environment of the biomacromolecule for the osteoblast and the new approach regarding the quantitatively evaluation of adhesion provides important contribution for the study of cell–material interaction, especially during the crucial first phase of cell attachment.     

Peptide-modified p(AAm-co-EG/AAc) IPNs grafted to bulk titanium modulate osteoblast behavior in vitro

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) (p(AAm-co-EG/AAc) applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cell-binding domain from rat bone sialoprotein (BSP) modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar p(AAm-co-EG/AAc) IPNs on clinically relevant titanium surfaces. Contact angle goniometry and X-ray photoelectron spectroscopy (XPS) data were consistent with the presence of the intended surface modifications. Cellular response was gauged by challenging the surfaces with primary rat calvarial osteoblast (RCO) surfaces in serum-containing media. IPN modified titanium and negative control (RGE-IPN) surfaces inhibit cell adhesion and proliferation, while RGD-modified IPNs on titanium supported osteoblast attachment and spreading. Furthermore, the latter surfaces supported significant mineralization despite exhibiting lower levels of proliferation than positive control surfaces. These results suggest that with the appropriate optimization, this approach may be practical for surface engineering of osseous implants.     

Antibacterial and osteogenic properties of silver-containing hydroxyapatite coatings produced using a sol gel process

Since bacterial infection is a rising complication following the wide use of implant, there is considerable attention on the effect of implant surface properties on bacterial adhesion. In this study, the effect of silver (Ag) doped hydroxyapatite (HA) coatings on initial antibacterial adhesion and osteoblast cell proliferation and differentiation was investigated. Using a sol-gel process, HA coatings doped with 1 wt % AgNO(3) (AgHA1.0) and 1.5 wt % Ag (AgHA1.5) were prepared. Coated surfaces were characterized using X-ray diffraction (XRD) and contact angles measurements. The initial bacteria adhesion was evaluated using a RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus, whereas osteoblast proliferation and differentiation were evaluated using human embryonic palatal mesenchyme cells (HEPM), an osteoblast precursor cell line. In this study, XRD analysis of all surfaces indicated peaks corresponding to HA. Contact angles for AgHA surfaces were observed to be significantly lower when compared to HA surfaces. In vitro initial bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on AgHA surfaces when compared to HA surface. The use of HEPM cells indicated no significant difference in double-stranded DNA (dsDNA) production between all surfaces. Additionally, no differences in alkaline phosphatase specific activity were observed between HA and AgHA1.0 surfaces. Overall, it was concluded that AgHA1.0 has the similar biological activity as HA, with respect to bone cell proliferation and differentiation. In addition, the AgHA1.0 was also concluded to have the ability to minimize the initial bacteria adhesion. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.     

In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating

Bacterial infection after implant placement is a significant rising complication. In order to reduce the incidence of implant-associated infections, several biomaterial surface treatments have been proposed. In this study, the effect of in vitro antibacterial activity and in vitro cytotoxicity of co-sputtered silver (Ag)-containing hydroxyapatite (HA) coating was evaluated. Deposition was achieved by a concurrent supply of 10 W to the Ag target and 300 W to the HA target. Heat treatment at 400 degrees C for 4 h was performed after 3 h deposition. X-ray diffraction, contact angles measurements, and surface roughness were used to characterize the coating surfaces. The RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus were used to evaluate the antibacterial activity of the Ag-HA coatings, whereas human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the in vitro cytotoxicity of the coatings. X-ray diffraction analysis performed in this study indicated peaks corresponding to Ag and HA on the co-sputtered Ag-HA surfaces. The contact angles for HA and Ag-HA surfaces were observed to be significantly lower when compared to Ti surfaces, whereas no significant difference in surface roughness was observed for all groups. In vitro bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on Ag-HA surface when compared to titanium (Ti) and HA surfaces. In addition, no significant difference in the in vitro cytotoxicty was observed between HA and Ag-HA surfaces. Overall, it was concluded that the creation of a multifunctional surface can be achieved by co-sputtering the osteoconductive HA with antibacterial Ag.     

The response of osteoblast-like cells towards collagen type I coating immobilized by p-nitrophenylchloroformate to titanium

The scaffold surface composition can be altered by the use of surface coatings. The use of thin coatings will give special surface properties, while the bulk properties of the scaffold are preserved. Collagen type I is known to play an important role during cell adhesion as well as osteoblast differentiation. A common way to coat surfaces is the adsorption method. An alternative way is the use of a protein immobilization method like p-nitrophenyl chloroformate. In this study, we investigated the effect of a collagen type I coating and p-nitrophenyl chloroformate as a protein immobilization method on osteoblast adhesion, proliferation, and differentiation. Titanium fiber meshes were treated with sodium hydroxide (NaOH), followed by p-nitrophenyl chloroformate, and coated with collagen type I. Osteoblast-like cells were seeded into the meshes and cultured for 24 days. The cell attachment, proliferation, and differentiation were measured by using Live and Dead assay, cell counting, DNA analysis, alkaline phosphatase activity assay, calcium content measurement, Real Time PCR (QPCR), and scanning electron microscopy (SEM). Results demonstrated that initially less cells were attached to the covalently bounded collagen meshes (NPC-Col) compared with titanium as control (Ti) and adsorbed collagen meshes (ABS-Col). Further, a decreased growth curve of cells cultured on the NPC-Col meshes was observed in comparison with Ti and ABS-Col meshes. The calcium measurements and SEM pictures revealed that all three surfaces showed differentiation of osteoblast-like cells after 8-24 days. On the basis of our results, we conclude that initially less cells were attached to the NPC-Col meshes and that they had a decreased proliferation rate. Further, we conclude that an adsorbed collagen type I coating stimulated the osteoblastic differentiation of rat bone marrow cells.     

The effect of polyelectrolyte multilayer coated titanium alloy surfaces on implant anchorage in rats

Advances have been achieved in the design and biomechanical performance of orthopedic implants in the last decades. These include anatomically shaped and angle-stable implants for fracture fixation or improved biomaterials (e.g. ultra-high-molecular-weight polyethylene) in total joint arthroplasty. Future modifications need to address the biological function of implant surfaces. Functionalized surfaces can promote or reduce osseointegration, avoid implant-related infections or reduce osteoporotic bone loss. To this end, polyelectrolyte multilayer structures have been developed as functional coatings and intensively tested in vitro previously. Nevertheless, only a few studies address the effect of polyelectrolyte multilayer coatings of biomaterials in vivo. The aim of the present work is to evaluate the effect of polyelectrolyte coatings of titanium alloy implants on implant anchorage in an animal model. We test the hypotheses that (1) polyelectrolyte multilayers have an effect on osseointegration in vivo; (2) multilayers of chitosan/hyaluronic acid decrease osteoblast proliferation compared to native titanium alloy, and hence reduce osseointegration; (3) multilayers of chitosan/gelatine increase osteoblast proliferation compared to native titanium alloy, hence enhance osseointegration. Polyelectrolyte multilayers on titanium alloy implants were fabricated by a layer-by-layer self-assembly process. Titanium alloy (Ti) implants were alternately dipped into gelatine (Gel), hyaluronic acid (HA) and chitosan (Chi) solutions, thus assembling a Chi/Gel and a Chi/HA coating with a terminating layer of Gel or HA, respectively. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bones’ response to polyelectrolyte surfaces in vivo. 48 rats were randomly assigned to three groups of implants: (1) native titanium alloy (control), (2) Chi/Gel and (3) Chi/HA coating. Mechanical fixation, peri-implant bone area and bone contact were evaluated by pull-out tests and histology at 3 and 8 weeks. Shear strength at 8 weeks was statistically significantly increased (p < 0.05) in both Chi/Gel and Chi/HA groups compared to the titanium alloy control. No statistically significant difference (p > 0.05) in bone contact or bone area was found between all groups. No decrease of osseointegration of Chi/HA-coated implants compared to non-coated implants was found. The results of polyelectrolyte coatings in a rat model showed that the Chi/Gel and Chi/HA coatings have a positive effect on mechanical implant anchorage in normal bone.     

Initial responses of human osteoblasts to sol-gel modified titanium with hydroxyapatite and titania composition

Sol-gel thin films of hydroxyapatite (HA) and titania (TiO(2)) have received a great deal of attention in the area of bioactive surface modification of titanium (Ti) implants. Sol-gel coatings were developed on Ti substrates of pure HA and TiO(2) and two composite forms, HA+10% TiO(2) and HA+20% TiO(2), and the biological properties of the coatings were evaluated. All the coating layers exhibited thin and homogeneous structures and phase-pure compositions (either HA or TiO(2)). Primary human osteoblast cells showed good attachment, spreading and proliferation on all the sol-gel coated surfaces, with enhanced cell numbers on all the coated surfaces relative to uncoated Ti control at day 1, as observed by MTT assay and scanning electron microscopy. Cell attachment rates were also enhanced on the pure HA coating relative to control Ti. The pure HA and HA+10% TiO(2) composite coating furthermore enhanced proliferation of osteoblasts at 4 days. Moreover, the gene expression level of several osteogenic markers including bone sialoprotein and osteopontin, as measured by RT-PCR at 24h, was shown to vary according to coating composition. These findings suggest that human primary bone cells show marked and rapid early functional changes in response to HA and TiO(2) sol-gel coatings on Ti.     

Anti-biofilm properties of chitosan-coated surfaces

Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria and yeast. Reductions in biofilm viable cell numbers ranging from 95% to 99.9997% were demonstrated for Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans on chitosan-coated surfaces over a 54-h experiment in comparison to controls. For instance, chitosan-coated surfaces reduced S. epidermidis surface-associated growth more than 5.5 (10)log units (99.9997%) compared to a control surface. As a comparison, coatings containing a combination of the antibiotics minocycline and rifampin reduced S. epidermidis growth by 3.9 (10)log units (99.99%) and coatings containing the antiseptic chlorhexidine did not significantly reduce S. epidermidis surface associated growth as compared to controls. The chitosan effects were confirmed with microscopy. Using time-lapse fluorescence microscopy and fluorescent-dye-loaded S. epidermidis, the permeabilization of these cells was observed as they alighted on chitosan-coated surfaces. This suggests chitosan disrupts cell membranes as microbes settle on the surface. Chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection.     

Differential regulation of osteoblasts by substrate microstructural features

Microtextured titanium implant surfaces enhance bone formation in vivo and osteoblast phenotypic expression in vitro, but the mechanisms are not understood. To determine the roles of specific microarchitectural features in modulating osteoblast behavior, we used Ti surfaces prepared by electrochemical micromachining as substrates for MG63 osteoblast-like cell culture. Cell response was compared to tissue culture plastic, a sand-blasted with large grit and acid-etched surface with defined mixed microtopography (SLA), polished Ti surfaces, and polished surfaces electrochemically machined through a photoresist pattern to produce cavities with 100, 30 and 10 microm diameters arranged so that the ratio of the microscopic-scale area of the cavities versus the microscopic-scale area of the flat region between the cavities was equal to 1 or 6. Microstructured disks were acid-etched, producing overall sub-micron-scale roughness (Ra=0.7 microm). Cell number, differentiation (alkaline phosphatase; osteocalcin) and local factor levels (TGF-beta1; PGE(2)) varied with microarchitecture. 100 microm cavities favored osteoblast attachment and growth, the sub-micron-scale etch enhanced differentiation and TGF-beta1 production, whereas PGE(2) depended on cavity dimensions but not the sub-micron-scale roughness.     

Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces

The protein adsorption behavior of thin films of calcium phosphate (CaP) bioceramic and titanium (Ti) was studied in this research. The thin films were produced with an ion beam sputter deposition technique using targets of hydroxyapatite (HA), fluorapatite (FA) and titanium (Ti). Fourier transform infrared spectroscopy (FTIR) with attenuated total internal reflectance (ATR) was used to evaluate protein adsorption on these surfaces. This study showed that surface composition and structure influenced the kinetics of protein adsorption and the structure of adsorbed protein. CaP surfaces adsorbed greater amount of protein than the Ti surface, and caused more alteration of the structure of adsorbed BSA than did the Ti surface. The differences in protein adsorption behavior could result in very different initial cellular behavior on CaP and Ti implant surfaces.     

Reduced medical infection related bacterial strains adhesion on bioactive RGD modified titanium surfaces: a first step toward cell selective surfaces

Ideally, implants should inhibit nonspecific protein adsorption, bacterial adhesion, and at the same time, depending on the final application be selective toward cellular adhesion and spreading for all or only selected cell types. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) polymers have been shown to adsorb from aqueous solution onto negatively charged metal oxide surfaces, reducing protein adsorption as well as fibroblast, osteoblast and epithelial cell adhesion significantly. PLL-g-PEG can be functionalized with bioligands such as RGD (Arg-Gly-Asp), which then restores host cell adhesion, but the surface remains resistant to nonspecific protein adsorption. Previously, it was also shown that both nonfunctionalized PLL-g-PEG and RGD-peptide functionalized PLL-g-PEG reduced the adhesion of Staphylococcus aureus to titanium (Ti) surfaces. The present study looked at the effect of other implant associated infection relevant bacteria, Staphylococcus epidermidis, Streptococcus mutans and Pseudomonas aeruginosa towards the same surface chemistries. The different surfaces were exposed to the bacteria for 1-24 h, and bacteria surface density was evaluated using scanning electron microscopy (SEM) and fluorescence light microscopy (FM). The adhesion of all bacteria strains tested was reduced on Ti surfaces coated with PLL-g-PEG compared to uncoated Ti surfaces even in the presence of RGD. The percentage reduction in bacterial adhesion over the 24-h culture time investigated was 88%-98%, depending on the bacteria type. Therefore, coating surfaces with PLL-g-PEG/PEG-RGD allows cells such as fibroblasts and osteoblasts to attach but not bacteria, resulting in a selective biointeractive pattern that may be useful on medical implants.     

RGDS peptides immobilized on titanium alloy stimulate bone cell attachment, differentiation and confer resistance to apoptosis

A major cause of implant failure in skeletal tissues is failure of osseointegration, often due to lack of adhesion of cells to the titanium (Ti) alloy interface. Since arginine-glycine-aspartic acid (RGD)-containing peptides have been shown to regulate osteoblast adhesion, we tested the hypothesis that, bound to a Ti surface, these peptides would promote osteoblasts differentiation, while at the same time inhibit apoptosis. RGDS and RGES (control) peptides were covalently linked to Ti discs using an APTS linker. While the grafting of both RGDS and RGES significantly increased Ti surface roughness, contact angle analysis showed that APTS significantly increased the surface hydrophobicity; when the peptides were tethered to Ti, this was reduced. To evaluate attachment, MC3T3-E1 osteoblast cells were grown on these discs. Significantly more cells attached to the Ti-grafted RGDS then the Ti-grafted RGES control. Furthermore, expression of the osteoblasts phenotype was significantly enhanced on the Ti-grafted RGDS surface. When cells attached to the Ti-grafted RGDS were challenged with staurosporine, an apoptogen, there was significant inhibition of apoptosis; in contrast, osteoblasts adherent to the Ti-grafted RGES were killed. It is concluded that RGD-containing peptides covalently bonded to Ti promotes osteoblasts attachment and survival with minimal changes to the surface of the alloy. Therefore, such modifications to Ti would have the potential to promote osseointegration in vivo.     

Effect of surface roughness of the titanium alloy Ti-6Al-4V on human bone marrow cell response and on protein adsorption

The effect of surface roughness of the titanium alloy Ti-6Al-4V (Ti alloy) on the short- and long-term response of human bone marrow cells in vitro and on protein adsorption was investigated. Three different values in a narrow range of surface roughness were used for the substrata (R(alpha): 0.320, 0.490 and 0.874 microm). Cell attachment, cell proliferation and differentiation (alkaline phosphatase specific activity) were determined past various incubation periods. The protein adsorption of bovine serum albumin and fibronectin, from single protein solutions, on rough and smooth Ti alloy surfaces was examined with two methods, X-ray photoelectron spectroscopy (XPS) and radiolabeling. Cell attachment and proliferation were surface roughness sensitive and increased as the roughness of Ti alloy increased. No statistically significant difference was observed in the expression of ALP activity on all three Ti alloy surfaces and culture plastic. Both methods, XPS and protein radiolabeling, showed that human serum albumin was adsorbed preferentially onto the smooth substratum. XPS technique showed that the rough substratum bound a higher amount of total protein (from culture medium supplied with 10% serum) and fibronectin (10-fold) than did the smooth one. The cell attachment may be explained by the differential adsorption of the two proteins onto smooth and rough Ti alloy surfaces.