Arachidonic Acid but not Eicosapentaenoic Acid (EPA) and Oleic Acid Activates NF-κB and Elevates ICAM-1 Expression in Caco-2 Cells

In patients with inflammatory bowel disease (IBD), intestinal activation of the transcription factor NF-κB as well as intercellular adhesion molecule (ICAM)-1 expression, which is involved in recruiting leukocytes to the side of inflammation is increased. Moreover, colonic arachidonic acid (ARA) proportions are increased and oleic acid (OA) proportions are decreased. Fish oils are protective in IBD patients however, a side-by-side comparison between effects of fish oils, ARA and OA has not been made. We therefore, compared effects of eicosapentaenoic acid (EPA) versus ARA and OA on ICAM-1 expression in Caco-2 enterocytes. To validate our model we showed that dexamethasone, sulfasalazine and PPARα (GW7647) or PPARγ (troglitazone) agonists significantly lowered ICAM-1 expression. ICAM-1 expression of non-stimulated and cytokine stimulated Caco-2 cells cultured for 22 days with ARA was significant higher as compared to EPA and OA. Furthermore, ARA increased NF-κB activation in a reporter cell-line as compared to EPA. Antibody array analysis of multiple inflammatory proteins particularly showed an increased monocyte chemotactic protein (MCP)-1 and angiogenin production and a decreased interleukin (IL)-6 and IL-10 production by ARA as compared to EPA. Our results showed that ARA but not EPA and OA activates NF-κB and elevates ICAM-1 expression in Caco-2 enterocytes. It suggests that replacement of ARA by EPA or OA in the colon mucosa might have beneficial effects for IBD patients. Finally, we suggest that the pro-inflammatory effects of ARA versus EPA and OA are not related to PPARγ activation and/or eicosanoid formation.
Introduction
Epidemiological studies have shown a low incidence of inflammatory bowel disease (IBD) in Eskimo’s as compared to West-European populations [1] and increasing incidences of IBD in Japan [2]. These findings suggest that an increased dietary intake of n-6 polyunsaturated fatty acids (PUFA) and a lower intake of n-3 PUFAs contribute to the development of IBD. Thus, n-3 fish oil PUFAs may have anti-inflammatory effects as compared to n-6 PUFA [3]. Indeed, elevated proportions of the n-6 PUFA arachidonic acid (ARA) in colon mucosa of both ulcerative colitis (UC) and Crohn's disease (CD) patients as compared to those of control subjects have been shown [4–7]. Although the proportion of the n-3 PUFA eicosapentaenoic acid (EPA) in the mucosa was in most studies not statistically different between IBD patients and healthy controls, one study reported a tendency towards lower EPA proportions in IBD patients [4]. In contrast to EPA, the proportion of the fish oil PUFA docosahexaenoic acid (DHA) was elevated in colonic mucosa of IBD patients as compared to control subjects [4, 5, 7]. Interestingly, the proportion of the n-9 monounsaturated fatty acid (MUFA) oleic acid (OA) was lower in colon mucosa of IBD patients [4, 7]. Because of these observations, and because EPA is a more important precursor of eicosanoids than DHA [8], we decided to compare the effects of EPA versus ARA and OA in an in vitro model of intestinal inflammation. OA is already the most abundant fatty acid present both in our diet [9] as well as in colon mucosa [4]. Therefore, and because of differences in ability to compete with ARA for incorporation in tissue phospholipids [10, 11], it is probably easier to lower mucosal ARA levels by increasing fish oil intake than by increasing OA intake. Indeed, by increasing their intake of fish oil, ARA in the colon mucosa of IBD patients was replaced by EPA and DHA [12], which was associated with significantly reduced corticosteroid requirements [12] and lower relapse rates [13]. It should be noted however, that not all intervention studies using fish oils were that positive, although the overall conclusion is that fish oil supplementation shows at least minor protective effects [14].
The intercellular adhesion molecule (ICAM)-1 plays an important role in the pathology of IBD. In IBD patients intestinal ICAM-1 expression [15] and plasma levels of soluble ICAM-1 (sICAM) are increased [16], and IBD is associated with polymorphisms in the gene encoding for ICAM-1 [17]. Moreover, animal models [18, 19] and a human intervention study [20] have shown that ICAM-1 blocking inhibited intestinal inflammation. The transcription factor NF-κB is a key regulator of the inflammatory response and activation of NF-κB seems to play a critical role in the initiation and perpetuation of intestinal inflammation in IBD [21, 22]. NF-κB activity in the colon is increased during active episodes in IBD patients and certain anti-inflammatory drugs commonly used for IBD appear to inhibit NF-κB [23–27]. In animal models, NF-κB blockade abolished experimental colitis [28, 29]. A side-by-side comparison of the n-3 PUFA EPA, the n-6 PUFA ARA and the n-9 MUFA OA on ICAM-1 expression and NF-κB activation of intestinal cells has, as far as we are aware of, never been performed. Therefore the aim of the present study was to compare the effects of EPA, ARA and OA on ICAM-1 expression and NF-κB activation in the human intestinal epithelial Caco-2 cell line in vitro.
Materials and Methods
Reagents
Bovine serum albumin (BSA; endotoxin and fatty acid-free), sulfasalazine, dexamethasone, GW7647, oleic acid (OA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and indomethacin were obtained from Sigma Chemical Company (St Louis, MO). Troglitazone was purchased from Biomol (Plymouth Meeting, PA). Recombinant human IL-1β and interferon (IFN)γ were purchased from Roche Molecular Biochemicals (Mannheim, Germany). DMEM, trypsin, penicillin streptomycin (PS), sodium pyruvate (SP) and non-essential amino acids (NEAA) were obtained from Invitrogen Corporation (Paisley, UK). Fetal calf serum (FCS; South-American) was obtained from Greiner Bio-one (Frickenhausen, Germany).
Intestinal Cell Cultures
The human cell line Caco-2 was purchased from the American Tissue Type Collection (ATTC). Caco-2 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS and 1% penicillin streptomycin (PS), 1% sodium pyruvate (SP) and 1% non-essential amino acids (NEAA). Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere, refreshed every second day and separated by trypsin–0.03% EDTA, when they had reached 70–90% confluence. To evaluate the immune-modulating effects of different interventions, Caco-2 cells were plated in six well tissue culture plates at an initial density of 0.5 × 106 cells/mL in a total volume of 1.5 mL. Medium was replaced every other day for 24 days. After 24 days Caco-2 wells were fully differentiated into small intestinal enterocytes [30]. First, effects of immune-suppressive pharmacological compounds (sulfasalazine, dexamethasone and troglitazone) were tested to validate the model. Although these pharmacologic compounds have known immune-suppressive effects, effects on ICAM-1 expression in Caco-2 cells have—as far as we know—not been reported before. Therefore, after Caco-2 cells were fully differentiated, medium was replaced by medium containing the compound of interest in combination with an inflammation-inducing cocktail consisting of the cytokines IFNγ (100 U/mL) and IL-1β (50 U/mL). The compounds of interest were pre-incubated 30 min (sulfasalazine) or 2 h (dexamethasone, troglitazone or GW7647) before stimulation with the cytokine cocktail. After 16 h of cytokine stimulation, cells were used to determine cell surface ICAM-1 protein expression.
Fatty Acid Experiments
The effects of various fatty acids were evaluated using the same Caco-2 cell model. For this, various fatty acids were added at indicated concentrations 2 days after plating the cells and again for the following 22 days each time when the medium was refreshed. We used OA as a control n-9 MUFA because this is the most abundant fatty acid in the diet [9]. We further compared the effects of the n-6 PUFA ARA versus the n-3 PUFA EPA. We have explicitly chosen to use EPA instead of DHA since the proportion DHA in the colon mucosa of IBD patients was already higher as compared to control subjects [4, 5, 7]. In addition, EPA is most likely a more important eicosanoid precursor [8]. The fatty acids were dissolved in ethanol up to a final ethanol concentration in the medium of maximal 0.5% (v/v). To prevent cytotoxicity of the fatty acids the FA were bound to albumin, by pre-incubating the fatty acids dissolved in ethanol for 30 min at 37 °C in full culture medium together with 10% FCS, which also contained 0.1% BSA. Caco-2 cells were cultured with respectively 160 μM OA [C18:1(n-9)] versus 130 μM ARA [C20:4(n-6)] plus 30 μM OA (in total 160 μM fatty acids) or 6 μM EPA [C20:5(n-3)] plus 154 μM OA (in total also 160 μM fatty acids). By this approach the total molarity of fatty acids supplied was similar in all experiments, while supplying different amounts of the fatty acid of interest (i.e., 130 μM ARA or 6 μM EPA). These relatively low concentrations of OA (160 μM), ARA (130 μM) and EPA (6 μM) were chosen because they are four times higher than normally present in culture medium of Caco-2 cells containing 10% FCS. We have deliberately chosen for this low EPA concentration since the EPA concentration is very low in FCS. However, the EPA concentration used is—as for all fatty acids used in these experiments—already four times higher than normally present in culture medium. We cultured the cells for 22 days with these relatively low concentrations of fatty acids to simulate a realistic long-term in vivo change in dietary fatty acid intake. After 22 days culture with fatty acids, medium was replaced by medium enriched with the different fatty acids plus the cytokine cocktail [IFNγ (100 U/mL) and IL-1β (50 U/mL)]. After 16 h stimulation, ICAM-1 expression on living cells was measured and culture medium was collected to determine inflammatory protein expression profiles. To evaluate the effects of the fatty acids on NF-κB activity and the role of cyclooxygenase (COX)-enzymes, the experiments with the fatty acids ARA and EPA were repeated but now in our NF-κB reporter Caco-2 cell line with and without indomethacin (20 μM) added 2 h before and during cytokine stimulation. Prostaglandin PGE2 levels in the supernatant were quantitated using a PGE2 Biotrak enzyme-immunoassay (EIA) system (Amersham Biosciences Ltd, Buckinghamshire, UK) according to the high sensitivity enzyme immunoassay protocol 2.
Flow Cytometry Analysis of ICAM-1
In order to quantify cell surface ICAM-1 protein expression on living Caco-2 cells, we developed a flow cytometry assay. After 16 h of stimulation, the cells were washed three times with PBS and detached with trypsin–0.03% EDTA. Next, medium was added and cell suspensions were centrifuged for 5 min at 1,200 rpm at room temperature, followed by resuspending the pellets in 500 μL PBS-1% BSA. Cells were counted and diluted to 106 cells/mL in PBS-1% BSA. Recombinant-phycoerythrin (R-PE)-conjugated mouse-anti-human CD-54 monoclonal antibody (anti-ICAM-1) or isotype-matched control antibody (Becton Dickinson Biosciences, San Diego, CA; 20 μL/106 cells) was added and incubated for 30 min on ice in the dark. Next, cell suspensions were centrifuged for 5 min at 1,500 rpm and pellets were resuspended in 500 μL PBS-1% BSA. The amount of fluorescence of 10,000 living cells was counted and analyzed with the FACSort and CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ).
Stable Transfection of NF-κB in Caco-2 Cells
For evaluating the effects of the various interventions on transcriptional activity of NF-κB, a stable NF-κB reporter Caco-2 cell line was created. The 6κB-TK-luciferase (NF-κB reporter) plasmid and neomycin resistance plasmid were both kindly provided by Dr. R.C. Langen (Department of Pulmonology, Maastricht University, The Netherlands). Cells were transfected using Lipofectamine 2000 (Invitrogen Corporation, Paisley, UK) according the manufacturers’ instructions. Positive clones were selected by culturing with geneticin (1 mg/mL). To determine luciferase activity, non-stimulated and 3 h cytokine (100 U/mL IFNγ and 50 U/mL IL-1β) stimulated cells were lysed in luciferase lysis buffer (Promega, Madison, WI) and stored at −80 °C. Luciferase (Promega) activity was measured according to the manufacturers’ instructions and expressed relative to total protein (Bio-rad assay; Bio-rad, Hercules, CA).
Peroxisome Proliferator-Activated Receptor (PPAR)γ and PPARα mRNA Expression of Differentiated Caco-2 Cells
Total RNA was extracted from differentiated Caco-2 cells with Trizol according to the manufacturers’ instructions (Gibco BRL, Gaithersburg, MD). Next, cDNA was made as described [31], and mRNA expression of PPARγ and PPARα was determined using commercially available Taqman gene expression assays (Applied Biosystems, Foster city, CA). Data were normalized against β-actin as housekeeping gene.
Fatty Acid Composition of Caco-2 Cells
Fatty acid incorporation into the Caco-2 cells was evaluated using extraction and analysis procedures as previously described [32]. Briefly, total lipids were extracted from 500 μL cell suspension in PBS-1% BSA according to the method of Bligh and Dyer [33]. Aminopropyl-bonded silica columns (Varian, Harbor City, CA) were used to separate phospholipids from the total lipid extract [34]. The phospholipids were then saponified, and the resultant fatty acids were methylated into their corresponding fatty acid methyl esters (FAMEs) [35]. Fatty acids were separated on an Autosystem (Perkin-Elmer, Norwalk, CT) gas chromatograph that was fitted with a silica-gel column (Cp-sil 88 for FAME, 50 m × 0.25 mm, 0.2-μm film thickness; Chrompack, Middelburg, The Netherlands) with helium gas (130 kPa) as the carrier gas. Both the injection and detection temperatures were set at 300 °C. The starting temperature of the column was 160 °C. Ten min after injection, the temperature was increased up to 190 °C at a rate of 2.5 °C/min. After 20 min at 190 °C, the temperature was increased up to 230 °C at a rate of 4 °C/min. The final temperature of 230 °C was maintained for 10 min. Data were analyzed by using CHROMCARD software (version 1.21; CE Instruments, Milan, Italy). The fatty acid compositions of the Caco-2 cells are expressed in relative amounts (% of total fatty acids identified; w/w).
Inflammatory Protein Expression Profiles Using an Antibody Array
Protein expression patterns of multiple cytokines, chemokines and growth factors, were detected simultaneously in Caco-2 cell culture media with the human cytokine antibody array III (Ray Biotech Inc., Norcross, GA) according to the manufacturers’ instructions. First, duplicates of cell culture media of Caco-2 cells cultured with ARA and EPA after cytokine stimulation were pooled. One millilitre of the pooled samples was added to the array membranes. After incubating and washing, the protein-bound membrane was incubated with a cocktail of biotin-labeled antibodies, followed by the addition of horseradish peroxidase-conjugated streptavidin. Array spot intensity was detected by using a LAS-3000 Lite Image reader (Raytest GmbH, Straubenhart, Germany) based on chemiluminecence imaging. Intensity of the spots was quantified in arbitrary units (a.u.) by densitometry using Aida software version 3.50 (Raytest GmbH), thereby correcting for background staining of the gel. Comparison of protein expression profiles was possible after normalization of each spot on an array using the positive controls, provided by the manufacturer. The sensitivity of the array is not the same for the various proteins. Differences in heights of bars from different proteins do therefore not necessarily represent differences in concentrations. The cytokines used for stimulation (IFNγ and IL-1β) were excluded from analysis.
Detection of ICAM-1 on Caco-2 Frozen Sections
To determine the localization of ICAM-1 in our in vitro Caco-2 cell model, Caco-2 cells were cultured and differentiated into small intestinal enterocyte on collagen-coated polyfluoroethylene transwell membrane inserts with a 0.4 μm membrane pore size (Corning Costar, Cambridge, MA). Differentiated Caco-2 cells were stimulated with IFNγ (100 U/mL) and IL-1β (50 U/mL) for 16 h, embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands) and rapidly frozen in 2-propanol (Fluka, Zwijndrecht, The Netherlands), dry-ice-cooled and stored at −80 °C. Serial cryosections (10 μm) were obtained using a Leica CM3050 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and thaw mounted on uncoated glass slides. Before processing or storage at −80 °C, the samples were air dried overnight. To detect ICAM-1 the sections were incubated 30 min in the dark at room temperature with recombinant-phycoerythrin (R-PE)-conjugated mouse–anti-human CD-54 monoclonal antibody or isotype-matched control antibody (Becton Dickinson Biosciences, San Diego, CA) 1:50 diluted in PBS-1% BSA. To detect cytokeratin (CK)-19 the sections were simultaneously incubated with a monoclonal antibody directed to CK-19, kindly obtained from Dr. E.B. Lane (University Dundee, Dundee, UK) 1:10 diluted. Then the sections were washed three times for 5 min in PBS. After that the secondary antibody goat anti-mouse IgG1 (ALEXA555) (Molecular Probes Europe, Leiden, The Netherlands) (1:500) against anti-CD54 (to evade fast quenching of the PE-label) and goat anti-mouse IgG2b (FITC) (Southern Biotech, Sanbio BV, Uden, The Netherlands) (1:50) against CK-19 diluted in PBS-1% BSA was added to the sections and incubated for 30 min. Again the sections were washed three times for 5 min with PBS. Finally, sections were mounted in Mowiol-TRIS pH 8.5 (Calbiochem, Omnilabo International, Etten-Leur, The Netherlands) containing 0.5 g/mL 4–6-diamino-2-phenylindole (DAPI; Molecular Probes Europe) to stain the nuclei. All sections were examined using a Nikon E800 fluorescence microscope (Uvikon, Bunnik, The Netherlands) coupled to a Basler A101C progressive scan colour CCD camera. By just a simple shift in filters, images were grabbed in fluorescence using the ALEXA excitation filter (540–580 nm), the FITC excitation filter (465–495 nm) and DAPI UV excitation filter (340–380 nm) in the red, green and blue channel, respectively. The images acquired were merged to examine the cellular localisation and level of expression of ICAM-1.
Statistical Analysis
Data were expressed as means and standard deviations (SD) for the non-stimulated condition, the cytokine stimulated condition and cytokine stimulated–non-stimulated (called net stimulated) condition. To determine statistical significance unpaired t-tests (comparison between two interventions) or ANOVA (comparison between three interventions) with a Bonferonni post-hoc test when differences between interventions were significantly different, were performed. First non-stimulated values between interventions were tested to examine differences in basal values. Then, net stimulated values between interventions were tested to examine differences in cytokine-induced changes. Furthermore cytokines stimulated values were tested to examine differences in “end” values. All statistical analyses were performed using SPSS 11 for Mac Os X (SPSS, Chicago, IL). P-values of less than 0.05 were considered statistically significant.
Results
Model Validation
Stimulation with IL-1β and IFNγ increased cell-surface ICAM-1 protein expression on Caco-2 cells, which resulted in a net stimulated ICAM-1 expression of about 50 a.u. (Fig. 1, panels a and b). After pre-treatment of the cells for 2 h with dexamethasone, a corticosteroid with known therapeutic effects in IBD patients, the cytokine stimulated ICAM-1 expression was significantly decreased as compared to control (P = 0.044). Also the net stimulated ICAM-1 expression (25 a.u.) was significantly reduced as compared to control (P = 0.019). Next, we examined the effect of another frequently used therapeutic drug for IBD patients, sulfasalazine. Effects of sulfasalazine were comparable to those observed for dexamethasone. However, also ICAM-1 expression without cytokine stimulation (P = 0.004) (Fig. 1, panel a) was lowered. Altogether, these results show that in our cell model ICAM-1 expression is related to the clinical outcomes of drugs proven to treat IBD and could therefore be used as the main outcome parameter in the following experiments. To further validate characteristics of our in vitro model, we also localized the site of ICAM-1 expression on the Caco-2 cells by means of immunohistochemistry on frozen sections. As shown in Fig. 1 (panels c–f), ICAM-1 was expressed on the apical (lumen) side of the polarized Caco-2 cells. Besides a low constitutive expression (panel d), there was a clear increase after stimulation with the cytokine cocktail (panel e). This localisation is in line with the apical ICAM-1 expression, as found in intestinal biopsies from IBD patients [36].
Fig. 1Model validation. a ICAM-1 expression (in arbitrary units a.u.) on living control Caco-2 cells and after 2 h pre-treatment with 1 μM dexamethasone or 30 min 5 mM sulfasalazine with and without cytokine stimulation for 16 h (100 U/mL IFNγ and 50 U/mL IL-1β) and b net stimulated (stimulated–non-stimulated) ICAM-1 expression. Results represent means ± SD; n = 2. Representative representation of two independent experiments. aP < 0.05 versus control non-stimulated, bP < 0.05 versus control cytokine stimulated, cP < 0.05 versus control net stimulated. (c–f) Immunohistochemistry of ICAM-1 (red) on frozen sections of Caco-2 cells on a transwell showed increased apical expression after cytokine stimulation (100 U/mL IFNγ and 50 U/mL IL-1β). c Hematoxylin staining d Non-stimulated Caco-2 cells e Apical cytokine stimulated Caco-2 cells f Isotype control staining of ICAM-1. Red staining ICAM-1, green staining cytoskeleton (cytokeratin-19), blue staining nucleus. Magnification 40X
Effects of PPAR Agonists on ICAM-1 Expression and NF-κB Activation
Since PPARs are known modulators of inflammation and fatty acids are natural ligands for PPARs, we first examined PPAR expression in our differentiated Caco-2 cells. We found that PPARα and PPARγ mRNA are expressed in equal amounts (data not shown). Next we examined the effects of the PPARγ agonist troglitazone [a thiazolidinedione (TZD)] and the PPARα agonist GW7647 on ICAM-1 expression. Although both troglitazone and GW7647 significantly lowered the cytokine stimulated ICAM-1 expression as compared to control (P < 0.001 and P = 0.009, respectively), only troglitazone significantly (P = 0.008) reduced the net stimulated ICAM-1 expression (Fig. 2, panels a and b). Troglitazone and GW7647 were used at a concentration where they are selective for their receptor subtypes [37, 38]. It is known that PPARs suppress inflammation by inhibiting the transcription factor NF-κB, which is a key regulator of inflammation [39]. Therefore, we also examined effects of troglitazone on NF-κB transactivation in our Caco-2 NF-κB reporter cell line. These results showed that the cytokine cocktail induced after already 3 h a more than 200% increase in NF-κB transactivation. This increase of NF-κB transactivation was significantly (P = 0.008) lower after 2 h pre-treatment with the PPARγ agonist troglitazone (Fig. 2, panels c and d). Moreover, the inhibitory effect of pre-treatment with the PPARγ agonist troglitazone on cytokine induced NF-κB transactivation was significantly (P = 0.001) larger as compared to pre-treatment with the PPARα agonist GW7647 (data not shown).
Fig. 2a, b ICAM-1 expression (in arbitrary units a.u.) on living control Caco-2 cells and after pre-treatment with PPAR agonists (2 h; PPARγ troglitazone 100 μM and PPARα GW7647 1 μM) and c, d NF-κB transactivation measured by luciferase activity (in RLU/mg) in a NF-κB reporter Caco-2 cell-line of control cells and after troglitazone pre-treatment with and without cytokine stimulation for 3 h (100 U/mL IFNγ and 50 U/mL IL-1β) and net stimulated (stimulated–non-stimulated). Results represent means ± SD; n = 2. Representative representation of two independent experiments. bP < 0.05 versus control cytokine stimulated, cP < 0.05 versus control net stimulated
Fatty Acid Incorporation in Phospholipids
Table 1 shows that the fatty acids kept for 22 days in the culture medium were incorporated into the phospholipid fraction (% of total fatty acids) of the Caco-2 cells. Fatty acid incorporation into phospholipids showed the same pattern as the fatty acid composition of total lipids (data not shown), although changes in the total lipids were more pronounced. Moreover, the fatty acid profiles of phospholipids (Table 1) and total lipids (data not shown) were similar in non-stimulated and stimulated Caco-2 cells. Cells cultured with ARA showed in particular an increase in the proportion of long chain n-6 PUFAs (mainly ARA) and a decrease in n-9 MUFAs (mainly OA) as compared with cells cultured with only OA. The cells cultured with EPA showed an increase in the proportion of n-3 fatty acids (mainly EPA) whereas the proportions of n-9 MUFAs and n-6 PUFAs did not change much as compared with cells cultured with only OA.
Table 1Fatty acid composition in phospholipids of Caco-2 cells without (−) and with (+) cytokine stimulation (IL-1β and IFNγ) supplemented with different fatty acids (% of total fatty acids)Fatty acidsOA (160 μM)ARA + OA (130 + 30 μM)EPA + OA (6 + 154 μM)−+−+−+16:0 (PA)16.016.118.719.516.315.818:0 (SA)5.96.19.610.56.76.518:1 trans 3.03.16.15.72.72.718:1 (n-7)3.43.42.62.53.33.318:1 (n-9) (OA)47.146.314.413.845.245.018:2 (n-6) (LA)1.71.71.11.11.71.620:1 (n-9)2.12.10.30.31.71.720:4 (n-6) (ARA)9.39.429.328.98.98.920:5 (n-3) (EPA)0.40.40.00.02.12.222:1 (n-9)0.80.81.51.30.80.824:1 (n-9)2.02.10.91.02.02.122:4 (n-6)0.40.47.77.80.30.322:5 (n-3)0.50.50.40.41.31.322:6 (n-3) (DHA)1.11.10.60.60.70.6∑93.793.593.293.493.792.8∑ SAFA21.922.228.330.023.022.3∑ MUFA58.457.825.824.655.755.6∑ PUFA12.312.438.538.214.314.3∑ n-32.02.01.01.04.14.1∑ n-611.411.538.137.810.910.8∑ n-952.051.317.116.449.749.6Data are representative for two independent experiments and the data are derived from pooled samplesARA arachidonic acid, EPA eicosapentaenoic acid, LA linoleic acid, MUFA monounsaturated fatty acids, OA oleic acid, PA palmitic acid, PUFA polyunsaturated fatty acids, SA stearic acid, SAFA saturated fatty acids, Σ sum, − non-stimulated, + cytokine stimulated
Effects of Fatty Acids on ICAM-1 Expression and NF-κB Activation
ICAM-1 expression was analyzed on living cells. FACS analysis did not show significant changes in cell populations cultured with the various fatty acids, suggesting no significant cell death. As shown in Fig. 3 (panel a), ARA significantly elevated ICAM-1 expression as compared to OA or EPA (non-stimulated P < 0.001 ARA versus OA and ARA versus EPA, and stimulated P = 0.010 ARA versus OA, and P = 0.013 ARA versus EPA). Interestingly, stimulated and non-stimulated ICAM-1 expression on cells cultured with EPA did not significantly differ from those cultured with OA (Fig. 3, panel a). Despite these clear differences in non-stimulated and stimulated ICAM-1 expression between ARA and EPA or OA, the net stimulated ICAM-1 expression did not differ between the groups (ANOVA P = 0.134). Since dietary interventions aiming at lowering mucosal ARA content are most likely more successful by dietary EPA enrichments than by OA enrichment–because OA is the most abundant fatty acid present in our diet [9], making higher intakes not realistic–in further experiments we have focused on effects of ARA versus EPA. First, we examined the effects on NF-κB activation in the stable NF-κB reporter Caco-2 cell line (Fig. 4). NF-κB transactivation in stimulated Caco-2 cells cultured with ARA was significantly increased as compared to that in stimulated cells cultured with EPA (Fig. 4, panels a and b) (stimulated P = 0.017 and net stimulated P = 0.001). To evaluate whether the effects were related to the production of different families of eicosanoids we measured PGE2 production and examined the effects of the different fatty acids in the presence of the cyclooxygenase (COX)-inhibitor indomethacin. As expected, PGE2 production by Caco-2 cells cultured with ARA was significantly higher than by cells cultured with EPA (data not shown). Stimulation of the cells with the cytokine cocktail did however, not influence PGE2 production. Since cytokine stimulation enhanced NF-κB activation, this suggests that PGE2 is not directly involved in NF-κB activation. Indomethacin decreased PGE2 production of Caco-2 cells by approximately 70% (data not shown), but had no effect on NF-κB transactivation (Fig. 4, panels c and d) as compared to untreated Caco-2 cells. Also ICAM-1 expression was not affected (data not shown). However, as shown in Fig. 4, the effects of ARA versus EPA in the presence of indomethacin treatment (panel e) showed an identical pattern as observed without indomethacin (panel a). Also in the presence of indomethacin the net cytokine stimulated NF-κB transactivation was significantly higher after ARA as compared to EPA (P = 0.047) (Fig. 4, panel f). This indicates that the increased basal PGE2 production by ARA cannot explain the different effects of ARA and EPA on NF-κB activation.
Fig. 3a–b ICAM-1 expression (in arbitrary units a.u.) on living Caco-2 cells cultured for 22 days with 160 μM oleic acid (OA) or 130 μM arachidonic acid (ARA) plus 30 μM OA or 6 μM eicosapentaenoic acid (EPA) plus 154 μM OA with and without cytokine stimulation for 16 h (100 U/mL IFNγ and 50 U/mL IL-1β) and net stimulated (stimulated–non-stimulated). Results represent means ± SD; n = 2. Representative representation of two independent experiments. ANOVA between groups, non-stimulated P < 0.001, stimulated P = 0.006 and net stimulated P = 0.134. aBonferroni P < 0.05 versus OA and EPA non-stimulated, bBonferroni P < 0.05 versus OA and EPA cytokine stimulatedFig. 4NF-κB transactivation with and without cytokine stimulation for 3 h (100 U/mL IFNγ and 50 U/mL IL-1β) (a, c, e) and net stimulated (stimulated–non-stimulated) (b, d, f) in a NF-κB reporter Caco-2 cell line measured by luciferase activity (in RLU/mg). a–b Reporter Caco-2 cells cultured for 22 days with 130 μM arachidonic acid (ARA) plus 30 μM oleic acid (OA) or 6 μM eicosapentaenoic acid (EPA) plus 154 μM OA. c–d Control reporter Caco-2 cells and after 2 h pre-treatment with the COX-inhibitor indomethacin (20 μM). e–f Reporter Caco-2 cells cultured for 22 days with 130 μM arachidonic acid (ARA) plus 30 μM OA or 6 μM eicosapentaenoic acid (EPA) plus 154 μM OA and 2 h pre-treatment with the COX-inhibitor indomethacin (20 μM). Results represent means ± SD; n = 2. bP < 0.05 versus EPA cytokine stimulated, cP < 0.05 versus EPA or EPA + indomethacin net stimulated
Effects of Fatty Acids on Inflammatory Proteins Expression Profiles
Finally, to explore the effects of ARA and EPA on a broader scale, we evaluated protein expression profiles consisting of various inflammatory mediators by using antibody arrays. Figure 5 shows that ARA treatment particularly increased expression of monocyte chemotactic protein (MCP)-1 and angiogenin, while EPA treatment increased IL-10, IL-6, macrophage inflammatory protein (MIP)-1δ, and growth regulated protein (GRO) expression.
Fig. 5Protein expression profile in the culture medium of Caco-2 cells cultured for 22 days with 130 μM arachidonic acid (ARA) plus 30 μM oleic acid (OA) or 6 μM eicosapentaenoic acid (EPA) plus 154 μM OA after 16 h cytokine stimulation (100 U/mL IFNγ and 50 U/mL IL-1β) measured with an antibody array. Results are represented as mean ± SD; n = 2. CSF colony stimulating factor, ENA epithelial-derived neutrophil activating protein, MCP monocyte chemotactic protein, MDC, macrophage derived chemokine, MIG monokine induced by gamma interferon, MIP macrophage inflammatory protein, SDF stromal cell-derived factor, GRO growth regulated protein, MCSF Macrophage colony stimulating factor, Tpo thrombopoietin, EGF epidermal growth factor, OSM oncostatin M, VEGF Vascular endothelial growth factor, SCF stem cell factor, IL interleukin, TNF tumor necrosis factor, Ang angiogenin
Discussion
ICAM-1 seems important in the pathology of IBD [15]. Whether fish oils, which have been shown to protect against relapses in IBD patients on remission [13], have effects on ICAM-1 expression, is however, unknown. In addition, a direct side-by-side comparison of fish oils with ARA, which is postulated to have pro-inflammatory effects [40] and is elevated in the colon mucosa of IBD patients [4, 7], has never been made. The transcription factor NF-κB is important in regulating intestinal inflammation and is elevated in IBD patients [21, 22, 26]. We have now shown that the n-3 PUFA EPA, as compared to the n-6 PUFA ARA, clearly reduced cytokine stimulated NF-κB activation and ICAM-1 expression in enterocytes in vitro. Moreover, effects of OA on ICAM-1 expression were comparable to those of EPA. Because EPA and OA resulted in comparable changes in the proportions of ARA in the phospholipids of the enterocytes, the reported effects may be ascribed to the increased ARA proportion in the ARA cultured cells. Thus, decreasing cellular ARA levels seems to be a crucial step. Since OA is already the most abundant fatty acid present in our diet [9] and in the colon mucosa [4], decreasing mucosal ARA levels may be easier by increasing fish oil intake than by increasing OA intake. Moreover, EPA does compete with ARA for incorporation into tissue phospholipids [10, 11].
Our data showed that replacing ARA for EPA or OA decreased ICAM-1 expression and NF-κB activation in Caco-2 enterocytes. In line with our observations in enterocytes, n-6 PUFAs also increased NF-κB activation as compared to n-3 PUFAs in monocytes [41] and macrophages [42]. Also, earlier in vitro studies have demonstrated that fish oils reduced cytokine stimulated ICAM-1 expression in endothelial cells [43] and monocytes [44] as compared to conditions without addition of fatty acids. Moreover, in vivo ICAM-1 expression (surface and mRNA) on peritoneal macrophages was reduced in mice fed fish oils compared to that in mice fed coconut oil [45]. In humans, dietary fish oil supplementation lowered expression of ICAM-1 on ex vivo stimulated monocytes as compared to no supplementation [46]. However, our study is the first that examined effects of EPA versus ARA on ICAM-1 expression and NF-κB activation in enterocytes. We however, realize that, although enterocytes play an important role in intestinal inflammation, immune modulating effects of fatty acids in CD patients will be influenced not only by enterocytes, but also the interaction with other intestinal immune and non-immune cells is important. Therefore, in future experiments effects on other cell types, e.g. isolated from mucosal biopsies of CD patients should be evaluated. Moreover, to validate if the effects of EPA versus ARA are also applicable in the pathogenesis or even the treatment of CD patients, these effects should be confirmed in appropriate animal models of IBD.
We used an approach of supplying different amounts of the fatty acids of interest (i.e., 130 μM ARA or 6 μM EPA), while the total molarity of fatty acids supplied was similar in all experiments by adding OA. These different concentrations of ARA and EPA were deliberately chosen because they were both four times the amount, in which the cells grow normally (i.e., the fatty acid composition of culture medium with 10% FCS). Since the EPA concentration is very low in FCS, we have deliberately chosen for a low EPA concentration. Higher concentrations of EPA would be interesting to examine, however, are difficult to be achieved with dietary interventions in vivo. Using iso-molaric total concentrations of fatty acids is essential, because an increase in total fat can be immune suppressive [47]. Therefore, we used OA as a reference fatty acid to make total fatty acid concentrations between experimental fatty acid conditions iso-molaric, i.e. OA was exchanged for ARA or EPA. In addition, we evaluated the condition of OA only. The results of this latter condition showed that decreasing ARA levels in the mucosa seems to be more important than increasing EPA levels.
Regarding the pathways underlying the anti-inflammatory effect of fish oils, several suggestions have been made. As compared to n-6 PUFAs, n-3 PUFAs may have different effects on (I) signal transduction pathways, and (II) the types and levels of eicosanoids synthesized [40]. Regarding the first mechanism, our finding that EPA lowered NF-κB activation and ICAM-1 expression as compared to ARA indeed showed that EPA and ARA differently affected the NF-κB signal transduction pathway. In this respect, two peroxisome proliferators-activated receptors (PPARs), PPARα and PPARγ, seem relevant, since fatty acids are natural ligands for these PPARs. Both PPARs seem to have anti-inflammatory effects by inhibiting NF-κB activation [48, 49]. In our in vitro model, the PPARγ agonist troglitazone inhibited NF-κB activation, and cytokine and net stimulated ICAM-1 expression, while the PPARα agonist GW7647 only inhibited cytokine-stimulated ICAM-1 expression. This suggests that both PPARγ and PPARα have anti-inflammatory effects on enterocytes, in which effects of PPARγ seem more pronounced. This is in agreement with the finding that PPARγ agonists but not a PPARα agonist inhibited IL-8 expression of Caco-2 cells and HT-29 cells [50]. Differences between fatty acids in activation of PPARs might explain the activation of NF-κB by ARA and not by EPA in our Caco-2 cells. However, our findings are not supported by the PPARγ binding affinities of EPA and ARA, which are about the same [51], while we used much lower EPA concentrations than ARA concentrations. Secondly, not only EPA but also OA lowered intestinal ICAM-1 expression while OA is a poor PPARγ ligand [51]. Therefore, although PPARγ activation certainly protects against inflammation, our results do not suggest that the protective effects of EPA and OA as compared to ARA on intestinal inflammation are PPARγ-mediated. Therefore, there should be another explanation why effects of OA and EPA were comparable but different from those of ARA.
The mechanism underlying a second possible explanation relates to the incorporation of fatty acids into cell membrane phospholipids and eicosanoids synthesis. Eicosanoids can modulate the intensity and duration of the inflammatory response. [8, 40] Replacement of ARA by EPA in the culture medium of the cells resulted in a decrease in the proportion of ARA in cell membranes and an increase in the EPA proportion and as expected the PGE2 production of Caco-2 cells cultured with EPA was also lower than PGE2 production of cells cultured with ARA. However, PGE2 production was not different between non-stimulated and stimulated cells, while stimulation resulted in an abundant elevation of NF-κB activation. To our opinion this rules out the role of PGE2 in the NF-κB activation of ARA and EPA. Moreover, although in our experiments indomethacin–a blocker of COX–indeed inhibited PGE2 production, it did not lower NF-κB activation. This is in line with findings of De Catherina et al. [43] showing that in endothelial cells the effects of DHA on VCAM-1 expression could not be inhibited by indomethacin, although prostacyclin production was completely suppressed. Therefore, we fully agree that in an in vivo situation pro-inflammatory effects of ARA are probably mediated by eicosanoids synthesized from ARA, however, these effects are not derived from a direct eicosanoid mediated activation of NF-κB as shown in our model.
Finally, we evaluated inflammatory protein signatures of Caco-2 cell culture medium after 22 days treatment with EPA versus ARA. Since these signatures were analyzed in pooled material we cannot draw conclusions based on statistical analysis. However, this array was intended to generate hypotheses about differences in immune modulating effect of ARA versus EPA based on the protein expression profiles. These profiles indicate, that both fatty acids induced specific changes in various inflammation mediators. Most of these proteins, but not all, are regulated by NF-κB. For example, IL-6 protein expression was higher after EPA as compared to ARA, whereas NF-κB activation was higher after ARA. It should however, be considered that although highly important, NF-κB is not the only transcription factor involved in the regulation of IL-6 and other inflammatory proteins. Thus, expression of individual proteins cannot be predicted by the level of NF-κB activation solely. In addition, we found clear differences between EPA and ARA, i.e., that some proteins were expressed higher after EPA (IL-6, IL-10, MIP-1δ and GRO), while others were higher expressed after ARA (MCP-1, and angiogenin). The precise role of these individual proteins in the process of intestinal inflammation is not clear. However, the increased MCP-1 expression after ARA treatment is in line with the finding that MCP-1 expression is upregulated in the mucosa of IBD patients and correlates with disease activity [52]. The increased expression of the Th2 cytokine IL-10 after EPA treatment fits with the anti-inflammatory role for IL-10 in IBD pathology. In this respect, IL-10 knockout mice develop chronic intestinal inflammation and delivery of recombinant IL-10 to the intestinal mucosa by the bacterium L. lactis attenuated mucosal inflammation in two mouse models [53]. In humans, a pilot study using these bacteria showed also promising effects [54].
In conclusion, we have shown that ARA but not EPA and OA activates NF-κB and elevates ICAM-1 expression in Caco-2 enterocytes and we hypothesize that the effects are not related to PPARγ activation or eicosanoid formation.

peroxisome proliferator-activated receptor (ppar)


Hepatitis C virus infection acquired in childhood

Hepatitis C virus (HCV) infection occurs less frequently in children than in adult patients, and the natural history, prognosis, and clinical significance of HCV infection in children are poorly defined. We report here a descriptive follow-up of the clinical course, biochemical data, and viral markers observed in 37 children with anti-HCV. Ten patients included in the study tested persistently negative for serum HCV-RNA (group 1) and 27 patients tested persistently positive (group 2). In group 1, serum alanine aminotransferase (ALT) was normal in all patients, while two patients had non-organ-specific autoantibodies. In group 2, serum ALT was elevated in 13 of 27 patients, and five patients had non-organ-specific autoantibodies. HCV genotype 1a and 1b were the most prevalent among HCV-RNA-positive patients. Twenty liver biopsies were carried out on 17 patients in our series (mean evolution time, 11.2 years; range, 3–21 years). The liver specimens showed mild necroinflammatory changes in most patients, and fibrosis was absent or low grade. Two HCV-RNA-positive patients became persistently HCV-RNA negative. Of the 26 children investigated, 7 (one in group 1, six in group 2) had a co-infection with hepatitis G virus. Conclusion Most children chronically infected with HCV were asymptomatic and presented only mild biochemical evidence of hepatic injury. Autoimmunity in the form of non-organ-specific autoantibodies was common. HCV in children induced mild changes in the liver with a low level of fibrosis and at a low rate of progression.
Introduction
HCV (hepatitis C virus) infection occurs less frequently in children than in adult patients. The natural history, prognosis and clinical significance of HCV infection are poorly defined in childhood [11]; in contrast, HCV infection in adults presents a high degree of chronicity, with up to 50% of all HCV-infected adults developing progressive liver disease [21]. Results from prospective studies show that 20–30% of chronically infected adults develop compensated and eventually decompensated cirrhosis or hepatocellular carcinoma, or both, within 20 years of the initial infection [1].
The lower prevalence of HCV infection in children and the fact that most patients undergo antiviral drugs treatment result in very limited knowledge of the natural outcome of chronic HCV infection acquired at early ages of life. Thus, immediate goals in the investigation of HCV infection should be to characterize current epidemiology and to describe the pathogenesis and course of hepatitis C in children and adults [20]. To understand the evolution of HCV infection could be itself an important surrogate end-point for the evaluation of infected young patients with prognostic implications and therapeutical consequences.
The aim of this study was to determine the clinical features and long-term evolution of HCV infection in a group of children who had never received treatment with antiviral drugs.
Patients and methods
Thirty-seven children (16 females, 21 males) with positive antibodies to hepatitis C (anti-HCV) were investigated retrospectively. These patients were followed-up for a period of 5 years. None had received treatment with antiviral drugs for viral hepatitis or had a history of intravenous drug abuse. All subjects made regular visits to our outpatient clinic, and the serum levels of alanine aminotransferase (ALT), albumin, prothrombin time, antinuclear antibodies (ANA), anti-mitochondrial antibodies (AMA), anti-smooth muscle antibodies (SMA), liver-kidney anti-microsomal antibodies type I (LKM), anti-gastric parietal cells antibodies (GPCA), rheumatoid factor, thyroxine (T4), thyroid stimulating hormone (TSH), anti-thyroid antibodies, anti-HCV and HCV-RNA were determined at least on five consecutive occasions at 1-year intervals. Hepatitis B virus (HBV) surface antigen (HBsAg), antibodies to HBV surface antigen (anti-HBs), antibodies to HBV core antigen (Anti-HBc), human immunodeficiency virus (HIV) and antibodies to hepatitis E (anti-HEV) were investigated in all patients during one visit. Hepatitis G virus-RNA (HGV-RNA) and antibodies to HGV (anti-HGV) were determined in 26 patients. Genotypes of HCV were performed in 21 viremic children.
The duration of infection was calculated as the interval between the presumed date of infection and the date of the last visit to the clinic. Liver biopsies were obtained in 17 patients. Repeated biopsies were performed in three patients.
Viral markers for HBV, HCV and HIV were tested by third generation ELISA (Axsym; Abbott Diagnostics, Chicago, Ill.). Anti-HEV was detected by a commercially available ELISA (Bioelisa HEV IgG; Biokit, Barcelona, Spain). HGV-RNA and anti-HEV antibodies were detected using commercial tests (Roche Diagnostics, Mannheim, Germany). HCV-RNA was detected by PCR (Amplicor HCV PCR test, Roche Diagnostics), and HCV genotyping was performed by a second generation line probe assay (INNO-LIPA HCV; Innogenetics, Ghent, Belgium).
Liver biopsies were performed percutaneously and under the ultrasound guidance of an experienced operator. All liver biopsy specimens (>12 mm long) were fixed in formalin, embedded in paraffin stained with hematoxylin-eosin and Masson’s trichrome and then observed by a single experienced pathologist (AM). Histological necroinflammatory activity and fibrosis were scored separately. The degree of histological necrosis with inflammatory activity was scored using the three parameters of the histological activity index (HAI) developed by Knodell et al. [12]; these consist of: (1) piecemeal necrosis and bridging necrosis (score: 0–10), (2) lobular cytolysis (score: 0–4) and (3) portal inflammation (score: 0–4). Fibrosis was scored independently using the following system: F0, absence of fibrosis; F1, fibrous expansion of portal areas; F2, portal to portal bridging fibrous tracts; F3, portal-central bridging fibrous septa; F4, cirrhosis (bridging fibrous septa with parenchymal nodules). This score is a modification of the fibrosis scoring system of Knodell’s method and introduces one additional degree of fibrosis. Macrovesicular steatosis was graded semiquantitatively using a modified score from Brunt et al. [5]. Alternative and additional diagnoses identified in the liver biopsy were also recorded.
Results
Mean chronological age for the 37 patients (21 males and 16 females) at the last follow-up evaluation was 20.1 years (range: 9–30 years). Most of the subjects were diagnosed among patients who were screened for HCV infection after receiving transfusions of blood products for heart surgery, hematological diseases or casual injuries before 1992. Thus, most of the patients included in the study presented with other diseases that were unrelated to HCV infection: 16 patients had a congenital heart disease, seven had various hematological disorders, three had neurological diseases, one had vesicoureteral reflux and one had Turner syndrome associated to IgA deficiency.
Ten patients were anti-HCV-positive and HCV-RNA-negative (Group 1). The presence of HCV-RNA was demonstrated in the other 27 patients (Group 2). Demographic characteristics and the main results obtained in these children are shown in Table 1.
Table 1Characteristics and biologic data of children with HCV infection Patients negative for HCV-RNA (n = 10)Patients positive for HCV-RNA (n = 27)Age (years) at the follow-up evaluationa18 (9–26)20 (9–30)Duration of the HCV infection (years) in 36 patients at the follow-up evaluationa14 (6–23)16 (5–30)Route of infection Parenteral1024 Vertical02 Unknown01Alanine aminotransferase (U/l)First visitLast visit Normal (n )10714 ≤2 × UNL (n )b067 2–4 × UNL (n )055 >4 × UNL (n )091Non-organ specific antibodies Antinuclear antibody02 Anti-mitochondrial antibodies10 Anti-smooth muscle antibodies01 Liver-kidney anti-microsomal antibody type 111 Gastric parietal cell antibodies01HCV genotype 1a8 1b12 2a1 Unknown6HBV00HIV10HGV-RNA/Anti-HGV (n = 26)1/04/2Anti-HEV00aValues are given as the mean, with the range presented in parenthesisbUNL, Upper normal level
Group 1
The once-yearly PCR analysis revealed that ten patients had positive anti-HCV and negative viremia, which reflected an ongoing or past infection with HCV. These patients were asymptomatic, and the liver function tests remained normal in all but two who had been diagnosed with congestive heart failure; the liver abnormalities of these two patients were normalized after their cardiac condition had improved.
One female patient presented persistent positive AMA for 3 years, which then normalized and remained negative during the subsequent 9-year follow-up period. Throughout this period the patient was asymptomatic, and liver tests were normal. Two years after the laboratory tests were negative for antibodies, the patient presented with psoriasis. Another patient had persistent high titers (1:620) of anti-LKM. Fifteen years after acquiring the infection this patient lost the anti-HCV but the anti-LKM remained positive for two more years. During the follow-up this patient did not present liver abnormalities or other diseases.
During the follow-up span of this study, another female patient became persistently anti-HCV negative 12 years after she was infected. At the time of writing, eight patients remain anti-HCV-positive and HCV-RNA-negative, and they show no evidence of liver disorders.
Group 2
HCV-RNA was detectable in the blood of 27 patients, two of whom became HCV-RNA-negative. The age of exposure to the infection and the type of exposure could not be established in one patient; following the detection of HCV in the blood serum, this patient was followed for 11 years and was positive for HCV up to and including the last follow-up visit.
All patients, with one exception, were asymptomatic. The exception had been diagnosed with leukemia in 1987 and received transfusions of blood products on several occasions. She underwent chemotherapy and had a cholestatic hepatitis; liver tests revealed persistent abnormalities. Anti-HCV and HCV-RNA were detected in 1991. The patient presented portal hypertension and progressive liver failure, and when she was 8 years old, in 1993, she received a liver transplant.
The evolution of serum ALT levels is shown in Table 1. Serum levels of albumin and prothrombin time were abnormal only in the patient who developed hepatic failure.
The presence of non-organ-specific autoantibodies (NOSAs) was detected in five patients. One patient (genotype1a) diagnosed with a single ventricle and Fontan surgery developed persistent anti-LKM (titers 1:160–1:620) and the rheumatoid factor throughout the evolution of the disease. Liver biopsy could not be obtained in this patient. Another male patient (genotype 1b) had positive ANA for 2 years, which subsequently normalized. This patient had increased ALT, and the liver biopsy showed minimal necrosis with inflammatory changes (HAI: 2) and no fibrosis. A third patient (genotype 1b) had persistent PGCA during her follow-up period (10 years), with slightly elevated ALT levels, mild necrosis with inflammatory changes (HAI: 4) and minimal fibrosis (stage 1). A fourth patient (genotype1b) with AST levels greater than fourfold the upper normal level presented SMA during the last 2 years of her follow-up (HAI: 6). The patient diagnosed with Turner syndrome and IgA deficiency (genotype 1b) developed throughout the years a seronegative poli-articular rheumatoid arthritis, psoriasis and celiac disease. She had intermittently positive ANA.
The levels of T4 and TSH were normal in all patients, and no patient developed antithyroid antibodies.
Abdominal ultrasounds did not show significant abnormalities. Only the patient who presented cirrhosis had changes associated to portal hypertension.
Liver biopsies were performed in viremic patients who presented altered hepatic function tests and for whom parental consent had been obtained. Seventeen patients underwent 20 liver biopsies. Mean evolution time of the infection at the time of biopsy was established in 16 patients (mean: 11.2 years; range: 3–21 years). The results on necroinflammatory activity and fibrosis are shown in Table 2.
Table 2Histopathological features of patients positive for HCV-RNALiver biopsyPatients (n)Necroinflammatory activity  1–26  3–47  5–64  6–180Fibrosis  F08  F16  F22  F30  F4 (Cirrhosis)1Steatosis0
A second hepatic biopsy was performed in three patients after 5, 12 and 14 years, respectively. The minimal necroinflammatory activity observed in the first biopsy specimens of these three children remained unchanged in the second biopsy. One of the patients progressed from F0 to F1 fibrosis. A second patient never presented with fibrosis, and both samples of the third patient had minimal fibrosis (F1).
During the follow-up period, 2/27 patients became persistently HCV-RNA-negative (for 9 and 10 years, respectively). Both patients showed minimal necrosis and inflammatory activity with minimal fibrosis (F1) in the biopsy performed before HCV-RNA clearance. One patient of Group 1 was HIV-positive, while one patient of Group 1 and six patients of Group 2 were HGV-positive.
Viral coinfections observed in both groups are expressed in Table 1.
Discussion
In this study, chronic hepatitis C infection was silent in most of the children, and there was little biochemical evidence of liver disease in these patients. Only one girl with portal hypertension and liver failure developed symptoms related to the infection. Consequently, our data suggest that HCV infection may be currently underdiagnosed in children and, moreover, that young patients could become a potential source of infection. We therefore strongly recommend that children falling in risk groups be screened for HCV infection.
Of the 37 pediatric patients in this series, seven (19%) had NOSAs. These antibodies are highly prevalent in subjects exposed to the HCV, and a positive test result for SMA, GPCA and ANA is part of the natural course of chronic HCV infection in adults and children [7]. Lenzi et al. [13] reported a higher prevalence of NOSAs in anti-HCV-positive adult patients than in normal controls (25 vs. 6%). Stroffolini et al. [22] found positive NOSAs in 36.9% of 502 subjects with HCV-RNA-positive chronic hepatitis. Muratori et al. [16] detected positive NOSAs in 16 of 47 children (34%) with chronic hepatitis C, and anti-LKM was present in 11% of the children.
Of the 37 children participating in our study who manifested past or active HCV infection, two had serum anti-LKM. This incidence is lower than that observed by Muratori et al. (11%) [16] and Bortolotti et al. (10.3%) [4] in children with HCV infection. A possible explanation for this finding is the higher age and longer mean duration of HCV infection in the patients included in our series in comparison to the children in the other two studies. The prevalence reported by Lenzi et al. (1.3% [13]), Stroffolini et al. (2.25% [22]) and Reddy et al. (0% [18]) in adults support this hypothesis. Anti-LKM is an immunomarker of type 2 autoimmune hepatitis [2] that tends to present at younger ages and to affect mainly children. Our data suggest that age may be a factor contributing to the presence of anti-LKM1 in children infected with HCV. Moreover, we observed that one of our non-viremic patients had persistent antibodies after the test for anti-HCV became negative.
Liver specimens from the children with hepatitis C showed mild necroinflammatory changes and a low level of fibrosis. In most patients, fibrosis was absent or low grade. Only one patient, as mentioned above, developed cirrhosis and liver failure over a period of 6 years. This leukemic patient had other additional risk factors for liver damage that may have affected this outcome. In this respect, Hoshiyama et al. [9] observed that chronic hepatitis C is more frequent among children with hepatitis C infection following blood transfusions for malignant disease than in patients with hepatitis C following blood transfusions for open heart surgery.
We have compared our results with those obtained from other pediatric series [6, 8, 9, 23]. Table 3 summarizes the data on fibrosis obtained from liver biopsies of 210 pediatric patients (above-mentioned studies). Fibrosis was not detected or was low grade in 200 patients (95%), and severe fibrosis or cirrhosis was detected in ten patients (5%). Minola et al. [15] and Poynard et al. [17] demonstrated an inverse correlation between age at HCV infection and progression to cirrhosis that suggests that chronic HCV infection in childhood induces mild changes in the liver with a low level of fibrosis and a low rate of progression. However, it has been observed that mild chronic hepatitis C can be a progressive disease in adult patients [3, 19] and that the long evolution of infection acquired in childhood could increase the risk of fibrosis. Thus, some children infected early in life, in whom chronic disease has a mild liver expression, might develop fibrosis in adulthood.
Table 3Hepatic fibrosis in biopsies from children with HCV infection Vogt et al. [23]Hoshiyama et al. [9]Guido et al. [8]El-Raziky et al. [6]This reportNumber of patients173811226a17Mean duration of the HCV infection (year)21.2 ± 4.67.1 ± 2.8 (n = 23)12.9 ± 3.1 (n = 15)8.04 ± 5.3Non-determined11.2 ± 5,6FibrosisNo/low grade14/2b231525/8120/47/9Severe/cirrhosis0/1c005/11/1d0/1eaSome patients were co-infected with HBV and /or had secondary iron overloadbTwo patients with congestive heart failurecPatient with Anti-HBs, anti-HBc and anti-HEV antibodiesdThis 12-year-old cirrhotic patient was not thalassaemic and HBV markers were negativeeThis leukemic patient was infected with HCV and was also receiving chemotherapy concurrently
It is noteworthy that 6/27 of the children with HCV infection in our series had a co-infection with HGV (HGV-RNA-positive) or had a past HGV infection (anti-HGV-positive). This prevalence of HGV infection is higher than that found in healthy children (6%) in Spain [10]. These data suggest that HCV-infected children must be considered to be a risk group for HGV infection since HCV and HGV are both parenterally transmitted. Moreover, no differences were observed in biochemical and histological findings in HCV-infected children with and without a concomitant infection with HGV, which suggests that superimposed HGV infection does not influence the course of HCV infection.
Conversely, our data do not support that HEV, which can be transmitted by a parenteral route [24], is prevalent in children who had undergone blood transfusions (prevalence rate: 0%). However, this low value may result from the low prevalence of HEV infection in Spain (2.8%) [14].
Conclusion
Most children chronically infected with HCV are asymptomatic and present only mild biochemical evidence of hepatic injury. Spontaneous clearing of the virus occurs occasionally. Autoantibodies are common in HCV patients. The natural history of chronic hepatitis C in children differs from that in adults since HCV infection is relatively benign, induces mild changes in the liver with a low level of fibrosis and a low rate of progression and is rarely associated with severe or decompensate liver disease.

hepatitis c virus
anti-liver/kidney microsomal antibody
infectious hepatitis
liver fibrosis
nonorgan-specific autoantibodies


Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein-2 (rhBMP-2). A pilot study in dogs

The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2-biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm2), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm2)] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependant manner.
Introduction
The adhesion of plasma proteins on the surface of titanium implants has been reported to play an essential role in the process of osseointegration [2, 4, 8, 9]. Each surface of a material is characterized by a unique composition of adsorbed proteins, which influences the type of cells that may adhere. Subsequently, the specific pattern of adsorbed proteins determines the type of tissue that will develop at the interface between the implanted material and the host [32, 41, 42]. In recent years, several modifications of specific surface properties such as structure, chemistry, surface charge, and wettability have been investigated to improve osseointegration of titanium implants [1]. Additionally, several growth factors and cytokines have also been suggested to stimulate a deposition of cells with the capacity of regenerating the desired tissue [27, 39, 46]. In case of endosseous titanium implants, an enhanced proliferation and differentiation of undifferentiated mesenchymal cells, osteoprogenitor cells, and preosteoblasts into osteoblasts may improve bone response and subsequently osseointegration [5]. One particular growth factor, bone morphogenetic protein (BMP), has shown considerable potential to stimulate bone formation both in extraskeletal sites [47, 48] and in defect models in different species [38, 50]. BMPs originate from the transforming growth factor-β family, including at least 18 proteins [29]. BMP-2, which has been described as an anthelix structure, seems to possess the highest osteoinductive potential among the BMPs [25]. In recent years, the regenerative potential of recombinant human BMP-2 (rhBMP-2) has been demonstrated in various experimental animal studies, including sinus floor augmentation, alveolar ridge preservation, bone augmentation procedures, and periodontal repair [3, 11–15, 31, 33, 49]. Most recently, the effects of rhBMP-2 on the osseointegration of titanium implants have also been investigated in experimental animal studies [14, 26, 33–37, 45]. Experimental titanium plasma-sprayed hollow cylinder implants were filled with a solution of rhBMP-2 soaked on an absorbable type-I collagen sponge before insertion. The histomorphometric analysis revealed a significantly increased bone regeneration in rhBMP-2-treated defects compared to controls. Furthermore, the level of osseointegration, as measured by direct bone-implant contact, was significantly higher for the rhBMP-2 implants compared to controls [34]. Recently, biologically active rhBMP-2 has also been covalently immobilized on metal surfaces [16, 19–21, 44]. Osseointegration of rhBMP-2-biocoated plasma spray-coated titanium–alloy cylinders, as evaluated histomorphometrically 4 weeks after implantation in the distal femur condylus in a gap healing model in sheeps, was predominantly characterized by circumferential bone formation and integration with minimal residual gaps. In contrast, control specimens generally exhibited a wide gap surrounding the implant cylinder [18]. The rationale for BMP immobilization was to avoid ectopic bone formation by a limited and targeted release of rhBMP-2 from the implant surface. Recently, treatment of titanium with chromosulfuric acid (CSA) has been reported to result in ultrahydrophilic bioadhesive surfaces, which in turn improves biocoating with rhBMP-2 [19]. However, there are currently no histological data evaluating osseointegration of CSA-modified and rhBMP-2-biocoated titanium implants.
Therefore, the aim of the present study was to investigate histomorphometrically bone formation to CSA-enhanced and rhBMP-2-biocoated titanium implants after implantation in the mandibula and tibia of dogs in comparison to control titanium implants.
Materials and methods
Animals
Two 3-year-old male mongrel dogs (approximate weight 25 kg) were used in the study. Both animals exhibited a fully erupted, healthy, permanent dentition. During the experiment, the dogs were fed ad libitum with soft-food diet and water. Animal selection, management, and surgery protocol were approved by the Animal Care and Use Committee of Belgrade University (ref. no. 2179). The experimental segment of the study started after an adaptation period of 4 weeks.
Study design
The study was performed in two surgical phases. In the first phase, extraction of the mandibular second, third, and fourth premolar and first molar (P2–M1) was performed bilaterally. After 4 months of healing, surgical implantation of rhBMP-2-biocoated and rhBMP-2-noncoated screw-typed implants was performed in a submerged healing procedure during the second phase. Throughout the study period, oral hygiene procedures were performed three times a week including tooth and implant brushing. Radiographs were obtained before and immediately after tooth extraction as well as immediately after implant installation. Both animals were killed after a healing period of 4 weeks.
Implant preparation
RhBMP-2 was prepared as previously described [21]. The biological activity of soluble rhBMP-2 was assessed with MC3T3-E1 cells by the induction of the de novo synthesis of alkaline phosphatase (AP) [43]. The half-activation constants (K0.5) were in the range of 20–75 nM [18]. Twenty-four screw-type implants (Camlog Screw Line, Wimsheim, Germany) were manufactured from commercially pure titanium. The core diameter of the implants was 3.3 mm and the total length was 11 mm. A total of six (n=6) implants were sand-blasted and acid-etched according to a standardized procedure (Promote, Altatec, Wimsheim, Germany) (C), while a total of 18 (n=18) implants were surface-enhanced by a novel procedure with CSA [21]. The treatment of metals with CSA (CSA–Ti–alloy) [21] leads to ultrahydrophilic (contact angles 0–10°, no hysteresis) bioadhesive surfaces [17]. A total of 12 (n=12) surface-enhanced implants were divided into two subgroups (A and B) and biocoated with rhBMP-2 [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm2), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm2)] [40]. RhBMP-2 was immobilized by covalent and noncovalent methods on these CSA-treated surfaces [18, 21, 40]. In brief, the implants were assigned to the following test and control groups: BMP-A (n=6), BMP-B (n=6), CSA (n=6), and C (n=6).
To control the produced surface, the following “sibling method” was employed: Parallel to the preparation of the above dental implants for in vivo experiments, miniplates (10×5×1 mm) with identical Promote surfaces were surface-enhanced with CSA and coated with 125I-rhBMP-2 under identical conditions as the dental implants. In this way, the corresponding contact angles, the amount of immobilized rhBMP-2, and the in vitro biological activity [6] could be tested before the implants were placed into the animals. Only those dental implants were released for implantation, whose sibling miniplates reached the standard as mentioned above and whose surfaces showed an intense in vitro bioactivity by fluorescence microscopy [6].
Surgical procedure for both phases
The dogs were anesthetized with 1 mg/kg sodium pentobarbital. To maintain hydration, both animals received a constant rate infusion of lactated Ringer’s solution while being anesthetized. Prophylactic antibiotics were administrated intraoperatively with a combination of 20,000 IU penicillin and 1.0 g streptomycin/10 kg body weight. In the first surgery, P2–M1 were carefully removed after reflection of full thickness mucoperiosteal flaps and tooth separation. After wound closure by means of mattress sutures, the sites were allowed to heal for 4 months. In the second surgery, the test and control implants were randomly allocated to both sides of the mandible (left and right sides, one implant each) and one implant each to either the posterior left or right tibia. In brief, bilateral vestibular incisions were made, and full thickness mucoperiosteal flaps were elevated to expose the respective sites for implant placement in the mandible. Surgical implant sites were prepared bilaterally, at a distance of 10 mm apart, according to the protocol suggested by the manufacturer. All implants in the mandible were sealed with cover screws (Camlog, Wimsheim, Germany). After irrigation, mucoperiosteal flaps were repositioned, and primary wound closure was achieved with consecutive polyglycolic acid 5.0 Polyester sutures (Resorba, Nürnberg, Germany). An area of approximately 15 cm in length and 4 cm in width was depilated on the respective side of the tibia using an electric shaver and a razor blade. After disinfection with polyvidone iodine (Betaisodona, Mundipharma, Limburg/Lahn, Germany), a skin incision was made and a flap was elevated to expose the respective sites of the tibia for implant placement. On the inner-posterior side of the tibias, implants were inserted at a distance of 30 mm apart. All titanium implants were inserted with good primary stability according to a low-trauma surgical technique under copious irrigation with sterile 0.9% physiological saline. After implant placement, the periosteum and fascia were sutured using 3.0 Polyester sutures (Resorba, Nürnberg, Germany).
Animal killing and retrieval of specimens
The animals were killed (overdose of sodium pentobarbital, 200 mg/kg i.v.) after 4 weeks. The jaws as well as the posterior tibias were dissected, and blocks containing the experimental specimens were obtained. Block sections of the anterior tibia served as additional control (UC). All specimens were fixed in 10% neutral buffered formalin solution for 4–7 days. The specimens were dehydrated using ascending grades of alcohol and xylol, and infiltrated and embedded in methylmethacrylate (MMA, Technovit 7200, Heraeus Kulzer, Wehrheim, Germany) for nondecalcified sectioning. After 18 to 24 h, the specimens were completely polymerized. Each implant site was cut in the mesiodistal direction and along with the long axis of the implant using a diamond wire saw (Exakt, Apparatebau, Norderstedt, Germany), resulting in four sections of approximately 500 μm in thickness [7]. Subsequently, all specimens were glued with acrylic cement (Technovit 7210 VLC, Heraeus Kulzer, Wehrheim, Germany) to opaque Plexiglas and ground to a final thickness of approximately 40 μm. All sections were stained with toluidine blue.
Histological and histomorphometric analysis
Histomorphometrical analyses as well as microscopic observations were performed by one experienced investigator masked to the specific experimental conditions. For histomorphometrical measurements, images were obtained using a light microscope (BX50, Olympus, Hamburg, Germany) at a magnification of 100×, associated with a video camera (SIS Color View3, Soft imaging System GmbH, Münster, Germany). Digital images were evaluated using a software program (SIS analySIS Auto Software 3.2, Soft imaging System GmbH, Münster, Germany). The percentage of direct contact between mineralized bone and the titanium surface (bone to implant contact—BIC) was measured at every thread on both sides of the implant [23]. BIC was subdivided into crestal and apical values (upper and lower halves of the intraosseous implant length, respectively). Furthermore, in the tibias, induced bone density (BD) adjacent to the titanium surfaces was measured at distances of <1 and 1 mm by assessing the ratio of mineralized bone vs bone marrow within the respective areas [28].
Results
Clinical observations
The postoperative healing was uneventful in both dogs. No complications such as allergic reactions, abscesses, or infections were observed throughout the study period.
Histological and histomorphometric analysis
BIC and BD values for each group and respective anatomical sites (mandible and tibia) are presented in Fig. 1a–c. In particular, all test and control implants inserted in the mandible generally exhibited new bone formation in direct contact with the implant interface. The formation of organized trabeculas of woven bone, recognized by osteon formation, could be observed in all groups. However, new bone formation in direct contact with the implant interface appeared to be higher in the BMP groups (Fig. 2a–d). In particular, BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C groups. No differences in BIC with respect to crestal and apical values were observed within or between groups (Fig. 1a).
Fig. 1Boxplots with outliners for the medians and Q1–Q3 quartiles of BIC and BD (%) in different groups after 4 weeks of healing with respective values in the crestal and apical portion of the implant (BIC) and at a distance of less than and greater than 1 mm adjacent to the implant surface (BD). Lines below and above box plots min, max. control c. a BIC mandibula. b BIC tibia. c BD tibiaFig. 2Histology of representative titanium implants after 4 weeks of healing in the mandible (original magnification ×40). BIC appeared to be highest for BMP-B, followed by BMP-A, CSA, and C. a C. b CSA. c BMP-A. d BMP-B
In comparison to the organized trabeculas of woven bone noted in the mandible, bone formation in the tibia seemed to be of a cancellous type, mainly characterized by tiny trabeculas (Fig. 3a–d). BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C groups. Again, with respect to crestal and apical BIC values, no differences were observed within groups (Fig. 1b). Histomorphometric analysis of BD in the tibia at a distance of <1 mm revealed obvious differences between groups. In particular, highest values were noted for both BMP groups (BMP-A=BMP-B). This was followed by CSA, also exhibiting higher BD values than C. No differences between groups were found at a distance of >1 mm (Fig. 2c).
Fig. 3Histology of representative titanium implants after 4 weeks of healing in the tibia (original magnification ×10). BIC and BD appeared to be highest for BMP-B, followed by BMP-A, CSA, and C. a C. b CSA. c BMP-A. d BMP-B
Discussion
The present histological study was designed to evaluate bone formation and direct bone apposition to rhBMP-2-biocoated, CSA surface-enhanced, and C titanium implants after implantation in the mandible and tibia of dogs. In particular, rhBMP-2 was immobilized by covalent and noncovalent methods on CSA surface-enhanced titanium implants [18, 21, 40]. Within its limits, histomorphometrical analysis of implants inserted in both mandible and tibia after 4 weeks revealed that BIC values appeared to be highest in the BMP groups, followed by CSA and C groups. Furthermore, it was observed that BD as measured at a distance of <1 mm in the tibia was obviously highest in the BMP-B group, followed by BMP-A, CSA, and C groups. However, no differences between groups were observed at a distance of >1 mm. In this context, it is important to realize that the present pilot study does not have the statistical power to rule out the possibility of a difference between groups. Further experimental studies of higher power are needed to support equivalence or superiority [10]. On the other hand, it needs also to be pointed out that these are the first histological data evaluating bone formation and apposition on rhBMP-2-biocoated and CSA surface-enhanced titanium implants in the mandible and tibia. However, the present findings corroborate, to a certain extent, previous results observed in an ectopic bone formation model [40]. Electropolished titanium miniplates were surface-enhanced by CSA and coated with a total amount of 150–200 ng rhBMP-2. Periosteal flaps were prepared from the anterior surface of the tibias of adult rabbits and wrapped around the titanium specimens. Additionally, some titanium miniplates were inserted to which nonimmobilized soluble rhBMP-2 was added. After 28 days of healing, noncoated specimens revealed bone formation in 2/12 implants, rhBMP-2-coated implants in 6/8, and implants with free rhBMP-2 in 8/8 cases. However, in the case of rhBMP-2-coated implants, the induced bone had direct contact to the implant in all cases. In contrast, titanium miniplates inserted with free administered rhBMP-2 revealed direct BIC in just six cases, whereas in two cases, the titanium surface was separated by a fibrous capsule [40]. The finding that rhBMP-2 may promote periimplant bone regeneration and osseointegration of titanium implants is in accordance with the previous studies [14, 26, 33–37, 45]. In all of these studies, however, rhBMP-2 was admixed with a carrier (i.e., collagen, calcium-phosphate cement carrier), acting as a slow delivery system, for instillation with the implant due to a rapid diffusion of BMP after implantation in vivo. In particular, Sigurdsson et al. [33] evaluated rhBMP-2- (2×0.43 mg/ml in a type-I bovine collagen carrier) induced bone regeneration and osseointegration in a supraalveolar periimplant defect model in dogs. At 16 weeks after healing, bone regeneration (height) was significantly larger for rhBMP-2 than control defects. However, the large amount of BMP-induced bone was poorly adapted to the implant surface. In contrast, the smaller amount of new bone in the control group seemed to be well adapted [33]. In contrast, Wikesjö et al. [45] reported similar BIC values 16 weeks after implantation of rhBMP-2 (0.4 and 0.75 mg/ml in a calcium-phosphate cement carrier) or carrier alone subsequent to a vertical alveolar ridge augmentation procedure and simultaneous implant installation in dogs. Furthermore, Howell et al. [14] applied rhBMP-2 using a collagen sponge carrier to stimulate bone formation in artificially created defects around endosseous implants in the canine mandible. Nonresorbable expanded tetrafluoroethylene (e-PTFE) membranes served as controls. Histological analysis revealed that the addition of rhBMP-2 resulted in a significantly greater amount of new bone area and BIC after 4 and 12 weeks of healing than e-PTFE. Although membrane-treated sites were reported to have less new bone formation after 4 weeks of healing, this difference seemed to be equalized after 12 weeks [14]. Similar results were also reported by Sykaras et al. [34]. RhBMP-2-induced bone regeneration and osseointegration was evaluated in mandibular bony defects created within the hollow chamber of endosseous dental implants in dogs. Before insertion, hollow chambers were filled with 20 μg of rhBMP-2 soaked on an absorbable type-I collagen sponge (0.4 mg/ml). Histological observation revealed statistically significant higher BIC values in the rhBMP-2 group at 8 and 12 weeks after implantation [34]. There might be several explanations for the discrepancies noted in these studies. First of all, it must be emphasized that little information is available on the interaction between rhBMP-2 and the individual carriers. Furthermore, the high concentrations of rhBMP-2 used in these studies (in the milligram per milliliter range) strongly indicate that an optimal method is still lacking. In this context, it is important to point out that BMP-2 has the potency to induce or modulate apoptosis [24], and that in vivo application of high doses of BMP-2 may inhibit bone formation [22, 30]. Furthermore, the results of a recent cell culture study have shown that the dose-dependent effect of rhBMP-2 on AP induction in MC3T3-E1 cells plateaus out into a maximal response at 300–1,000 nM BMP-2 (i.e., 8–25 μg/ml) [43]. Indeed, the results of the present study have shown that BIC and BD values seemed to be ameliorated after application of rhBMP-2 at far lower concentrations. Furthermore, it must also be noted that the effects of rhBMP-2 were limited to a range of 1 mm, outlining that both covalent and noncovalent methods of immobilization seemed to be stable. This finding may also be supported by the observation that no differences with respect to BIC values were observed in crestal and apical areas of the implant surface in all groups. In accordance, it might be hypothesized that from a clinical point of view, both methods of immobilization are suitable to avoid ectopic bone formation due to a limited and targeted release of rhBMP-2 from the implant surface. However, further studies are needed to clarify this issue.
Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependent manner.

titanium implant
rhbmp-2
animal study
bioactive surface
ultrahydrophilic surface