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.     

Increased osteoblast adhesion on nanograined Ti modified with KRSR

Peptide sequences such as lysine-arginine-serine-arginine (KRSR) selectively bind transmembrane proteoglycans (e.g. heparin sulfate) of osteoblasts (bone-forming cells) and are, therefore, actively being investigated for orthopedic applications. Further, nanophase materials (or materials with grain or particle sizes less than 100 nm) are promising new materials that promote new bone growth more than compared to conventional (that is, micron grain or particle size) materials. To combine the above two promising approaches for improving orthopedic implants, the objective of this in vitro study was to functionalize titanium (Ti) surfaces (both nanophase and conventional) with KRSR peptides and study their osteoblast cell adhesive properties. Materials were characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. Results of this in vitro study provided evidence of increased osteoblast adhesion on nanophase compared to conventional Ti whether functionalized with KRSR or not. Results further showed that the immobilization of KRSR onto Ti (both nanophase and conventional) increased osteoblast adhesion compared to respective nonfunctionalized Ti and those functionalized with the negative control peptide KSRR. Most importantly, osteoblast adhesion on nonfunctionalized nanophase Ti increased compared to conventional Ti functionalized with KRSR. Further, select initial osteoblast adhesion was observed to occur at particle boundaries for any type of nanophase and conventional Ti formulated in this study. In summary, results provided evidence that not only should nonfunctionalized nanophase Ti be further studied for improved orthopedic applications but so should nanophase Ti functionalized with KRSR.     

Increased osteoblast adhesion on nanograined hydroxyapatite and tricalcium phosphate containing calcium titanate

Depending on the coating method utilized and subsequent heat treatments (such as through the use of plasma-spray deposition), inter-diffusion of atomic species across titanium (Ti) and hydroxyapatite (HA) coatings may result. These events may lead to structural and compositional changes that consequently cause unanticipated HA phase transformations which may clearly influence the performance of an orthopedic implant. Thus, the objective of the present in vitro study was to compare the cytocompatibility properties of chemistries that may form at the Ti:HA interface, specifically HA, tricalcium phosphate (TCP), HA doped with Ti, and those containing calcium titanate (CaTiO(3)). In doing so, results of this study showed that osteoblast (bone-forming cells) adhesion increased with greater CaTiO(3) substitutions in either HA or TCP. Specifically, osteoblast adhesion on HA and TCP composites with CaTiO(3) was almost 4.5 times higher than that over pure HA. Material characterization studies revealed that enhanced osteoblast adhesion on these compacts may be due to increasing shrinkage in the unit lattice parameters and decreasing grain size. Although all CaTiO(3) composites exhibited excellent osteoblast adhesion results, Ca(9)HPO(4)(PO(4))(5)OH phase transformation into TCP/CaTiO(3) increased osteoblast adhesion the most; because of these reasons, these materials should be further studied for orthopedic applications.     

Additive effect of RGD coating to functionalized titanium surfaces on human osteoprogenitor cell adhesion and spreading

Titanium-based biomaterials for endosseous implants have found widespread applications in the orthopedic, maxillofacial, and dental domains. Indeed, the surface characteristics such as their chemical modification control considerably the cellular response and, subsequently, the quality and the quantity of new-formed bone around the implant. In this study, human osteoprogenitor (HOP) cell adhesion on different titanium surfaces functionalized with hydroxyapatite (HA), type I collagen, or Arg-Gly-Asp (RGD)-containing peptides is investigated by the quartz crystal resonators and by confocal laser scanning microscopy (CLSM) for the imaging of focal contact formation. Data obtained by quartz crystal resonator technique revealed that RGD-containing peptides alone increase HOP cell adhesion in early time period of culture. Moreover, association of RGD-containing peptides with either type I collagen or with HA layers induces an additive effect on HOP cell adhesion compared to Ti-Coll or Ti-HA. CLSM shows both the area of focal contact by cell unit and the cytoskeleton network organization to differ according to the surfaces. Interestingly, association of RGD-containing peptides with HA layers induces an additive effect on focal contact formation on HOP cells compared to Ti-HA alone. These data confirm that an RGD peptide effect occurs in the early time of culture, which is beneficial for osteoblast to spreading, differentiation, and survival.     

In vivo study of the effect of RGD treatment on bone ongrowth on press-fit titanium alloy implants

Early bone ongrowth is known to increase primary implant fixation and reduce the risk of early implant failure. Arg-Gly-Asp (RGD) peptide has been identified as playing a key role in osteoblast adhesion and proliferation on various surfaces. The aim for this study is to evaluate the effect of RGD peptide coating on the bony fixation of orthopaedic implants, to justify its further evaluation in clinical applications. Sixteen unloaded cylindrical plasma sprayed Ti6Al4V implants coated with cyclic RGD peptide were inserted as press-fit in the proximal tibia of 8 mongrel dogs for 4 weeks. Uncoated control implants were inserted in the contralateral tibia. Results were evaluated by histomorphometry and mechanical push-out test. A significant two-fold increase was observed in bone ongrowth for RGD-coated implants. Also, fibrous tissue ongrowth was significantly reduced for RGD-coated implants. Bone volume was significantly increased in a 0-100 microm zone around the implant. The increased bony anchorage resulted in moderate increases in mechanical fixation as apparent shear stiffness was significantly higher for RGD-coated implants. Increases in median ultimate shear strength and energy to failure were also observed. This study demonstrates that cyclic RGD coating increases early bony fixation of unloaded press-fit titanium implants.     

Collagen-infiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study

Bone-implant interface is critical for the early fixation of orthopedic implants. In this study, porous hydroxyapatite (HA) coatings were prepared through a liquid precursor plasma spraying process and were infiltrated with the collagen, alone and with the additional incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) and RGD peptide (RGD). The results showed significantly improved mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation on collagen-modified HA coatings, partially benefited from the formation of a fibrous network due to the self-reconstitution of collagen on the HA surface. Further enhancements on MSC proliferation and differentiation were generally observed through the additional incorporation of bone morphogenetic protein (BMP) and RGD. The osteoinductive and osteoconductive properties of the collagen/BMP-modified HA coatings were studied in vivo. Clear ectopic bone formation and significantly accelerated bone growth rate (29% increase, p < 0.05) have been observed after 1-month implantation of HA-collagen/rhBMP-2-coated Ti alloy samples into the rabbit muscle and dog femora, respectively. Overall, our results suggest that collagen-modified HA coating surface is a far superior substrate for cell attachment, proliferation, and differentiation, and collagen can be used an efficient carrier for BMP in vivo. Therefore, modification of HA coating with collagen is a simple but effective biomimetic approach to enhancing the osteointegration and early fixation of bone-implant interface.     

In vitro assessment of bioactive coatings for neural implant applications

Recent efforts in our laboratory have focused on developing methods for immobilizing bioactive peptides to low cell-adhesive dextran monolayer coatings and promoting biospecific cell adhesion for biomaterial implant applications. In the current study, this dextran-based bioactive coating technology was developed for silicon, polyimide, and gold, the base materials utilized to fabricate our prototype neural implants. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). We observed that surface-immobilized dextran supported very little cell adhesion in vitro (24-h incubation with serum-supplemented medium) on all base materials. Inactive nonadhesion-promoting Gly-Arg-Ala-Asp-Ser-Pro peptides immobilized on dextran-coated materials promoted adhesion and spreading at low levels not significantly different from dextran-coated substrates. Arg-Gly-Asp (RGD) peptide-grafted surfaces were observed to promote substantial fibroblast and glial cell adhesion with minimal PC12 (neuronal cell) adhesion. In contrast, dextran-coated materials with surface-grafted laminin-based, neurite-promoting Ile-Lys-Val-Ala-Val (IKVAV) peptide promoted substantial neuron cell adhesion and minimal fibroblast and glial cell adhesion. It was concluded that neuron-selective substrates are feasible using dextran-based surface chemistry strategies. The chemical surface modifications could be utilized to establish a stable neural tissue-implant interface for long-term performance of neural prosthetic devices.     

Attachment of hyaluronan to metallic surfaces

Metal implants are in general not compatible with the tissues of the human body, and in particular, blood exhibits a severe hemostatic response. Herein we present results of a technique to mask the surface of metals with a natural biopolymer, hyaluronan (HA). HA has minimal adverse interactions with blood and other tissues, but attachment of bioactive peptides can promote specific biological interactions. In this study, stainless steel was cleaned and then surface-modified by covalent attachment of an epoxy silane. The epoxy was subsequently converted to an aldehyde functional group and reacted with hyaluronan through an adipic dihydrazide linkage, thus covalently immobilizing the HA onto the steel surface. Fluorescent labeling of the HA showed that the surface had a fairly uniform covering of HA. When human platelet rich plasma was placed on the HA-coated surface, there was no observable adhesion of platelets. HA derivatized with a peptide containing the RGD peptide sequence was also bound to the stainless steel. The RGD-containing peptide was bioactive as exemplified by the attachment and spreading of platelets on this surface. Furthermore, when the RGD peptide was replaced with the nonsense RDG sequence, minimal adhesion of platelets was observed. This type of controlled biological activity on a metal surface has potential for modulating cell growth and cellular interactions with metallic implants, such as vascular stents, orthopedic implants, heart valve cages, and more.     

Influence of RGD-loaded titanium implants on bone formation in vivo

Little is known about the ability of peptide-coated surfaces to influence cell responses in vivo. Many studies have demonstrated that peptide-modified surfaces influence cell responses in vitro. Integrins, which bind specifically short peptide sequences, are responsible for these cell responses. In this way, information can be transmitted to the nucleus through several cytoplasmic signaling pathways. The peptide sequence Arg-Gly-Asp (RGD peptide) plays an important role in osteoblast adhesion. The present study was designed to investigate new bone formation in a porous titanium (Ti) fiber mesh implant, which was coated with cyclic RGD peptide. The RGD-Ti implants were inserted into the cranium of a rabbit and were compared with porous titanium fiber mesh disks without RGD sequence (Ti) and with an open control defect. Histologic and histomorphometric examinations were performed 2, 4, and 8 weeks postoperatively. A significant increase in bone formation, or bone ingrowth, was seen in the RGD-Ti group compared with the Ti group after 4 and 8 weeks. All control defects stayed open in all three periods. It was concluded that the use of cyclic RGD peptide in combination with titanium fiber mesh has a positive effect on bone formation in vivo in a rabbit animal model.     

Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD

Better materials are needed to promote bone growth. For this reason, the present study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate compacts functionalized with the arginine-glycine-aspartic acid (RGD) peptide sequence. Crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by a hydrothermal treatment for 2 h at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of conventional HA particles (or those with micron diameters), the aforementioned pressed compacts were sintered at 1,100 degrees C for 2 h. Peptide functionalization was conducted by means of a three step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The three step reaction procedure was characterized by a novel 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) fluorescence technique. For all materials, results showed that the immobilization of the cell adhesive RGD sequence increased osteoblast (bone-forming cell) adhesion compared to those non-functionalized and those functionalized with the noncell adhesive control peptide (RGE) after 4 h. However, surprisingly, results also showed that the adhesion of osteoblasts on non-functionalized amorphous nanoparticulate calcium phosphate was similar to conventional HA functionalized with RGD. Osteoblast adhesion on nanocrystalline HA (unfunctionalized and functionalized with RGD) was below that of the respective functionalized amorphous calcium phosphate but above that of the respective functionalized conventional HA. In this manner, results of this study suggest that decreasing the particulate size into the nanometer regime and reducing crystallinity of calcium phosphate based materials may promote osteoblast adhesion to the same degree as the well-established techniques of functionalizing conventional HA with RGD.     

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.     

Increased osteoblast adhesion on nanograined hydroxyapatite and partially stabilized zirconia composites

To improve the mechanical properties of hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)) for orthopedic applications, numerous investigators have proposed combining HA with high strength materials, specifically zirconia. Despite the fact that, compared to pure HA, it is now well-established that zirconia and HA composites have improved mechanical properties, the cytocompatibility properties of this composite remain largely uninvestigated. For these reasons, the objective of the present in vitro study was to synthesize HA and partially stabilized zirconia composites for osteoblast (bone-forming cell) adhesion assays. Various sintering temperatures and amounts of zirconia in HA composites were used in order to ascertain their influence on osteoblast adhesion. Results demonstrated increased interactions between HA and partially stabilized zirconia, when either higher sintering temperatures (between 900 and 1,300 degrees C for 1 h) or higher zirconia contents (between 10 and 40 wt %) were used during material synthesis. More importantly, greater osteoblast adhesion was measured on HA-zirconia composites sintered either at lower temperatures (specifically, 900 degrees C) or with lower amounts of zirconia added to HA composites (specifically, 10 wt %). Results further indicated that when sintered at lower temperatures the composites possessed smaller nanometer grain sizes with increased surface roughness and a more stable HA phase. For these reasons, this study suggests that to optimize osteoblast adhesion on HA and partially stabilized zirconia composites for orthopedic applications, low sintering temperatures and low amounts of zirconia should be used. This suggests that a delicate balance must be reached between increasing mechanical properties of HA without decreasing osteoblast cytocompatibility properties through zirconia addition.     

Surface coatings for improvement of bone cell materials and antimicrobial activities of Ti implants

Ti surface was modified to simultaneously improve bone cell materials and antimicrobial activities. Titanium surface was first anodized in sodium fluoride and sulfuric acid electrolytic solution to form titania nanotube on the surface to improve the biocompatibility of the surface. Silver was electrodeposited on the titania nanotube surface at 5 V. Silver added titania nanotube surface was tested for compatibility with bone-cell materials interactions using human osteoblast bone cells. The antibacterial effect was studied using Pseudomonas aeruginosa. Our results show that silver-treated titania nanotube surface may provide antibacterial properties to prevent implants against postoperative infections without interference to the attachment and proliferation of bone tissue on titanium, which is commonly used in dental and orthopedic surgical procedures.     

Synthesis, analytical characterization, and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin (RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.     

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.     

Synthesis,analytical characterization,and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin(RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.     

Novel osteoblast-adhesive peptides for dental/orthopedic biomaterials

Next generation dental/orthopedic biomaterials must be designed to enhance and support osteoblast adhesion. The osteoblasts use different ways to adhere, that is, integrin- and proteoglycan-mediated mechanisms. The present study reports on the synthesis and osteoblast-adhesive properties of peptides carrying RGD motifs and of sequences mapped on human vitronectin. Our data suggest that osteoblast adhesion on polystyrene plates modified with a linear peptide, in which the GRGDSP sequence is repeated four times, was significantly higher when compared to the adhesion obtained using branched peptides, interestingly containing the same motif. Osteoblast adhesion assays on acellular bone matrix using this active peptide gave very promising results. We also demonstrated that a novel peptide, carrying the X-B-B-B-X-B-B-X motif (where B is a basic amino acid and X is a nonbasic residue), promotes proteoglycan-mediated osteoblast adhesion more efficiently with respect to the KRSR sequence that was recently proposed as heparan-sulfate binding peptide.     

Silver/hydroxyapatite composite coatings on porous titanium surfaces by sol-gel method

Hydroxyapatite (HA) coatings loaded with nanosilver particles is an attractive method to impart the HA coating with antibacterial properties. Producing Ag/HA coatings on porous Ti substrates have been an arduous job since commonly used line-of-sight techniques are not able to deposit uniform coatings on the inner pore surfaces of the porous Ti. In this study, porous Ti scaffolds with high porosity and interconnected structures were prepared by polymer impregnating method. A sol-gel process was used to produce uniform Ag/HA composite coatings on the surfaces of porous Ti substrates. Ca(NO(3) )(2) ·4H(2) O and P(2) O(5) in an ethyl alcohol based system was selected to prepare the sol, which ensured the homogeneous distribution of Ag in the sol. The characterization revealed that silver particles uniformly distributed in the coatings without agglomeration. High antibacterial ratio (>95%), against E. coli and S. albus was expressed by the silver-containing coatings (Ag/HA 0.8 and 1.6 wt %). The biocompatibility of the Ag/HA 0.8 surfaces was as good as that of pure HA surface, as revealed by culturing osteoblasts on them. The results indicated that Ag/HA 0.8 had the good balance between the biocompatibility and antibacterial properties of the coatings.     

TiO2 nanotubes functionalized with regions of bone morphogenetic protein-2 increases osteoblast adhesion

Titanium (Ti) and its alloys are widely used in orthopedic and dental applications. However, the native TiO2 layer is not bioactive enough to form a direct bond with bone, which sometimes translates into a lack of osseointegration into juxtaposed bone that might lead to long term implant failure. In this study, the 20 amino acid peptide sequence (the so-called "knuckle epitope") of bone morphogenetic protein-2 (BMP-2) was immobilized onto Ti nanotubes created by electrochemical anodization. Further, human osteoblast (bone-forming cell) responses to such anodic Ti oxides functionalized with the BMP-2 knuckle epitope was examined in vitro. Materials were characterized by scanning electron and atomic force microscopy. Results of this in vitro study continued to provide evidence of increased osteoblast adhesion on Ti anodized to possess nanotubes compared to unanodized Ti. However, for the first time, results also showed that the immobilization of the BMP-2 knuckle epitope onto Ti anodized to possess nanotubes increased osteoblast adhesion compared to non-functionalized anodized Ti, anodized Ti functionalized with amine (NH2) groups, and unanodized Ti after 4 h. Results also showed increased osteoblast adhesion on amine terminated anodized Ti compared to respective non-functionalized anodized Ti and unanodized Ti. In summary, results of this in vitro study provided evidence that Ti anodized to possess nanotubes and then further functionalized with the BMP-2 knuckle epitope should be further studied for improved orthopedic applications.     

Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. II. Mechanisms of osteoblast adhesion.

The present in vitro study investigated osteoblast adhesion on hydroxylapatite (HA) doped with either cadmium (Cd), zinc (Zn), magnesium (Mg), or yttrium (Y). Compared with any other dopant tested in the present study, osteoblast adhesion was significantly (p < 0.05) greater on HA doped with Y after 4 h; in addition, osteoblast adhesion increased with concentration (2-7 mol%) of Y in HA. The findings that HA doped with greater amounts of Y adsorbed higher concentrations of calcium and, subsequently, of vitronectin and collagen (proteins known to mediate osteoblast adhesion), but not of albumin, laminin, and fibronectin, may explain the observed enhanced adhesion of osteoblasts on these substrates. Interactions (i.e., adsorption and configuration/bioactivity) of vitronectin and collagen may have been promoted by increased porosity of doped HA. Through doping with Y, the present study provided the first evidence that HA can be synthesized and processed with improved cytocompatibility properties for osteoblast adhesion, and thus offered essential information for the design of novel proactive bioceramics. Proactive bioceramics which elicit specific, timely, and desired responses from surrounding cells and tissues are necessary for improving bonding of orthopaedic/dental implants to juxtaposed bone; such osseointegration will, undoubtedly, enhance implant efficacy.