Kaili Anier

Kristina Malinovskaja

Anu Aonurm-Helm

Alexander Zharkovsky

Dr Anti Kalda
email: anti.kalda@ut.ee

DNA Methylation Regulates Cocaine-induced Behavioural Sensitization in Mice

Keywords: DNA methylation, DNA methyltransferase, cocaine, behavioural sensitization, nucleus accumbens, zebularine

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INTRODUCTION

Repeated administration of psychostimulants, such as cocaine or amphetamine, induces an enhanced behavioural response to subsequent drug exposure, a phenomenon known as psychomotor or behavioural sensitization that can persist for months [START_REF] Robinson | The neural basis of drug craving: an incentivesensitization theory of addiction[END_REF][START_REF] Pierce | A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants[END_REF]. Psychostimulant-induced behavioural sensitization in rodents provides a model for addictive behaviours such as those associated with craving and relapse, as well as for psychotic complications of psychostimulant abuse [START_REF] Koob | Cellular and molecular mechanisms of drug dependence[END_REF][START_REF] Robinson | The neural basis of drug craving: an incentivesensitization theory of addiction[END_REF].

Persistent behavioural sensitization after repeated psychostimulant treatment indicates that drug-induced short-and long-term changes in gene expression may be involved [START_REF] Nestler | Molecular and cellular basis of addiction[END_REF]. Recently, new concepts have emerged about chromatin modification and gene regulation in dividing and non-dividing cells. Chromatin-related modifications of gene expression patterns have been shown to be important for the long lasting effects of psychostimulants on the brain [START_REF] Kumar | Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum[END_REF][START_REF] Brami-Cherrier | Parsing molecular and behavioral effects of cocaine in mitogen-and stressactivated protein kinase-1-deficient mice[END_REF][START_REF] Renthal | Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli[END_REF]Renthal et al, 2008;Renthal and Nestler, 2008;[START_REF] Shen | Additive effects of histone deacetylase inhibitors and amphetamine on histone H4 acetylation, cAMP responsive element binding protein phosphorylation and DeltaFosB expression in the striatum and locomotor sensitization in mice[END_REF][START_REF] Stipanovich | A phosphatase cascade by which rewarding stimuli control nucleosomal response[END_REF][START_REF] Renthal | Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins[END_REF].

Over the past few years much attention has been paid to the role of histone acetylation and phosphorylation on chromatin modification in psychostimulant-induced neuroadaptations, however there is no data concerning DNA methylation. The most common form of DNA alteration is the addition of a methyl group to the 5 position of cytosine, catalysed by DNA methyltransferases (DNMTs) [START_REF] Bestor | The DNA methyltransferases of mammals[END_REF]. Cytosine methylation takes place almost exclusively at a cytosine located 5' to guanine; cytosine-phospho-guanine (CpG) [START_REF] Singal | DNA methylation[END_REF]. The CpG dinucleotides are unevenly distributed: in mammalian genomes they are clustered in so-called CpG islands -short regions of 0.5 to 4 kb in length having a rich (60-70%) cytosine-guanine content. Over 50% of all dinucleotides in these islands are CpG, compared to the rest of the genome where the CpG content is ≤ 20% [START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF]. About 50% of CpG islands are located in the promoter regions and around the transcription start sites and are unmethylated in normal cells. The change from unmethylated to methylated CpG islands is recognized as an essential contributor to transcriptional silencing [START_REF] Robertson | DNA methylation in health and disease[END_REF][START_REF] Jones | The fundamental role of epigenetic events in cancer[END_REF].

In mammalian cells, two classes of DNMTs catalyse DNA methylation. DNAmethyltransferase-1 (DNMT1) is essential for maintaining DNA methylation patterns in proliferating cells and is also involved in establishing new DNA methylation patterns (de novo methylation) [START_REF] Bestor | The DNA methyltransferases of mammals[END_REF][START_REF] Goll | Eukaryotic cytosine methyltransferases[END_REF][START_REF] Siedlecki | Mammalian DNA methyltransferases[END_REF].

Members of the second class of methyltransferases, DNMT3A and DNMT3B, are also required for de novo methylation.

In the current study, we investigated whether cocaine treatment changes DNMT expression in the nucleus accumbens (NAc), whether DNA methylation regulates protein phosphatase-1 catalytic subunit (PP1c) and fosB gene transcription in the NAc after acute and repeated cocaine treatment, and whether a pharmacological inhibition of DNMT alters the development of behavioural sensitization in mice.

MATERIALS AND METHODS

Animals

All experiments were performed in accordance with EU guidelines (directive 86/609/EEC) on the ethical use of animals using the experimental protocol approved by the Faculty of Medicine of the University of Tartu. Male C57BL/6 mice (weight, 25-30 g) were obtained from Scanbur BK (Sweden) and were maintained in temperature and humidity-controlled rooms with 12 h light-dark cycle (light from 7:00 a.m. to 7:00 p.m.). Animals were allowed access to rodent chow and water ad libitum. Animals were allowed to acclimate to laboratory conditions and were handled at least four days before use in behavioural experiments (which were conducted during the light phase of the light-dark cycle).

Behavioural procedures

Horizontal locomotor activity was assessed in standard polypropylene cages (15 x 25 cm) uniformly illuminated with dim lighting. A light-sensitive video camera, connected to a computer, was mounted about 1.5 m above the observation cage and locomotor activity of eight animals in time was monitored and analysed using VideoMot2 software (TSE Systems,

Germany).

For experiments investigating the effect of acute and repeated cocaine treatment, animals were randomly assigned to the following treatment groups: (1) "SAL", mice were treated for seven days with sterile saline 0.1 ml/10 g body weight intraperitoneally (ip); (2) acute cocaine "AC", mice were treated for six days with saline and on the seventh day with cocaine hydrochloride (15 mg/kg, ip, Oriola Oy, Finland); (3) repeated cocaine "RC", mice were treated for seven days with cocaine (15 mg/kg, ip). On the first and seventh treatment days, locomotor activity was recorded for 60 min after the last injection. On the second through to the sixth treatment days, mice were injected with the drugs and placed in the test cages for 60 min without locomotion recording.

For experiments investigating the effect of inhibition of DNMTs, intracerebroventricular (icv) infusions of zebularine (300 ng/0.5 μl, Tocris Bioscience, UK) or saline (0.5 μl) were performed 20 min before cocaine (15 mg/kg ip) or saline (0.1 ml/10 g body weight, ip) treatment. Zebularine and cocaine were freshly prepared in sterile saline.

Mice were randomly assigned to the following treatment groups: (1) saline (icv) + saline (ip) "S+S"; (2) zebularine (icv) + saline (ip) "Z+S"; (3) saline (icv) + cocaine (ip) "S + C" and ( 4) zebularine (icv) + cocaine (ip) "Z + C". Locomotor activity was recorded for 60 min after ip injection, daily for seven days.

Intracerebroventricular cannula implantation

For cannula implantation, mice were anaesthetized with Hypnorm (VetaPharma, Leeds, UK) and Dormicum (Roche, Basel, Switzerland). During surgery each mouse was implanted with a 26-gauge bilateral stainless steel guide cannula (Plastics One, Roankoke, USA) from which the injector extended 0.5 mm to end in the ventricle. Stereotaxic coordinates used were as follows: antero-posterior, -0.4 mm from bregma, -1.25 mm lateral from the midline, and -2.0 mm from bregma measured from the tip of the cannula guide [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF].

Animals were habituated to dummy cannula removal and given four days of recovery and handling before the start of the experimental procedure. At the beginning of the study, target coordinates and proper cannula placement were verified by slowly injecting 1 μl of methylene blue dye into the cannulas of the first four mice, while they were under anaesthesia. After waiting 5 min for the dye to circulate through the ventricles, the animals were euthanized.

Brains were removed, sliced at the point of cannula entry and target coordinates were verified.

Tissue isolation

Dissection of the nucleus accumbens (NAc) was performed using a round-shape puncher. The NAc and hippocampus were immediately frozen in liquid nitrogen and stored at -80 °C until RNA or DNA extracts were prepared.

Measuring mRNA levels by real-time PCR

Total RNA was extracted from the NAc using RNeasy Mini Kit (QIAGEN, Hilden, Germany). Oligo-dT first-strand cDNA was synthesized from 0.4 μg of total RNA using First Strand cDNA Synthesis Kit (Fermentas Inc, Canada). Real-time PCR was performed using an ABI PRISM 7000 Sequence Detection System equipped with ABI PRISM 7000 SDS Software (Applied Biosystems Inc, USA). Primers for mouse PP1c were from SABiosciences (In Vitro Sweden AB, Sweden) and primers designed for mouse DNMT1, DNMT3A, DNMT3B and fosB were as listed in Table 1. PCR amplification was performed in a total reaction volume of 25 μl in three parallels. The reaction mixture consisted of 1 μl First Strand cDNA diluted template, 12.5 μl 2x Master SYBR Green RT-PCR Master Mix (Applied Biosystems Inc, USA), 10.5 μl H 2 0 and 1 μl gene-specific 10 μM PCR primer pair stock. Amplification specificity was controlled by a melting curve analysis and a gel electrophoresis of the PCR product. Serial dilutions (5-fold) from one wild type sample total RNA were analysed for each target gene and allowed to construct linear standard curves from which the concentrations of the test sample and efficiency of PCR reaction were calculated. Results were normalized to β-actin or GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) using the ΔΔCT method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[END_REF].

Methylated DNA Immunoprecipitation Assay

Methylated DNA immunoprecipitation (MeDIP) was performed using EpiQuik Tissue Methylated DNA Immunoprecipitation Kit (Epigentek Group Inc, USA). Genomic DNA was K Anier et al 8 extracted from frozen mouse NAc, sonicated into fragments ranging in size from 200-1000 bp and divided into immunoprecipitated (IP) and input (IN) portions. IP DNA incubated with anti-5-methylcytosine monoclonal antibody to bind methylated DNA, negative control was normal mouse IgG from the EpiQuik MeDIP Kit. Methylated DNA was subjected to quantitative real-time PCR using primers from SABiosciences for mouse PP1c and fosB. To evaluate the relative enrichment of target sequences after MeDIP, we calculated the ratios of the signals in the immunoprecipitated DNA versus input DNA. The resulting values were standardized against the unmethylated control sequence GADPH, the results of control group (SAL or S+S) was given the value 1 and fold changes calculated.

Methylation-specific real-time PCR analysis

DNA was isolated from mouse NAc using QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany) and processed for bisulphite modification using Epitect Bisulfite Kit (QIAGEN, Hilden, Germany). Quantitative real-time PCR was used to determine the DNA methylation status of the PP1c and fosB genes. Methylation-specific PCR primers were designed using Methprimer software (www.urogene.org/methprimer). Methylation-specific and unmethylated PCR primers were designed to target putative CpG islands detected in silico in promoter or non-promoter regions of the PP1c and fosB genes. Primer sequences are listed in Table 1.

PCR reactions were performed as described above. Ct values were chosen within the linear range and the comparative Ct method was used to calculate differences in methylation between samples.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) of genomic DNA associated with a methyl CpG binding protein 2 (MeCP2) was carried out according to the manufacturer's protocol (Millipore Inc, USA). Mouse NAc was minced and cross-linked in 1% formaldehyde (10 μl/mg) for 15 min at 37 °C. Adding glycine stopped the cross-linking reaction and the tissue was washed twice in ice-cold PBS containing a protease inhibitor cocktail. The minced, fixed tissue was homogenized in SDS lysis buffer and the homogenate was sonicated to produce 200-1000 bp genomic fragments. Following this, the resulting homogenate was centrifuged for 15 min at 13,000 x g and the supernatant was used for ChIP assay. Immunoprecipitations were carried out at 4 °C overnight with 5 μg of rabbit polyclonal MeCP2 (Abcam plc, Cambridge, UK), negative control was anti-Rat IgG (Vector Laboratories Inc, Burlingame, CA, USA) and positive control was anti-RNA Polymerase II from the EZ-ChIP Kit. A portion of the sonicated DNA was left untreated to serve as input control. Immune complexes were collected with protein A beads and, according to the manufacturer's protocol, sequentially washed two times with low salt buffer, high salt buffer, LiCl immune complex buffer and TE buffer. Immunoprecipitated DNA was subjected to quantitative real-time PCR using ChIP primers from SABiosciences for mouse PP1c and fosB.

Western immunoblotting

The NAc was lysed in 10 vol (w/vol) of lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X 100 and protease inhibitors, homogenized with ultrasound homogenizer, incubated 1 h at +4 °C and centrifuged (13,000 x g for 10 min at 4 °C). The protein concentration was determined using RCDC Protein Assay (BioRad, Hercules, CA, USA). The supernatants (50 μg of protein) were resolved by electrophoresis on 8% SDS-polyacrylamide gels. The membranes were blocked with 0.1% (w/w) Tween 20/TBS (T-TBS) containing 5% (w/w) non-fat dried milk at room temperature for 1 h. After blocking, the membranes were incubated overnight with the anti-PP1γ1 polyclonal antibody (1:1,000, Millipore, Temecula, CA, USA) followed by incubation with biotinylated anti-rabbit IgM secondary antibody K Anier et al 10

(1:1,000) for 2 h and ABC solution for 30 min. Mouse monoclonal anti-β-actin antibody (Sigma-Aldrich Inc, St. Louis, MO, USA), was used to normalize PP1 immunoreactivity on the same blot and PP1/β-actin optical density (OD) ratio was calculated.

Statistical analysis

Behavioural data of all experiments were analysed using ANOVA for repeated measures, followed by Bonferroni post-test, as appropriate. Expression level of genes, MeDIP and ChIP data were analysed using one-way ANOVA with Bonferroni post-test. Differences in methylation-specific real-time PCR data were analysed by two-way ANOVA with Bonferroni post-test. GraphPad Prism software was used for statistical analysis of the data. All data expressed as mean±SEM, significance were set at p < 0.05.

RESULTS

Cocaine-induced Upregulation of DNMT3A and DNMT3B mRNA Levels in Nucleus

Accumbens and Hippocampus

Firstly, the effect of acute and repeated cocaine (15 mg/kg/day) on adult mice was evaluated.

Acute treatment with cocaine (AC) significantly increased the locomotor activity (Fig. 1; twoway ANOVA repeated measures, interaction F 2,35 = 7.43, p = 0.002, treatment effect F 2,35 = 18.9, p < 0.0001, days effect F 1,35 = 28.81, p < 0.0001; Bonferroni post-test, p < 0.01). After a daily injection of cocaine for seven days (RC), all of the mice displayed a significantly enhanced locomotion in response to the same dose of cocaine on day seven compared with day one (p < 0.001), indicating a behavioural sensitization. The saline-treated mice (SAL) did not show any sensitization across test sessions (p > 0.05).

It was recently reported that DNMT3A and DNMT3B gene expression was upregulated in the adult rat hippocampus following contextual fear conditioning suggesting that DNMTs are dynamically regulated in the adult nervous system [START_REF] Miller | Covalent modification of DNA regulates memory formation[END_REF]. Therefore, we investigated whether or not DNMTs mRNA levels are altered by acute and repeated cocaine treatment. We assayed mRNA levels of DNMT1, DNMT3A and DNMT3B 1.5 h and 24 h after acute and repeated cocaine treatment in the NAc (Fig. 2A-C). A single administration of cocaine displayed an increase in DNMT3A mRNA level 1.5 h after treatment, relative to animals treated with saline control (DNMT3A; one-way ANOVA, Bonferroni post-test, p < 0.01 SAL vs AC, n = 12) and DNMT3A and DNMT3B mRNA levels 24 h after treatment (DNMT3A; one-way ANOVA, Bonferroni post-test, p < 0.001 SAL vs AC, n = 12; DNMT3B, p < 0.05 SAL vs AC, n = 12). However, the repeated treatment with cocaine did not change significantly mRNA levels of DNMT3A and DNMT3B genes compared with saline control. Both acute and repeated cocaine treatment did not alter the mRNA level of DNMT1 gene.

In a parallel study, the effect of acute and repeated cocaine treatment on DNMT1, DNMT3A and DNMT3B mRNA levels in the hippocampus 1.5 h and 24 h after treatment was determined (Fig. S1 A-C). We found that the acute and repeated cocaine treatment increased DNMT3A mRNA levels 1.5 h after treatment (one-way ANOVA, Bonferroni post-test, p < 0.001 SAL vs AC or RC, n = 7) and DNMT3B expression level was increased in the hippocampus only after acute cocaine treatment (one-way ANOVA, Bonferroni post-test, p < 0.001 SAL vs AC, n = 7). There were no changes in DNMT3A and DNMT3B mRNA levels 24 h after cocaine treatment compared with the saline control. Both acute and repeated cocaine treatment did not alter the mRNA level of DNMT1 gene in the hippocampus. Thus, the time-course analysis demonstrated that DNMT3A and DNMT3B expression is dynamically regulated in the adult NAc and hippocampus in response to cocaine treatment.

Altered Patterns of Gene Expression after Cocaine Treatment

Increased expression of DNMT3A and DNMT3B might cause hypermethylation of certain gene promoters associated with CpG islands and consequently downregulate the expression of these genes. Therefore, a search was conducted for genes which show a diminished expression following cocaine treatment. Several genes were tested, such as brain-derived neurotrophic factor (BDNF), protein phosphatase-1 catalytic subunit (PP1c), fosB, and adenosine A 2A receptor (A 2A R), which participate in cocaine-induced neuroadaptations in the NAc and in silico analysis revealed CpG islands located within promoter regions.

Using quantitative real-time PCR we found that PP1c mRNA levels in the NAc were not altered 1.5 h after both acute and repeated cocaine treatment (Fig. 3A). However, 24 h after treatment in both acute and repeated cocaine groups the PP1c mRNA levels were significantly decreased compared to the saline control (one-way ANOVA, Bonferroni post-test, p < 0.05 AC or RC vs SAL, n = 12). To further confirm PP1c mRNA downregulation in the NAc, western blot analysis was performed. We found that both acute and repeated cocaine treatment resulted in a significantly lower PP1c protein level 24 h after treatment (Fig. 3B; one-way ANOVA, Bonferroni post-test, p < 0.05 AC or RC vs SAL, n = 9).

As expected, it was found that acute and repeated cocaine treatment resulted in upregulation of fosB mRNA level 1.5 h after treatment (Fig. 3C; one-way ANOVA, Bonferroni post-test, p < 0.001 AC or RC vs SAL, n = 12). However, there were no significant changes 24 h after acute and repeated cocaine treatment. Acute and repeated cocaine treatment also resulted in significant downregulation of A 2A R mRNA level 1.5 h after treatment (Fig. 3D; one-way ANOVA, Bonferroni post-test, p < 0.0001 AC or RC vs SAL, n = 7). However, there were no significant changes of mRNA level of BDNF gene after cocaine treatment in the NAc (Fig. S2). As we were interested in studying cocaine-induced longlasting effects, for future DNA methylation studies, PP1c and fosB (as reference) genes were selected.

Cocaine Treatment Changed DNA Methylation at PP1c and fosB Promoter Regions

To shed further light on the role of DNA methylation in cocaine-induced behavioural sensitization, we next looked for an aberrant DNA methylation during acute and repeated cocaine treatments. First, we evaluated cocaine-induced changes of DNA methylation patterns at the PP1c promoter region using methylated DNA immunoprecipitation (MeDIP) assay. We found that both acute and repeated cocaine treatment resulted in PP1c promoter-associated CpG island hypermethylation 24 h after treatment (Fig. 4B; one-way ANOVA, Bonferron post-test, p < 0.001 AC or RC vs SAL, n = 6).

To verify cocaine-induced hypermethylation at the PP1c promoter region, methylation-specific quantitative real-time PCR (MSP qPCR) was employed. Data generated using MSP qPCR demonstrated that acute and repeated cocaine treatment elicited DNA hypermethylation associated with the PP1c promoter region 24 h after treatment (Fig. 4A,C; two-way ANOVA, interaction F 2,66 = 8.34, p = 0.0006, treatment effect F 2,66 = 2.77, p = 0.0699, methylation effect F 1,66 = 31.24, p < 0.0001; Bonferroni post-test, p < 0.05 methylated AC vs methylated SAL, and p < 0.001 methylated RC vs methylated SAL). As a DNA methylation control, levels of unmethylated DNA within the PP1c promoter after acute and repeated cocaine treatment was measured, using primer sequences designed to amplify unmethylated CpG island sites. Thus, MeDIP and MSP qPCR methods demonstrated that acute and repeated cocaine treatment resulted in hypermethylation at the PP1c promoter region 24 h after treatment.

In correlation with upregulated fosB mRNA levels, we found hypomethylation at the fosB promoter 1.5 h after acute and repeated cocaine treatment, using MeDIP assay (Fig 5A,B; one-way ANOVA; Bonferroni post-test, p < 0.001 AC or RC vs SAL, n = 6). To further confirm that acute and repeated cocaine administration altered DNA methylation at the promoter regions of PP1c and fosB genes, chromatin immunoprecipitation (ChIP) assay was performed. Consistent with the MeDIP and MSP qPCR results, ChIP analysis demonstrated that acute and repeated cocaine treatment increased by 1.8 to 2-fold the PP1c promoterassociated methyl CpG binding protein 2 (MeCP2) binding 24 h after treatment in the NAc (Fig. 6A; one-way ANOVA, Bonferroni post-test, p < 0.01 AC or RC vs SAL, n = 6).

However, in the fosB promoter acute and repeated cocaine treatment was associated with a significant decrease of MeCP2 binding compared with the saline control 1.5 h after treatment (Fig. 6B; one-way ANOVA, Bonferroni post-test, p < 0.001 AC or RC vs SAL, n = 6). Taken together, these data support the idea that alteration of DNA methylation in promoter regions controls gene expression, because there is a correlation of increased PP1c promoter-associated CpG island hypermethylation with increased MeCP2 binding and with decreased PP1c gene transcription and opposite effect at the fosB promoter.

Zebularine Inhibited DNA Hypermethylation at PP1c Promoter and Delayed Cocaineinduced Behavioural Sensitization

As we found that cocaine-induced DNMT3A and DNMT3B expression and PP1c promoter hypermethylation, the next goals in this study were to assess the effect of DNMT inhibitor on cocaine-induced molecular changes and development of behavioural sensitization. We selected the DNMT inhibitor zebularine for testing based on reported data [START_REF] Miller | Covalent modification of DNA regulates memory formation[END_REF][START_REF] Lubin | Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory[END_REF]. To correlate the molecular changes with cocaine-induced locomotor sensitization, a dose of zebularine (300 ng/0.5 μl, icv) that did not affect basal locomotor activity and decreased DNA methylation at the PP1c promoter region 1.5 h after acute treatment (Fig. 7A; t-test, p = 0.034, n = 4) were selected from our pilot study.

For the co-treatment experiment, all mice received icv infusion of saline or zebularine followed, after 20 min, by intraperitoneal (ip) saline or cocaine (15 mg/kg). Locomotor activity was recorded for 60 min immediately after the last ip injection (see Materials and Methods). Repeated saline and cocaine (S+C) treatment for seven days displayed a significantly enhanced locomotion on day seven compared with that of day one (Fig. 7B; twoway ANOVA with repeated measures, interaction F 3,39 = 5.69, p = 0.0025, treatment effect F 3,39 = 15.98, p < 0.0001, days effect F 1,39 = 10.26, p = 0.0027; Bonferroni post-test, p < 0.001 S+C day 1 vs day 7). However, zebularine and cocaine (Z+C) co-treated mice did not show any sensitization on day seven compared with day one (p > 0.05). There was also no behavioural sensitization after repeated zebularine and saline (Z+S) treatments (p > 0.05). Therefore, our results suggest that DNMTs inhibition with zebularine did not affect acute cocaine-induced locomotor activity, but instead delayed cocaine-induced behaviour sensitization in mice.

Finally, we studied for molecular evidence to confirm that zebularine infusions (icv) before cocaine treatment altered DNA methylation level at the PP1c promoter region in the NAc.

Using MeDIP assay we found that repeated cocaine treatment (S+C) induced, at the PP1c promoter region, DNA hypermethylation 24 h after treatment compared with the saline control group (S+S), and repeated zebularine and cocaine (Z+C) co-treatment avoided this effect (Fig. 8A; one-way ANOVA, Bonferroni post-test, p < 0.001 S+S vs S+C, p < 0.001 S+C vs Z+C, n = 6). Similarly, MSP qPCR analysis demonstrated that there was a significant decrease of DNA methylation associated with the PP1c promoter region in the Z+C group compared with the S+C group (Fig. 8B; two-way ANOVA, interaction F 3,45 = 80.8, p < 0.0001, treatment effect F 3,45 = 39.0, p < 0.0001, methylation effect F 1,45 = 42.8, p < 0.0001; Bonferroni post-test, p < 0.001 methylated S+C vs methylated Z+C). Interestingly, in both studies we found significant changes of DNA methylation between the zebularine (Z+S) and the saline (S+S) control groups (p < 0.05).

In correlation with the PP1c promoter-associated CpG island methylation results, we observed that zebularine attenuated cocaine-induced downregulation of PP1c mRNA level (Fig. 8C; one-way ANOVA; Bonferroni post-test, p < 0.001 S+S vs S+C, p < 0.05 S+C vs Z+C, n = 6). These results indicate that DNMTs inhibition by zebularine reverses cocaineinduced PP1c gene hypermethylation and mRNA downregulation in the NAc.

DISCUSSION

In this study we have produced four main findings. Firstly, it was found that acute cocaine treatment results in an upregulated mRNA of DNMT3A and DNMT3B in the NAc. Secondly, it was observed that acute and repeated cocaine treatment is associated with DNA hypermethylation and transcriptional downregulation of the PP1c gene, and hypomethylation and transcriptional activation of the fosB gene in the NAc. Thirdly, it was found that intracerebroventricular infusions of DNMT inhibitor zebularine, before cocaine treatment, decreased levels of DNA hypermethylation at the PP1c promoter-associated CpG island and attenuated PP1c mRNA downregulation in the NAc. Finally, zebularine and cocaine cotreatment were found to delay development of cocaine-induced behavioural sensitization.

These observations suggest that DNA hyper-and hypomethylation may be important processes during the development of cocaine-induced behavioural sensitization.

Psychostimulants such as cocaine and amphetamine induce neuroadaptations through regulation of gene expression. New concepts have emerged concerning the role of chromatin modification as a requirement for the regulation of gene expression in neurons. In this study we focused on the function of DNA methylation in cocaine-altered gene expression in the NAc. Previous findings have implied that the function of DNMTs in the CNS might be involved in DNA repair and neurodegeneration [START_REF] Brooks | DNA mismatch repair and DNA methylation in adult brain neurons[END_REF][START_REF] Endres | DNA methyltransferase contributes to delayed ischemic brain injury[END_REF]Fan et al, 2001;[START_REF] Endres | Effects of cerebral ischemia in mice lacking DNA methyltransferase 1 in post-mitotic neurons[END_REF] and that misregulation of DNA methylation and DNMTs might be involved in cognitive disorders such as schizophrenia, Rett syndrome and Fragile X mental retardation [START_REF] Veldic | DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains[END_REF][START_REF] Amir | Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2[END_REF][START_REF] Sutcliffe | DNA methylation represses FMR-1 transcription in fragile X syndrome[END_REF].

Recently, Miller and Sweatt showed that DNA methylation levels were rapidly and dynamically regulated in the hippocampus following the associative training paradigm of contextual fear conditioning [START_REF] Miller | Covalent modification of DNA regulates memory formation[END_REF]. They also found that mRNAs for conditioning. The results of the present study were comparable in that they demonstrated that acute cocaine treatment was associated with an upregulation of DNMT3A and DNMT3B genes in the NAc and hippocampus (Fig. 2 B,C; Fig. S1 B,C respectively). However, no significant changes of DNMT1 mRNA levels in the NAc and hippocampus were found, suggesting that cocaine induces rather de novo than maintenance of DNA methylation. It was also found that DNMT3A gene expression achieved the highest level after acute cocaine treatment and it diminished after repeated cocaine exposure. In relation to DNMT3A and DNMT3B expression levels in the NAc, cocaine-induced changes in the hippocampus were more dynamic as there were no significant changes 24 h after treatment.

The signal transduction processes that might control cocaine-induced DNMT expression in the adult CNS are unknown at present. While DNA methylation is a part of the regulation of gene expression, it infers that there may be some overlap of intracellular signalling pathways with other chromatin modification mechanisms, such as histone acetylation and phosphorylation. In the adult, CNS chromatin modification is governed by various intracellular signalling pathways, including the Ras-mitogen-activated protein kinase (MAPK/ERK) cascade (Renthal and Nestler, 2008;[START_REF] Brami-Cherrier | Role of the ERK/MSK1 signalling pathway in chromatin remodelling and brain responses to drugs of abuse[END_REF]. Several studies demonstrated that targeting the Ras signalling pathway with drugs such as methotrexate and inhibitors of MAPK/ERK, decreases DNA methylation in malignant haematological diseases and colon cancer cells, indicating a causal relationship between Ras signalling and DNA methylation [START_REF] Philips | Methotrexate and Ras methylation: a new trick for an old drug?[END_REF][START_REF] Morgan | Targeting the RAS signaling pathway in malignant hematologic diseases[END_REF][START_REF] Lu | Inhibition of extracellular signal regulated kinase mitogen-activated protein kinase pathway decrease DNA-methylation in colon cancer cells[END_REF][START_REF] Diaz-Flores | Targeting oncogenic Ras[END_REF].

In recent years, the MAPK/ERK signalling cascade has been implicated in responses to most drugs of abuse [START_REF] Berke | Addiction, dopamine, and the molecular mechanisms of memory[END_REF][START_REF] Nestler | Molecular basis of long-term plasticity underlying addiction[END_REF][START_REF] Fasano | Cellular mechanisms of striatum-dependent behavioral plasticity and drug addiction[END_REF][START_REF] Girault | ERK2: a logical and gate critical for druginduced plasticity?[END_REF]. Several studies have demonstrated that in response to cocaine, the MAPK/ERK and the downstream mitogen-and stress-activated protein kinase 1 (MSK1) controls an early phase of histone H3 phosphorylation at the c-fos promoter in striatal neurons [START_REF] Brami-Cherrier | Parsing molecular and behavioral effects of cocaine in mitogen-and stressactivated protein kinase-1-deficient mice[END_REF]. MSK1 action may be potentiated by the concomitant inhibition of PP1 by nuclear translocation of dopamine and cAMP-regulated phosphoprotein Mr = 32,000 (DARPP-32) [START_REF] Stipanovich | A phosphatase cascade by which rewarding stimuli control nucleosomal response[END_REF]. Cocaine also phosphorylates the transcription factor cAMP response element binding protein (CREB) via the MAPK/ERK-MSK1 cascade [START_REF] Swank | Increased histone acetyltransferase and lysine acetyltransferase activity and biphasic activation of the ERK/RSK cascade in insular cortex during novel taste learning[END_REF][START_REF] Levine | CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum[END_REF]. This phosphorylation and activation of CREB recruits CREB binding protein (CBP), which is a histone acetyltransferase that regulates local chromatin structure as part of CREB-dependent activation of nuclear gene transcription.

Therefore, we speculate that cocaine-induced activation of the Ras-signalling pathway might trigger both DNA methylation and histone modifications.

Using MeDIP and MSP qPCR methods we demonstrated that DNMT activation results in increased methylation of the PP1c promoter region in the NAc after acute and repeated cocaine treatment (Fig. 4B,C). It was also found that PP1c promoter region hypermethylation corresponded with decreased expression levels, both of mRNA and protein (Fig. 3A). These changes also related to an overall increase in methyl-CpG-binding domain proteins, such as MeCP2 binding in the PP1c promoter region (Fig. 6A). It has been shown experimentally that methylation of DNA brings about general deacetylation of histones H3 and H4, prevents methylation at histone H3 K4 and induces methylation of histone H3 K9 [START_REF] Shahbazian | Functions of site-specific histone acetylation and deacetylation[END_REF][START_REF] Li | The role of chromatin during transcription[END_REF][START_REF] Kouzarides | Chromatin modifications and their function[END_REF][START_REF] Patra | DNA methylation-mediated nucleosome dynamics and oncogenic Ras signaling. Insights from FAS, FAS ligand and RASSF1A[END_REF].

Therefore, we speculate that MeCP2 can recruit co-repressor complexes to methylated PP1c promoter regions, including histone-modifying enzymes such as histone deacetylases (HDAC) and histone methyltransferases.

Repression of PP1c may result in several effects on the intracellular signal transduction level, for example inhibition of PP1c has been shown to increase phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 and Ca2+/calmodulin-dependent protein kinase II [START_REF] Genoux | Protein phosphatase 1 is a molecular constraint on learning and memory[END_REF]. Reduced PP1 may have important effects for transcriptional regulation by CREB through PP1's ability to complex with HDAC. Canettieri and colleagues demonstrated that a HDAC1-PP1 complex represses CREB activation under basal conditions and dephosphorylates CREB to return the system to baseline after a stimulus [START_REF] Canettieri | Attenuation of a phosphorylation-dependent activator by an HDAC-PP1 complex[END_REF]. This suggests that repression of PP1 is crucial in providing phosphorylated CREB with the capacity to recruit CBP to the promoter, at which time histones become acetylated and help to drive the transcription of particular genes. Taken together, repression of the PP1c gene via methylation of promoter regions should allow aberrant phosphorylation of crucial receptors, protein kinases and transcription factors during cocaine-induced neuroadaptations.

Previous studies have shown that expression of Fos-family proteins (c-Fos, FosB, Fra-1 and Fra2) is rapidly induced in the NAc after acute exposure to drugs of abuse [START_REF] Hope | Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments[END_REF]. This induction is transient, lasting only 4-12 h after drug exposure. Because acute cocaine treatment caused in fosB promoter-associated CpG island hypomethylation (Fig. 5B), decreased MeCP2 binding (Fig. 6B) and fosB mRNA upregulation (Fig. 3C), this suggests that cocaine may also induce DNA demethylation and increase expression in certain genes. At present there is no clearly identified DNA demethylase in mammals and the biochemical mechanism for active DNA demethylation is unknown (Ooi and Bestor, 2008). Several reports discovered that the demethylation process is initiated by DNMT3A and DNMT3B [START_REF] Kangaspeska | Transient cyclical methylation of promoter DNA[END_REF][START_REF] Métivier | Dynamics of estrogen receptormediated transcriptional activation of responsive genes in vivo: apprehending transcription in four dimensions[END_REF]. These studies proposed that DNMT3A and DNMT3B possess deaminase activity and that both enzymes are involved in a dynamic demethylation-methylation pathway which operates during gene transcription [START_REF] Kangaspeska | Transient cyclical methylation of promoter DNA[END_REF][START_REF] Métivier | Dynamics of estrogen receptormediated transcriptional activation of responsive genes in vivo: apprehending transcription in four dimensions[END_REF]. Recently [START_REF] Ma | DNA excision repair proteins and Gadd45 as molecular players for active DNA demethylation[END_REF] proposed that active DNA demethylation can be potentially carried out through three mechanisms: direct removal of the methyl group from the 5-methylcytosine base; base excision of 5-methylcytosine through DNA glycosylases; nucleotide excision of 5-methylcytosine through endonucleases. Base and nucleotide excisions are followed by similar subsequent fill-in repair with unmethylated cytosine.

In this study we demonstrate that DNMT inhibition by zebularine, blocked hypermethylation on the PP1c promoter region and delayed the development of cocaineinduced behavioural sensitization. The decrease of PP1c hypermethylation was triggered using icv infusion of zebularine and it was sufficient to re-establish PP1c gene expression in the NAc. Zebularine is a potent inhibitor, affecting all DNMTs subtypes [START_REF] Weisenberger | Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation[END_REF][START_REF] Marquez | Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy[END_REF]. However, we were unable to determine which subtype of DNMT is important for the development of behavioural sensitization. It is possible that both DNMT3A and DNMT3B work together to hypermethylate the promoter associated CpG islands and to then transcriptionally repress these genes, aiding in the development of behavioural sensitization.

In Figure 7A we show that a single dose zebularine caused hypomethylation at the promoter of PP1c 1.5 h after treatment. However, the effect of repeated zebularine (Z+S) administration resulted with PP1c promoter region hypermethylation 24 h after last treatment (Fig. 8A,B). Based on zebularine pharmacokinetics [START_REF] Holleran | Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine[END_REF], we speculate that 24 h after the last administration of zebularine (Z+S) the DNMT inhibition effect is ended. Therefore, it is possible that a slight increase of methylation in PP1c promoter-associated CpG island in Z+S groups (Fig. 8A,B) is caused by a compensatory increase of DNMT activity after inhibition by zebularine. Despite slight hypermethylation at PP1c we found 1.5fold increased mRNA level in the Z+S group compared with the saline control (S+S, Fig. 8C), suggesting that smaller changes of DNA methylation may not alter gene transcription. Other DNA methylation associated epigenetic mechanisms (histone acetylation, phosporylation, methylation) may also alter gene transcription activity. Our results demonstrated that zebularine blocked hypermethylation at the PP1c promoter region and downregulation PP1c mRNA level. We speculate that in addition to PP1c, zebularine may also inhibit other genes promoter-associated CpG island hypermethylation, re-establishes these genes transcription and therefore inhibit cocaine-induced neuroplasticity in the brain.

At the behavioural level, we found that repeated co-administration of zebularine with cocaine markedly delayed the development of behavioural sensitization while zebularine by itself did not affect locomotor activity. Interestingly, zebularine did not modify acute cocaine effect, suggesting that the promoter region hypermethylation might be an important underlying mechanism for altered gene expression during development of behavioural sensitization. Therefore we hypothesize that zebularine via normalization of gene transcription at cellular level, stabilized neuronal network function and therefore delayed the development of psychostimulant-induced behavioural sensitization. Since DNA methylation plays an important role in learning and memory formation [START_REF] Miller | Covalent modification of DNA regulates memory formation[END_REF], zebularine may delay cocaine-induced behavioural sensitization also by inhibiting DNA methylation in the hippocampus and altering associated learning and memory formation.

In conclusion, our study demonstrated that cocaine treatment might cause a dynamic increase of DNMT3A and DNMT3B expression and consequently hypermethylation of promoter-associated CpG islands in the NAc. It is not known which subtype of DNMT is important for DNA methylation during the development of behavioural sensitization. However, our results suggest that cocaine induces rather de novo than maintenance DNA methylation. The results of DNMT inhibitor zebularine and cocaine co-treatment experiments demonstrated that zebularine effectively inhibited cocaine-induced PP1c hypermethylation and delayed the development of behavioural sensitization. Therefore, DNMT3A and DNMT3B may be important molecular targets for modifying psychostimulant-induced neurochemical and behavioural effects.

Figure Legends Figure 1 .

 Legends1 Figure Legends

Figure 2 .

 2 Figure 2. Acute cocaine treatment was associated with an upregulation of DNMT3A and

Figure 3 .

 3 Figure 3. Altered patterns of PP1c, fosB, and A2AR genes expression after cocaine treatment

Figure 4 .

 4 Figure 4. Cocaine treatment was associated with altered DNA methylation at PP1c promoters

Figure 5 .

 5 Figure 5. Cocaine treatment was associated with altered DNA methylation at fosB promoters

Figure 6 .

 6 Figure 6. Altered patterns of MeCP2 binding at PP1c and fosB promoter regions in the NAc,

Figure 7 .

 7 Figure 7. Inhibition of DNMT by zebularine delayed cocaine-induced behavioural

Figure 8 .

 8 Figure 8. Inhibition of DNMT by zebularine altered DNA methylation at the PP1c promoter

ACKNOWLEDGMENTS

We thank Dr. G. Tasa for help with methylation-specific real-time PCR and MeDIP analysis and A. Harrison for critical reading of the paper. This study was supported by the Estonian Science Foundation (grant ETF7585), University of Tartu (grant PARFR08903) and European Regional Development Fund.

DISCLOSURE/CONFLICTS OF INTEREST

None of the authors have anything to disclose. No conflicts of interest.


Vojislav ; Petrovic
email: vpetrovic@aimme.es

Juan Vicente Haro
email: jvharo@aimme.es

Olga Jordá
email: ojorda@aimme.es

Javier Delgado
email: jdelgado@aimme.es

Jose Ramón Blasco

Luis Portolés

Delgado

Ramón

Javier Delgado Gordillo

Additive Layer Manufacturing: State of the art in industrial applications through case studies

Keywords: ADVANCED MANUFACTURING PROCESSES, PRODUCT DEVELOPMENT, RAPID MANUFACTURING, RAPID PROTOTYPING, RAPID additive layered manufacturing, customized product

come

In recent years, European industry has been facing the challenge of losing competitiveness in mass production. Due to important factors such as lower labour costs, lower taxes or in-site access to raw materials, mass production has migrated to third world countries. However, European industry is more advanced in technological aspects and is in need of a qualitative advantage in the development of new technologies. One of the efforts of European companies is directed towards the production of short series of customized products with added value. A great effort has been done in order to customize products and give them an added value by developing new fabrication technologies. Additive Fabrication (AF) is a powerful tool that offers the necessary competitiveness to European companies.

There are many terms commonly used for AF, such as Solid Free Form Fabrication (SFF), Rapid Manufacturing (RM), Additive Layered Manufacturing (ALM) and 3D Printing (3DP). The latter may be the most descriptive for those not familiar with additive technologies. Unfortunately, this term may produce a wrong idea, since AF machinery is much more than any kind of printer. Anyhow, AF comprises the use of layer-by-layer manufacturing in order to build a part by addition of material.

The physical process of addition

Additive fabrication is performed directly from a 3-D CAD file in which a part's model is stored. The usual file format is STL. The STL file is imported into a specific software (such as Magics RP ® by Materialise, Viscam ® by Marcam and Netfabb ® by FIT which are available in the market), where it is pre-processed. The part is oriented for building and a support structure is made for the part's surfaces that are facing down. Afterwards, cross-sections of a given thickness, known as 'slices', are generated from 3-D CAD descriptions of the part and support structures. The slices are saved in a 'slice file' (SLI, SLC or any other format that may depend on patented technology). After pre-processing, the slice file is sent to the machine to be 'printed' slice-by-slice.

Insert Figure 1 about here The fabrication process consists of two basic steps: coating and fusing. The coating step is the process in which material is laid over the working surface forming a very thin layer. The thickness of the layer depends on the chosen technology and is in the range of 0.03 mm to 0.20 mm. The fusing step refers to the process of printing the part slice by the action of a source of energy. The active principle can be a light source, laser beam or electron beam. It acts over the layer of material and produces fusion, which fuses the part slice's footprint to the layer below it (Figure 1). The power of the source of energy depends on the chosen technology: in the low end, we have Stereolithography that uses 100 W, and in the high end, we have Electron Beam Melting that uses 3000 W. Only the contour of a part slice and its interior are fused; the rest of the material is left untouched and may be recycled (depending on the process and material). The actions of coating and slice printing follow each other until all the slices are printed correctly, forming the final threedimensional part. Once fabrication is finished, the part is taken out to be cleaned of material that is not fused and, if necessary, to be given further post-processing. The most common post-processing techniques are sanding, polishing, homogenization, thermal treatment, etc.

It is important to note that, in general, material that is not fused (be it powder or liquid) is not capable of supporting fused structures. If a part has overhanging zones, they may need to be supported by a firm structure. Additionally, the support structure acts as a conductor of excess heat created in the process of fusing. Finally, in some technologies, the support structure prevents warping of the part due to the thermal stress created during the process. Hence, the first step of pre-processing actually consists of generating an efficient support structure, which is then sliced and fabricated together with the part.

Advantages and challenges of AF

The most important advantages of Additive Fabrication are:

1. Time-to-market reduction due to high speed of the process. A complex race car upright (Figure 2) whose dimensions are 280 mm x 180 mm x 120 mm was designed and fabricated by EBM in just 28 hours (Arcam (2006));

Insert Figure 2 about here 2. Product customization with complete flexibility in design & construction of a product. Unlike conventional processes, AF can produce parts of almost any desired form and can almost be free from geometrical manufacturing constraints.

3. Maximum material savings. Material is added and not subtracted. For some applications, especially in the metal sector, case studies show that the waste of raw material is reduced by up to 40% when using additive technologies instead of subtractive (machining) technologies [START_REF] Reeves | Manufacturing a Low Carbon Footprint: Zero Emission Enterprise Feasability Study[END_REF]). Also, 95% to 98% of the remaining material (powder that is not fused) may be recycled;

4. No tools, moulds or punches are needed. The part is obtained directly from its 3-D CAD model; an additional advantage is the almost absolute absence of human errors in production.

5. Full-density of final parts. Unlike other powder-bed processes, additive technologies produce parts without residual porosity.

6. Fabrication of free form enclosed structures. Additive technologies are capable of fabricating free form channels as well as different forms of latticed structures.

However, additive technologies must face some pending challenges in the near future.

1. Remove the 'stigma' of its original name 'Rapid Prototyping'. Although ALM has evolved in a way that it delivers ready-to-use products made from a wide variety of metals and polymers, it is still wrongly considered that additive fabrication is valid only for prototype fabrication.

Validation of mechanical and thermal properties of existing materials and ALM technologies.

Unlike conventionally produced parts, AF parts do not behave identically in all directions. They are 'stronger' when the load is applied along the direction of the layer as compared to the build-up direction. At the moment no characterization has been done to quantify the difference. Hence, a client will fully trust ALM technologies when the proper part characterization is done.

Development and characterization of new materials for ALM.

Additive technologies are already dealing with a wide variety of metals (stainless steel, titanium, cobalt chromium, etc.) and polymers. However, there are many interesting materials that need further research (such as magnesium, copper, biodegradable polymers, etc.).

4. Change in the designers' way of thinking. ALM has enabled a new "free form fabrication" that is free from manufacturing constraints and must be introduced and advertised.

5. Automation design and process planning. The overall tendency in production processes (no matter if subtractive, forming or additive) is the development of automatic assessment tools. There are several result parameters that are considered when one designs for AF and all of them can be put together in order to automate decision making in ALM.

6. The stair stepping effect is created by the placement of one layer on top of another, affecting surface quality and roughness. Depending on how the model is oriented for slicing and fabrication, roughness will be better or worse in different zones.

7. Support structure. As the material used for the support structure cannot be recycled, it should be minimized by a good build-up orientation.

In this paper, the authors review the main AF technologies and their actual industrial applications, offering a global view of the state-of-the-art in additive fabrication. The most relevant and commonly used additive technologies are briefly commented. These technologies are capable of processing a wide variety of materials (polymers and metals).

After a brief introduction of all technologies, the most attractive fields of application are highlighted. Finally, the authors describe their experience in the application of additive fabrication in the tooling, biomedicine, aerospace and ceramic industries, underlining the qualitative advantages offered by AF. The Results section shows various case studies in the framework of AIMME's projects related to biomedicine (Fabio), tooling (Heat Transfer Tooling and Custom Ceramic) and lattice structure (Mepess).

Additive technologies

Polymer processing

Polymer processing technologies have been present in the global industry market for almost two decades. These technologies are well known in all industrial sectors because they have been widely used for prototype fabrication. In this section, only the most widely used will be mentioned.

Stereolithography (SLA)

SLA technology uses a laser beam to perform the photopolymerization of a material. This way, the material that is initially in a liquid state becomes hard and consistent. SLA is capable of processing different resins that emulate the performance of thermoplastic material. Actually there are many types of resins that are capable of resisting different loads and temperatures. Some of them are for general purpose and others have been developed for a specific use: high thermal resistance, better surface quality, elasticity, dimensional precision or other.

Both the technology provider (3D Systems) and material providers (such as SOMOS) offer a wide range of materials that are capable of being processed successfully. There are materials made for general purpose use (Somos 7100), for the good emulation of the properties of thermoplastic materials (Somos 8100 and Somos 9100), to provide stiffness and resistance to high temperatures (Somos ProtoTool TM and Somos NanoForm TM) and to provide high precision (3D Systems Accura Amethyst TM or 3dSystems Accura Bluestone TM).

The main application fields for SLA are the construction of functional and aesthetic prototypes, construction of models for VC and models with non-crystalline nanoparticles as mould inserts in the injection of certain polymers.

The main advantages of SLA are the fabrication of very complex geometries, very thin wall features, SLA parts with resistance to high temperatures and high reliability. The main disadvantage of SLA is that the operation of changing from one type of resin to another requires a substantial amount of time.

Selective Laser Sintering (SLS)

This technology uses a laser beam to fuse powder and create a part layer by layer. It normally uses powdered polyamide that can sometimes be reinforced with a filler of glass fibre or aluminium. The main advantages of polyamide are its good mechanical properties and stiffness which make it possible to fabricate a model without the need of a support structure which is generally needed for the downfacing surfaces of the model.

One of the most representative technologies for SLS is provided by Electro Optical Systems. This company has a variety of machines such as Formiga P 100, EOSINT P 390, etc. EOS offers a variety of materials for use in their lasersintering systems. These materials are based on PA 12 or polystyrene. Polyamide has a good resistance to most chemicals, and the material itself is not critical with regard to environmental and health aspects. There is a wide range of variants of this material to satisfy the special requirements of different applications. These materials can be distinguished, among other things, by the type of filler, for example with aluminium (Alumide), glass or carbon fibre (CarbonMide).

SLS technology is mostly used for prototypes, models and final products. Regarding the technology providers, apart from EOS, 3D Systems (www.3dsystems.com) is also present in the world market. The main advantages of SLS technology are the use of polyamide materials with good mechanical characteristics, the possibility of fabricating parts with up to 30% of glass fibres, the capability of resisting higher temperatures than SLA parts (up to 140ºC), good performance at increased levels of humidity, the absence of a support structure and a higher rate of fabrication. The main disadvantages of SLS are porous surfaces, possible warping of the part due to high temperature gradients and dimensional tolerances that are difficult to control.

Digital Light processing (DLP)

The technology known as DLP uses a UV-light lamp to expose a photo-sensible resin which will solidify. The Perfactory Xede and Xtreme machines use DLP-based projectors patented by Texas Instruments to produce multiple parts in a continuous Z build mode. This technology allows for the production of highly accurate parts without the trade-off between precision and building speed.

There are different variants in materials that can be used on DLP machines. The most commonly used are R5 and R11 for high-precision models, PIC 100 and 100G for jewellery and vacuum casting models, and the E-Shell 100 and 200 biocompatible materials for external use.

The main advantages of DLP technology are the fabrication of very complex geometries, very thin and precise details, and an excellent surface finish. The main disadvantages of this technology are its limitation to fabricating only relatively small parts and the difficulty of eliminating the support structure without causing damage to the part's surface. FDM technology consists of the deposition of fused material which was previous stored in the form of a plastic thread on a roll. Materials used in FDM are usually based on polycarbonate (PC) and ABS or a mixture of these two. Known as PC-ABS, PC-ISO or ABSi, these materials have properties that are similar to thermoplastic material for injection moulding. Also, nylon-based materials and other polymers can be used. The main advantage of FDM technology is in the use of low-cost materials.

Regarding available materials, ABS (P400 and P500) is suitable for functional prototype construction. Also, E20 is an elastomeric material available for highly flexible prototypes.

The main advantages of FDM technology are the fabrication of low cost parts, the use of water to dissolve the support structure and the possibility of coating the surface to improve its quality. The main disadvantages are a poor surface quality with grainy appearance and poor dimensional precision.

Metal processing

The raw material in metal processing with additive technologies is powder (powder bed technologies). The working principle is similar in all technologies and consists of coating and fusing. A thin layer of powder is coated over the working platform and a slice is printed by fusion. The platform descends along the Z-axis an amount equal to the thickness of a slice and a new layer of powder is coated over the working platform so that the next slice may be printed.

The actions of coating and slice printing are repeated until all the slices are printed correctly, forming the final part.

The source of energy used for fusion can be of the laser (laser based technologies) or electron beam (electron beam melting, EBM ® ) types. All metal processing technologies are capable of producing fully-dense and functional parts. These parts are offering complete reliability and are used in many industrial sectors such as biomedical, tooling, aerospace, automotive, etc.

Laser based technologies

In laser based technologies, a focused beam of laser light is used to produce fusion. There are several variants of laser technology. All of these laser technology variants are available in their respective machines. Generally, the parts fabricated with these metal processing technologies are prone to oxidation which can diminish their mechanical properties. This is why laser-based machines work with a protective atmosphere in the build chamber. When working with steel powder, nitrogen is commonly used, while for processing titanium and cobalt-chromium, argon is the best option to create an inert atmosphere.

Selective Laser Melting (SLM) is currently found in two variants, developed respectively by Electro Optical Systems and MTT Technologies. EOSINT M 270 is capable of processing a huge variety of metals such as steel, cobalt chromium, titanium alloys (Ti6Al4V), bronze-based alloys, nickel-based alloys, etc.

MTT machines, SLM 100 and SLM 250 are capable of processing stainless steel, tool steel (H13), titanium alloys (TiAl6V4 and TiAl6Nb7), cobalt chromium, AlSi12Mg, etc.

Another patented laser technology is Laser Cusing, property of Concept Laser. This technology is available on three machines, M1, M2 and M3. While M1 and M3 are capable of processing stainless and tool steel, M2 is capable of processing non ferrous alloys as well, such as titanium (Ti6AlV4), aluminium (AlSi12 and AlSi10Mg) and nickel based alloys.

Also available in the market is Selective Laser Sintering, property of 3D Systems, and a type of Laser Sintering developed by Phoenix Systems.

Electron beam based technology

In electron beam melting, the energy is provided by the impact of a focused beam of electrons. The electrons are emitted from a filament made of tungsten. A series of electromagnetic coils are used to focus and direct the beam towards the working platform. The particularity of EBM technology is its use of a vacuum in the build chamber. The vacuum prevents the beam from being deflected and maintains it well focused. This technological characteristic is wisely used to obtain parts of high purity.

There is only one EBM technology available, it is patented by Arcam and it is available on two machines: Arcam S12 and Arcam A 2 . These machines can process any material that has an adequate electrical conductivity. However, up to the moment, only three types of titanium (Ti6Al4V, Ti6Al4V ELI and Ti Grade 2) and cobalt chromium (CrCo ASTM F75) materials are officially released for EBM.

Application sectors

Additive fabrication is able to offer an added value to products and increase their quality as can be seen in the following section. The original purpose of SLA and DLP parts was to use them as prototypes for visual validation and validation of assemblies. There is a large number of applications for polymer prototypes. Some of them can be shown on Figure 3.

Insert Figure 3 about here

Biomedical parts

Polymer materials are very important for the biomedical sector. There are three different types of polymers that can be used in the biomedical sector. These are biocompatible polymers for external use, biocompatible polymers for implants and biodegradable polymers for implants as well.

Biocompatible polymers for external use are those that can be used for medical or prosthetic devices. An example is the EnvisionTEC e-Shell 200 series material that has been designed specially for applications in the Hearing Aid industries and is distinguished for its rigidity and durability. It is suitable for thin-walled Hearing Aid shells and otoplastic products. The material is used on all Perfactory ® and Vanquish ® systems. An example is shown at Figure 4(a).

Biocompatible polymer materials have been used in the surgical implants industry. These materials are not aggressive to the human body and do not cause rejection. Figure 4(b) shows a coronary bypass implant with very small internal channels. This model was fabricated with the photopolymer R11 material. Additionally, guides for maxillofacial surgery have been fabricated with transparent photopolymers.

Insert Figure 4 about here Hard tissue lumbar implants made with Stereolithography for vertebral fixation have been implanted on cadavers and used in clinical studies [START_REF] Goffin | Three dimensional computed tomography-based, personalized drill guide for posterior cervical stabilization at C1-C2[END_REF]). Also, various research works have been conducted to obtain scaffolds for tissue engineering using polylactic glycolic acid (PLGA) and fibrin gel [START_REF] Liu | Multinozzle low-temperature deposition system for construction of gradient tissue engineering scaffolds[END_REF]) or a combination of ether, epoxy resin and a photoinitiator [START_REF] Quadrani | High-resolution 3D scaffold model for engineered tissue fabrication using a rapid prototyping technique[END_REF]).

The next step in implant fabrication is the use of biodegradable polymer materials. Implants with the most desired features in implant research would have structures that have an osteoinductive function that permits bone growth and would also degrade with time. Additionally, flexible forms of these polymers would permit real tissue engineering, being a base for the construction of human organs.

Artistic design

Due to its extraordinary capacity of obtaining free forms, AF is frequently used in artistic design. Parts such as the one shown in Figure 5(a) can only be fabricated by additive technologies. One of the special fields in which additive manufacturing has been used is jewellery design. Unique designs become a reality with polymer processing. Polymer models can serve as models for vacuum investment casting (Figure 5(b)) or they can serve as jewels themselves (Figure 5(c)).

Insert Figure 5 about here

Metal parts

Tooling

The tooling sector has already been converted into a stronghold of AF. Additive technologies offer the possibility of free form design. One of the principal tasks in mould making is to provide adequate cooling. Post-injection warping and contraction of parts, as well as overall quality, are strongly influenced by the heat transfer between the injected mass and the cooling liquid that flows through the mould's walls. Cooling channels are normally obtained by drilling, once the mould has been machined. Hence, in conventional mould fabrication, the cooling channels are normally straight (Figure 6(a)).

Insert Figure 6 about here Additive technologies enable the fabrication of free form cooling channels. These channels are designed to follow the mould cavity surface and provide a completely homogeneous heat transfer, adaptive cooling, which helps to provide better quality to injected parts. Another important issue is the injection cycle time since adaptive cooling enables significant reduction of cycle time.

Metal additive technologies, laser or electron beam-based, are capable of fabricating hybrid moulds. In other words, they can build mould inserts over pre-fabricated mould blocks. Hence, only the mould cavity zone is fabricated by AF, saving time & costs comparing with full mould fabrication by AF (Figure 6

(b)).

There are many research works that support this claim. [START_REF] Godec | Influence of hybrid moulds on moulded parts properties[END_REF] emphasize an experimental comparative analysis of the influence of hybrid and classic moulds on the properties of moulded parts and their processing parameters, showing the advantages of tool inserts made with DMLS. [START_REF] Wong | Selective Laser Melting of heat transfer devices[END_REF] conducted a study that demonstrates the performance enhancements that can be achieved by using novel heat sink designs, fabricated with Selective Laser Melting, as compared to conventional pin fin arrays. [START_REF] Rännar | Efficient cooling with tool inserts manufactured by electron beam melting[END_REF] shows that efficient mould inserts with conformal cooling channels can be fabricated with EBM.

Although this subsection deals with metal parts, when speaking about Rapid Tooling, a use of Stereolithography in mould inserts fabrication must be mentioned. Silica moulds for aluminium casting have been successfully used (Esposito ( 2006)), and SLA has been used to fabricate PEK mould inserts [START_REF] Harris | Thermal conditions in Stereolithography injection mould tooling and their use for PEK moulding[END_REF]).

Biomedical parts

The main field of application for additive technologies may be resumed in the following statement: customized parts with short fabrication series. Biomedical parts are the target group that fits perfectly into this definition. Implants are highly personalized and as unique as the human body. The advantages of AF in biomedical applications are numerous.

The most important can be summarized as follows:

• Complex and personalized forms can be obtained: the human body, especially its osseous structure, has a very complex shape full of details. AF is the best option to reproduce these forms.

• The overall product development is much faster than conventional processes: a CT scan of a zone to be reconstructed is accomplished and converted to a model. The model is then exported to a STL file, sliced and sent to the machine: the part is fabricated. Figure 7 shows the overall scheme of the 'scan-to-part' process.

Apart from being faster, this product development process is automated and the possibility of human error is considerably reduced.

Insert Figure 7 about here

• Surgical intervention time is reduced: a CT scan of the zone to be reconstructed is used also to reconstruct the whole zone, not only the implant. A polymer model of the surrounding zone where the implant must fit is fabricated with AF (normally with Stereolitography). This way, all the necessary adjustments of the implant can be made using the model, before the surgery begins, which avoids the risk of having more than one surgical intervention. This is essential for the post-operative period since the patient will recover in a shorter time.

Insert Figure 8 about here

• Gradual structures can be fabricated easily with AF. Both the exterior and interior of an implant can be fabricated employing a lattice structure (Figure 8). The option of having a fully-dense core and a gradually formed lattice structure towards the exterior is favourable for osteoinduction, osteoconduction and osteointegration. The bone grows "inside" the implant, which is then integrated completely into the body's osseous structure. On the other hand, an implant fabricated as an external shell with an interior lightweight structure is much lighter and offers the same strength in use.

Due to these unquestionable advantages, the fabrication of implants with additive technologies is emerging rapidly. It can be predicted that in the near future AF will occupy a major share of the implant fabrication market. However, since it is considered a novel technique, it is submitted to thorough processes of testing and certification. A first step on this road is the use of customized implants upon the approval of the surgeon and acceptance of the patient. The pre-clinical and clinical tests are performed before the customized implants are fabricated and implanted. There have been several successful interventions placing implants, made with AF, into the human body (Arcam (2007)).

However, some implants are suitable for serial production. Before they can be placed on the market, the fabrication process must be certified. In Spain, the fabrication of implants must be done according to Royal Decree RD 414/96, which regulates the fabrication of all sanitary products. The certification includes traceability of raw materials and the certification of the production process. Regarding the first aspect, all technology providers have already completed the process of standardization and certification of the raw materials that they provide together with their machines. However, the second part of the certification is left to the medical device manufacturer who will actually produce the implants. Several steps must be completed in order to get certified for implant fabrication. Anyhow, there are already enterprises which have completed this process such as Adler Ortho for an acetibular cup [START_REF] Cremascoli | New orthopaedic implants produced with Rapid Manufacturing improve people's life[END_REF]) and Leader Italy for dental implants [START_REF] Traini | Direct laser metal sintering as a new approach to fabrication of an isoelastic functionally graded material for manufacture of porous titanium dental implants[END_REF]).

Lightweight structures in aerospace and automotive parts

One of the main target sectors for AF is the fabrication of lattice or spatial structures. The idea of having a shell of a defined thickness with a lattice structure in its interior in order to reduce its weight is not new. However, with conventional processes it is difficult to fabricate this structure. Additive technologies enable direct fabrication of lattice structures with gradual and controlled porosity.

Weight reduction and the increase of the strength-to-weight ratio are important in many industrial sectors. One of the sectors where weight reduction can be very beneficial is the aerospace industry. Aerospace parts are very expensive because conventional processes are highly wasteful in their operations. Using AF in the aerospace sector, parts are redesigned and their weight is reduced, which means that the costs of fabrication will decrease significantly. Also, a lower weight of aerospace structural parts has repercussions on fuel consumption which decreases significantly as well.

The advantages offered by AF may be summarized as follows:

• it reduces the use of raw material;

• it reduces buy-to-fly ratios;

• it provides freedom from geometrical constraints;

• it reduces the use of energy.

Some empirical studies [START_REF] Reeves | Manufacturing a Low Carbon Footprint: Zero Emission Enterprise Feasability Study[END_REF]) compare traditional manufacturing processes for aerospace and automotive parts (gravity die casting, cross drilling, electrochemical deburring and hole blanking) with Selective Laser Melting. It was shown that the same part, redesigned for Selective Laser Melting, had the same mechanical properties and 40% less material. When the lightweight parts are introduced into aircrafts and vehicles, a lot of energy is saved by using less material in part fabrication and the lower fuel consumption in the useful life of the vehicle. The cited study shows that the overall effect of saving 100 kg in material is 4.3 M€ per aircraft and 770€ per car. Here, both lower price of the vehicle and lower consumption during its useful life is considered.

Results and conclusions.

AIMME is an institute that is constantly trying to convert research efforts into industrial applications. ALM technologies and applications are brought to SME users and the advantages of ALM are converted to reality by specific industrial case studies. In the Results section, the authors discuss several case studies deriving from various industrial projects for tooling (HTT, Custom Cooling), biomedical (FABIO) and lightweight structures (MEPESS) applications. These results make obvious the previously mentioned advantages of ALM technologies.

Conformal Cooling in mould fabrication (Cooling Casting 1 , HTT) 2

One of the strongholds of AF is mould construction. The possibility of building free-form channels offers an important added value to injection moulds. The HTT and Cooling Casting projects were conceived to respond to the tooling sector's demands and make the most of the main advantages in adaptive cooling: cycle time reduction and part quality improvement.

The Cooling Casting project was developed with the goal of improving heat transfer in non-ferrous alloy (ZAMAK) injection moulds and reducing their cooling time so as to improve productivity. This was done by using hybrid moulds consisting of a machined mould block and mould inserts made with Laser CUSING which contain the adaptive cooling channels (Figure 9).

Insert Figure 9 about here In this project two production cycles were compared: one cycle included conformal cooling and the other cycle did not. The production cycle that did not included conformal cooling resulted in a cycle time of 6.7 seconds, whereas the cycle that included conformal cooling inserts resulted in a cycle time of 5 seconds. This reduction of 1.7 seconds in cycle time translates into an improvement of 180 parts per hour which represents a 33% increase in productivity.

The HTT project was developed with the goal of improving the quality of die-casting parts and eliminating defects caused by bad cooling. Parts were made of aluminium and, due to their particular design, had pores in the zone shown in Figure 10(a). These defects were caused by bad cooling and damaged the quality of the part.

Insert Figure 10 about here

The male and female parts of the mould were redesigned to include conformal cooling channels (shown in Figure 10(d)) as well as an interior honeycomb structure employed to reduce the use of material in the fabrication of the mould (Figure 10(c)). The redesigned male and female parts are shown in Figure 10(e). Thanks to the use of the shell and structure concept in the fabrication of the mould there was a 34% and 33% reduction in the quantity of material required to fabricate the male and female parts, respectively. The final cost of tool inserts was 1800 € and 1700 €, respectively. Also, the simulation shows that the cycle time was reduced by 18% which means that the productivity was increased. Finally, and maybe the most important, the part defects were eliminated due to homogeneous cooling.

Development of a new generation of biomedical materials and implants (FABIO) 3

This project included improvements and applications of new bio-materials and AF technologies to obtain a new generation of innovative customized health products (orthosis, osseous substitutes and prosthesis). The project involved three main lines of investigation (shown in three study cases): • Trans-tibial prosthesis and ankle/foot orthosis (AFO): Development of a new design process for a new generation of "scan-to-part" biomedical products, being able to obtain complex shapes and personalized forms. Definition of the procedure for the integration of several stages of the value chain for customized health products to minimize cost and delivery time.

• Mandible implant: Improvement of polymers for the direct fabrication of biomedical products using additive technologies. The goal is to match, as closely as possible, the mechanical properties of natural bone material (biomimetic properties) by improving the material. Similarly, another goal is to match, as closely as possible, the thermal properties of natural bone material (biomimetic properties) through a new design concept.

• Hip prosthesis: Incorporation of a new design procedure that is capable of generating added value for product design. Generation of 3D structures from AF technologies to obtain controlled-porosity scaffolds.

Figure 11 shows an example, which is actually used by a patient, of a customized socket of a trans-tibial prosthesis made with SLA and the Somos 18420 material.

Insert Figure 11 about here

Regarding the development of new materials, a biocompatible material is being developed and has passed the biocompatibility and citotoxicity tests. The next step in the development of the project will be the fabrication and use in implant surgery of several custom-made implants (mandible, cranial, etc.) fabricated with the newly developed biocompatible material. Figure 12 shows a part of a mandible that was reconstructed and fabricated with the Envision DLP machine and the newly developed biocompatible material.

Insert Figure 12 about here

Selective Space Structures in Rapid manufacturing of ferrous and non-ferrous parts (MEPESS) 4

This project has been defined so as to integrate knowledge of selective space structures (SSS) and Additive Fabrication applications so as to produce complex and high value-added ferrous and non-ferrous parts. The idea is to offer a feasible solution to the fabrication of lightweight structures (especially in the aerospace and biomedical sectors) as well as to improve the performance of products intended to support dynamic loads by incorporating SSS.

For this purpose, different types of structures have been analyzed and used to create the interior structure of a part.

Regarding their structural behaviour, the selected parts can be divided into two groups. The first group includes parts that are redesigned using SSS in order to improve their strength to weight ratio. The second group includes parts that are redesigned using special dynamic-cell SSS which enables the transmission of movement or displacement.

Working with structured parts is complicated due to excessively large size of their CAD models. Conventional 3D modellers have difficulties managing these files. Hence, special software is used to create and operate SSS parts. The development of this project will require the used of Netfabb by Fruth Innovative Technologies GmbH.

Actual results of this project include a test of the capacity of different technologies to fabricate SSS parts. Some of these parts are shown in the following figures. Figure 13 shows the types of structures accomplished by fabrication with Laser CUSING. These structures are meant to be used in scaffold cooling and weight reduction of automotive and aerospace parts. Figure 14 shows a free form SSS structure made with EBM technology with a pore size of 500 µm (this image was obtained from a Starret HB-350 profile projector). Such a fine structure is desirable in osteoconductive implant structures. As can be seen in the figure, this structure is completely regular and controlled, and has a smooth cell transition. Finally, Figure 15 shows one of the first demonstrators fabricated with EBM: a solid femur bone with a SSS zone.

Insert Figure 13

Conclusions

Additive Layered Manufacturing is a set of emerging technologies that are becoming a serious competitor to machining and forming technologies. Due to their obvious advantages in product customization, maximum material savings, and gradual and controlled porous structures, additive technologies are becoming more interesting to many industrial sectors such as biomedical, aerospace, automotive and tooling. With the introduction of new materials and active principles, the ALM technologies that originally were only used for prototypes and models have now been converted into technologies that can fabricate fully-dense functional parts with high added value, as shown in the previous figures.

References.

Figure 1 .Figure 2 .

 12 Figure 1. Fusing on an EBM machine (a); scheme of an additive machine (b).

Figure 3 .

 3 Figure 3. Prototypes and models fabricated with SLA and DLP.

Figure 4 .

 4 Figure 4. Hearing Aid apparatus shell (a) and coronary bypass (b).

Figure 5 .

 5 Figure 5. Artistic design supported by AF: artistic model (a), jewel model fabricated with DLP and (c) vacuum casted silver ring.

Figure 7 .Figure 8 .Figure 9 .Figure 10 .Figure 11 .Figure 12 .Figure 13 .

 78910111213 Figure 6. Conventional (a) and conformal (b) cooling channels.

Figure 14 .Figure 15 .

 1415 Figure 14. Fine pore space structure fabricated with an EBM machine.

http://mc.manuscriptcentral.com/tprs Email: ijpr@lboro.ac.ukInternational Journal of Production Research

Financed by European Regional Development Fund

Financed by Spanish Government's Ministry of Industry, Tourism and Commerce

Financed by Spanish Government's Ministry of Industry, Tourism and Commerce