Bruce E. Rapuano

Surface oxide net charge of a titanium alloy: Modulation of fibronectin-activated attachment and spreading of osteogenic cells

In the current study, we have altered the surface oxide properties of a Ti6Al4V alloy using heat treatment or radiofrequency glow discharge (RFGD) in order to evaluate the relationship between the physico-chemical and biological properties of the alloys surface oxide. The effects of surface pretreatments on the attachment of cells from two osteogenic cell lines (MG63 and MC3T3) and a mesenchymal stem cell line (C3H10T1/2) to fibronectin adsorbed to the alloy were measured. Both heat and RFGD pretreatments produced a several-fold increase in the number of cells that attached to fibronectin adsorbed to the alloy at a range of coating concentrations (0.001-10nM FN) for each cell line tested. An antibody (HFN7.1) directed against the central integrin binding domain of fibronectin produced a 65-70% inhibition of cell attachment to fibronectin-coated disks, indicating that cell attachment to the metal discs was dependent on fibronectin binding to cell integrin receptors. Both treatments also accelerated the cell spreading response manifested by extensive flattening and an increase in mean cellular area. The treatment-induced increases in the cell attachment activity of adsorbed fibronectin were correlated with previously demonstrated increases in Ti6Al4V oxide negative net surface charge at physiological pH produced by both heat and RFGD pretreatments. Since neither treatment increased the adsorption mass of fibronectin, these findings suggest that negatively charged surface oxide functional groups in Ti6Al4V can modulate fibronectins integrin receptor activity by altering the adsorbed proteins conformation. Our results further suggest that negatively charged functional groups in the surface oxide can play a prominent role in the osseointegration of metallic implant materials.

Tumor necrosis factor-α stimulates phosphatidylinositol breakdown by phospholipase C to coordinately increase the levels of diacylglycerol, free arachidonic acid and prostaglandins in an osteoblast (MC3T3-E1) cell line

The effects of (human recombinant) tumor necrosis factor-alpha on phosphatidylinositol breakdown, release of 1,2-diacylglycerols, mobilization of arachidonate from diacylglycerol and prostaglandin synthesis were examined in a model osteoblast cell line (MC3T3-E1). Tumor necrosis factor-alpha (10 nM) caused a specific (30%) decrease in the mass of phosphatidylinositol (and no other phospholipids) within 30 min of exposure. Tumor necrosis factor-alpha doubled the rate of incorporation of [32P]orthophosphoric acid into phosphatidylinositol, indicating that the turnover of inositol phosphate was enhanced, and increased the content of diacylglycerol in parallel with phosphatidylinositol breakdown. The cytokine (10-50 nM; 4 h) also promoted a specific release of 24-34% of the [3H]arachidonate from prelabeled phosphatidylinositol, a release of 80% of the 3H-fatty acid from the diacylglycerol pool, and a 30-fold increase in the synthesis of prostaglandin E2. The tumor necrosis factor-alpha induced liberation of [3H]arachidonate from diacylglycerol, cellular arachidonate release and the synthesis of prostaglandin E2 were each blocked by an inhibitor of diacylglycerol lipase, the compound RHC 80267 (30 microM). Therefore, we conclude that, in the MC3T3-E1 cell line, tumor necrosis factor-alpha activates a phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3) to release diacylglycerol, and increases the metabolism of diacylglycerol to liberate arachidonate for prostaglandin synthesis.

Surface oxide net charge of a titanium alloy: Comparison between effects of treatment with heat or radiofrequency plasma glow discharge

In the current study, we have compared the effects of heat and radiofrequency plasma glow discharge (RFGD) treatment of a Ti6Al4V alloy on the physico-chemical properties of the alloys surface oxide. Titanium alloy (Ti6Al4V) disks were passivated alone, heated to 600 °C, or RFGD plasma treated in pure oxygen. RFGD treatment did not alter the roughness, topography, elemental composition or thickness of the alloys surface oxide layer. In contrast, heat treatment altered oxide topography by creating a pattern of oxide elevations approximately 50-100 nm in diameter. These nanostructures exhibited a three-fold increase in roughness compared to untreated surfaces when RMS roughness was calculated after applying a spatial high-pass filter with a 200 nm-cutoff wavelength. Heat treatment also produced a surface enrichment in aluminum and vanadium oxides. Both RFGD and heat treatment produced similar increases in oxide wettability. Atomic force microscopy (AFM) measurements of metal surface oxide net charge signified by a long-range force of attraction to or repulsion from a (negatively charged) silicon nitride AFM probe were also obtained for all three experimental groups. Force measurements showed that the RFGD-treated Ti6Al4V samples demonstrated a higher net positive surface charge at pH values below 6 and a higher net negative surface charge at physiological pH (pH values between 7 and 8) compared to control and heat-treated samples. These findings suggest that RFGD treatment of metallic implant materials can be used to study the role of negatively charged surface oxide functional groups in protein bioactivity, osteogenic cell behavior and osseointegration independently of oxide topography.

Titanium alloy surface oxide modulates the conformation of adsorbed fibronectin to enhance its binding to α5β1 integrins in osteoblasts

Our laboratory has previously demonstrated that heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of a titanium alloy (Ti6Al4V) increased the net negative charge of the alloys surface oxide and the attachment of osteoblastic cells to adsorbed fibronectin. The purpose of the current study was to investigate the biological mechanism by which these surface pretreatments enhance the capacity of fibronectin to stimulate osteoblastic cell attachment. Each pretreatment was found to increase the binding (measured by ELISA) of a monoclonal anti-fibronectin Ig to the central integrin-binding domain of adsorbed fibronectin, and to increase the antibodys inhibition of osteogenic cell attachment (measured by hexosaminidase assay). Pretreatments also increased the binding (measured by ELISA) of anti-integrin IgGs to the α(5) and β(1) integrin subunits that became attached to fibronectin during cell incubation. These findings suggest that negatively charged surface oxides of Ti6Al4V cause conformational changes in fibronectin that increase the availability of its integrin-binding domain to α(5) β(1) integrins.

Heat or radiofrequency plasma glow discharge treatment of a titanium alloy stimulates osteoblast gene expression in the MC3T3 osteoprogenitor cell line

Purpose The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat (600℃) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line. Methods Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (α1), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix. Results Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectins bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression. Conclusions The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectins bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.

Role of Protein Kinase A in Collagenase-1 Gene Regulation by Prostaglandin E1: Studies in a Rabbit Synoviocyte Cell Line, HIG-82

Gene expression of the matrix‐degrading enzyme collagenase‐1 in rabbit synoviocytes and human fibroblasts is down‐regulated by prostaglandin E1 (PGE1) through a cyclic adenosine monophosphate (cAMP)–dependent pathway. In the current study, we examined the role of protein kinase A (PKA) in the PGE1‐mediated effect on collagenase‐1 gene expression. Collagenase‐1 gene expression was rapidly induced several‐fold above control both by a phorbol ester, 12‐o‐tetradecanoyl phorbol 13 acetate, and interleukin‐1β (IL‐1β) in HIG‐82 synoviocytes. Treatment with PGE1 and forskolin increased PKA activity in the HIG‐82 cells within 15 minutes of adding the stimulating agents. Two inhibitors of PKA, the isoquinoline‐sulfonamide derivative, H‐89 and a cAMP analog, RpcAMP, blocked the ability of PGE1 to down‐regulate collagenase‐1 gene expression. However, if PGE1 was added from 6 h to 30 minutes before the PKA inhibitor H‐89, collagenase‐1 gene expression was inhibited. Constitutive PKA activity was increased in HIG‐82 synoviocytes stably transfected with an expression vector pCMV.Cα that caused the HIG‐82 cells to overexpress an active catalytic subunit of PKA. Cells stably transfected with an inactive, mutated C‐α‐variant showed no change in PKA activity. Collagenase‐1 mRNA levels in TPA‐stimulated cells were reduced to baseline levels in the pCMV.Cα but not in the mutated C‐α–transfected cells. These data show the importance of PKA in regulating collagenase‐1 gene expression in a synoviocyte cell line.

Pertussis toxin-sensitive and insensitive G proteins mediate ALFL4-stimulated release of inositol phosphates, diglycerides and arachidonic acid in MC3T3-E1 osteoblasts

In Ihis study, we have measured the effects of the G protein activator AIF;on the formation of inositol phosphates. diglycerides and arachidonic acid in MC3T3-El osteoblasth. We have shown that AIF, induces a similar fold elevation in the levels of all three of these second messengers. AIF,also decreased the specific radioactivity of [‘Hlarachidonic acid in the diglyceride pool. These results suggest that A1F4activated a phosphatidylinositolspecific phospholipase C to liberate diglycerides and stimulated the metabolism of diglycerides to release arachidonic acid. The G protein inhibitor pertussis toxin was demonstrated to reduce the AIF,-induced release of inositol phosphates or [jH]arachidonic acid by 20-30%. However, incubation of cells with cholera toxin, a G protein activator, resulted in an amplification of the hasal and AIF,--stimulated liberation of inositol phosphates, diglycerides and [“Hlarachidonic acid (following deesterification from diglycerides). Therefore, this \tudy provides evidence that arachidonic acid availability for prostaglandin synthesis in osteoblasts may be regulated by the activity of a G protein coupled to a phosphatidylinositol-specific phospholipase C. Prostaglandins may play important role(s) in the regulation of bone metabolism and remodeling, since they have been demonstrated to stimulate both the formation and resorption of bone (1). However, during the invasion of bone by malignant cells or immune cells that mediate inilammatory responses. elevated levels of prostaglandins art: released from bone cells in reponse to cytokines like PDGF or monokines such as tumor necrosis factor ITNF) or interleukin 1 (11-l). Abnormally high levels of prostaglandins are thought to be at least partially responsible for the accelerated resorption of bone that is caused by cancer or inflammation (2-7). Therefore. the attenuation of prostaglandin synthesis in bone may be central to the therapeutic control of some forms of pathological osteolysis. Recently, we have demonstrated that TNFalpha stimulates prostaglandin synthesis in the osteoblast cell line MC3T3-El by increasing the availability of arachidonic acid substrate (8). TNF-alpha was shown to activate both a phospholipasr C which selectively hydrolyzes phosphatidylinositol (over other phospholipids) to release diglyceride and diglyceride-metabolizing lipases to release arachidonic acid (8). These findings suggested that changes in the activity of a PI-specific phospholipase C may modulate the levels of free arachidonic acid and availability for prostaglandin synthesis in bone cells. The activity of PI-specific phospholipase C has been shown to be regulated by GTP-binding proteins (9-1 I ). We have used the compound AIF.,-. which has been shown to activate a G protein coupled to a PI-specific phospholipase C ( 12), to study the role of G protein in the regulation of arachidonic acid release via this pathway in the mouse osteoblast cell line. MC3T3El. We present evidence that an activator of G proteins stimulates arachidonic acid release by increasing the metabolism of PI by phospholipase C and that of diglyceride by diglyceride lipases. These findings provide evidence for a new and potentially important regulatory site in the pathogenesis 01‘ prostaglandindependent bone resorption.

Protein kinases A and C positively regulate G protein‐dependent activation of phosphatidylinositol‐specific phospholipase C by tumor necrosis factor‐alpha in MC3T3‐E1 osteoblasts

The role(s) of protein kinases in the regulation of G protein‐dependent activation of phosphatidylinositol‐specific phospholipase C by tumor necrosis factor‐alpha was investigated in the osteoblast cell line MC3T3‐E1. We have previously reported the stimulatory effects of tumor necrosis factor‐alpha and A1F4−, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F4− ‐promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor‐alpha was found to elicit a 4–5‐fold increase in the formation of [3H]inositol‐1,4‐phosphate and [3H]inositol‐1,4,5‐phosphate; and a 36% increase in [3H]inositol‐1‐phosphate within 5 min in prelabeled cells. [3H]inositol‐4‐phosphate, a metabolite of [3H]inositol‐1,4‐phosphate and [3H]inositol‐1,4,5‐phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor‐alpha and A1F4− ‐activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor‐induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et‐18‐OMe and H‐7, dramatically decreased the formation of [3H]inositol phosphates stimulated by either tumor necrosis factor‐alpha or A1F4− by 90–100% but did not affect basal formation. The activation of cAMP‐dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8‐BrcAMP augmented basal, tumor necrosis factor‐alpha and A1F4−‐induced [3H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor‐alpha‐initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G‐protein and phosphatidylinositol‐specific phospholipase C. J. Cell. Biochem. 65:198–208.

Heat and radiofrequency plasma glow discharge pretreatment of a titanium alloy promote bone formation and osseointegration

Orthopedic and dental implants manifest increased failure rates when inserted into low density bone. We determined whether chemical pretreatments of a titanium alloy implant material stimulated new bone formation to increase osseointegration in vivo in trabecular bone using a rat model. Titanium alloy rods were untreated or pretreated with heat (600°C) or radiofrequency plasma glow discharge (RFGD). The rods were then coated with the extracellular matrix protein fibronectin (1 nM) or left uncoated and surgically implanted into the rat femoral medullary cavity. Animals were euthanized 3 or 6 weeks later, and femurs were removed for analysis. The number of trabeculae in contact with the implant surface, surface contact between trabeculae and the implant, and the length and area of bone attached to the implant were measured by histomorphometry. Implant shear strength was measured by a pull‐out test. Both pretreatments and fibronectin enhanced the number of trabeculae bonding with the implant and trabeculae‐to‐implant surface contact, with greater effects of fibronectin observed with pretreated compared to untreated implants. RFGD pretreatment modestly increased implant shear strength, which was highly correlated (r2 = 0.87–0.99) with measures of trabecular bonding for untreated and RFGD‐pretreated implants. In contrast, heat pretreatment increased shear strength 3–5‐fold for both uncoated and fibronectin‐coated implants at 3 and 6 weeks, suggesting a more rapid increase in implant‐femur bonding compared to the other groups. In summary, our findings suggest that the heat and RFGD pretreatments can promote the osseointegration of a titanium alloy implant material. J. Cell. Biochem. 114: 2363–2374, 2013.

Heat and radiofrequency plasma glow discharge pretreatment of a titanium alloy: Eveidence for enhanced osteoinductive properties†

It is believed that orthopedic and implant longevity can be improved by optimizing fixation, or direct bone‐implant contact, through the stimulation of new bone formation around the implant. The purpose of this study was to determine whether heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of Ti6Al4V stimulated calcium‐phosphate mineral formation in cultures of attached MC3T3 osteoprogenitor cells with or without a fibronectin coating. Calcium‐phosphate mineral was analyzed by flame atomic absorption spectrophotometry, scanning electron microscopy (SEM)/electron dispersive X‐ray microanalysis (EDAX) and Fourier transformed infrared spectroscopy (FTIR). RFGD and heat pretreatments produced a general pattern of increased total soluble calcium levels, although the effect of heat pretreatment was greater than that of RFGD. SEM/EDAX showed the presence of calcium‐and phosphorus‐containing particles on untreated and treated disks that were more numerous on fibronectin‐coated disks. These particles were observed earliest (1 week) on RFGD‐pretreated surfaces. FTIR analyses showed that the heat pretreatment produced a general pattern of increased levels of apatite mineral at 2–4 weeks; a greater effect was observed for fibronectin‐coated disks compared to uncoated disks. The observed findings suggest that heat pretreatment of Ti6Al4V increased the total mass of the mineral formed in MC3T3 osteoprogenitor cell cultures more than RFGD while the latter pretreatment hastened the early deposition of mineral. These findings help to support the hypothesis that the pretreatments enhance the osteoinductive properties of the alloy. J. Cell. Biochem. 114: 1917–1927, 2013.