Enhancing bioactivity, physicochemical, and pharmacokinetic properties of a nano-sized, anti-VEGFR2 Adnectin, through PASylation technology.

The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli-expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.

A nano-scaled and multi-layered recombinant fibronectin/cadherin chimera composite selectively concentrates osteogenesis-related cells and factors to aid bone repair.

Easily accessible and effective bone grafts are in urgent need in clinic. The selective cell retention (SCR) strategy, by which osteogenesis-related cells and factors are enriched from bone marrow into bio-scaffolds, holds great promise. However, the retention efficacy is limited by the relatively low densities of osteogenesis-related cells and factors in marrow; in addition, a lack of satisfactory surface modifiers for scaffolds further exacerbates the dilemma. To address this issue, a multi-layered construct consisting of a recombinant fibronectin/cadherin chimera was established via a layer-by-layer self-assembly technique (LBL-rFN/CDH) and used to modify demineralised bone matrix (DBM) scaffolds. The modification was proven stable and effective. By the mechanisms of physical interception and more importantly, chemical recognition (fibronectin/integrins), the LBL-rFN/CDH modification significantly improved the retention efficacy and selectivity for osteogenesis-related cells, e.g., monocytes, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), and bioactive factors, e.g., bFGF, BMP-2 and SDF-1α. Moreover, the resulting composite (designated as DBM-LBL-rFN/CDH) not only exhibited a strong MSC-recruiting capacity after SCR, but also provided favourable microenvironments for the proliferation and osteogenic differentiation of MSCs. Eventually, bone repair was evidently improved. Collectively, DBM-LBL-rFN/CDH presented a suitable biomaterial for SCR and a promising solution for tremendous need for bone grafts. STATEMENT OF SIGNIFICANCE: There is an urgent need for effective bone grafts. With the potential of integrating osteogenicity, osteoinductivity and osteoconductivity, selective cell retention (SCR) technology brings hope for developing ideal grafts. However, it is constrained by low efficacy and selectivity. Thus, we modified demineralized bone matrix with nano-scaled and multi-layered recombinant fibronectin/cadherin chimera (DBM-rFN/CDH-LBL), and evaluate its effects on SCR and bone repair. DBM-rFN/CDH-LBL significantly improved the efficacy and selectivity of SCR via physical interception and chemical recognition. The post-enriched DBM-rFN/CDH-LBL provided favourable microenvironments to facilitate the migration, proliferation and osteogenic differentiation of MSCs, thus accelerating bone repair. Conclusively, DBM-rFN/CDH-LBL presents a novel biomaterial with advantages including high cost-effectiveness, more convenience for storage and transport and can be rapidly constructed intraoperatively.

Irisin, a unique non-inflammatory myokine in stimulating skeletal muscle metabolism

Exercise offers several benefits for health, including increased lean body mass and heightened energy expenditure, which may be partially attributable to secretory factors known as myokines. Irisin, a recently identified myokine, was shown to increase metabolic rate and mitochondrial content in both myocytes and adipocytes; however, the mechanism(s) of action still remain largely unexplained. This work investigated if irisin functions by acting as an inflammatory myokine leading to cellular stress and energy expenditure. C2C12 myotubes were treated with various concentrations of irisin, TNFAlpha, or IL6 for various durations. Glycolytic and oxidative metabolism, as well as mitochondrial uncoupling, were quantified by measurement of acidification and oxygen consumption, respectively. Metabolic gene and protein expression were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting, respectively. Mitochondrial content was assessed by fluorescent imaging. NFkappaB activity was assessed using an NFkappaB GFP-linked reporter system. Consistent with previous findings, irisin significantly increased expression of several genes including peroxisome proliferator-activated receptor Alpha coactivator-1Alpha (PGC-1Alpha) leading to increased mitochondrial content and oxygen consumption. Despite some similarities between TNFAlpha and irisin treatment, irisin failed to activate the NFkappaB pathway like TNFAlpha, suggesting that irisin may not act as an inflammatory signal. Irisin has several effects on myotube metabolism which appear to be dependent on substrate availability; however, the precise mechanism(s) by which irisin functions in skeletal muscle remain unclear. Our observations support the hypothesis that irisin does not function through inflammatory NFkappaB activation like other myokines (such as TNFAlpha)

A novel bispecific antibody targeting tumor necrosis factor α and ED-B fibronectin effectively inhibits the progression of established collagen-induce arthritis.

Specific delivery of TNF-α antagonist to the inflamed site can increase its efficacy and reduce the side effects. In this study, we constructed a bispecific diabody (BsDb) that targets TNF-α and ED-B-containing fibronectin (B-FN), a fibronectin isoform specifically expressed in the pannus of the inflamed joint in rheumatoid arthritis. BsDb was produced in Escherichia coli as inclusion bodies, purified to homogeneity, and refolded to the functional form. Our data demonstrate that BsDb could simultaneously bind to human TNF-α and B-FN and neutralize TNF-α action. In the collagen-induced arthritis mouse model, we compared the biodistrubtion and therapeutic efficacy of BsDb with those of the anti-TNF-α scFv (TNF-scFv). Similar to TNF-scFv, BsDb penetrated into the synovial tissue quickly, showing a rapid clearance from blood and normal organs. In contrast, BsDb could selectively accumulate and retain in arthritic joints of mice for a long period time, resulting in a much stronger inhibition of arthritis progression in mice than TNF-scFv. The findings described herein indicate that BsDb has a good specificity to the inflamed joint, with low toxicity to normal organs and seems to be an ideal biological agent for the treatment of RA and other chronic inflammatory disease.

Nanoengineered particles for enhanced intra-articular retention and delivery of proteins.

Localized intra-articular delivery of anti-inflammatory proteins can reduce inflammation in osteoarthritis but poses a challenge because of raid clearance within few hours of injection. A new class of polymer is developed that forms self-assembled nanoparticles ranging from 300 to 900 nm and demonstrates particle size dependent prolonged retention in intra-articular joint spaces compared to bolus protein over a period of 14 d.

Cell membrane deformation induced by a fibronectin-coated polystyrene microbead in a 200-MHz acoustic trap.

The measurement of cell mechanics is crucial for a better understanding of cellular responses during the progression of certain diseases and for the identification of the cell's nature. Many techniques using optical tweezers, atomic force microscopy, and micro-pipettes have been developed to probe and manipulate cells in the spatial domain. In particular, we recently proposed a two-dimensional acoustic trapping method as an alternative technique for small particle manipulation. Although the proposed method may have advantages over optical tweezers, its applications to cellular mechanics have not yet been vigorously investigated. This study represents an initial attempt to use acoustic tweezers as a tool in the field of cellular mechanics in which cancer cell membrane deformability is studied. A press-focused 193-MHz single-element lithium niobate (LiNbO3) transducer was designed and fabricated to trap a 5-µm polystyrene microbead near the ultrasound beam focus. The microbeads were coated with fibronectin, and trapped before being attached to the surface of a human breast cancer cell (MCF-7). The cell membrane was then stretched by remotely pulling a cell-attached microbead with the acoustic trap. The maximum cell membrane stretched lengths were measured to be 0.15, 0.54, and 1.41 µm at input voltages to the transducer of 6.3, 9.5, and 12.6 Vpp, respectively. The stretched length was found to increase nonlinearly as a function of the voltage input. No significant cytotoxicity was observed to result from the bead or the trapping force on the cell during or after the deformation procedure. Hence, the results convincingly demonstrated the possible application of the acoustic trapping technique as a tool for cell manipulation.

Fibronectin and vitronectin promote human fetal osteoblast cell attachment and proliferation on nanoporous titanium surfaces.

Improvements in osteoconduction of implant biomaterials require focusing on the bone-implant interface, which is a complex multifactorial system. Surface topography of implants plays a crucial role at this interface. Nanostructured surfaces have been shown to promote serum protein adsorption and osteoblast adhesion when compared to micro-structured surfaces for bone-implant materials. We studied the influence of the serum proteins fibronectin and vitronectin on the attachment and proliferation of osteoblasts onto nanostructured titania surfaces. Human fetal osteoblastic cells hFOB 1.19 were used as model osteoblasts and were grown on nanoporous TiO2 templates, using Ti6AI4V and commercially pure Ti substrates as controls. Results show a significant increase in cell proliferation'on nanoporous TiO2 over flat substrates. Initial cell attachment data exhibited a significant effect by either fibronectin or vitronectin on cell adhesion at the surface of any of the tested materials. In addition, the extent of cell adhesion was significantly different between the nanoporous TiO2 and both Ti6AI4V and commercially pure Ti substrates, with the first showing the highest surface coverage. There was no significant difference on osteoblast attachment or proliferation between the presence of fibronectin or vitronectin using any of the material substrates. Taken together, these results suggest that the increase in osteoblast attachment and proliferation shown on the nanoporous TiO2 is due to an increase in the adsorption of fibronectin and vitronectin because of the higher surface area and to an enhanced protein unfolding, which allows access to osteoblast binding motifs within these proteins.

Kinetics and isotherm of fibronectin adsorption to three-dimensional porous chitosan scaffolds explored by ¹²5I-radiolabelling.

In this study, (125)I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using (125)I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

Wettability versus electrostatic forces in fibronectin and albumin adsorption to titanium surfaces.

OBJECTIVES: Although the enhancement of plasma protein adsorption to titanium ( Ti ) following wetting has been recognized, the relationship between wettability and electrostatic forces has remained unclear. Thus, we have carried out a series of studies to determine the role of wettability and electrostatic forces on protein adsorption. METHODS: Titanium disks with different surfaces were wetted with a range of solutions, two of which contained divalent positive ions ( Ca and Mg ). Unwetted disks served as a control. Subsequently, the wetted disks were subjected to three treatment regimes: (1) incubation in human serum albumin (HSA) or human serum fibronectin (HSF); (2) drying the wetted disks, followed by incubation in HSA or HSF; and (3) following protein adsorption, the Ca originating in the wetting solutions was removed by divalent positive ions chelator treatment (EGTA), and the remaining quantities were assessed. The quantity of the adsorbed proteins was determined by ELISA. RESULTS: It was found that in the case of HSA, adsorption was enhanced by the wettability, the presence of Ca and Mg in the wetting solution, and the existence of rough surfaces. For HSF, the wettability and rough surfaces enhanced adsorption. CONCLUSION: The results demonstrate that in addition to wettability, the composition of the wetting solution affects the protein adsorption. While wetting reduces the time for the HSA and HSF adsorption to reach saturation, the electrostatic forces enhance the amount of HSA adsorption. Thus, the protein adsorption capacity of titanium rough surfaces can be selectively manipulated by changing of the wetting solution.

Designed hydrophilic and charge mutations of the fibronectin domain: towards tailored protein biodistribution.

Engineered proteins are attractive affinity scaffolds for molecular imaging and drug delivery. Although exquisite binding specificity and affinity can be engineered, many proteins exhibit off-target uptake, particularly in the kidneys and liver, from physiologic effects. We quantified the ability to alter renal and hepatic uptake via hydrophilic and charge mutations. As a model protein, we used the 10th type III domain of human fibronectin, which has been engineered to bind many targets and has been validated for molecular imaging. We screened rational mutants, identified by structural and phylogenetic analyses, to yield eight mutations that collectively substantially increase protein hydrophilicity. Mutation of two parental clones yielded four domains with a range of hydrophilicity. These proteins were labeled with (64)Cu, injected intravenously into nu/nu mice (n = 3-5 each) and evaluated by positron emission tomography. Renal uptake strongly correlated with hydrophilicity (Pearson's correlation coefficient = 0.97), ranging from 29 ± 11 to 100 ± 22% ID/g at 1 h. Hepatic uptake inversely correlated with hydrophilicity (Pearson's correlation coefficient = -0.92), ranging from 30 ± 7 to 3 ± 1% ID/g. Thus, renal and hepatic uptake are directly tunable through hydrophilic mutation, identifiable by structural and phylogenetic analyses. To investigate charge, we mutated acidic and basic residues in both parental clones and evaluated (64)Cu-labeled mutants in nu/nu mice (n = 5-7). Selected charge removal reduced kidney signal: 78 ± 13 to 51 ± 8%ID/g (P < 0.0001) for the hydrophilic clone and 32 ± 10 to 21 ± 3 (P = 0.0005) for the hydrophobic clone. Elucidation of hydrophilicity and charge enabled modulation of background signal thereby enhancing the utility of protein scaffolds as translatable targeting agents for molecular imaging and therapy.

Development of a carbon-14 labeling approach to support disposition studies with a pegylated biologic.

Although it is widely accepted that one can extend the pharmacokinetic half-life of a therapeutic protein by covalent conjugation with polyethylene glycol (PEG), the disposition properties of such biologics have not yet been fully evaluated. Therefore, a novel [¹4C]-labeling method was developed that can be applied to a biologic conjugated with PEG through a maleimide-cysteine reaction. The method was used to study the tissue and tumor distribution of a PEGylated Adnectin, a recombinant protein derived from the 10th type III domain of fibronectin, in nude mice bearing human xenograft tumors. The PEGylated Adnectin contained a 40-kDa branched PEG (P40B) that was labeled with [¹4C] at the linker region between the PEG and Adnectin, without compromising cellular activity and plasma half-life in mice. After a single intravenous or intraperitoneal dose (33 mg/kg, 1.7 µCi per mouse) of [¹4C]-P40B-Adnectin, quantitative whole-body autoradiography analysis revealed that the liver had the highest uptake of the radioactivity among nontumor tissues, followed by the kidneys and lung. The muscle and brain showed the least penetration of the radioactivity among all tissues examined. In addition, the [¹4C]-P40B-EI-tandem penetrated into the tumor tissue, although the extent of accumulation was largely dependent on tumor type. Therefore, it was possible to assess the tissue distribution of a PEGylated biologic after it had been [¹4C] labeled using the novel method described herein.

The development of fibronectin-functionalised hydroxyapatite coatings to improve dermal fibroblast attachment in vitro.

The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls. Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively.

Phase I and pharmacokinetic study of CT-322 (BMS-844203), a targeted Adnectin inhibitor of VEGFR-2 based on a domain of human fibronectin.

PURPOSE: To determine the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary antitumor activity of CT-322 (BMS-844203), a VEGFR-2 inhibitor and the first human fibronectin domain-based targeted biologic (Adnectin) to enter clinical studies. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were treated with escalating doses of CT-322 intravenously (i.v.) weekly (qw), or biweekly (q2w). Plasma samples were assayed for CT-322 concentrations, plasma VEGF-A concentrations, and antidrug antibodies. RESULTS: Thirty-nine patients completed 105 cycles of 0.1 to 3.0 mg/kg CT-322 i.v. either qw or q2w. The most common treatment-emergent grade 1/2 toxicities were fatigue, nausea, proteinuria, vomiting, anorexia, and hypertension. Grade 3/4 toxicities were rare. Reversible proteinuria, retinal artery, and vein thrombosis, left ventricular dysfunction, and reversible posterior leukoencephalopathy syndrome were dose limiting at 3.0 mg/kg. The MTD was 2 mg/kg qw or q2w. CT-322 plasma concentrations increased dose proportionally. Plasma VEGF-A levels increased with dose and plateaued at 2 mg/kg qw. Anti-CT-322 antibodies developed without effects on pharmacokinetics, VEGF-A levels, or safety. Minor decreases in tumor measurements occurred in 4 of 34 evaluable patients and 24 patients had stable disease. CONCLUSIONS: CT-322 can be safely administered at 2 mg/kg i.v. qw or q2w and exhibits promising antitumor activity in patients with advanced solid tumors. The absence of severe toxicities at the MTD, demonstration of plasma drug concentrations active in preclinical models, and clinical pharmacodynamic evidence of VEGFR-2 inhibition warrant further development of CT-322 and suggest strong potential for Adnectin-based targeted biologics.

Protein adsorption under electrical stimulation of neural probe coated with polyaniline.

Polyaniline (PANI) nanoparticles were successfully polymerized on the surface of Pt electrode to form nanostructured films. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) micrographs showed that the just-synthesized films were compact with diameter of nanoparticles on films about 50 and 30nm for HClO(4) and citric acid doped PANI films, respectively. And the surface of the electrode was coated completely with PANI films. After electrical stimulation for 1 month in 0.9% sodium chloride solution, there were no fissures appearing on PANI films. The compact film would act as a protecting membrane of the Pt surface, and is suitable to be used as the electrode coating for long-time performance. The time frame of human plasma fibronectin (FN) and bovine serum albumin (BSA) adsorption illustrated that electrical stimulation enhanced the amount of protein adsorption on PANI films up to 1.7-fold increase as compared to that without electrical stimulation. The SEM images of BSA adsorption for 120min indicated that electrical stimulation might initiate the aggregation of BSA, and the nanostructure of the PANI films could inhibit the aggregation. We also found that the protein adsorption decreased conductivity of PANI films, which maybe due to the protein barriers formed on them. These results provided a good reference for the use of conducting polymers as neural probe coating.

Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy.

Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews recent developments in the synthesis and characterization of SELP hydrogels and their use for matrix-mediated gene delivery.

Absorption of bioactive molecules into OASIS wound matrix.

OBJECTIVE: To examine the ability of OASIS Wound Matrix to absorb, retain, and protect bioactive molecules from solution. DESIGN: Samples of OASIS Wound Matrix were incubated in solutions of bioactive molecules, specifically heparin, albumin, fibronectin, basic fibroblast growth factor 2, and platelet-derived growth factor (PDGF). Half of the samples were then rinsed, and all of the samples were evaluated using enzyme-linked immunosorbent assays (ELISAs) and dye-mediated spectrophotometric methods for absorption and retention of the bioactive molecules. Protection of PDGF was measured by placing PDGF-incubated and control samples into a degradation solution containing plasmin. Intact PDGF levels were then evaluated using a PDGF-specific ELISA. MAIN OUTCOME MEASURES: The main outcome measures were the amount of each bioactive molecule that was absorbed after incubation in solutions and retained after rinses as well as the amount of PDGF remaining after plasmin degradation. MAIN RESULTS: OASIS Wound Matrix absorbed bioactive molecules from solution, selectively absorbed PDGF from serum, and protected PDGF from protease degradation. CONCLUSIONS: Although OASIS Wound Matrix potentially has multiple functions in wound healing, it likely promotes wound healing, in part, by absorbing, retaining, and protecting bioactive molecules from the wound environment.

Preparation and characterization of novel biocompatible cryogels of poly (vinyl alcohol) and egg-albumin and their water sorption study.

Polyvinyl alcohol (PVA) and egg albumin are water-soluble, biocompatible and biodegradable polymers and have been widely employed in biomedical fields. In this paper, novel physically cross-linked hydrogels composed of poly (vinyl alcohol) and egg albumin were prepared by cyclic freezing/thawing processes of aqueous solutions containing PVA and egg albumin. The FTIR analysis of prepared cryogels indicated that egg albumin was successfully introduced into the formed hydrogel possibly via hydrogen bonds among hydroxyl groups, amide groups and amino groups present in PVA and egg albumin. The gels were also characterized thermally and morphologically by DSC and SEM-techniques, respectively. The prepared so called 'cryogels' were evaluated for their water uptake potential and influence of various factors such as chemical architecture of the spongy hydrogels, pH and temperature of the swelling bath were investigated on the degree of water sorption by the cryogels. The effect of salt solution and various simulated biological fluids on the swelling of cryogel was also studied. The in vitro biocompatibility of the prepared cryogel was also judged by methods such as protein (BSA) adsorption, blood clot formation and percentage hemolysis measurements.

Effect of magnetic field on the fibronectin adsorption, cell attachment and proliferation on titanium surface.

OBJECTIVES: We studied the effect of various static magnetic fields (SMFs) on the adsorption of specific recombinant fibronectin (FN) peptide (hFNIII9-10) on the titanium surface. Furthermore, the responses of human osteosarcoma TE-85 cells in the SMF were observed. MATERIAL AND METHODS: Various magnetic fields--1, 2, 3, 5, 7, 10 mT--were established by controlling the distance from Nd-Fe-B magnet to the disks. For FN adsorption experiment, machined titanium disks were incubated in 1 microM hFNIII9-10 at 37 degrees C overnight under magnetic field. The adsorbed hFNIII9-10 was measured as optical density (OD). For attachment study, TE-85 cells were incubated for 2 h on the hFNIII9-10 coated machined titanium disks and OD values were measured. As for proliferation study, titanium disks were incubated for 48 h after washing unattached cells in 2 h. The amount of proliferated TE-85 cell was also measured as OD value. Attachments of TE-85 cells under various intensities of magnetic field were observed using a scanning electron microscope. RESULTS: The amount of adsorbed hFNIII9-10 showed no significant difference between control (0 mT) and six experimental groups (1, 2, 3, 5, 7, 10 mT). However, TE-85 cells attached significantly higher in groups of 1, 2, 5, 10 mT than in control group (P=0). Cell attachment in groups of 3, 7 mT showed no significant difference with that of control group. TE-85 cells were observed to attach through filopodia. Especially in 1 mT, flattened cells were predominant. In proliferation assay, 1 mT stimulated TE-85 cells showed significantly higher proliferation than those in 2, 3 and 7 mT (P=0). CONCLUSION: Magnetic fields under 10 mT did not influence FN adsorption on the titanium surface. However, a significant effect was found on cell attachment and proliferation.

Fibronectin preadsorbed on hydroxyapatite together with rough surface structure increases osteoblasts' adhesion "in vitro": the theoretical usefulness of fibronectin preadsorption on hydroxyapatite to increase permanent stability and longevity in spine implants.

OBJECTIVE: The aim of this study was to investigate the contribution of fibronectin (FN) preadsorption to enhance osteoblast adhesion and strength on hydroxyapatite (HA) used either as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. METHODS: HA substrata with two different surface roughness values (rough HA180 and smooth HA1200) were produced, and human osteoblasts were seeded after culturing on them. Prior to osteoblast seeding, the HA substrata were immersed in FN solution. Osteoblast attachment on each of the two HA substrata was evaluated by recording the number of cells, whereas osteoblast adhesion strength was determined by measuring the shear stress required to detach the cells from the HA substrates. RESULTS: FN preadsorption increased the number of attached osteoblasts on smooth and rough HA substratum at 40% and 62%, respectively, whereas it increased osteoblast attachment strength on the smooth and rough substratum at 165% and 73%, respectively. CONCLUSIONS: This study showed that FN preadsorption and rough HA surface texture synergistically increased "in vitro" both the number and the adhesion strength of human osteoblasts. Further studies in primates and humans should be carried out to disclose the clinical relevance (increase implant's stability and longevity) of the above-mentioned observations.