Bacterial adhesion and osteoblast function on titanium with surface-grafted chitosan and immobilized RGD peptide

Biomaterials-associated infections remain a source of serious complications in modern medicine. When a biomaterial is implanted in the body, the result of successful tissue integration or implant infection depends on the race for the surface between bacteria and tissue cells. One promising strategy to reduce the incidence of infection is the functionalization of the biomaterial surface to inhibit bacterial adhesion and encourage the growth of cells. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted chitosan (CS). The cell-adhesive Arg-Gly-Asp (RGD) peptide was then immobilized on the CS-grafted surface through covalent binding of peptide to the free NH(2) groups of CS. Both these functionalized surfaces showed a decrease in adhesion of Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) compared with the pristine substrate. A significant increase in osteoblast cell attachment, proliferation, and alkaline phosphatase activity was observed on the surface with the immobilized Arg-Gly-Asp peptide. Thus, utilizing surface-grafted chitosan in conjunction with the cell-adhesive peptide to modify the metal surface provides a promising means for enhancing tissue integration of implants by reducing bacterial adhesion and promoting osteoblast functions.     

Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces

The demand for orthopedic and dental implants will continue to grow, and for these applications, titanium and its alloys have been used extensively. While these implants have achieved high success rates, two major complications may be encountered: the lack of bone tissue integration and implant-centered infection. The surface of the implant, through its interactions with proteins, bacteria and tissue cells, plays a determining role in the success or failure of the implant. Ideally, to enhance the success of implants, their surfaces should inhibit bacterial colonization and concomitantly promote osteoblast functions. In this article, we discuss strategies for tailoring implant surfaces by exploiting the differences in the response of bacteria and osteoblasts to proteins and surface structures. Nevertheless, limitations still exist in the quest for an ideal implant surface. Further advances in this field will require concurrent development in surface modification techniques and a better understanding of the complex and highly inter-related events occurring at the implant surface after implantation.     

Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO2

Many engineers and surgeons trace implant failure to poor osseointegration (or the bonding of an orthopedic implant to juxtaposed bone) and/or bacteria infection. By using novel nanotopographies, researchers have shown that nanostructured ceramics, carbon fibers, polymers, metals, and composites enhance osteoblast adhesion and calcium/phosphate mineral deposition. However, the function of bacteria on materials with nanostructured surfaces remains largely uninvestigated. This is despite the fact that during normal surgical insertion of an orthopedic implant, bacteria from the patient's own skin and/or mucosa enters the wound site. These bacteria (namely, Staphylococcus epidermidis) irreversibly adhere to an implant surface while various physiological stresses induce alterations in the bacterial growth rate leading to biofilm formation. Because of their integral role in determining the success of orthopedic implants, the objective of this in vitro study was to examine the functions of (i) S. epidermidis and (ii) osteoblasts (or bone-forming cells) on ZnO and titania (TiO(2)), which possess nanostructured compared to microstructured surface features. ZnO is a well-known antimicrobial agent and TiO(2) readily forms on titanium once implanted. Results of this study provided the first evidence of decreased S. epidermidis adhesion on ZnO and TiO(2) with nanostructured when compared with microstructured surface features. Moreover, compared with microphase formulations, results of this study showed increased osteoblast adhesion, alkaline phosphatase activity, and calcium mineral deposition on nanophase ZnO and TiO(2). In this manner, this study suggests that nanophase ZnO and TiO(2) may reduce S. epidermidis adhesion and increase osteoblast functions necessary to promote the efficacy of orthopedic implants.     

Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion

It would be ideal to have implants which can simultaneously inhibit bacterial adhesion and promote osteoblast functions. In this work, titanium surfaces were modified with poly(methacrylic acid) (P(MAA)) followed by immobilization of silk sericin. Firstly a trichlorosilane coupling agent, which is an atom transfer radical polymerization (ATRP) initiator, was immobilized on the oxidized titanium surface to facilitate the surface-initiated ATRP of methacrylic acid sodium salt (MAAS). The pendant carboxyl end groups of the grafted and partially protonated MAA chains were subsequently coupled with silk sericin via carbodiimide chemistry. The functionalized Ti surfaces were characterized by X-ray photoelectron spectroscopy, and assayed for osteoblast cell functions and bacterial adhesion. The covalently immobilized MAA brushes significantly reduce the adhesion of the two bacterial strains (Staphylococcus aureus and Staphylococcus epidermidis) tested. The silk sericin-immobilized surfaces, at the same time, promote osteoblast cells' adhesion, proliferation, and alkaline phosphatase activity. Thus, the P(MAA) and silk sericin functionalized Ti surfaces have potential applications combating biomaterial-centered infection and promoting osseointegration.     

Bioactive titanium implant surfaces with bacterial inhibition and osteoblast function enhancement properties

Infection in orthopedic implant surgery is a serious complication and a major cause of implant failure. Upon implant insertion, a contest between microbial colonization and tissue integration of the implant surface ensues. This race for the surface determines the probability of tissue integration or infection, and the surface properties of the substrate have an important role to play in determining the outcome. A number of strategies have been developed for the modification of implant surfaces to promote bone cell (osteoblast) functions and inhibit bacterial adhesion and growth. In this article, a review is given of these surface modification strategies, in particular those which can achieve the dual aim of bacterial inhibition and simultaneous enhancement of osteoblast functions.Surfaces of these types can be expected to have excellent potential for orthopedic applications.     

Immobilization strategy for optimizing VEGF's concurrent bioactivity towards endothelial cells and osteoblasts on implant surfaces

Orthopedic implant failure is mainly due to defective osseointegration and bacterial infection. Hence, a promising strategy to overcome these two problems is to functionalize the implant surface with both growth factors (GFs) and anti-infective agents. Covalent immobilization is widely used for such functionalization, but few studies have investigated the possible decrease in the GF's bioactivity as a result of conformational changes upon immobilization. In our study, vascular endothelial growth factor (VEGF) was immobilized on titanium surface via either covalent binding or heparin-VEGF interaction, and its bioactivity on endothelial cells (ECs) was compared. Although a similar surface density of immobilized VEGF was achieved by these two strategies, the bioactivity of the covalently immobilized VEGF on EC functions is significantly lower than that of the heparin-bound VEGF. The heparin-bound VEGF also enhanced mineralization in an osteoblast/endothelial cell co-culture to a much greater extent than in an osteoblast monoculture, illustrating the importance of crosstalk between osteoblasts and endothelial cells. In addition, the surface of the substrates with heparin-bound VEGF is highly hydrophilic and negatively-charged, which significantly inhibits Staphylococcus aureus adhesion. These results suggest that our strategy of immobilizing VEGF on titanium via heparin-VEGF interaction can preserve the GF's bioactivity on both osseous and vascular components and concomitantly reduce bacterial infection, which is promising to enhance the long-term stability of implants.     

A comparison of the adhesion of mammalian cells and Staphylococcus epidermidis on fibronectin-modified polymer surfaces

The modification of polymer surfaces to mimic the extracellular matrix, and therefore, stimulate cell growth via receptor-mediated interactions, is one approach used to promote the integration of tissue-engineering scaffolds and biomaterials into the body. However, by optimizing surfaces for tissue integration it is likely that bacterial adhesion may also be affected, resulting in a greater risk of biomaterial-related infection. This could be detrimental to both the implant and the patient because biomaterial related infections are particularly resistant to host defenses and antibiotics. In this study, we analyzed the adhesion of a Staphylococcus epidermidis clinical isolate and 3T3 rat fibroblasts to tissue culture plastic coated with varying concentrations of fibronectin (Fn). Bacterial adhesion was always lower than tissue culture plastic and appeared to decrease with increasing Fn concentrations. Mammalian cell adhesion to Fn exceeded adhesion to tissue culture plastic but did not differ significantly over the range of protein concentrations or between 1 and 4 h of incubation. In most cases, the total surface coverage did not vary with time or Fn concentration, indicating that maximal cell adhesion and spreading occurred rapidly and at low protein concentrations. This study suggests that, by controlling the density of proteins or ligands on a surface, we can potentially optimize mammalian cell adhesion without stimulating bacterial adhesion, hence reducing the likelihood of infection.     

Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells

Hydrophobins are fungal proteins with the ability to form immunologically inert membranes of high stability, properties that makes them attractive candidates for orthopaedic implant coatings. Cell adhesion on the surface of such implants is necessary for better integration with the neighbouring tissue; however, hydrophobin surfaces do not mediate cell adhesion. The aim of this project was therefore to investigate whether the class I hydrophobin DewA from Aspergillus nidulans can be functionalized for use on orthopaedic implant surfaces. DewA variants bearing either one RGD sequence or the laminin globular domain LG3 binding motif were engineered. The surfaces of both variants showed significantly increased adhesion of mesenchymal stem cells (MSCs), osteoblasts, fibroblasts and chondrocytes; in contrast, the insertion of binding motifs RGD and LG3 in DewA did not increase Staphylococcus aureus adhesion to the hydrophobin surfaces. Proliferation of MSCs and their osteogenic, chondrogenic and adipogenic differentiation potential were not affected on these surfaces. The engineered surfaces therefore enhanced MSC adhesion without interfering with their functionality or leading to increased risk of bacterial infection.     

The influence of BMP-2 and its mode of delivery on the osteoconductivity of implant surfaces during the early phase of osseointegration

Osteogenic agents, such as bone morphogenetic protein-2 (BMP-2), can stimulate the degradation as well as the formation of bone. Hence, they could impair the osteoconductivity of functionalized implant surfaces. We assessed the effects of BMP-2 and its mode of delivery on the osteoconductivity of dental implants with either a naked titanium surface or a calcium-phosphate-coated one. The naked titanium surface bore adsorbed BMP-2, whilst the coated one bore incorporated, adsorbed, or incorporated and adsorbed BMP-2. The implants were inserted into the maxillae of adult miniature pigs. The volume of bone deposited within a defined "osteoconductive" (peri-implant) space, and bone coverage of the implant surface delimiting this space, were estimated morphometrically 1-3 weeks later. After 3 weeks, the volume of bone deposited within the osteoconductive space was highest for coated and uncoated implants bearing no BMP-2, followed by coated implants bearing incorporated BMP-2; it was lowest for coated implants bearing only adsorbed BMP-2. Bone-interface coverage was highest for coated implants bearing no BMP-2, followed by coated implants bearing either incorporated, or incorporated and adsorbed BMP-2; it was lowest for uncoated implants bearing adsorbed BMP-2. Hence, the osteoconductivity of implant surfaces can be significantly modulated by BMP-2 and its mode of delivery.     

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.     

A multifunctional streptococcal collagen-mimetic protein coating prevents bacterial adhesion and promotes osteoid formation on titanium

The major barriers to the clinical success of orthopedic and dental implants are poor integration of fixtures with bone tissue and biomaterial-associated infections. Although multifunctional device coatings have long been considered a promising strategy, their development is hindered by difficulties in integrating biocompatibility, anti-infective activity and antithrombotic properties within a single grafting agent. In this study, we used cell adhesion assays and confocal microscopy of primary murine osteoblasts and human osteoblast cell lines MG-63 and Saos-2 to demonstrate that a streptococcal collagen-like protein engineered to display the α1 and α2 integrin recognition sequences enhances osteoblast adhesion and spreading on titanium fixtures. By measuring calcium deposition and alkaline phosphatase activity, we also showed that selective activation of α2β1 integrin induces osteoblast differentiation, osteoid formation and mineralization. Moreover, cell adhesion assays and scanning electron microscopy of clinical isolates Staphylococcus aureus Philips and Staphylococcus epidermidis 9491 indicated that streptococcal collagen-mimetic proteins inhibit bacterial colonization and biofilm formation irrespective of their interaction with integrins. Given that streptococcal collagenous substrates neither interact with platelets nor trigger a strong immune response, this novel bioactive coating appears to have desirable multifaceted properties with promising translational applications.     

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.     

Osteointegration of titanium implant is sensitive to specific nanostructure morphology

An important aspect of orthopedic implant integration is the enhancement of functional activity of osteoblasts at the tissue-implant interface without any fibrous tissue intervention. Nanostructured implant surfaces are known to enhance osteoblast activity. Previously, we have reported a simple hydrothermal method for the fabrication of non-periodic nanostructures (nanoscaffold, nanoleaves and nanoneedles) on titanium implants showing good biocompatibility and a distinct osteoblast response in vitro in terms of osteoblast adhesion to the surface. In the present work, these nanostructures have been evaluated for their detailed in vitro cellular response as well as in vivo osteointegration. Our studies showed that a specific surface nanomorphology, viz. nanoleaves, which is a network of vertically aligned, non-periodic, leaf-like structures with thickness in the nanoscale, provided a distinct increase in osteoblast cell proliferation, alkaline phosphatase (ALP) activity and collagen synthesis compared to several other types of nanomorphology, such as nanotubes, nanoscaffold and nanoneedles (rods). Gene expression analysis of ALP, osteocalcin, collagen, decorin and Runx2 showed ~20- to 40-fold up-regulation on the leaf-like topography. Cytoskeletal arrangement studies on this substrate again revealed a unique response with favorable intracellular protein expressions of vinculin, FAK and src. In vivo osteointegration study over 12 weeks on rat model (Sprague-Dawley) showed early-stage bone formation (60% bone contact by week 2 and ~85% by week 8, p<0.01) in the leaf-like nanopattern, without any inflammatory cytokine production.     

Biocompatible polymer coating of titania nanotube arrays for improved drug elution and osteoblast adhesion

Bacterial infection, extensive inflammation and poor osseointegration have been identified as the major reasons for [early] orthopaedic implant failures based on titanium. Creating implants with drug-eluting properties to locally deliver drugs is an appealing way to address some of these problems. To improve properties of titanium for orthopaedic applications, this study explored the modification of titanium surfaces with titaniananotube (TNT) arrays, and approach that combines drug delivery into bone and potentially improved bone integration. A titania layer with an array of nanotube structures (∼120 nm in diameter and 50 μm in length) was synthesized on titanium surfaces by electrochemical anodization and loaded with the water-insoluble anti-inflammatory drug indomethacin. A simple dip-coating process of polymer modification formed thin biocompatible polymer films over the drug-loaded TNTs to create TNTs with predictable drug release characteristics. Two biodegradable and antibacterial polymers, chitosan and poly(lactic-co-glycolic acid), were tested for their ability to extend the drug release time of TNTs and produce favourable bone cell adhesion properties. Dependent on polymer thickness, a significant improvement in the drug release characteristics was demonstrated, with reduced burst release (from 77% to >20%) and extended overall release from 4 days to more than 30 days. Excellent osteoblast adhesion and cell proliferation on polymer-coated TNTs compared with uncoated TNTs were also observed. These results suggest that polymer-modified implants with a TNT layer are capable of delivering a drug to a bone site over an extended period and with predictable kinetics. In addition, favourable bone cell adhesion suggests that such an implant would have good biocompatibility. The described approach is broadly applicable to a wide range of drugs and implants currently used in orthopaedic practice.     

In vivo effects of RGD-coated titanium implants inserted in two bone-gap models

RGD (Arg-Gly-Asp) coating has been suggested to enhance implant fixation by facilitating the adhesion of osteogenic cells to implant surfaces. Orthopedic implants are unavoidably surrounded partly by gaps, and these regions represent a challenging environment for osseointegration. We examined the effects of cyclic RGD-coated implants on tissue integration and implant fixation in two cancellous bone-gap models. In canines, we inserted loaded RGD-coated implants with 0.75-mm gap (n = 8) and unloaded RGD-coated implants with 1.5-mm gap (n = 8) into the distal femur and proximal tibia, respectively. Control gap implants without RGD were inserted contralaterally. The titanium alloy (Ti-6Al-4V) implants were plasma sprayed and cylindrical. The observation period was 4 weeks and the fixation was evaluated by push-out test and histomorphometry. Mechanical implant fixation was improved for RGD-coated implants. Unloaded RGD-coated implants showed a significant increase in bone whereas both loaded and unloaded implants showed a significant reduction in fibrous tissue anchorage. The results are encouraging, because RGD had an overall positive effect on the fixation of titanium implants in regions where gaps exist with the surrounding bone. RGD peptide coatings can potentially be used to enhance tissue integration in these challenging environments.     

Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion

Titanium (Ti) and its alloys are used extensively in orthopedic implants due to their excellent biocompatibility and mechanical properties. However, titanium-based implant materials have specific complications associated with their applications, such as the loosening of implant-host interface owing to unsatisfactory cell adhesion and the susceptibility of the implants to bacterial infections. Hence, a surface which displays selective biointeractivity, i.e. enhancing beneficial host cell responses but inhibiting pathogenic microbial adhesion, would be highly desirable. This present study aims to improve biocompatibility and confer long-lasting antibacterial properties on Ti via polyelectrolyte multilayers (PEMs) of hyaluronic acid (HA) and chitosan (CH), coupled with surface-immobilized cell-adhesive arginine-glycine-aspartic acid (RGD) peptide. The HA/CH PEM-functionalized Ti is highly effective as an antibacterial surface but the adhesion of bone cells (osteoblasts) is poorer than on pristine Ti. With additional immobilized RGD moieties, the osteoblast adhesion can be significantly improved. The density of the surface-immobilized RGD peptide has a significant effect on osteoblast proliferation and alkaline phosphatase (ALP) activity, and both functions can be increased by 100-200% over that of pristine Ti substrates while retaining high antibacterial efficacy. Such substrates can be expected to have good potential in orthopedic applications.     

Healing of complement activating Ti implants compared with non-activating Ti in rat tibia

Recent studies have revealed that ozone ultraviolet (UVO) illumination of titanium (Ti) implants improves bone-implant anchorage by altering the physico-chemical and immune activating properties of the titanium dioxide (TiO(2)) layer. In the present rat tibia model, the authors compared the early events of inflammation and bone formation around UVO-treated Ti and complement activating immunoglobin g (IgG)-coated Ti. Machined Ti and machined Ti coated with a physical vapour-deposited Ti layer were used as references. Screw-shaped test and reference implants were implanted into rat tibia and harvested after 1, 7 and 28 days. Messenger RNA expression of implant adhered cells and peri-implant tissue ~250 μm from the surface were subsequently analysed with regard to IL-1β, TNF-α, osteocalcin, cathepsin K, BMP-2 and PDGF. Separate implants were retrieved after 7 and 28 days for removal torque measurements, and histological staining and histomorphometric analysis of bone area and bone-to-implant contact. While enhanced expression of inflammatory markers, TNF-α and IL-1β, was observed on IgG-coated surfaces throughout the observation time, UVO-treated surfaces indicated a significantly lower early inflammatory response. In the early phases (1 and 7 days), the UVO-treated surfaces displayed a significantly higher expression of osteoblast markers BMP-2 and osteocalcin. In summary, complement activating Ti implants elicited a stronger inflammatory response than UVO-treated Ti, with low complement activation during the first week of healing. In spite of this, the UVO-treated Ti induced only marginally more bone growth outside the implants.     

Titanium crystal orientation as a tool for the improved and regulated cell attachment

Cell adhesion is a fundamental process that controls cell proliferation, migration, and differentiation and is crucial for biomaterial-tissue integration. Osteoblast attachment on the surfaces of implant materials is, therefore, essential for the proper function of any implant in which osseointegration is required. Although many reports are available on osteoblast attachment using different surface modification, there is no specific report, so far, that investigates the effect of atomic order of specific crystallographic orientation of substrates on cell behavior. A novel coculture system is proposed to show the differential response of preosteoblast and fibroblast cell lines to the titanium single-crystal substrates. Our investigation has shown that surface recognition by the cell is influenced by the atomic structure of the surface leading to cell-type-specific adhesion. The degree of preosteoblast attachment is significantly higher on the Ti-(1120), whereas the fibroblast adhesion is increased on the Ti-(1010). This demonstrates that the three distinct faces of titanium substrates differ greatly in their capacity to serve as cell adhesive substrates. It also provides clear evidence for the role of crystal structure in regulating and improving cell-substrate interactions relevant for the optimal function of bone implant materials.     

Primary human marrow stromal cells and Saos-2 osteosarcoma cells use different mechanisms to adhere to hydroxylapatite

One important step in bone formation on hard tissue implants is adhesion of osteoblast precursors to the implant surface. In this study, we used function-blocking antibodies against integrin subunits to characterize the mechanisms used by human marrow stromal cells and Saos-2 osteosarcoma cells to adhere to protein-coated hydroxylapatite (HA). We found that Saos-2 use both alpha5- and alphav-containing integrins, whereas stromal cells use alphav-containing integrins but not alpha5-containing integrins, despite the presence of alpha5-containing integrins on cell surfaces. On the basis of this difference, we examined binding of these cell types to HA coated with fibronectin (FN) or vitronectin (VN), to determine whether these ligands for alpha5 and alphav integrins could enhance the numbers or morphology of cells adhered to them. We also examined the adhesion of cells to HA coated with RGD peptides designed to bind to FN or VN receptors. Morphology and number of adherent stromal cells were markedly enhanced on serum-coated surfaces compared with FN or VN alone, whereas, surprisingly, Saos-2 cells failed to spread on serum-coated HA and displayed superior spreading and stress fiber formation on FN-coated [corrected] HA. Collectively, these results have important implications for the design of protein coatings to enhance the performance of HA implants.