M-C Kastrinaki

P Sidiropoulos

S Roche

J Ringe

Sylvain Lehmann

H Kritikos

V-M Vlahava

B Delorme

G D Eliopoulos

Christian Jorgensen

P Charbord

T Ha

D T Boumpas

H A Papadaki
email: epapadak@med.uoc.gr

Marrow Mesenchymal Stem Cells in Rheumatoid Arthritis

Adult multipotential stem cells isolated from a variety of tissues have been demonstrated to participate in tissue homeostasis and repair under the influence of appropriate signals. Among the most well characterised types of adult stem cells are the bone marrow (BM) derived mesenchymal stem cells (MSCs). 1 2 These cells reside in BM stroma providing the supporting feeder cells necessary for the haematopoietic progenitor cell growth but they may also differentiate into cells of connective tissue such as osteocytes, chondrocytes, tenocytes, adipocytes and smooth muscle cells. 3 4 There is currently great interest in exploring the potential use of MSCs in regenerative medicine with particular target the bone and cartilage defects. [START_REF] Deans | Mesenchymal stem cells: biology and potential clinical uses[END_REF][START_REF] Barry | Mesenchymal stem cells: clinical applications and biological characterization[END_REF][START_REF] Otto | Tomorrow's skeleton staff: mesenchymal stem cells and the repair of bone and cartilage[END_REF][START_REF] Jorgensen | Tissue engineering through autologous mesenchymal stem cells[END_REF][START_REF] Mankani | In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape[END_REF][START_REF] Tsuchida | Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats[END_REF] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by cartilage and bone destruction associated with local production of inflammatory mediators such as tumour necrosis factor a (TNFa) and interleukin (IL)1b. Joint destruction renders RA a candidate disease for cartilage and bone repair using MSCs. There are, however, some specific issues concerning the reserves and function of BM MSCs in RA that need to be addressed before proceeding to clinical application. In addition to the concept that BM MSCs may be primarily involved in joint damage in RA, [START_REF] Nakagawa | Bone marrow stromal cells contribute to synovial cell proliferation in rats with collagen induced arthritis[END_REF][START_REF] Suzuki | Osteoclast-like cells in murine collagen induced arthritis[END_REF][START_REF] Sen | Expression and function of wingless and frizzled homologs in rheumatoid arthritis[END_REF] there are studies demonstrating that increased local production of TNFa may injure the BM microenvironment and may affect the reserves of BM haematopoietic progenitor cells. [START_REF] Papadaki | Bone marrow progenitor cell reserve and function and stromal cell function are defective in rheumatoid arthritis: evidence for a tumor necrosis factor alpha-mediated effect[END_REF] Accordingly, a critical issue is whether BM MSCs are also depleted or functionally altered in RA.

This study evaluates the reserves and function of BM MSCs in patients with RA in terms of their proliferative, clonogenic and differentiation potential. Molecular and proteomic characteristics of patient MSCs with special focus on inflammatory cytokine gene and protein expression were also studied.

PATIENTS AND METHODS Patients

We studied 26 patients with active RA [START_REF] Arnett | The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis[END_REF] [START_REF] Pincus | Quantitative measures for assessing rheumatoid arthritis in clinical trials and clinical care[END_REF] and 21 healthy individuals, age-and sex-matched with the patients (table 1). The Institutional ethics committee approved the study and informed consent according to the Helsinki Declaration was obtained from all subjects.

MSC culture and differentiation

BM mononuclear cells (BMMCs) obtained from posterior iliac crest aspirates were cultured in Dulbecco modified Eagle medium, low glucose (DMEM-LG; Gibco/Invitrogen, Paisley, Scotland)/ 10% foetal calf serum (FCS; Hyclone, Logan, Utah, USA)/100 IU/ml penicillin-streptomycin (MSC medium) and MSCs were grown as previously described. 17 18 Cell-free supernatants were stored (- 72uC) for cytokine measurements by ELISA (Quantikine; R&D Systems, Minneapolis,

Extended report

Table 1 Clinical and laboratory data of the patients studied {Recombinant human soluble p75 TNF receptor fusion protein (25 mg/biweekly).

1Humanised anti-TNF monoclonal antibody (40 mg every 2 weeks). "Interleukin 1b receptor antagonist (100 mg/day). **Human/murine chimeric anti-CD20 monoclonal antibody (2 doses of 1 g within 2 weeks).

CSA, ciclosporine-A (150 mg/day) ; D-pen, D-penicillamine (200 mg/day); DAS, Disease Activity Score; DMARDs, disease-modifying antirheumatic drugs; HCQ, hydroxychloroquine (400 mg/day); HHQ, hexahydroquinoline; LEF, leflunamide (10-20 mg/day); MTX, methotrexate; NSAIDs, non-steroidal anti-inflammatory drugs; PDN, prednisone; SSA, sulphasalazine-A (2 g/day); TNF, tumour necrosis factor; UPN, unique patient number.

Extended report

Minnesota, USA). Trypsinised MSCs from passage (P)-2 were induced for differentiation (fig 1A -F). MSC adipogenic and osteogenic differentiation was induced as previously described and assessed by Oil Red O and alkaline phosphatase (ALP)/Von Kossa stains, respectively. [START_REF] Gronthos | Isolation, purification and in vitro manipulation of human bone marrow stromal precursor cells[END_REF] For chon- drogenic induction, MSCs were pelleted and cultured in DMEM high glucose (DMDM-HG, Gibco), supplemented with 6.25 mg/ ml insulin, 6.25 mg/ml transferrin, 1.33 mg/ml linoleic acid, 1.25 mg/ml bovine serum albumin, 1 mM sodium pyruvate, 0.17 mM ascorbate-2-phosphate, 0.1 mM dexamethasone, 0.35 mM L-proline, 6.25 ng/ml selenous acid and 0.01 mg/ml transforming growth factor (TGF)-b 3 (R&D Systems). [START_REF] Lisignoli | Chondrogenic differentiation of murine and human mesenchymal stromal cells in a hyaluronic acid scaffold: differences in gene expression and cell morphology[END_REF] Chondrogenic differentiation was identified with Alcian blue and Masson trichrome stains. Reagents were purchased from Sigma (St Louis, Minnesota, USA) unless otherwise indicated.

MSC immunophenotypic and survival characteristics

Trypsinised MSCs from passages 1-6 (P1-P6) were immunophenotypically characterised by flow-cytometry, using anti-CD29 (4B4; Cyto-Stat, Beckman-Coulter, Fullerton, California, USA), anti-CD44 (J173; Immunotech/Coulter, Marseille, France), anti-CD73 (AD2; Pharmingen, San Diego, California, USA), anti-CD90 (F15.42; Immunotech/Coulter), anti-CD105 (SN6; Caltag, Burlingame, California, USA), anti-CD146 (P1H12; Pharmingen), anti-CD45 (IMMU19.2; Immunotech/ Coulter), anti-CD14 (RMO52; Immunotech/Coulter) and anti-CD34 (QBend10; Beckman-Coulter) monoclonal antibodies.

Apoptosis was studied by flow-cytometry using 7-aminoactinomycin-D (7-AAD; Calbiochem-Novabiochem, La Jolla, California, USA). 14 21 Results were expressed as 7-AAD neg (live), 7-AAD dim (early apoptotic) and 7-AAD bright (late apoptotic) cells (fig 2A ,B).

MSC quantification in the BMMC fraction

A limiting dilution assay (LDA) was used to evaluate MSC frequency within BMMCs following 6-week culture of seven different BMMC concentrations (250-10 000 cells/well) in 96well plates. [START_REF] Anker | Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential[END_REF] Wells with .50 adherent spindle shaped cells were considered positive and MSC frequency corresponded to the dilution resulting in 37% negative wells. [START_REF] Rizzo | Stem cell defect in aplastic anemia: reduced long term culture-initiating cells (LTC-IC) in CD34+ cells isolated from aplastic anemia patient bone marrow[END_REF]

Clonogenic potential and proliferative potential of MSCs

A colony forming unit fibroblastic assay (CFU-F) was also used to evaluate MSC frequency within BMMCs. Briefly, day-0 BMMCs were seeded at three different concentrations, expanded for 14 days and CFU-F number was estimated using linear regression analysis. The clonogenic potential of trypsinised MSCs from P1-P6 was also evaluated by CFU-F. MSC proliferative potential was evaluated by a methyl triazolyl tetrazolium (MTT) based assay in P2 and also by estimating population doubling time through P1-P6.

RT-PCR

Total RNA isolated from P2 and differentiated MSCs (RNeasy mini kit; Qiagen GmbH, Hilden, Germany) was reverse transcribed (SUPERSCRIPT II; Gibco) and amplified by RT-PCR. Products were normalised according to b 2 -microglobulin (b 2 m) using the ImageJ densitometry analysis system (http:// rsb.info.nih.gov/ij/). Primer sequences are listed in supplementary table 1.

Real time quantitative PCR IL1b and TNFa expression

PlatinumHqPCR SuperMix-UDG (Invitrogen) and TaqManH Gene Expression Assays (Applied Biosystems; Foster City, California, USA) were used for IL1b, TNFa and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) identification. Measurements were performed using the ABI Prism 7000 System (Applied Biosystems). Results were normalised according to GAPDH quantity.

Telomere length measurement

DNA was extracted from P2 MSCs (Qiagen). b-Globin was used as control single copy gene. 23 24 iTaq SYBR Green Supermix with ROX (Biorad, Hercules, California, USA) was used for the reactions. Primer sequences are listed in supplementary table 1. Telomere length was reflected by the relative telomere/singlecopy-gene ratio (T/S): T/S = 2 2DCt (DCt = Ct telomere 2Ct b-globin ).

Two-dimensional gel electrophoresis

Whole cell lysates were obtained in 8 M urea, 2 M thiourea, 4% (CHAPS), 50 mM dithiothreitol (DTT) (Sigma). For the first dimension, 30 mg proteins from P2 MSCs were added to rehydration buffer. Immobilised pH gradient (IPG) strips (pH 3-10) were re-hydrated and focalised (70 000 v/h). For the second dimension, strips were equilibrated and proteins were separated by SDS-PAGE. Gels were stained with silver nitrate [START_REF] Shevchenko | Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels[END_REF] and scanned using the Labscan-3 software (GE Healthcare; Little Chalfont, Buckinghamshire, UK) after calibration by the Kaleidoscope LaserSoft Imaging software (Kodak ref: R020123; Eastman Kodak, Rochester, New York, USA). Spot detection and quantitation were performed by ImageMaster 2D Platinum software (GE Healthcare).

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

Microarray experiments

Hybridisation on HG-U133 Plus 2.0 microarrays was performed according to manufacturer (Affymetrix; Santa Clara, California, USA). Arrays were washed, stained and scanned on a Hewlett Packard Genearray Scanner (Affymetrix). Affymetrix GCOS 1.2 software was used to control washing and scanning, generate DAT/CEL/EXP files and process data. Group comparison and gene retrieval was performed by SiPaGene (BioRetis GmbH; Berlin, Germany). Cluster analysis was calculated with Genesis (A. Sturn; Graz University, Graz, Austria).

Statistical analysis

Data were analysed by Mann-Whitney U test, one-way and two-way analysis of variance (ANOVA) tests (GraphPad Software; San Diego, California, USA). Two-dimensional gel electrophoresis data were analysed by the Hierarchical Clustering Explorer 3.0 software (http://www.cs.umd.edu/ hcil/multi-cluster/) and Pearson correlation coefficient. [START_REF] Seo | Interactively optimizing signal-to-noise ratios in expression profiling: project-specific algorithm selection and detection p-value weighting in Affymetrix microarrays[END_REF] Grouped data are expressed as mean (SD).

RESULTS

MSC immunophenotype and differentiation potential

Immunophenotypic analysis at the end of each passage demonstrated that cultures constituted of a homogenous cell population positive for CD73, CD90, CD146, CD105, CD29, CD44 and negative for CD45, CD14, CD34 surface antigens (fig 1G). [START_REF] Gronthos | Isolation, purification and in vitro manipulation of human bone marrow stromal precursor cells[END_REF] Culture-expanded MSCs from patients were able to differentiate into osteogenic/chondrogenic/adipogenic lineages as shown by the respective cytochemical staining (fig 1B -F). Furthermore, the osteogenic/chondrogenic/adipogenic differentiating capacity assessed by the relative expression of ALP and CBF-1 mRNA, COLLII and AGR (aggrecan) mRNA, and aP2 (adipose fatty-acid binding protein 2) and peroxisome proliferator-activated receptor (PPAR)-c mRNA expression, respectively, did not differ between patients and controls, suggesting normal differentiation potential of RA MSCs (fig 1H).

MSC frequency in the BMMC fraction

The estimated frequency of MSCs in the BMMC fraction did not differ significantly between patients (20.74 (12.41)/10 5 BMMCs) and controls (23.90 (15.65)/10 5 BMMCs) (p = 0.8894) by LDA. Quantification of BM MSCs by CFU-F analysis at day 0 was in accordance with LDA data. CFU-F number obtained by day-0 MSCs did not differ significantly between patients (18.42 (13.75)/10 5 BMMCs) and controls (19.85 (18.96)/10 5 BMMCs) (p = 0.3464), further suggesting normal numbers of MSCs in RA. No statistically significant difference was documented between the LDA and CFU-F assays in the estimation of MSC frequency in either patients (p = 0.210) or healthy controls (p = 0.199).

MSC clonogenic and proliferative potential

The growth potential of BM MSCs in patients with RA was studied by evaluating their clonogenic and proliferative capacity over time. CFU-F recovery by culture-expanded MSCs was significantly lower in patients with RA compared to controls through P1-P6 (p,0.01) (fig 3A). In association to the lower clonogenic potential, patient MSCs displayed also defective proliferative capacity compared to controls as estimated by the population doubling time over P1-P6 (p,0.001) (fig 3B). These findings were further substantiated by the MTT assay; as shown in fig 3C depicting results from a representative passage (P2), the number of live cells remained significantly lower in patients with RA compared to controls (p,0.001).

The majority of patients had previously been treated with cytotoxic and/or immune suppressant or anti-cytokine agents. To exclude the possibility of a drug-related damage of MSCs, a subset analysis was performed in the group of previously treated (n = 20) and untreated (n = 6) patients. Compared to healthy controls, treated and untreated patient groups displayed lower CFU-F recovery through P1-P6 (p,0.001 and p,0.05, respectively). Similarly, treated and untreated patients displayed increased MSC doubling time (p,0.001 and p,0.05, respectively) and defective proliferative potential in the MTT assay (p,0.001 and p,0.05, respectively) compared to controls. However, no statistically significant differences were found between the two patient groups in the above parameters. Thus drug-induced damage is unlikely to be the cause of the defective clonogenic/proliferative potential of MSCs in RA.

MSC survival characteristics

The impaired clonogenic and proliferative ability of MSCs in patients with RA might have resulted from increased cell apoptosis. The total proportion of apoptotic cells ranged from 7.68 (3.70)% (P1) to 8.24 (2.01)% (P6) in the patients and from 8.25 (4.15)% (P1) to 8.24 (2.85)% (P6) in the controls. Overall, no statistically significant differences were found between patients and controls in the proportion of early (F = 0.8206,F [START_REF] Owen | Marrow stromal cells[END_REF] 84 , not significant) or late apoptotic (F = 0.9977,F [START_REF] Owen | Marrow stromal cells[END_REF] 84 , not significant) cells throughout P1-P6 culture period, suggesting that the impaired clonogenic and proliferative potential of MSCs in RA is not due to accelerated cell apoptosis (fig 2C ,D).

Cytokine production by MSCs

To investigate whether BM MSCs from patients with RA display abnormal cytokine production similar to RA synovial fibroblasts [START_REF] Takeuchi | Establisment and characterization of nurse-like stromal cell lines from synovial tissues of patients with rheumatoid arthritis[END_REF] we initially assessed IL1b and TNFa production. The relative IL1b/GAPDH and TNFa/GAPDH mRNA levels at P2 did not differ significantly between patients and controls (0.059 (0.029) and 0.040 (0.022), respectively). Similar to the mRNA data, protein levels of IL1b and TNFa determined by ELISA in culture supernatants did not differ significantly between patients and controls through P2-P6 (F = 0.1474,F 1 48 and F = 1.2373,F [START_REF] Owen | Marrow stromal cells[END_REF] 48 , respectively). The levels of the MSC growth-promoting cytokines vascular endothelial growth factor and stromal-derived factor-1 and the values of the inhibitory/inflammatory cytokines IL6, TGF-b 1 , IL8 and IL15 in culture supernatants through P2-P6 did not differ significantly between patients and controls. These data indicate that abnormal cytokine production by MSC per se seems unlikely to be the cause of the impaired growth potential of MSCs in RA.

Extended report Proteomic analysis

Two-dimensional electrophoresis was performed using P2 MSCs in patients with RA (n = 10) and healthy controls (n = 6) (fig 4A ,B). By using the Hierarchical Clustering method, we could not define any cluster that might discriminate patient and control cells (fig 4C). The Pearson correlation coefficient was not significantly different between patient and control cells (r = 0.933 (0.022) and r = 0.929 (0.020), respectively). These data corroborate the lack of significant changes in cytokine production between patients and controls.

MSC telomere length

Decreased cellular growth associated with premature telomeric loss has previously been described in periarticular osteoblasts in RA. [START_REF] Yudoh | Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients[END_REF] We therefore evaluated the relative telomere length (RTL) of MSCs from P2 in 12 patients and 12 age-and sex-matched healthy controls. An age-related decrease of the RTL as estimated by the relative T/S ratio was observed in patients (r = 20,899, p,0.001) and controls (r = 20.906, p,0.001) (fig 3D ,E). To characterise patient MSC RTLs appropriate or inappropriate for a given age, we defined the observed/predicted RTL ratio (O/P ratio) for each sample, according to the equation derived from the linear regression analysis of the correlation between RTL and age (years) of the controls. We found that the mean O/P ratio of the patients (0.83 (0.20)) was out of the 95% confidence limits of the controls (mean O/P ratio 0.67 (0.17), p = 0.0226), suggesting inappropriate MSC telomeric loss by age. By analysing the mean RTL per decade of years in patients and controls, a highly significant difference was obtained (p,0.001) (fig 3F), indicating further premature telomeric loss of MSCs in RA that might account, at least in part, for the defective clonogenic and proliferative potential of cells in culture.

Gene expression profiling of MSCs

Genes encoding for cell surface molecules, cytokines and differentiation markers were analysed in particular. Selection for genes differentially expressed in RA compared to normal MSCs allowed separation of the RA from normal samples by hierarchical clustering (fig 5). Affymetrix probe sets that were differentially expressed in more than 80% of the pairwise comparisons between three RA and six normal MSC samples were selected to identify objects in canonical pathways of the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The most differentially expressed genes were those encoding for proteins implicated in focal adhesions (supplementary fig 1 and supplementary table 2). Specifically, several genes involved in cytoskeleton regulation and therefore linked to cell motility, proliferation and survival, were found to be differentially expressed between patients and controls. Of particular interest was the downregulation of cyclin-D, which plays an important role in the transition from G1 to S-phase in cell cycle progression, as well as the upregulation of cyclin-D inhibitors Ink4a-d and Kip1,2 and their upstream inductors Smad2/3 and TGF-b (supplementary table 3).

Figure 3 Clonogenic/proliferative potential and relative telomere length of mesenchymal stem cells (MSCs). The left panel depicts the mean (SEM) number from colony forming unit fibroblastic assay (CFU-F) (A) and days for population doubling (B) over passages 1-6 (P1-P6) as well as the mean (SEM) optical density corresponding to the number of live cells over 13-day culture using the methyl triazolyl tetrazolium (MTT) assay in P2 (C). The right panel illustrates the correlation (regression line within 95% confidence limits) between age and relative MSC telomere/single-copy-gene values (diagrams D and E, respectively) and the mean (SEM) relative telomere length per decade of years (F) in RA (solid squares) and normal (open squares) subjects. Analyses have been performed by two-way analysis of variance (ANOVA).

DISCUSSION

To explore the potential use of MSCs for cartilage and bone repair, we conducted the current study to evaluate the reserves, functional, molecular, proteomic characteristics, and differentiation potential of BM MSCs in patients with RA, a candidate disease characterised by destruction of articular structures. Characterisation of autologous MSCs in patients with RA is important since emerging data suggest that allogeneic MSCs may not be as ''immune privileged'' as previously considered. [START_REF] Nauta | Donorderived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting[END_REF] We found that the frequency of MSCs within BMMCs did not differ between patients and controls. Our findings are in accordance with previously reported data on the frequency of MSCs in normal BM suggesting that patients with RA have normal BM MSC reserves. 4 30 Furthermore, culture-expanded MSCs from patients with RA were morphologically and immunophenotypically indistinguishable from the controls.

Culture-expanded MSCs from our patients with RA displayed normal osteogenic, chondrogenic and adipogenic differentiation. Interestingly, reduced chondrogenic activity of BM MSCs has been reported for osteoarthritis patients and this defect has been associated with the disturbed homeostatic maintenance of the articular cartilage. [START_REF] Murphy | Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis[END_REF] Normal differentiation potential of BM MSCs however, has been described in other autoimmune diseases such as in systemic sclerosis. [START_REF] Larghero | Phenotypical and functional characteristics of in vitro expanded bone marrow mesenchymal stem cells from systemic sclerosis patients[END_REF] MSC expansion through passages, assessed by the population doubling time, was significantly reduced in patients with RA, suggesting defective proliferative capacity. This finding was further substantiated by the MTT assay. The defective proliferative potential was in accordance with the impaired clonogenic capacity of patient MSCs as demonstrated by the low CFU-F recovery timecourse. In addition, the impaired growth of MSCs in RA is not likely to be due to accelerated cell apoptosis, since apoptosis in culture was not found to be significantly different between patients and controls.

The majority of our patients had been treated with methotrexate and/or corticosteroids, anti-cytokine or diseasemodifying anti-inflammatory agents. It has been reported that methotrexate does not affect the survival and functional characteristics of MSCs, while corticosteroids may damage their proliferative potential. [START_REF] Li | Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents[END_REF] No available data on the effect of anti-cytokine and anti-inflammatory agents on MSCs are presently known. To exclude the possibility of drug-mediated damage, we have separately analysed MSC clonogenic/proliferative potential in untreated patients. Cellular growth and Figure 4 Proteomic analysis of mesenchymal stem cells (MSCs). Two-dimensional gel electrophoresis was performed using whole protein cell extracts from P2 MSC cultures of patients with rheumatoid arthritis (RA) (n = 10) (A) and healthy controls (n = 6) (B). After scanning, spot detection, quantification and normalisation, gels were compared using Hierarchical Clustering Software and Pearson test (C). No cluster could be detected using these proteomic profiles.

CFU-F recovery were significantly lower even in this group of patients compared to controls, whereas no significant differences were documented between treated and untreated patients with RA. These findings suggest that treatment with antirheumatic agents is not the major factor affecting MSC growth in RA.

It has been suggested that BM derived MSCs with altered properties may repopulate the synovial membrane in RA, whereas abnormal expression of inflammatory cytokines has been reported for RA synovial fibroblasts. 12 13 MSCs from our patients displayed normal cytokine expression in array profiling and ELISA. We have specifically focused on IL1b and TNFa since these cytokines have been found overexpressed in synovial fluid and BM culture supernatants. 14 34 35 No differences were found between patients and controls in the inflammatory cytokine expression or the growth promoting cytokine levels, suggesting that the impaired growth potential found in patient MSCs is not due to altered cytokine expression.

It has been previously shown that aging may affect the proliferative potential of MSCs. [START_REF] Mareschi | Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow[END_REF] Since patients with RA and controls in our study were age-matched, an age-related defect in the clonogenic and proliferative capacity of patient MSCs is unlikely. Patient MSCs, however, displayed age-inappropriate relative telomere loss. Since there is evidence suggesting that the replicative capacity of cells correlates with the initial cellular telomere length 37 38 we may speculate that the defective growth potential of patient MSCs is due to inappropriate telomere loss. Although telomerase activity was not assessed in our study, we hypothesise that it is under the influence of the inflammatory BM milieu. [START_REF] Papadaki | Bone marrow progenitor cell reserve and function and stromal cell function are defective in rheumatoid arthritis: evidence for a tumor necrosis factor alpha-mediated effect[END_REF] MSCs in RA undergo accelerated proliferation resulting in premature replicative exhaustion as previously described for RA periarticular osteoblasts. [START_REF] Yudoh | Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients[END_REF] The recently described accelerated telomere shortening under the influence of chronic mild oxidative stress further corroborates this assumption. [START_REF] Martin-Ruiz | Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span[END_REF] In the search for direct molecular mechanisms by array profiling, differentially expressed genes were found to be involved especially in cell adhesion processes and matrix (collagen) synthesis. In particular, downregulation of cyclin-D was identified. Thus, a repressive influence on the transition from G1 to S-phase could provide a direct molecular explanation for the altered proliferative capacity of MSCs from patients with RA. This could be related to the increased local production of TGF-b in patients' BM microenvironment (data not shown) that may result in increase of TGF-b signalling in MSCs and consecutive expression of cyclin-dependent kinase inhibitors, as suggested from the ''cell cycle'' pathway from KEGG. Thus, MSCs from patients with RA may present in vitro with a phenotype of cells that are more adherent, produce more matrix and are less active in proliferation. However, functional data are needed to substantiate this hypothesis.

In conclusion, our data have shown that patients with RA display a normal number, molecular and proteomic profile and differentiation potential of BM MSCs, encouraging, therefore, the use of autologous MSCs for the repair of cartilage and bone damage associated with long-standing RA. The proliferative and clonogenic potential of patient BM MSCs, however, was reduced compared to normal MSCs, and this defect was associated with decreased cellular telomere length and altered expression of genes implicated in focal adhesion and cell cycle pathways. This altered MSC activity may potentially influence the preparation of the cells for therapeutic usage in RA. In summary, this is the first study characterising BM MSCs in patients with RA under defined conditions in the context of potential clinical use for the repair of tissue injury associated with RA.

*

  Patients had discontinued any medication for at least 24 h prior to BM aspiration. {Human/murine chimeric anti-TNFa monoclonal antibody (3 mg/kg every 4-8 weeks).

Figure 1

 1 Figure 1 Differentiation potential and immunophenotypic characteristics of mesenchymal stem cells (MSCs). The upper panel shows undifferentiated MSCs from passage 2 (P2) (A) and differentiated cells towards the adipogenic (B and C), osteogenic (D) and chondrogenic (E and F) lineages from a representative patient with rheumatoid arthritis (RA). Cell differentiation was identified with Oil Red O (C), alkaline phospahatse (ALP)/Von Kossa (D), Masson (E) and Alcian blue (F) staining. G. Immunophenotypic characteristics of patient MSCs at P2. The red and blue histograms depict the positive and negative markers, respectively, compared to the isotype-matched negative controls (open histograms). H. Specific gene mRNA expression (on the left) of P2 BM MSCs upon differentiation towards the osteogenic, chondrogenic and adipogenic lineages in representative RA (n = 3) and control (n = 3) subjects as well as cumulative data (mean relative values (SEM)) of specific gene mRNA expression from all RA (solid squares) and controls (open squares) studied (on the right). Comparison has been performed by means of the non-parametric Mann-Whitney U test.

Figure 2

 2 Figure 2 Survival characteristics of mesenchymal stem cells (MSCs). MSCs from passages 1-6 (P1-P6) were stained with 7-aminoactinomycin-D (7-AAD) and analysed by flow cytometry for the study of apoptosis. Scattergram B shows the proportion of 7-AAD neg (R2; live), 7-AAD dim (R3; early apoptotic) and 7-AAD bright (R4; late apoptotic) cells in the gate of R1 (scattergram A) representing MSCs from P2. Bars in the lower panel represent the mean (SEM) proportion of live, early and late apoptotic cells timecourse in patients (C) and healthy controls (D) analysed as above. No statistically significant differences were documented between patients and controls by means of two-way analysis of variance (ANOVA).

Figure 5

 5 Figure 5 Gene expression profiling of mesenchymal stem cells (MSCs). RNA from P2 bone marrow (BM) MSCs from patients with rheumatoid arthritis (RA) (n = 3) and healthy controls (n = 6) was hybridised to the GeneChips and arrays were processed for signal calculation and pairwise chip comparison. Selection of genes differentially expressed in RA compared to non-differentiated (ND) bone marrow mononuclear cells (BMMCs) allowed us to distinguish RA MSCs from normal MSCs by hierarchical clustering.

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Ann Rheum Dis 2008;67:741-749. doi:10.1136/ard.2007.076174 group.bmj.com on December 20, 2017 -Published by http://ard.bmj.com/ Downloaded from

Ann Rheum Dis 2008;67:741-749. doi:10.1136/ard.2007.076174

Acknowledgements:

The authors wish to thank Mrs Helen Koutala, Mrs Claudia Gemetzi and Mrs Athina Damianaki (University of Crete, Greece) for their valuable technical assistance.

/week No LEF, CSA, SSA Etanercept{, Adalimumab1 MTX mg/week No LEF, SSA Kineret" Kineret 5 71/M 6 4.SSA, HCQ None MTX, HCQ, SSA /week 5 mg/day LEF, CSA, SSA, D-Pen None LEF, PDN /week 7.5 mg/day LEF, CSA, SSA, D-pen Adalimumab MTX, LEF, PDN LEF, CSA, SSA, HCQ LEF, CSA, SSA, HCQ, D-pen

Funding: This study was supported by the 6th European Union Framework Program (FP6, GENOSTEM #503161).

Competing interests: None declared.

Ethics approval:

The Institutional ethics committee approved the study and informed consent according to the Helsinki Declaration was obtained from all subjects.


Georgios Zardalidis

Ioannis Karafyllidis

Fundamental Building Blocks for The Design of A Single-electron Nanoelectronic Processor

A single-electron random access memory array (RAM) and a single-electron universal Fredkin gate are designed and simulated. The universality of the Fredkin gate in combination with the RAM gives the potential of the realization of an elementary singleelectron nanoelectronic processor.

I.

INTRODUCTION

Single-electronics is one of the emerging nanoelectronic technologies that deal with the control of transport and position of a single or a small number of electrons. The fundamental physical principle of single-electronics is the tunnelling effect and the phenomenon of Coulomb blockade.

Several single-electron circuits have been recently proposed in the literature: single-electron memories [START_REF] Stone | Silicon single-electron memory structure[END_REF], inverters and pumps [START_REF] Fukui | Simple and stable single-electronics logic utilizing tunnel junction load[END_REF], majority gates [START_REF] Iwamura | Single-electron majority logic gates[END_REF], logic gates [4, [START_REF] Dasigenis | A single-electron XOR gate[END_REF], half adders [START_REF] Zardalidis | A single-electron half-adder[END_REF] and adders [START_REF] Zardalidis | Design and simulation of a single-electron fulladder[END_REF]. The need for computer-aided design and simulation of single-electron circuits has long been recognized [START_REF] Karafyllidis | Determination of lowest energy state in single-electron circuits[END_REF]. Several simulators have been implemented to support single-electron circuit design [START_REF] Chen | Single-electron transistor logic[END_REF], [START_REF] Karafyllidis | A simulator for single-electron devices and circuits based on simulated annealing[END_REF]. Nevertheless the development and fabrication of single electron devices has already taken place [11-[13]. More about single electron circuits can be found in [START_REF] Averin | Theory of single electron charging of quantum wells and dots[END_REF], [START_REF] Wasshuber | SIMON-A Simulator for Single-Electron Tunnel Devices and Circuits[END_REF].

One of the promising single-electron memory cells is the electron trap [START_REF] Nakazato | The multiple-tunnel junction and its application to single-electron memory and logic circuits[END_REF]. This cell presents a high energy barrier to electron transport because of thermal fluctuations, is not sensitive to random background charge, and has been recently fabricated [START_REF] Stone | Silicon single-electron memory cell[END_REF].

The most difficult hurdle for constructing single-electron memory arrays is the selective read and write operations. One of the aims of this work is to tackle this problem.

The Fredkin gate, known also as Fredkin-Toffoli gate, has been proposed by Fredkin and Toffoli in 1982 [START_REF] Fredkin | Conservative logic[END_REF] and performs conditional crossover of two data bits according to the values of a control bit. The Fredkin gate is a computationally universal gate, i.e. any Boolean function can be implemented using only by Fredkin gates.

A Single-electron Fredkin gate (SEF-gate) would be very useful, because digital single-electron circuits that use only one type of gate would be very much easier to fabricate than single-electron circuits that use various types of gates.

II.

OPERATION OF THE BASIC SINGE-ELECTRON MEMORY CELL

Figure 1(a) shows the circuit of the basic single-electron memory cell that will be used throughout this work, i.e. the electron trap. The electron trap circuit comprises six islands N, bounded by one capacitor, C 1 , and six tunnel junctions J. The tunnel junctions are identical. When a positive voltage V g is applied, an electron or a small number of electrons is transported from the ground to island N 1 . The presence of an excess electron or a small number of excess electrons at N 1 corresponds to logical "1" and the absence to logical "0". Figure 1(b) shows the symbol of the electron trap. Here junctions J 1 -J 6 and islands N 2 -N 6 are absorbed into the symbol MTJ, that stands for "multiple-tunnel junction". The simulated operation of the basic memory cell of Figure 1 can be described as follows. Initially, the voltage V g is zero and there are no excess electrons at the island N 1 . After some time V g becomes positive and reaches the value of 0.6 V. At this time an electron is transported from the ground to N 1 and causes the negative current pulse. Although V g remains at 0.6 V for some time, no more electrons are transported to N 1 , because of the Coulomb blockade. After that, the value of V g becomes zero again and remains zero for some time, but the electron remains in N 1 , because it can not surmount the potential barrier imposed by junctions J 1 -J 6 and escape to ground. Thus the logical "1" has been written in the memory cell and this logical value is kept at the cell although the value of V g has returned to zero.

To write the logical value "0" in the cell, which currently keeps the logical value "1", V g becomes negative and reaches the value of -0.4 V. At this time the excess electron is transported from N 1 to the ground and causes the positive current pulse. After that the value of V g remains at -0.4 V for some time and then becomes zero again. Thus the logical "0" has been written in the memory cell.

From the simulation becomes clear that the electron trap has two stable states, corresponding to energy levels E A , (logical "0") and E F (logical "1"). To change the state of the electron trap from "0" to "1" the electron has to surmount an energy barrier equal to (E D -E A ), whereas to change the state from "1" to "0" the electron has to surmount an energy barrier equal to (E D -E F ). Since (E D -E A ) > (E D -E F ) a smaller absolute value of V g is required (i.e. |0.4| V) in order to drive the circuit from state "1" to state "0" than the absolute value required for driving the circuit from state "0" to state "1" (i.e. |0.6| V).

To read the content of the memory cell an electrometer, which detects the presence or absence of excess electrons at the island N 1 , is used. This electrometer has already been fabricated [START_REF] Stone | Silicon single-electron memory cell[END_REF]. The most difficult hurdle for constructing single-electron memory arrays is the selective read and write operations. The aim of this work is the design and the simulation of the operation of a single-electron random access memory array using the electron trap as the basic memory cell. The basic cells will be organized as a xy matrix and two voltages, V X and V Y , will be used to control the horizontal (x-direction) row cells and the vertical (y-direction) column cells, respectively.

III. THE SINGLE-ELECTRON RANDOM ACCESS MEMORY ARRAY

A. Selective writing

Figure 2(a) shows an electron trap in which the access of V g to the island N 1 is controlled by two voltages, V X and V Y , which are applied to nodes N X and N Y through junctions J X and J Y , respectively. The tunnel junctions J 7 , J 8 , J X , and J Y are identical. The resistance of each one of these junctions is 10 5 Ohm and the capacitance 10 -18 F. V X and V Y can take only two values, 0.0 V and -0.4 V. V g can access the island N 1 to write the logical "0" or "1" only if V X = V Y = 0.0 V. In any other case (i.e. V X =V Y = -0.4V, or V X = -0.4V and V Y = 0.0 V, or V X = 0.0V and V Y = -0.4 V) the memory cell can not be accessed. If any of the two selection voltages is equal to -0.4 V, the circuit free energy increases when electrons tunnel in or out of the island and thus, this tunneling event is suppressed. This is shown in Figure 3 where the simulation results of the memory cell access are presented.

Initially the memory cell stores the logical "0", i.e. no excess electrons are present at island N 1 . As shown in Figure 3, all possible combinations of the V X and V Y values are applied to islands N X and N Y . An attempt to write the logical "1" to the memory cell takes place at each one of the four possible combinations of V X and V Y values, by raising the value of V g to 1.2 V. This value is larger than the 0.6 V of the case of Figure 1, because V g now drives a larger capacitance. The simulation results of Figure 3 show that this attempt is successful only in the case of V X = V Y = 0.0 V.

B. The single-electron memory array

A single-electron memory array in which selective writing and reading is possible can be constructed using as basic cell the cell presented in Figure 4. The selective writing operation has been shown in the last section, whereas, the reading operation is performed by sensing the charge at memory islands using electrometers. Figure 4 shows an (n x m) single-electron memory array in which the symbol of Figure 4(b) has been used. Voltage lines V Xi and V Yj with i = 1, 2, 3, …n and j = 1, 2, 3, …m, control the access to the memory cells. To access the (k, l) cell, all voltages V Xi , and V Yj are set to -0.4 V except V Xk and V Yl which are set to 0.0 V.

IV. THE SINGLE -ELECTRON FREDKIN GATE

A. The single electron circuit

A SEF-gate is a single-electron logic circuit that has the same logic operation as the classical F-gate but is constructed using single-electron circuitry. The SEF-gate presented here, is reversible in the sense that the input data can be deduced if the output data are known, but inputs cannot be physically regenerated by applying outputs as inputs, because of the non-reversibility of the electron tunneling process.

The circuit of the single-electron SEF-gate is shown in Figure 5. The circuit comprises sixteen islands Na, Nb, Nc and N1 through N13. The islands Na, Nb and Nc, are the output islands. Na is the output coming from the controlling input and Nb and Nc are the exchange outputs. Twenty-two junctions J1 through J22 bound the islands. Junctions J4 and J13 are less transparent in order to prevent electron transport from the ground (Vss) to islands Na and N8 and vise versa, while junctions J8 and J19 are less transparent in order to prevent electron transport and charge oscillation to islands Nb and Nc. The voltages Vdd1, Vdd2, Vdd3 and Vdd4 are constant at -0.1V, 0.5V, -0.1V, and -0.11V respectively. The voltage source V1 is the controlling input of the SEF-gate, while the voltage sources V2 and V3 are the target inputs. These input voltages can take only two values 0.0Volt which corresponds to the logic "0", and 0.1Volt which corresponds to the logic "1". The input voltage V1 is applied to islands N2, N4, N5 and N13 through capacitors C2, C3, C4 and C11 respectively. The input voltage V2 is applied to islands N6 and N12 through capacitors C6 and C10 respectively. Finally, the input voltage V3 is applied to islands N3 and N10 through capacitors C5 and C8 respectively. The capacitors C1 through C11 have a capacitance of 10 -18 F. The output signals of the F-gate are taken from islands Na, which corresponds to the controlling input V1, and Nb and Nc, which are the outputs that correspond to the exchanging inputs V2 and V3, respectively. The presence of positive charge at the output islands corresponds to logic "1", whereas no charge corresponds to logic "0". The logic operation of the SEF-gate is shown in Figure 6. Figures 6(a), 6(b) and 6(c) show the time variation of the input voltages V1, V2 and V3, respectively. The inputs are piece-wise constant and apply all possible combinations of logic "0" and "1" to the circuit. Figures 6(d) 6(e) and 6(f) show the time variation of the charge q a , q b and q c at the output islands Na, Nb and Nc respectively. The results from the graphs of Figure 6, compose the truth table of the SEF-gate. The output transition from logic "0" to logic "1" and vice versa does not drive the circuit to instability. The presence of charge on the output islands can be detected and transferred to circuits connected to the SEF-gate output using a sense amplifier

B. Gate operation

VI. CONCLUSIONS

The design of a single-electron random access memory array and a Singleelectron Fredkin gate have been presented. The memory array utilizes a basic singleelectron memory cell that has been recently fabricated. Simulation, using a Monte Carlo simulator, showed that selective writing is possible in this array, whereas, the reading operation can be performed by sensing the charge at memory islands using electrometers. The SEF-gate would be very useful, because digital single-electron circuits that use only one type of gate would be very much easier to fabricate than single-electron circuits that use various types of gates.

It's universal nature gives the SEF gate the ability and the advantage of being used as a fundamental building block for the implementation of a variety of logical circuits, which in combination with the SE RAM can lead to an elementary processor.

Figure 1 .

 1 Figure 1. (a) The circuit of the electron trap, (b) the symbol of this circuit.

Figure 2 .

 2 Figure 2. (a) Electron trap in which the access of V g to the island N 1 is controlled by two voltages, V X and V Y , which are applied to nodes N X and N Y through junctions J X and J Y , respectively, (b) the symbol of this circuit.The most difficult hurdle for constructing single-electron memory arrays is the selective read and write operations. The aim of this work is the design and the simulation

Figure 2 (

 2 Figure2(a) shows an electron trap in which the access of V g to the island N 1 is controlled by two voltages, V X and V Y , which are applied to nodes N X and N Y through junctions J X and J Y , respectively. The tunnel junctions J 7 , J 8 , J X , and J Y are identical. The resistance of each one of these junctions is 10 5 Ohm and the capacitance 10 -18 F. Figure 2(b) shows the symbol of the electron trap, which comprises junctions J 7 and J 8 , and islands N X and N Y .

Figure 3 .

 3 Figure 3. Simulation results for selective writing of logical "1" to the memory cell.

Figure 4 .

 4 Figure 4. An (n x m) single-electron memory array in which the symbol of Figure 2(b) has been used.

Figure 5 .

 5 Figure 5. The circuit of the single-electron F-gate.

Figure 6 .

 6 Figure 6. Operation of the SEF-gate. (a) time variation of controlling input voltage V1, (b) time variation of target input voltage V2, (c) Time variation of target input voltage V3 (d) time variation of the charge at the controlling output island Na, (e)-(f) time variation of the charge at the exchange output islands Nb, Nc respectively.

© TIMA Editions / ENS 2005 ISBN: 2-916187-02-2


Konstantin K Likharev

CMOL: SECOND LIFE FOR SILICON?

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

It is now generally accepted [1] that the current VLSI paradigm (based on a combination of lithographic patterning, CMOS circuits, and Boolean logic) can hardly be extended into a-few-nm region. The most fundamental reason is that at gate length below 10 nm, the sensitivity of parameters (most importantly, the gate voltage threshold) of silicon MOSFETs to inevitable fabrication spreads grows exponentially [START_REF] Frank | Device scaling limits of Si MOSFETs and their application dependencies[END_REF][START_REF] Likharev | Electronics below 10 nm[END_REF]. As a result, the gate length should be controlled with a few-angstrom accuracy, far beyond even the long-term projections of the semiconductor industry [1]. (Similar problems are faced by the lithography-based single-electron devices [START_REF] Likharev | Electronics below 10 nm[END_REF].) Even if such accuracy could be technically implemented using a sophisticated patterning technology, this would send the fabrication facilities costs (growing exponentially even now) skyrocketing, and lead to the end of the Moore's Law some time during the next decade. This is why there is a rapidly growing consensus that the impending crisis of the Moore Law may be deferred only by a radical paradigm shift from the lithography-based fabrication to the "bottom-up" approach based on nanodevices with Nature-fixed size, e.g., specially designed molecules. Since the functionality of such nanodevices is relatively low [START_REF] Likharev | Electronics below 10 nm[END_REF], they almost necessarily should be used just as an add-on to a CMOS subsystem. Such combination allows the functionality, flexibility, and reliability of CMOS circuits (and all the enormous infrastructure created by the electronic industry for their design and fabrication) to be used in full extent.

Several proposals of such hybrid CMOS/nanodevice circuits were put forward recently (for their reviews, see Refs. [START_REF] Stan | Molecular electronics: From devices and interconnect to circuits and architecture[END_REF][START_REF] Das | Introducing Molecular Electronics[END_REF]. Essentially, all of these proposals are based on the use of two-terminal nanodevices with the functionality illustrated by Fig. 1a. At low voltages, such devices may behave as usual diodes, but the application of a higher voltage may switch them between lowresistive (ON) and high-resistive (OFF) states. (This means that the device incorporates single-bit internal memory.) Such "programmable diode" functionality may be achieved in several ways, for example, by switching between two atomic configurations of a molecule -see, e.g., Refs. 6-8. However, nanosecond-scale operation speed requires electron switches, e.g., single-electron latches (Fig. 1b) [START_REF] Fölling | Single-electron latching switches as nanoscale synapses[END_REF] whose low-temperature prototypes have already been demonstrated [START_REF] Ji | Fabrication and characterization of single-electron transistors and traps[END_REF].

(a) Room-temperature versions of such devices may be readily implemented using relatively simple organic molecules [START_REF] Likharev | CrossNets: High-performance neuromorphic architectures for CMOL circuits[END_REF]. (Their main components, molecular single-electron transistors, have already been demonstrated by several research groups [START_REF] Park | Nanomechanical oscillations in a single-C-60 transistor[END_REF][START_REF] Gubin | Molecular clusters as building blocks for nanoelectronics: The first demonstration of a cluster single-electron tunnelling transistor at room temperature[END_REF][START_REF] Zhitenev | Conductance of small molecular junctions[END_REF][START_REF] Park | Coulomb blockade and the Kondo effect in single-atom transistors[END_REF][START_REF] Kubatkin | Singleelectron transistor of a single organic molecule with access to several redox states[END_REF].)

I ON state OFF → ON V - V OFF state V + 0 ON → OFF ON state (b) single-electron trap C s tunnel junction C c V s V d single-electron transistor
The decisive advantage of using two-terminal nanodevices like programmable diodes is that reproducible fabrication of their molecular versions seems rather feasible -see, e.g., the recent experiments [START_REF] Jiang | Structure and bonding issues at the interface between gold and self-assembled conjugated dithiol monolayers[END_REF].

CMOL

Our group is working on a particular type of hybrid CMOS/nanodevice circuits, dubbed CMOL, which look more plausible for implementation [START_REF] Likharev | Electronics below 10 nm[END_REF][START_REF] Likharev | CMOL: Devices, circuits, and architectures[END_REF]. As in several earlier proposals [START_REF] Stan | Molecular electronics: From devices and interconnect to circuits and architecture[END_REF][START_REF] Das | Introducing Molecular Electronics[END_REF][START_REF] Heath | Molecular electronics[END_REF][7][START_REF] Kuekes | The crossbar latch: Logic value storage, restoration, and inversion in crossbar circuits[END_REF], nanodevices in CMOL circuits are formed (e.g., self-assembled) at each crosspoint of a "crossbar" array, consisting of two levels of nanowires (Fig. 2). However, in order to overcome the CMOS/nanodevice interface problems pertinent to earlier proposals, in CMOL circuits the interface is provided by pins that are distributed all over the circuit area, on the top of the CMOS stack. (Silicon-based technology necessary for fabrication of pins with nanometer-scale tips has been already developed in the context of fieldemission arrays [START_REF] Jensen | Field emitter arrays for plasma and microwave source applications[END_REF].) As Fig. 2c shows, pins of each type (reaching to either the lower or the upper nanowire level) are arranged into a square array with side 2βF CMOS , where F CMOS is the half-pitch of the CMOS subsystem, and β is a dimensionless factor larger than 1 that depends on the CMOS cell complexity. The nanowire crossbar is turned by angle α = arcsin(F nano /βF CMOS ) relative to the CMOS pin array, where F nano is the nanowiring half-pitch. By activating two pairs of perpendicular CMOS lines, two pins (and two nanowires they contact) may be connected to CMOS data lines (Fig. 2b). As Fig. 2c illustrates, this approach allows a unique access to any nanodevice, even if F nano « F CMOS -see Ref. [START_REF] Likharev | CMOL: Devices, circuits, and architectures[END_REF] for a detailed discussion of this point. If the nanodevices have a sharp current threshold, like the usual diodes, such access allow to test each of them. Moreover, if the nanodevice may be switched between two internal states (e.g. is a programmable diode), switching between the states may be achieved by applying voltages ±V W to the selected nanowires, so that voltage V = ±2V W applied to the selected nanodevice exceeds the corresponding switching threshold, while half-selected devices (with V = ± V W ) are not disturbed.

Two advantages of CMOL circuits over other crossbar-type hybrids look most important. First, due to the uniformity of the nanowiring/nanodevice levels of CMOL, they do not need to be precisely aligned with each other and the underlying CMOS stack, thus allowing the use for nanowire formation of advanced patterning techniques [START_REF] Zankovych | Nanoimprint lithography: Challenges and prospects[END_REF], [START_REF] Brueck | There are no fundamental limits to optical lithography[END_REF] which lack precise layer alignment. Second, CMOL circuits may work with two-terminal nanodevices (e.g., single-electron latching switches). Still, CMOL, similarly to all other nanodevice-based technologies, requires defect-tolerant circuit architectures, since the fabrication yield of such devices will hardly ever approach 100% as closely as that achieved for the semiconductor transistors.

MEMORIES

The most straightforward potential application of CMOL circuits are embedded memories and stand-alone memory chips, with their simple matrix structure. In such memories, each nanodevice would play the role of a single-bit memory cell, while the CMOS subsystem may be used for coding, decoding, line driving, sensing, and input/output functions.

We have carried out [START_REF] Strukov | Prospects for terabitscale nanoelectronic memories[END_REF][START_REF] Strukov | Architectures for defect-tolerant CMOL memories[END_REF] a detailed analysis of CMOL memories with global and quasi-local ("dash") structure of matrix blocks, including the application of two major techniques for increasing their defect tolerance: the memory matrix reconfiguration (the replacement of several rows and columns, with the largest number of bad memory cells, for spare lines), and advanced error correction codes. Figure 3 shows the final result of that analysis: the optimized total chip area per useful bit, as a function of the nanodevice yield. The results show that the memories may be rather defect-tolerant: a 90% yield may be achieved with 8 to 10% of bad nanodevices (depending on the required access time). When such yield has been achieved, the performance of CMOL memories will be extremely impressive. For example, the normalized cell area a ≡ A/N(F CMOS ) 2 = 0.4 (red dashed line on Fig. 3) at F CMOS = 32 nm means that a memory chip of a reasonable size (2 × 2 cm 2 ) can store about 1 terabit of data -crudely, one hundred Encyclopedia Britannica's.

DIGITAL LOGIC CIRCUITS

The reconfiguration is the most efficient technique for coping with defective nanodevices in hybrid circuits. This is why the most significant published proposals for the implementation of logic circuits using CMOL-like hybrid structures had been based on reconfigurable regular structures like the field-programmable gate arrays (FPGA). Before our recent work, two FPGA varieties had been analyzed, one based on look-up tables (LUT) and another one using programmable-logic arrays (PLA). Unfortunately, all these approaches run into substantial problems -see Ref. [START_REF] Likharev | CMOL: Devices, circuits, and architectures[END_REF] for discussion.

Recently, D. Strukov suggested [START_REF] Strukov | CMOL FPGA: A cellbased, reconfigurable architecture for hybrid digital circuits using two-terminal nanodevices[END_REF] an alternative approach to Boolean logic circuits based on CMOL concept, that is close to the so-called cell-based FPGA [START_REF] Rabaey | Digital Integrated Circuits: A Design Perspective[END_REF]. In this approach an elementary CMOS cell includes two pass transistors and an inverter, and is connected to the nanowire/nanodevice subsystem via two pins (Fig. 4a). Disabling the CMOS inverters allows to carry out the circuit reconfiguration via cell's pass transistors. On the other hand, enabling the inverter turns the cell into a NOR gate (Fig. 4b), generally with an almost arbitrary fan-in. Fraction q of bad bits

Ideal CMOL 1 mm 2 8 Gb
First results for CMOL FPGA have been obtained using a simple, two-step approach to reconfiguration, in which the desired circuit is first mapped on the apparently perfect (defect-free) CMOL fabric, and then is reconfigured around defective components using a simple algorithm [START_REF] Strukov | CMOL FPGA: A cellbased, reconfigurable architecture for hybrid digital circuits using two-terminal nanodevices[END_REF]. The Monte Carlo simulation (so far only for the "stack-on-open"-type defects which are expected to dominate in CMOL circuits) has shown that even this simple configuration procedure may ensure very high defect tolerance. For example, the reconfiguration of a 32-bit Kogge-Stone adder, mapped on the CMOL fabric with realistic values of parameters, may allow to achieve the 99% circuit yield (sufficient for a ~90% yield of properly organized VLSI chips), with as may as 22% of defective devices, while the defect tolerance of another key circuit, a fully-connected 64-bit crossbar switch, is about 25%.

Our most striking result was that such high defect tolerance may coexist with high density and performance, at acceptable power consumption. For example, our estimates have shown [START_REF] Strukov | CMOL FPGA: A cellbased, reconfigurable architecture for hybrid digital circuits using two-terminal nanodevices[END_REF] that for the total power of 200 W/cm 2 (planned by the ITRS for the long-term CMOS technology nodes), an optimization may bring the logic delay of the 32-bit Kogge-Stone adder down to just 1.9 ns, at the total area of 110 µm 2 , i.e. provide an area-delay product of 150 ns-µm 2 , for realistic values F CMOS = 32 nm and F nano = 8 nm (Fig. 5). A minor error in that work (which was found later [START_REF] Strukov | A reconfigurable architecture for hybrid CMOS / nanodevice circuits[END_REF]) has led to underestimation of the actual delay by a factor close to 2.5. Still, the areadelay product compares very favorably with the estimated 70,000 ns-µm 2 (with 1.7 ns delay and 39,000 µm 2 area) for a fully CMOS FPGA implementation of the same circuit (with the same F CMOS ). Fig. 5. CMOL FPGA performance as a function of nanowire half-pitch F nano (at the total power fixed at 200 W/cm 2 ) for two digital circuits, each for three long-term CMOS technology nodes [START_REF] Strukov | CMOL FPGA: A cellbased, reconfigurable architecture for hybrid digital circuits using two-terminal nanodevices[END_REF].

Very recently, these calculations were extended [START_REF] Strukov | A reconfigurable architecture for hybrid CMOS / nanodevice circuits[END_REF] to all 20 circuits of the so-called Toronto benchmark set [START_REF] Betz | FPGA place-and-route challenge[END_REF]. In order to accomplish this task, the first CMOL CAD tool has been developed using a combination of known CMOS FPGA design algorithms such as SIS, T-VPack and VPR [START_REF] Sentovich | SIS: A system for sequential circuit synthesis[END_REF][START_REF] Betz | VPR: A new packing, placement and routing tool for FPGA research[END_REF] with a CMOL-specific, custom routing program. There is still a lot of place for improvement in this tool, so that the preliminary results for area and (especially) logic delay for CMOL FPGA, presented in the right part of Table 1, should be considered just as upper bounds. Still, even these preliminary results show almost a spectacular density advantage (on the average, about two orders of magnitude) over the purely CMOS circuits and a considerable leading edge over a competing (and less feasible) hybrid circuit concept, so-called nanoPLA [START_REF] Dehon | Design of programmable interconnect for sublithographic programmable logic arrays[END_REF].

MIXED-SIGNAL NEUROMORPHIC CIRCUITS

One more possible application of CMOL circuits is neuromorphic networks (see, e.g., Ref. 32). We have explored a specific architecture of such networks, called Distributed Crossbar Networks ("CrossNets") [START_REF] Likharev | CrossNets: High-performance neuromorphic architectures for CMOL circuits[END_REF][START_REF] Türel | Neuromorphic architectures for nanoelectronic circuits[END_REF], which are uniquely suitable for CMOL implementation.

In each CrossNet (Fig. 6), relatively sparse neural cell bodies ("somas") are implemented in the CMOS subsystem, while the much denser latching switches are used as elementary synapses. The mutually perpendicular nanowires of the CMOL crossbar naturally implement the axons and dendrites which carry signals between the cells, allowing one cell to be directly connected to a virtually unlimited number M of other cells. Due to this parallelism, CrossNets can be spectacularly resilient, with visible performance degradation at as many as ~50% of bad nanodevices [START_REF] Türel | Neuromorphic architectures for nanoelectronic circuits[END_REF].

Fig. 6. The simplest feedforward CrossNet.

CrossNet functionality is strongly dependent on the distribution of the somas over the axon/dendrite/synapse field (Fig. 7). For example, "FlossBars" (Fig. 7a) have a layered topology and hence can be used to implement multi-layer perceptron (MLPs), while "InBars" (Fig. 7b) have an "interleaved" structure which is natural for the implementation of recurrent networks -see, e.g., Ref. [START_REF] Hertz | Introduction to the Theory of Neural Computation[END_REF].

CrossNet training faced several challenges including the binary character of the elementary synapse (latching switch) and a certain statistical uncertainty of its switching. In our recent work [START_REF] Türel | Neuromorphic architectures for nanoelectronic circuits[END_REF] we have proved that, despite these limitations, CrossNets can be taught, by at least two different methods, to perform virtually all the major functions demonstrated earlier with usual neural networks, including the corrupted pattern restoration in the recurrent quasi-Hopfield mode and pattern classification in the feedforward MLP mode. The importance of this result is in the CrossNet's potential unparalleled density and speed [START_REF] Likharev | CrossNets: High-performance neuromorphic architectures for CMOL circuits[END_REF][START_REF] Türel | Neuromorphic architectures for nanoelectronic circuits[END_REF]: for realistic parameters, the cell density may exceed that of cerebral cortex (above 10 7 cells per cm 2 ), while the average cell-to-cell communication delay may be as low as ~10 ns (i.e., about six orders of magnitude lower than that in the brain), at acceptable power. Even putting aside the exciting long-term prospects of creating high-speed artificial brain-like systems [START_REF] Türel | Neuromorphic architectures for nanoelectronic circuits[END_REF], CMOL CrossNet chips of modest size might be used for important present-day problems, e.g., online recognition of a person in a large crowd [START_REF] Lee | CMOL CrossNets as pattern classifiers[END_REF].

CONCLUSIONS

I believe there is a chance for the development, within perhaps the next 10 to 15 years and maybe substantially earlier, of hybrid "CMOL" integrated circuits that will allow to extend Moore's Law to the few-nm range. Estimates show that such circuits could be used for several important applications, notably including terabitscale memories, reconfigurable digital circuits with multi-teraflops-scale performance, and mixed-signal neuromorphic networks that may, for the first time, compete with biological neural systems in areal density, far exceeding them in speed, at acceptable power dissipation.

The major challenges on the way toward practical CMOL circuits include the development of high-yield techniques for formation (e.g., chemically-directed molecular self-assembly) of single-electron latching switches, even better architectures for digital CMOL

Fig. 1 .

 1 Fig. 1. Programmable diode ("latching switch"): (a) I-V curve (schematically) and (b) possible implementation using single-electron devices.

Fig. 2 .

 2 Fig.2. Low-level structure of the generic CMOL circuit: (a) schematic side view; (b) the idea of addressing a particular nanodevice, and (c) zoom-in on several adjacent interface pins to show that any nanodevice may be addressed via the appropriate pin pair (e.g, pins 1 and 2 for the leftmost of the two shown devices, and pins 1 and 2' for the rightmost device). On panel (b), only the activated CMOS lines and nanowires are shown, while panel (c) shows only two devices. (In reality, similar nanodevices are formed at all nanowire crosspoints.)

Fig. 3 .

 3 Fig.3. The optimized area per useful bit as a function of single nanodevice yield, for two different CMOL memories (with "dash" and global block structure) and for several values of access time.

Fig. 4 .

 4 Fig. 4. CMOL FPGA: (a) logic cell schematics, and (b) implementation of a fan-in-two NOR gate.

Fig. 7 .

 7 Fig. 7. Two particular CrossNet species: (a) FlossBar (shown for the connectivity parameter M equal to 10) and (b) InBar (for M = 9). For clarity, the figures show only the axons, dendrites, and synapses providing connections between one soma (indicated by the dashed red circle) and its direct signal recipients (inside the dashed blue lines), for the simplest (non-Hebbian feedforward) networks.

  training mixed-signal neuromorphic networks to perform more advanced, eventually intellectual information processing tasks.

TABLE 1 .

 1 Results of performance calculations for circuits of the Toronto 20 benchmark set implemented using CMOS and CMOL FPGAs, for F CMOS = 45 nm and F nano = 4.5 nm. Delay results for CMOL are just an upper bound rather than the critical path latency.

		CMOS FPGA		CMOL FPGA
	Circui
	t	Area	Delay	CMOS	Nano-	Area	Delay
		(µm 2 )	(ns)	cells	devices	(µm 2 )	(ns)
	Alu4 137700 5.1	1854	9788 1004 18.4
	apex2 166050 6.0	1928 11365 914 20.7
	apex4 414619 5.5	1176	7781	672 15.2
	bigkey 193388 3.1	2065 10207 829 16.0
	clma 623194 13.1 7585 48746 9308 59.9
	des 148331 4.2	2321 12610 1097 22.3
	diffeq 100238 6.0	2004 10799 1194 58.3
	dsip 148331 3.2	1615	9905	829 20.7
	elliptic 213638 8.6	4799 25415 4581 64.6
	ex1010 391331 9.0	2986 28746 3486 34.3
	ex5p 100238 5.1	902	6875	829 21.5
	frisc 230850 11.3 4715 25869 4199 91.0
	misex3 124538 5.3	1397	9211 1004 19.2
	pdc 369056 9.6	4752 14841 4979 43.1
	s298 166050 10.7 1030 10161 829 35.9
	s38417 462713 7.3	8289 53156 9308 41.5
	s38584 438413 4.8	6502 50275 9872 51.1
	seq 151369 5.4	1832 11027 1296 18.4

ACKNOWLEDGMENTS

Useful discussions of the issues considered in this paper with P. Adams, J. Barhen, W. Chen, S. Das, J. Ellenbogen, D. Hammerstrom, R. Karri. P. Kuekes, J. H. Lee, X. Liu, J. Lukens, X. Ma, A. Mayr, V. Protopopescu, M. Reed, M. Stan, D. Strukov, Ö. Türel, and S. R. Williams are gratefully acknowledged. The work has been supported in part by AFOSR, ARDA, NSF, and MARCO via FENA Center. This paper is an updated and extended version of the invited talk [36].


J A Dumas

De Guise

J Clément

T Cresson

N Hagemeister

R Dumas

J A De Guise

Estimating joint space of the knee during weight-bearing squatting activity using motion capture -Preliminary results of a new method

Keywords: joint space, articular contact kinematics, motion capture, optimization, squatting activity

come

Estimating joint space of the knee during weight-bearing squatting activity using motion capture -preliminary results of a new method

Introduction

Over the last 10 years, several studies analyzed 3D joint space of the knee and deduced articular contact kinematics during various weight-bearing activities [START_REF] Li | Articular contact kinematics of the knee before and after a cruciate retaining total knee arthroplasty[END_REF]. Joint space and articular contact kinematics provide relevant data on how the intrinsic biomechanics of the knee is altered after a disease or a surgical procedure, such as knee osteoarthritis (OA) or total knee arthroplasty [START_REF] Li | Articular contact kinematics of the knee before and after a cruciate retaining total knee arthroplasty[END_REF].

Although very accurate, the methods employed by these studies require complex acquisition protocols, implying 3D/2D registration techniques or MRI-based methods, which turn out unusable in clinical routine. The goal of the present study is to assess the feasibility of a new method to estimate 3D joint space of the knee during dynamic weight-bearing squats, which are the 2 nd most studied activity in orthopedics [START_REF] Fukagawa | Age-related changes in kinematics of the knee joint during deep squat[END_REF], performed by healthy and OA subjects. To our knowledge, this method based on motion capture has never been proposed before.

Methods

Subjects

This preliminary study involves two subjects: one healthy woman (60 years, 58.3 kg, 156 cm) and one OA woman (64 years, 77.8 kg, 150 cm).

Kinematic acquisition and optimization

3D knee kinematics was recorded on the two subjects while performing dynamic weight-bearing squats (0°-60°-0° of knee flexion-extension during 10 seconds) with the KneeKG™ (Emovi Inc., Laval, QC, Canada) and Polaris Spectra ® camera (60-Hz, NDI, Waterloo, ON, Canada). The dynamic squats were standardized with a proprioceptive jig [START_REF] Clément | Comparison of quasi-static and dynamic squats: A three-dimensional kinematic, kinetic and electromyographic study of the lower limbs[END_REF]. The KneeKG™ provides repeatable and reliable measurements [START_REF] Lustig | The KneeKG system: a review of the literature[END_REF]), but they are still influenced by soft tissue artefacts (STA) with kinematics errors up to 7° and 11 mm [START_REF] Südhoff | Comparing three attachment systems used to determine knee kinematics during gait[END_REF]). These STA errors were therefore corrected with a multi-body optimization method (MBO) [START_REF] Gasparutto | Validation of a multi-body optimization with knee kinematic models including ligament constraints[END_REF] The lower limb model used in MBO consisted of 4 segments (pelvis, femur, tibia, foot) imposing spherical joint constraints at the hip and ankle, and parallel mechanism constraints at the knee. The parallel mechanism was composed of 2 sphere-onplane contacts and 4 deformable ligaments and was personalized from the subject-specific 3D knee bone models. These 3D models were obtained from the upright low-dose biplane radiographic imaging EOS ® system (EOS Imaging Inc., Paris, France) with an accuracy around 2 mm [START_REF] Cresson | 3D shape reconstruction of bone from two x-ray images using 2D/3D non-rigid registration based on moving least-squares deformation[END_REF].

Knee joint space calculation

Fusion of the 3D bones models of subjects with the optimized knee kinematics allowed computing the 3D joint space during a descent phase of the recorded squats. We established a distance map between the two meshes of the femur and tibia at each instant of the movement, according to the symmetric Hausdorff distance definition [START_REF] Aspert | MESH: measuring errors between surfaces using the Hausdorff distance[END_REF].

Validation

The validation process was done by comparing the distance maps obtained using our method with those obtained during a quasi-static squat recorded with the EOS ® system. The quasi-static squat consisted of 4 positions of knee flexion and was standardized with the same proprioceptive jig used during the dynamic squat. The method used to obtain the 3D knee models for these 4 positions is detailed in [START_REF] Kanhonou | A method to study 3D knee pseudo-kinematics using low-dose stereoradiography during static squat[END_REF] and presents an accuracy and a precision about 0.3° and 0.3 mm in terms of positions and orientations of knee bones. Calculation of distance maps was the same as detailed above.

Results and discussion

The two figures below show the distance maps of the healthy and OA subjects obtained from the recordings of the KneeKG™ and EOS ® systems. *Corresponding author. Email: clement.julien.24@gmail.com On average, measurements based on the KneeKG™ recordings overestimates of about 2 mm the distance between the articular surfaces of the femur and tibia compared to the measurements based on the EOS ® system. However, the patterns of the distance maps and the positions of the points of closest contact remain qualitatively similar between the two methods. This could be very interesting to assess the joint reaction forces and torques in the context of a musculoskeletal modeling of the lower limb for example.

Conclusions

The estimation of joint space using a new method based on motion capture showed very encouraging results. These results nevertheless remain very preliminary. Further investigations on a greater number of subjects, i.e. 10 healthy and 10 OA, and exhibiting more quantitative data, i.e. the area covered by the distance maps on the bones and the position of the points of closest contact between the bones, are ongoing.

Figure 1

 1 Figure 1 Knee distance maps of a healthy subject

Acknowledgements

This work was funded in part by the FRQNT, FRQ-S, NSERC, the CIHR MENTOR program, and grant from Region Rhone-Alpes.


Thomas Hielscher

ULTRASONIC PRODUCTION OF NANO-SIZE DISPERSIONS AND EMULSIONS

Ultrasound is a well-established method for particle size reduction in dispersions and emulsions. Ultrasonic processors are used in the generation of nano-size material slurries, dispersions and emulsions because of the potential in the deagglomeration and the reduction of primaries. These are the mechanical effects of ultrasonic cavitation. Ultrasound can also be used to influence chemical reactions by the cavitation energy. This is sonochemistry. As the market for nano-size materials grows, the demand for ultrasonic processes at production level increases. At this stage, energy efficiency becomes important. Since the energy required per weight or volume of processed material links directly to the equipment size required, optimization of the process efficiency is essential to reduce investment and operational costs. Furthermore it is required to scale the lab and bench top configurations to this final level without any variations in the process achievements. Scale up by power alone will not do this.

INTRODUCTION

Ultrasound is a very effective processing method in the generation and application of nano-size materials. In general, ultrasonic cavitation in liquids may cause fast and complete degassing: initiate various chemical reactions by generating free chemical ions (radicals); accelerate chemical reactions by facilitating the mixing of reactants; enhance polymerization and depolymerization reactions by temporarily dispersing aggregates or by permanently breaking chemical bonds in polymeric chains; increase emulsification rates; improve diffusion rates; produce highly concentrated emulsions or uniform dispersions of micron-size or nano-size materials; assist the extraction of substances such as enzymes from animal, plant, yeast, or bacterial cells; remove viruses from infected tissue; and finally, erode and break down susceptible particles, including micro-organisms [START_REF] Kuldiloke | Effect of Ultrasound, Temperature and Pressure Treatments on Enzyme Activity an Quality Indicators of Fruit and Vegetable Juices[END_REF]. Ultrasound can be tested in lab and bench-top scale before the results are scaled up to the commercial level.

ULTRASONIC CAVITATION

Low-intensity or high-frequency ultrasound is mainly used for analysis, non-destructive testing and imaging.

High-intensity ultrasound is used for the processing of liquids such as mixing, emulsifying, dispersing and deagglomeration, or milling. When sonicating liquids at high intensities, the sound waves that propagate into the liquid media result in alternating high-pressure (compression) and low-pressure (rarefaction) cycles, with rates depending on the frequency. During the lowpressure cycle, high-intensity ultrasonic waves create small vacuum bubbles or voids in the liquid. When the bubbles attain a volume at which they can no longer absorb energy, they collapse violently during a highpressure cycle. This phenomenon is termed cavitation. Cavitation, that is "the formation, growth, and implosive collapse of bubbles in a liquid. Cavitational collapse produces intense local heating (~5000 K), high pressures (~1000 atm), and enormous heating and cooling rates (>10 9 K/sec)" and liquid jet streams (~400 km/h)" [START_REF] Suslick | Kirk-Othmer Encyclopedia of Chemical Technology[END_REF]. Cavitation can be produced in different ways: e.g. by high-pressure nozzles, rotor-stator mixers, or ultrasonic processors. In all those systems the input energy is transformed into friction, turbulences, waves and cavitation. The fraction of the input energy that is transformed into cavitation depends on several factors describing the movement of the cavitation generating equipment in the liquid. The intensity of acceleration is one of the most important factors influencing the efficient transformation of energy into cavitation. Higher acceleration creates higher-pressure differences. This in turn increases the probability of the creation of vacuum bubbles instead of the creation of waves propagating through the liquid. Thus, the higher the acceleration the higher is the fraction of the energy that is transformed into cavitation. In case of an ultrasonic transducer, the amplitude of oscillation describes the intensity of acceleration. Higher amplitudes result in a more effective creation of cavitation. In addition to the intensity, the liquid should be accelerated in a way to create minimal losses in terms of turbulences, friction and wave generation. For this, the optimal way is a unilateral direction of movement.

MATERIALS

Nanomaterials have become integral components of products as diverse as sunscreens, electrically conductive coatings, and strong, lightweight plastic composites or paints and inks. The production and processing of these materials is a quickly progressing technology. Indeed, it is advancing rapidly and creating new possibilities for many Thomas Hielscher Ultrasonic Production of Nano-Size Dispersions and Emulsions industrial processes. Solid nanomaterials fall into three broad categories: metal oxides, nanoclays, and carbon nanotubes. Metal-oxide nanoparticles include nanoscale zinc oxide, titanium oxide, iron oxide, cerium oxide and zirconium oxide, as well as mixed-metal compounds such as indium-tin. In general, nanomaterials are defined as materials of less than 100nm in size. When matter is reduced in size it changes its characteristics, such as color and interaction with other matter such as chemical reactivity. The change in the characteristics is caused by the change of the electronic properties. By the particle size reduction, the surface area of the material is increased. Due to this, a higher percentage of the atoms can interact with other matter, e.g. with the matrix of resins. Surface activity is a key aspect of nanomaterials. Agglomeration and aggregation blocks surface area from contact with other matter. Only well-dispersed or singledispersed particles allow to utilize the full beneficial potential of the matter. In result good dispersing reduces the quantity of nanomaterials needed to achieve the same effects. As most nanomaterials are still fairly expensive, this aspect is of high importance for the commercialization of product formulations containing nanomaterials. Today, many nanomaterials are produced in a dry process. As a result, the particles need to be mixed into liquid formulations. This is where most nanoparticles form agglomerates during the wetting. Especially carbon nanotubes are very cohesive making it difficult to disperse them into liquids, such as water, ethanol, oil, polymer or epoxy resin. Therefore effective means of deagglomerating and dispersing are required to overcome the bonding forces after wettening the micronpowder or nano-powder.

EFFECTS

Ultrasound is used in a wide range of physical, chemical and biological processes. Homogenizing, emulsifying, and dispersing are examples for physical processes. Most of the applications of high-intensity ultrasound are based on cavitational effects. The physical effects of cavitation are being used in a top-down generation of nano-particles. Here, particles are reduced in size by the forces of cavitation. This includes the breaking of agglomerates and aggregates. The physical effects are used in combination with the chemical effects (sonochemistry) in the bottom-up production of nano-particles and crystals, i.e. during precipitation or crystallization. Here, ultrasound serves a number of roles in the initiation of seeding and subsequent crystal formation and growth.

Dispersing and Deagglomeration

Dispersion by ultrasound is a consequence of microturbulences caused by fluctuation of pressure and cavitation. Investigations at different materials, such as aqueous solutions of nanoparticulate siliciumdioxid powder and spray frozen agglomerates with a variable solid content have demonstrated the considerable advantage of ultrasound when compared with other technologies, such as rotor-stator mixers (e.g. turrax) or colloid mills. In particular for small matter from several nanometers to couple of microns, ultrasonic cavitation is very effective in breaking agglomerates, aggregates and even primaries. When ultrasound is being used for the milling of high concentration batches, the liquid jets streams resulting from ultrasonic cavitation, make the particles collide with each other at velocities of up to 1000km/h. This breaks van der Waals forces in agglomerates and even primary particles. Large particles are subject to surface erosion (via cavitation collapse in the surrounding liquid) or particle size reduction (due to fission through interparticle collision or the collapse of cavitation bubbles formed on the surface).

Emulsifying

If a cavitation bubble implodes near the phase boundary of two immiscible liquids the resultant shock wave can provide a very efficient mixing. Stable emulsions produced by sonication can be used in the textile, cosmetic, pharmaceutical, food, and petrochemical industry. Ultrasonically generated emulsions are often more stable and require less, if any surfactant than those produced conventionally. Since ultrasound is fully controllable and adaptable by the choice of amplitude, pressure and temperature, sonication is an effective instrument to obtain emulsions with smaller droplet sizes within a narrow size distribution.

Sonochemistry

Recent studies on the effect of sonication on suspended powders have shown that the particles can be forced into violent collision that, in the case of metals, fusion can occur. In some cases the colliding particles undergo chemical reactions. Thus, when copper and sulphur are sonicated together in hexane for 1 hour, 65% Cu 2 S is generated [START_REF] Goh | Investigations of the effect of ultrasound on some metal and nonmetal systems[END_REF]. The chemical effects include the formation of OH -and H + species and hydrogen peroxide. The ultrasonic effects to chemical reactions -sonochemistryinclude hydrolysis, oxidation, and depolymerization processes, too.

PROCESSING

Ultrasound describes a wide range technology. From cleaning tanks to sensors, from submersible transducers to nebulizers, from dental tools to baby images. These applications differ from each other because the Thomas Hielscher Ultrasonic Production of Nano-Size Dispersions and Emulsions parameters of the ultrasound differ a lot. For each specific application and process there is a specific optimal parameter configuration. For new processes and formulations this configuration has to be identified. In the following the parameters important to the liquid processing are examined.

Parameters

Ultrasonic liquid processing is described by a number of parameters. Most important are amplitude, pressure, temperature, viscosity, and concentration. The process result, such as particle size, for a given parameter configuration is a function of the energy per processed volume. The function changes with alterations in individual parameters. Furthermore, the actual power output per surface area of the sonotrode of an ultrasonic unit depends on the parameters.

Surface intensity

The power output per surface area of the sonotrode is the surface intensity (I). The surface intensity depends on the amplitude (A), pressure (p), the reactor volume (V R ), the temperature (T), viscosity (η) and others. ) ,... , , ,

[ ] [ ] [ ] [ ²] / [ . + - - + + °= cP C ml bar m mm W T V p A f I R η µ , ( ] [ ]
The impact of the generated cavitation depends on the surface intensity. So does the process result. The total power output of an ultrasonic unit is the product of surface intensity (I) and surface area (S):

²] [ ²] / [ ] [ * mm mm W W S I P L =

Amplitude

The amplitude of oscillation describes the way the sonotrode surface travels in a given time (e.g. 1/20,000s at 20kHz). The larger the amplitude, the higher is the rate at which the pressure lowers and increases at each stroke. In addition to that, the volume displacement of each stroke increases resulting in a larger cavitation volume (bubble size and/or number). When applied to dispersions, higher amplitudes show a higher destructiveness to solid particles. Table 1 shows general values for some ultrasonic processes.

5.1.3

Pressure Elevated pressure allows cavitation at temperatures close to or above the boiling point. It also increases the intensity of the implosion, which is related to the difference between the static pressure and the vapor pressure inside the bubble [START_REF] Vercet | Inactivation of heat-resistant pectinmethylesterase from orange by manothermosonication[END_REF]. Since the ultrasonic power and intensity changes quickly with changes in pressure, a constant-pressure pump is preferable. When supplying liquid to a flow-cell the pump should be capable of handling the specific liquid flow at suitable pressures. Diaphragm or membrane pumps; flexible-tube, hose or squeeze pumps; peristaltic pumps; or piston or plunger pump will create alternating pressure fluctuations. Centrifugal pumps, gear pumps, spiral pumps, and progressive cavity pumps that supply the liquid to be sonicated at a continuously stable pressure are preferred.

5.1.4

Power and Intensity vs. Energy Surface intensity and total power do only describe the intensity of processing. The sonicated sample volume and the time of exposure at a certain intensity have to be considered to describe a sonication process in order to make it scalable and reproducible. For a given parameter configuration the process result, e.g. particle size or chemical conversion, will depend on the energy per volume (E/V).

) / ( Result V E f =
Where the energy (E) is the product of the power output (P) and the time of exposure (t).

] [ ] [ ] [ * s W Ws t P E =
Changes in the parameter configuration will change the result function. This in turn will vary the amount of energy (E) required for a given sample value (V) to obtain a specific result value. For this reason it is not enough to deploy a certain power of ultrasound to a process to get a result. A more sophisticated approach is required to identify the power required and the parameter configuration at which the power should be put into the process material.

THREE STEP APPROACH

By dividing the process development into three stages, the costs and risks in the development can be reduced. Therefore the three steps are introduced: Feasibility Study, Optimization and Scale-Up. Because the sample volume is kept low, the development process can be speeded up while generating reproducible and scalable results based on the energy/volume.

Thomas Hielscher Ultrasonic Production of Nano-Size Dispersions and Emulsions

Feasibility Study

The objective of the feasibility study is the identification of the potential for a specific product formulation or process. Unless there is data available from similar formulations and processes, this incorporates a trial-anderror testing of various parameter configurations. When starting with a random parameter configuration, the likelihood to be at the optimal configuration is fairly low. Also the energy per volume required to obtain a result at this random or the optimal configuration is still to be discovered. Based on the premises that the process result for a given parameter configuration is a function of the energy per processed volume, if any result can be obtained, it will be better for a higher energy input. For this reason, the first tests should use a very high energy per volume level at high intensity levels. In this stage, trials using small sample volumes are preferred to shorten the time using a given equipment size. Lower volumes reduce the sample amount, too. This can be important for more precious materials. Most important is the even distribution of the sonication energy in the sample. Because the sonic intensity decreases with distance from the emitting surface, short distances are preferred. When a small liquid volume is exposed, the distance from the sonotrode can be kept short. Table 2 shows typical energy/volume levels for sonication processes when being optimized. Since the first trials are not at optimum configuration, sonication at 10 to 50 times the typical value will show if there is any effect to the sonicated material or not. As energy is transmitted into the liquid, the liquid will heat up. In order to allow for the input of high energy/volume levels cooling is required. Picture 1 shows a simple setup for the conduct of the feasibility trials. A cold water (or other coolant) bath is used to dissipate the heat generated in the small sample tube (green).

Picture 1 -100W Setup for Feasibility Testing

By recording the actual power input, via a PC-interface or powermeter, the time of sonication and the actual amplitude and temperature in combination with the results of each trial a bottom line for the energy/volume can be established. If by chance an optimal configuration has been chosen, this configuration performance could be verified during optimization and could be scaled up to commercial level. During feasibility study, the limits of sonication, e.g. temperature, amplitude or energy/volume for specific formulations should be examined, too. As ultrasound could generate negative effects to chemicals or particles, the critical levels for each parameter need to be examined in order to limit the following optimization to the parameter range where the negative effects are not observed.

For the feasibility study small lab or bench-top units are recommended to limit the expenses for equipment and samples in such trials. Generally 100 to 1,000 Watts units serve the purposes of the feasibility study very well.

Optimization

The results achieved during the feasibility study may show high energy/volume levels which when scaled up may be not cost effective. As these trials were to show the general impact of ultrasound only, optimization is needed to evaluate the cost effectiveness of ultrasound for the process. To identify the most energy and cost efficient way to deploy the ultrasonic energy in order to obtain the Thomas Hielscher Ultrasonic Production of Nano-Size Dispersions and Emulsions potential benefits observed in the feasibility study, a systematic optimization is required. In this step, the correlations between the relevant parameters -in particular amplitude, pressure and liquid formulationare investigated. The objective is to find the optimal parameter configuration in which a minimum of energy is utilized to obtain the required benefit. This is the most efficient configuration. As this energy per volume value is the basis for the scale up to commercial level, efficiency gains from thorough optimization result in savings in operation and maintenance cost. In this stage, systematic batch samples may help to identify the most important parameters for the specific process. Batch trials may also serve as indicators for the correlation between the specific parameter and the process result. Picture 2 shows the results of trials using different intensity and mass concentration values. The results indicate, that a higher Aerosil concentration improves the process efficiency.

Picture 2 -Influence of solid concentration on dispersion (UIP1000) [3]

In a batch testing as shown in Picture 1 the pressure is limited to ambient pressure. For this reason, flow cells are required to determine the correlation between pressure and process efficiency.

Picture 3 -Flow Chart for Optimization Setup

For the optimization, versatile ultrasonic units of 500 to 2,000 watts are suitable for this stage. In particular, if there is no information available on the sonication of similar materials the systems should be capable of covering a wide range for each parameter. For nanomaterials amplitudes from 10 to 100 micron should be tested. For most applications pressures from ambient to 2 or 3 bars above ambient are sufficient. For strong materials higher pressures of up to 50 bars above ambient may show better performance. Again, the actual power input, the time of sonication, and the amplitude, pressure and temperature used are recorded together with the results of each trial. In the end the trial using the lowest energy per volume achieving a satisfying result will be chosen for scale up. It may be recommended to validate this particular trial by a second run at the same configuration.

Scale Up

Ultrasound is reproducible. When ultrasound is applied to an identical liquid formulation at an identical processing parameter configuration, the same energy per volume is required to obtain an identical result independent of the scale of processing. This allows for a linear scale up of the optimized parameter configuration to the full commercial scale. Based upon the optimal parameter configuration found during optimization and the related energy/volume required the power capacity required for scale up can be calculated. The parameter configuration of the final system is to be identical to the optimal configuration. Commercial systems of up to 16,000 watts per single device are available. Such systems can be widely adapted to amplitude and pressure requirements in order to perform at the optimal efficiency. Picture 4 shows a complete 16,000 watts with generator, transducer, sonotrode and flow cell. For larger industrial setups, several such units can be used in parallel or in series. Such clusters are almost unlimited in total power. As the adaptation of such ultrasonic units is mostly limited to boosters, sonotrodes and flow cells, it mostly possible to re-adapt the units to new parameter configurations if necessary. This my be required, if the product formulation or the required result changes.

Thomas Hielscher Ultrasonic Production of Nano-Size Dispersions and Emulsions

CONCLUSIONS

By a systematic approach to the development of ultrasonic processes, the costs and risks involved in the development and commercialization can be reduced. Because feasibility and optimization tests can be done in small lab or bench-top scale requiring small to medium size equipment, the time and expenses for this systematic identification of the optimal parameter configuration are low compared to the savings when scaling up to the production level. This approach does also help to find the important parameters behind the process result. This in turn can be used to obtain better results and a high reproducibility.

Picture 4 -

 4 Commercial 4,000 Watts (left) and 16,000 Watts (right) System for Inline Operation

Table 1 -General Recommendations for Amplitudes

 1

	Process	Amplitude
	Cleaning	0.5 to 2 micron
	Intensive Cleaning	10 to 20 micron
	Dispersing/Deagglomeration 10 to 30 micron
	Emulsifying	20 to 60 micron
	Primary Particle Reduction 40 to 120 micron

Table 2 -Typical Sonication Values

 2

	Process Energy/	Sample	Power Time
		Volume	Volume
	Simple	< 100Ws/ml	10ml	50W	<20s
	Medium 100Ws/ml to	10ml	50W	20 to
		500Ws/ml			100s
	Hard	> 500Ws/ml	10ml	50W	>100s