Rolled-Up Metal Oxide Microscaffolds to Study Early Bone Formation at Single Cell Resolution.

Titanium and its alloys are frequently used to replace structural components of the human body due to their high mechanical strength, low stiffness, and biocompatibility. In particular, the use of porous materials has improved implant stabilization and the promotion of bone. However, it remains unclear which material properties and geometrical cues are optimal for a proper osteoinduction and osseointegration. To that end, transparent tubular microscaffolds are fabricated, mimicking the typical pores of structural implants, with the aim of studying early bone formation and cell-material interactions at the single cell level. Here, a ß-stabilized alloy Ti-45Nb (wt%) is used for the microscaffold's fabrication due to its elastic modulus close to that of natural bone. Human mesenchymal stem cell migration, adhesion, and osteogenic differentiation is thus investigated, paying particular attention to the CaP formation and cell-body crystallization, both analyzed via optical and electron microscopy. It is demonstrated that the developed platform is suited for the long-term study of living single cells in an appropriate microenvironment, obtaining in the process deeper insights on early bone formation and providing cues to improve the stability and biocompatibility of current structural implants.

Heparin Enriched-WPI Coating on Ti6Al4V Increases Hydrophilicity and Improves Proliferation and Differentiation of Human Bone Marrow Stromal Cells.

Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure.

Glycosaminoglycan derivatives: promising candidates for the design of functional biomaterials.

Numerous biological processes (tissue formation, remodelling and healing) are strongly influenced by the cellular microenvironment. Glycosaminoglycans (GAGs) are important components of the native extracellular matrix (ECM) able to interact with biological mediator proteins. They can be chemically functionalized and thereby modified in their interaction profiles. Thus, they are promising candidates for functional biomaterials to control healing processes in particular in health-compromised patients. Biophysical studies show that the interaction profiles between mediator proteins and GAGs are strongly influenced by (i) sulphation degree, (ii) sulphation pattern, and (iii) composition and structure of the carbohydrate backbone. Hyaluronan derivatives demonstrate a higher binding strength in their interaction with biological mediators than chondroitin sulphate for a comparable sulphation degree. Furthermore sulphated GAG derivatives alter the interaction profile of mediator proteins with their cell receptors or solute native interaction partners. These results are in line with biological effects on cells relevant for wound healing processes. This is valid for solute GAGs as well as those incorporated in collagen-based artificial ECM (aECMs). Prominent effects are (i) anti-inflammatory, immunomodulatory properties towards macrophages/dendritic cells, (ii) enhanced osteogenic differentiation of human mesenchymal stromal cells, (iii) altered differentiation of fibroblasts to myofibroblasts, (iv) reduced osteoclast activity and (v) improved osseointegration of dental implants in minipigs. The findings of our consortium Transregio 67 contribute to an improved understanding of structure-function relationships of GAG derivatives in their interaction with mediator proteins and cells. This will enable the design of bioinspired, functional biomaterials to selectively control and promote bone and skin regeneration.

Sulfated hyaluronan containing collagen matrices enhance cell-matrix-interaction, endocytosis, and osteogenic differentiation of human mesenchymal stromal cells.

Inorganic-organic composite implant materials mimicking the environment of bone are promising applications to meet the increasing demands on biomaterials for bone regeneration caused by extended life spans and the concomitant increase of bone treatments. Besides collagen type I (Col-I) glycosaminoglycans (GAG), such as hyaluronan, are important components of the bone extracellular matrix (ECM). Sulfated GAGs are potential stimulators of bone anabolic activity, as they are involved in the recruitment of mesenchymal stromal cells (MSCs) to the site of bone formation and support differentiation to osteoblasts. Nevertheless, no consecutive data is currently available about the interaction of hyaluronan or sulfated hyaluronan derivatives with hMSCs and the molecular processes being consequently regulated. We applied quantitative proteomics to investigate the influence of artificial ECM composed of Col-I and hyaluronan (Hya) or sulfated hyaluronan (HyaS3) on the molecular adaptation of osteogenic-differentiated human MSCs (hMSCs). Of the 1,370 quantified proteins, the expression of 4-11% was altered due to both aECM-combinations. Our results indicate that HyaS3 enhanced multiple cell functions, including cell-matrix-interaction, cell-signaling, endocytosis, and differentiation. In conclusion, this study provides fundamental insights into regulative cellular responses associated with HyaS3 and Hya as components of aECM and underlines the potential of HyaS3 as a promising implant-coating-material.

Response of human bone marrow stromal cells, MG-63, and SaOS-2 to titanium-based dental implant surfaces with different topography and surface energy.

OBJECTIVES: Osseointegration is dependent on different parameters of the implant surface like surface roughness and physicochemical properties. In vitro studies using a wide variety of surface parameters and cell lines make it difficult to address the influence of a single parameter. With this study the influence of surface topography and energy on different osteoblast derived cell lines, namely MG-63 and SaOS-2 and of human mesenchymal stromal cells (hMSC) were investigated. MATERIAL AND METHODS: Cells were cultured on polished (POL) and sandblasted/hot acid etched (SBA) titanium surfaces which were partly alkaline treated (SBA NaOH). Cell morphology, metabolic activity, tissue non-specific alkaline phosphatase (TNAP) activity and prostaglandin E(2) (PGE(2) ) formation were determined. RESULTS: Impaired spreading was found on both SBA surfaces. Proliferation after 4 and 7 days increased on POL compared to both SBA surfaces. TNAP activity of hMSC and MG-63 was increased on POL compared to both SBA surfaces whereas SaOS-2 did not discriminate between the three surfaces. PGE(2) formation of hMSC and MG-63 was on both SBA surfaces after 2 days significantly higher than on POL. CONCLUSIONS: The results of this study show that surface roughness has a distinct influence on proliferation and differentiation of osteoblasts. However, variations in physicochemical properties seem to have little influence under the used experimental conditions. It is suggested that more sever and long-lasting modifications of surface chemistry would have an influence on osteoblastic cells.

Response of osteoblast-like SAOS-2 cells to zirconia ceramics with different surface topographies.

OBJECTIVES: Zirconia is a suitable biomaterial for use in medicine (stomatology, orthopaedics) due to its good biocompatibility and outstanding mechanical properties. This study compares the effect of (i) zirconia to the widely used titanium and (ii) zirconia with two different surface topographies (sandblasted and sandblasted/etched) on the adhesion, proliferation and differentiation of SAOS-2 osteoblasts. METHODS: SAOS-2 cells were cultured on either sandblasted or sandblasted/etched zirconia and compared with sandblasted/etched titanium. 2 and 24 h after plating, cell morphology was investigated by scanning electron microscope (SEM) and fluorescence imaging. At 24 and 48 h, cell number-relevant parameters were determined. Alkaline phosphatase (ALP) activity and mineral accumulation were measured at days 8, 11, 15 and day 22 of culture, respectively. RESULTS: SEM and fluorescence images revealed a faster spreading as well as higher number of adherent cells after 24 h incubation on zirconia compared with titanium. Also, the cellular metabolic activity after 24 h and the proliferation rate after 48 h is higher with zirconia compared with titanium. Zirconia had a more pronounced effect compared with titanium on the differentiation of SAOS-2 cells: ALP activity, an early differentiation marker increased earlier and mineralization, a late differentiation marker was increased. Only minor differences were found between zirconia with two different surface topographies; etched zirconia promoted slightly greater the differentiation of SAOS-2 cells. CONCLUSIONS: These data indicate that zirconia mediates a pronounced stronger effect on the adhesion, proliferation and differentiation compared with titanium; and that topographical differences of zirconia have minor effects on osteoblast biology.

Metal release and cell biological compatibility of beta-type Ti-40Nb containing indium.

Small indium (In) additions up to 5 wt % to the beta-type Ti-40Nb alloy effectively improve its mechanical biofunctionality. The impact on its biocompatibility is addressed in this work. Comparative electrochemical polarization studies and inductively coupled plasma optical emission spectrometry analyses were conducted in Tris-buffered saline (on the basis of 150 mM NaCl) with pH 7.6 and 2.0 at 310 ± 1 K with Ti-6Al-4V as reference. The metal ion releases from beta-type alloys were generally very low, for example, those of In3+ ions from (Ti-40Nb)-4In specimens were below 6 × 10-7 mmol/cm2 . X-ray photoelectron spectroscopy revealed the passivation mainly by Ti- and Nb-oxides with traces of In-oxides as the dominating surface process. In vitro studies demonstrate a better human bone marrow stromal cells (hBMSC) activity on the beta-type alloys in comparison to CP-Ti (grade 2), which is mainly due to their high Nb content. At 24 h after seeding on (Ti-40Nb)-4In the metabolic activity of hBMSC was 1.5-fold higher and after 11 days, the tissue non-specific alkaline phosphatase activity was 1.8-fold higher relative to values for CP-Ti. Surface treatments, like chemical etching or plasma oxidation, change the surface topography and the thickness and composition of the oxide layers, but they are not effective in further improving the cell response. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1686-1697, 2018.

Response of human endothelial cells to oxidative stress on Ti6Al4V alloy.

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H(2)O(2) and compared this with the response of endothelial cells grown on cell culture polystyrene (PS). We could show that although the cell number was the same on both surfaces, metabolic activity of endothelial cells grown on Ti6Al4V alloy was reduced compared to the cells on PS and further decreased following prototypic oxidative stress (H(2)O(2)-treatment). The analysis of H(2)O(2)-induced oxidative stress showed a higher ROS formation in endothelial cells on Ti6Al4V than on PS. This correlated with the depletion of reduced glutathione (GSH) in endothelial cells grown on Ti6Al4V surfaces and indicated permanent oxidative stress. Thus, endothelial cells in direct contact with Ti6Al4V showed signs of oxidative stress and higher impairment of cell vitality after an additional oxidative stress. However, the exact nature of the agent of oxidative stress generated from Ti6Al4V remains unclear and requires further investigation.

The effect of electrochemically simulated titanium cathodic corrosion products on ROS production and metabolic activity of osteoblasts and monocytes/macrophages.

Nowadays aseptic loosening is the most common cause of orthopaedic implant failure. Some of its reasons have already been described up to now; however, others remain still hypothetical. Besides the inflammatory response to wear particles originating at different sources, the role of reactive oxygen species as products of cellular reactions and/or as a result of the process of corrosion of an implant leading to implant failure has recently been discussed too. In the present study, we used a galvanostatic polarization to simulate the cathodic partial reaction of the corrosion process at a titanium alloy surface. With respect to cells occurring at the interface of a metal implant, the behaviour of osteoblasts and monocytes/macrophages was investigated. It has been found that cathodic polarization of Ti6Al4V induces an increase in the level of intracellular reactive oxygen species as well as suppressing the metabolic activity of cells in a dose-dependent manner. This is in agreement with the results obtained with cells after external addition of hydrogen peroxide as another kind of oxidative stress. In both approaches, monocytes/macrophages show a higher tolerance to oxidative stress than osteoblasts. It could be concluded that the electrochemical setup developed induced intracellular changes occurring during oxidative stress and it could be used for future detailed analysis of the consequences of corrosion processes for cellular reactions.

Influence of extracellular matrix coatings on implant stability and osseointegration: an animal study.

Aim of the present study was to test the hypothesis that the application of components of the extracellular matrix such as glycosaminoglycans used as implant surface coatings in combination with collagen, with and without growth factor, can lead to enhanced ossification and thus improve implant stability compared with collagen coatings alone. Twenty miniature pigs received 120 experimental titanium implants in the mandible. Three types of surface coatings were created: (1) collagen type I (coll), (2) collagen type I/chondroitin sulphate (coll/CS), (3) collagen type I/chondroitin sulphate/BMP-4 (coll/CS/BMP). Periimplant bone formation was assessed within a defined recess along the length axis of the implant. Bone-implant contact (BIC) and bone volume density (BVD) were determined, using both histomorphometry and synchrotron radiation micro computed tomography (SRmicroCT). To measure implant stability, resonance frequency analysis was applied after implantation and 1, 3, 7, and 22 weeks after placement. BIC was highest for coll/CS coated implants, followed by coll, p = 0.082. Histomorphometric BVD did not significantly change for any coating. SRmicroCT analysis showed an increased BVD for collagen coated implants, compared with the other two surface coatings. Implant stability showed a decrease for all coatings up to the third week. At 22 weeks, all coatings showed an increase in stability without reaching their initial level. Highest stability was reached for coll coated implants, p = 0.051. It was concluded that collagen and coll/CS implant coatings have advantageous characteristics for peri-implant bone formation, compared with the further integration of BMP-4.

Drug delivery and cell interaction of adhesive poly(ethyleneimine)/sulfated polysaccharide complex particle films.

Herein, the authors report and review polyelectrolyte complex (PEC) nanoparticles (NPs) loaded with zoledronate (ZOL) and simvastatin and their effects on bone cells. PEC NPs are intended for modification of bone substitute materials. For characterization, they can be solution casted on germanium (Ge) substrates serving as analytically accessible model substrate. PEC NPs were generated by mixing poly(ethyleneimine) (PEI) either with linear cellulose sulfate (CS) or with branched dextransulfate (DS). Four important requirements for drug loaded PEC NPs and their films are addressed herein, which are the colloidal stability of PEC dispersions (1), interfacial stability (2), cytocompatibility (3), and retarded drug release (4). Dynamic light scattering measurements (DLS) showed that both PEI/CS and PEI/DS PEC NP were obtained with hydrodynamic radii in the range of 35-170 nm and were colloidally stable up to several months. Transmission FTIR spectroscopy evidenced that films of both systems were stable in contact to the release medium up to several days. ZOL-loaded PEI/CS nanoparticles, which were immobilized on an osteoblast-derived extracellular matrix, reduced significantly the resorption and the metabolic activity of human monocyte-derived osteoclasts. FTIR spectroscopy at cast PEC/drug films at Ge substrates revealed retarded drug releases in comparison to the pure drug films.

Modification of Ti6Al4V surfaces using collagen I, III, and fibronectin. I. Biochemical and morphological characteristics of the adsorbed matrix.

Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). Aiming at load-bearing applications in bone contact, in this study the modification of titanium surfaces with the collagen types I and III in combination with fibronectin was undertaken; fibrillogenesis, fibril morphology and adsorption of type I, III and I/III-cofibrils onto titanium were assessed. Increasing the collagen type III amount resulted in a decrease of fibril diameter, while no significant changes in adsorption could be detected. The amount of fibronectin bound to the heterotypic fibrils depended on fibrillogenesis parameters such as ionic strength or concentration of phosphate, and varied with the percentage of integrated type III collagen.

Modification of Ti6AL4V surfaces using collagen I, III, and fibronectin. II. Influence on osteoblast responses.

Responses of osteoblastic cells are influenced by morphology and composition of the extracellular matrix, and this fact has been used to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). In this study, the effect of the collagen types I and III on adhesion, proliferation, and differentiation was studied, using primary osteoblastic cells from rat calvariae. Differences in alkaline phosphatase activity (ALP) and collagen synthesis were observed between differently composed collagen coatings. An increase in collagen type III resulted in an increase in collagen synthesis and a concomitant decrease in ALP activity and Ca deposition. Initial adhesion mechanism of the cells depended on the substrate (titanium, collagen, fibronectin).

Interaction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells.

BACKGROUND: Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis). For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles' composition and surface net charge. MATERIALS AND METHODS: Polyelectrolyte complex nanoparticles (PECNPs) composed of the polycations poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) in combination with the polyanions dextran sulfate (DS) or cellulose sulfate (CS) were prepared. PECNPs' physicochemical properties (size, net charge) were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs) cultured on immobilized PECNP films (5-50 nmol·cm(-2)) by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay, as well as cell morphology (phase contrast microscopy). RESULTS: PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC(+)NP) or negative (PEC(-)NP) net charge were obtained. The PECNP composition significantly affected cell morphology and metabolic activity, whereas the net charge had a negligible influence. Therefore, we classified PECNPs into "variant systems" featuring a significant dose dependency of metabolic activity (DEAE/CS, PEI/DS) and "invariant systems" lacking such a dependency (DEAE/DS, PEI/CS). Immunofluorescence imaging of fluorescein isothiocyanate isomer I (FITC)-labeled PECNPs suggested internalization into hMSCs remaining stable for 8 days. CONCLUSION: Our study demonstrated that PECNP composition affects hMSC behavior. In particular, the PEI/CS system showed biocompatibility in a wide concentration range, representing a suitable system for local drug delivery from PECNP-functionalized bone substitute materials.

Physicochemical and cell biological characterization of PMMA bone cements modified with additives to increase bioactivity.

Polymethylmethacrylate (PMMA) bone cement is the most widely used material in surgery to fix joint replacements in the bone. In this study, we propose a new approach to generate bioactive PMMA surfaces directly at the site of implantation by adding the amphiphilic molecule phosphorylated 2-hydroxyethylmethacrylate (HEMA-P) to commercial PMMA bone cement, both with or without addition of 1-5% soluble calcium and carbonate salts. The setting behavior as well as the mechanical properties, the bonding quality at the metal/cement interface, mineral deposition, and cellular response for different cement modifications were investigated in vitro. The addition of HEMA-P resulted in entirely positive effects with respect to proliferation and differentiation of osteoblast-like cells (SaOs-2) and a very tight contact at the metal/cement interface. No detrimental changes of other properties were detected. The additional incorporation of salts provoked an increased deposition of calcium phosphate minerals but no further improvement in SaOs-2 cell differentiation. A significant decrease in polarization resistance for cements with high salt content (5%) was attributed to debonding between metal and cement. The results suggest an improved clinical performance of PMMA/HEMA-P composites, which might be further enhanced by small amounts of the soluble salts.

Artificial extracellular matrices composed of collagen I and sulfated hyaluronan with adsorbed transforming growth factor ß1 promote collagen synthesis of human mesenchymal stromal cells.

Sulfated glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix and are involved in the regulation of adhesion, proliferation and differentiation of cells. The effects of GAG are mediated in general by their interactions with cations and water, and in particular by their binding to growth factors. The aim of this study was to generate artificial extracellular matrices (aECM) containing collagen I and hyaluronan sulfate (HyaS), which are capable of adsorbing and releasing transforming growth factor ß1 (TGF-ß1), and to promote collagen synthesis of cultured human mesenchymal stromal cells (hMSC). For the preparation of aECM, monosulfated Hya (HyaS1) or trisulfated Hya (HyaS3) were used; the natural chondroitin-4-sulfate was used as a control. As applied for the in vitro experiments, the resulting matrices were composed of 93-98% collagen I and 2-7% GAG derivative. Adsorption of TGF-ß1 to the aECM and release from the aECM was dependent on the degree of sulfation of hyaluronan. Collagen synthesis of hMSC was promoted only by aECM with adsorbed TGF-ß1; the bare aECM had a slightly inhibitory effect on collagen synthesis. The promoting effect did not correlate either to the amount of adsorbed TGF-ß1 nor to the release of TGF-ß1, indicating that the correct presentation of TGF-ß1 to the cells might be critical. The results indicate that sulfated hyaluronan-containing aECM have the potential to control both the adsorption and release of TGF-ß1, and thereby promote collagen synthesis of hMSC. Thus, these aECM might be a useful tool for different tissue-engineering applications to enhance bone formation when used for biomaterial coating.

Influence of collagen-fibril-based coatings containing decorin and biglycan on osteoblast behavior.

Collagen is used as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration by mimicry of the bone extracellular matrix in vivo. The biomimicry strategy can be taken further by incorporating the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, which are expressed in bone. Both bind to fibrils during fibrillogenesis in vitro. In this study, the ability of collagen types I, II, and III to bind decorin and biglycan was compared. Collagen type II bound significantly more SLRPs in fibrils than collagen I and III, with more biglycan than decorin bound by all three collagen types. Therefore, type II fibrils with bound decorin or biglycan or neither were used to coat titanium surfaces. Bioavailability of SLRPs was confirmed by direct ELISA after SLRP biotinilation. The in vitro behavior of osteoblasts from rat calvaria (rOs) and human knee (hOs) cultured on different surfaces was compared. Proliferation and collagen synthesis were determined. Also, the influence of SLRPs on the formation of focal adhesions by rO was investigated. Biglycan enhanced the formation of focal adhesions after 2 and 24 h. Decorin and biglycan affected rO and hO proliferation and collagen synthesis differently. Biglycan stimulated hO proliferation significantly but had no effect on rO proliferation, and also inhibited rO collagen synthesis significantly while not affecting hO collagen synthesis. Decorin promoted hO proliferation slightly but did not influence rO proliferation. The results could be relevant when designing implant coatings or tissue engineering scaffolds.

Characterization of collagen II fibrils containing biglycan and their effect as a coating on osteoblast adhesion and proliferation.

Collagen has been used as a coating material for titanium-based implants for bone contact and as a component of scaffolds for bone tissue engineering. In general collagen type I has been used, however very little attention has been focussed on collagen type II. Collagen-based coatings and scaffolds have been enhanced by the incorporation of the glycosaminoglycan chondroitin sulphate (CS), however the proteglycan biglycan, which is found in bone and contains glycosaminoglycan chains consisting of CS, has not been used as a biomaterial component. The study had the following aims: firstly, five different collagen II preparations were compared with regard to their ability to bind CS and biglycan and the changes in fibril morphology thereby induced. Secondly, the effects of biglycan on the adhesion of primary rat osteoblasts (rO) as well as the proliferation of rO, primary human osteoblasts (hO) and the osteoblast-like cell line 7F2 were studied by culturing the cells on surfaces coated with collagen II fibrils containing biglycan. Fibrils of the collagen II preparation which bound the most biglycan were used to coat titanium surfaces. Bare titanium, titanium coated with collagen II fibrils and titanium coated with collagen II fibrils containing biglycan were compared. It was found that different collagen II preparations showed different affinities for CS and biglycan. In four of the five preparations tested, biglycan reduced fibril diameter, however the ability of a preparation to bind more biglycan did not appear to lead to a greater reduction in fibril diameter. Fibrils containing biglycan promoted the formation of focal adhesions by rO and significantly enhanced the proliferation of hO but not of rO or 7F2 cells. These results should encourage further investigation of biglycan as a component of collagen-based scaffolds and/or coatings.