WO2005098032A1 - Method of diagnosing osseointegration of artificial dental implants by means of gene arrays - Google Patents

Abstract

The present invention relates to clinically relevant pre-implantation analysis and diagnosis of artificial implant materials, preferably artificial implants which can be used in dentistry as dental prosthesis. The diagnostic approach according to the invention is based on preparing arrays of specific genes or gene transcripts found to be predictive of regular or optimized integration of the artificial implants, or, on the other hand of misdirected or failed osseointegration caused by general or individual inflammatory effects occurring after implantation of artificial, non-natural or natural exogenous implants.

Description

METHOD OF DIAGNOSING OSSEOINTEGRATION OF ARTIFICIAL DENTAL IMPLANTS BY MEANS OF GENE ARRAYS

FIELD OF THE INVENTION The present invention relates to clinically relevant pre-implantation analysis and diagnosis of artificial implant materials, preferably artificial implants which can be used in dentistry as dental prosthesis. The diagnostic approach according to the invention is based on preparing arrays of specific genes or gene transcripts found to be predictive of regular or optimized integration of the artificial implants, or, on the other hand of misdirected or failed osseointegration caused by general or individual inflammatory effects occurring after implantation of artificial, non- natural or natural exogenous implants.

BACKGROUND OF THE INVENTION The loosening of implants from bone tissues has been a cause of problems in reconstructive surgery and joint replacement. The thought for decades has been that the layer of fibrous tissue that develops around the implant diminishes the integrity and mechanical stability of the implant/bone interface {Southam JC, Selwyn R. Structural changes around screws used in the treatment of fractured human mandibles. Br J Oral Surg 1971 8:211-21.). During the 1950s it had been shown by Branemark and others that chambers made of the metal titanium could become permanently incorporated with bone. That is, the living bone could become so fused with the titanium oxide layer of the implant that the two could not be separated without fracture (Branemark PL. Osseointegration and its experimental studies. J Prosthetic Dentistry 1983 50:399-410.). Osseoimplant surgery for prostethic reconstruction in the toothless jaws has been growing each year as patients become increasingly familiar with its advantages. However, despite the fact that dental titanium implants are becoming more predictable, several studies have demonstrated that osseointegration success rates do not exceed 78% to 92% over a period of 5 to 10 years. (Lekholm et al, Int J Oral Maxillofac Implants 1994 9:627-635; {Branemark et al, Clin Oral Implants Res. 1995 Dec;6(4): 227-31). Implants, titanium as well as implants of other materials, fail for a variety of reasons. Some studies have related failures to biological or microbiological factors (e.g Koutsonikos A. Ann R Australas Coll Dent Surg. 1998 14: 75- 80) while others attribute dental implant failure to biomechanical factors, biomaterial factors, or implant surface treatments and characteristics (e.g. Takeshita et al, Int J Oral Maxillofac Implants. 1995 10(3): 367-72). It has been reported (Jaffinet al, J Periodontol 1991 62(1): 2- 4.) that a 5-year success rate of 1,054 Branemark (screw type) titanium implants cab be obtained with regards to bone quality. Type I, II, and III bone may offer sufficient strength, in contrast to Type IV bone, which has a thin cortex, low trabecular density, and poor medullary strength. Only 3% of fixtures placed in Types I, II, and III bone were lost compared to a 35% failure rate of implants placed in Type IV bone. However, bone quality may be less of a concern when cylinder type implant fixtures are utilized. In addition, patients that smoke cigarettes have demonstrated an increase in implant failures. Overall failure rates have been reported as 11.28% for smokers compared to 4.76% for non-smokers (Bainet al, Int J Oral Maxillofac Implants. 1993 8(6): 609-15.).

Additionally, failure rates were higher in the posterior maxilla (19.1%) and the anterior maxilla (16.82%), while mandibular failure rates were between 4% and 5%. Taken together, factors delineating mechanisms of acute or chronic inflammation depending as well on the presence of microbial agents as on an exaggerated immune response or vascularization of bone and bone stability have been found to be the main reason for implant failure. Dental implants are the preferred replacement for the lost teeth. As anchors in the jaw bone, dental implants form a stable foundation for permanent attachment of the crown; grinding of adjacent teeth does not take place, and since implants act as "artificial dental roots" by transmitting the chewing forces into the jar, implants help to preserve the bone. It has been estimated that 300,000 - 450,000 endosseous dental implants are placed annually in the US. The success of these implants has been attributed to their firm bone anchorage, referred to as osseointegration, or functional ankylosis. If an dental implant is osseointegrated, no soft connective tissue or periodontal ligament-like interface is detectable between the bone and the implant. At the electron microscopic level, bone has been shown to be approximately 20 nm from the implant surface, or in contact with the implant surface. An oxide layer (3 to 5A°), formed by the oxidation of titanium and its alloys, is found on metal implant surfaces. The oxide layer, like ceramic, is hydrophilic, corrosion resistant, and biocompatible. Initial studies focused on commercially pure titanium implants with a relatively smooth surface created by the machining process. Subsequent investigations have indicated that implants made of titanium alloy and/or with relatively rougher surfaces also become integrated with bone. These include, but are not limited to, the following types of implants: titanium plasma sprayed, acid- etched, grit blastedacid-etched, and hydroxyapatite coated. The bonding of hydroxyapatite to bone is different from titanium and has been termed biointegration. Biointegration denotes a direct biochemical bond of the bone to the surface of an implant at the electron microscopic level and is independent of any mechanical interlocking mechanism. Several kinds of dental implant systems are available. These are classified according to their shape and relation to the bony housing. They include subperiosteal, transosteal, and endosseous implants. The most frequently used implants are endosseous implants. Endosseous implant systems include a range of sizes, shapes, coatings, and prosthetic components. Implant length and width can be chosen to fit the available bone and prosthetic components can be selected in a size and angle to accommodate the final restoration. Implant shape is usually a screw-type or cylindrical press-fit design. Unfortunately, little is known on the specific composition of biological mechanisms involved in ordinary versus misdirected wound healing in context with implants, which eventually causes the failure of osseoimplants. In the past several years, a new technology, called DNA microarray, has attracted tremendous interests among biologists. This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously. Base-pairing (i.e., A-T and G-C for DNA; A-U and G-C for RNA) or hybridization is the underlining principle of DNA microarray technology. An array is an orderly arrangement of samples. It provides a medium for matching known (probes) and unknown DNA samples (targets) based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots. Microarrays require specialized robotics and imaging equipment that generally are not commercially available as a complete system. DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously - a dramatic increase in throughput. There are two major application forms for the DNA microarray technology: 1) Identification of sequence (gene / gene mutation); and 2) Determination of expression level (abundance) of genes. There are two variants of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity: Format I probe cDNA (500-5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally" called DNA microarray, is widely considered as developed at Stanford University. A recent article by R. Ekins and F. . Chu (Microarrays: their origins and applications. Trends in Biotechnology, 1999, 17, 217-218) provides some general facts. Format II: an array of ohgonucleotide (20~80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labelled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined. This method, "historically" called DNA chips, was developed at Affymetrix, Inc., which sells its photolithographically fabricated products under the GeneChip® trademark. Many companies are manufacturing ohgonucleotide based chips using alternative in-situ synthesis or depositioning technologies. DNA microarray technology can be used in the following applications: • Gene discovery • Disease diagnosis • Drug discovery: Pharmacogenomics • Toxicological research: Toxicogenomics DNA microarrays have served a variety of purposes, including gene expression profiling, de novo gene sequencing, gene mutation analysis, gene mapping and genotyping. cDNA microarrays are printed with distinct cDNA clones isolated from cDNA libraries. Therefore, each spot represents an expressed gene, since it is derived from a distinct mRNA. Typically, a method of monitoring gene expression involves providing a pool of sample polynucleotides comprising RNA transcripts of target genes or nucleic acids derived from the RNA transcripts; hybridizing the sample polynucleotides or nucleic acids derived from the RNA transcripts to an array of probes (for example, polynucleotides obtained from a polynucleotide library including control probes, and detecting the hybridized double-stranded polynucleotides/nucleic acids. The label used to label polynucleotide samples is selected from the group consisting of radioactive, colorimetric, enzymatic, molecular amplification, bioluminescent or fluorescent label.

SUMMARY AND DETAILS OF THE INVENTION

The present invention relates to molecular methods for diagnosing proper osseointegration of dental osseoimplants using arrays of candidate polynucleotides by means of gene expression profiling images from human bone materials. The invention provides genes or gene transcripts in form of a polynucleotide library which are specifically responsible for a regular and most favorable integration of artificial implants into the corresponding natural tissue environment. By means of this library a gene chip or gene array can be designed which is specific for osseointegration of the artificial dental implant, whereby an array can be generated deriving from an optimum osseointegration of a specific implant under defined normal or pathological conditions. Such a dental implant or the gene library thereof, generated from tissue in the immediate environment or vicinity of said implant, can be used as standard or reference implant against any other implant or gene image deriving from dental implants under suboptimum, misdirected, failed or pathological conditions. It is also possible to use as reference library or implant an implant or library that derives from tissue under specific suboptimum, misdirected, failed or pathological conditions of an individual, and compares these results to any testing implant showing improved osseointegration.

The methods according to the invention additionally enables to define the actual or potential influence of specific diseases or disorders, such as, for example, diabetes, or cell toxins, such as nicotine or other pollution agents, on the osseointegration of dental implants in individuals or groups of individuals with respect to a differentiating specific genetic profiles of up- or downregulated genes or gene transcripts, which can be deduced to said influence. The invention, furthermore, enables, to define the actual or potential influence of and to screen for specific agents, drugs, compounds, medicaments, which are effective in improving osseointegration of specific implants under specific conditions in specific individual or groups of individuals by means of said gene array-based analysis. In the context of this disclosure, a number of terms shall be utilized. The term "polynucleotide" refers to a polymer of RNA or DNA that is single-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. The term "immobilized on a support" means bound directly or indirectly thereto including attachment by covalent binding, hydrogen bonding, ionic interaction, hydrophobic interaction or otherwise. The term "osseointegration" means a stable and apparently immobile support of a prosthesis or implant under functional loads, without significant pain, inflammation or loosening. The term "probe" means a tethered or fixed nucleic acid with known sequence, whereas the term "target" means a free nucleic acid sample whose identity/abundance is being detected. The term "artificial implant" includes implants of non-natural origin, such as implants made of plastic, like polyethylene, metals especially noble metals, metal alloys, ceramic materials; and natural exogenous implants. The term "reference implant" or "reference sample" means a dental implant or a tissue sample, respectively, deriving from the direct environment of this implant, providing a gene profile which is used as reference gene profile compared to gene profile generated from the tissue ^testing sample") in the immediate vicinity of a "testing implant". Usually the reference implant essentially shows optimum osseointegration under specific "reference conditions", whereas the testing implant shows a comparably decreased osseointegration related to the reference implant under specific "testing conditions". Preferably reference and testing conditions are the same ones, or at least essentially the same ones, however, it is possible, as mentioned above, to choose different conditions. An aspect of the invention relates to the use of polynucleotide arrays, which allows to quantitatively study mRNA expression levels of selected candidate genes in human bone biopsies. The present invention relates to a novel specific polynucleotide library, wherein the polynucleotides included are immobilized on a solid support in order to form a polynucleotide array. Arrays, suitable according to this invention, including the solid supports or matrices, are principally well known in the art. Preferably the support is selected from the group consisting of a nylon membrane, glass slide, glass beads, or a silicon chip. The polynucleotide libraries provided herewith are highly specific for the genetic condition of osseointegration of dental implants in context with their implantation into the human jaw bone and are useful in the molecular characterization of a failed or suboptimal or pathological osseointegration process in a human individual compared to a regular osseointegration in the same individual. The libraries include a pool of polynucleotide sequences or subsequences thereof, as given in the Tables 1, la an 2, as shown below, wherein the sequences or subsequences are either underexpressed or overexpressed in cells taking part in pathological osseoinegration. Each of these oligo- or polynucleotide probes is obtainable on the basis of structural information mediated by the sequence data provided by the respective accession number set out for each candidate gene. Preferably the pool of polynucleotide sequences or subsequences correspond substantially to the polynucleotide sequences useful in differentiating a normal cell, or a cell from a patient suffering from failing osseointegration. The method for detecting differentially expressed polynucleotide sequences according to this invention is used for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating conditions associated with failing osseointegration, and namely all forms of failing osseointegration with all forms of osseoimplants.

Table 1 : Induced genes caused by a polyethylene implant in comparison to a titanium implant (polishes titanium according Branemark) F* = factor against reference implant (polyethylene) V* = Expression rate y* F* Description Homo sapiens small inducible cytokine subfamily B (Cys-X-Cys), member 14 8942,3 2,04 (BRAK) (SCYB 14), mRNA Consensus includes Homo sapiens cytochrome P450, subfamily I (dioxin-inducible), 8864,4 2,04 polypeptide 1 8169,0 6,24 Homo sapiens matrix metalloproteinase 7 (matrilysin, uterine) (MMP7), mRNA Consensus includes Homo sapiens collagen, type X, alpha 1 (Schmid metaphysea! 7628,4 2,04 chondrodysplasia) 7517,6 2,04 Human connective tissue growth factor, complete cds 7262,4 2,06 Homo sapiens hemoglobin, gamma G (HBG2), mRNA 7171,8 2,04 Homo sapiens CYR61 mRNA, complete cds (Angiogenic inducer) 7111,6 2,04 Homo sapiens hexabrachion (tenascin C, cytotactin) (HXB), mRNA 6491,2 2,06 Homo sapiens hemoglobin, gamma A (HBG1), mRNA 6484,3 2,18 Homo sapiens metallothionein 2A (MT2A), mRNA 6290,2 2,08 Homo sapiens matrix metalloproteinase 12 ( acrophage elastase) (MMP12), mRNA V* F* Description

5733,9 2,06 Homo sapiens interleukin 8 (IL8), mRNA

5364,2 4,12 Human microfibril-associated glycoprotein-2 MAGP-2 mRNA, complete cds. Homo sapiens latent transforming growth factor beta binding protein 2 (LTBP2),

5353,5 2,04 mRNA

5142,8 2,04 Human nerve growth factor (HBNF-1) mRNA

5121,5 2,04 Homo sapiens cysteine-rich, angiogenic inducer, 61 (CYR61), mRNA Consensus includes Homo sapiens myosin, light polypeptide 4, alkali; atrial,

5035,0 2,06 embryonic

4804,9 2,24 Homo sapiens early growth response 1 (EGR1), mRNA

4690,1 2,18 Consensus includes Homo sapiens activated leucocyte cell adhesion molecule Homo sapiens platelet derived growth factor C (PDGFC), mRNA. PROD=secretory

4461,2 2,04 growth factor-like protein fallotein Human glycoprotein mRNA, complete cds. Homo sapiens 75184 chitinase 3-like 1

4444,9 2,06 (cartilage glycoprotein-39) Human glycoprotein mRNA, complete cds. Homo sapiens chitinase 3-like 1 (cartilage

4386,3 2,06 glycoprotein-39) Consensus includes Homo sapiens collagen, type X, alpha 1 (Sch id metaphyseal

4088,0 2,04 chondrodysplasia)

4041,9 4,22 Homo sapiens tissue inhibitor of metalloproteinase 3 (TIMP3), mRNA Homo sapiens, pleiotrophin (heparin binding growth factor 8, neurite growth-

4021,8 2,04 promoting factor 1) mRNA, complete cds.

3843,8 2,06 Consensus includes Homo sapiens 278503 regulated in glioma

3727,1 4,18 Homo sapiens integrin, beta-like 1 (with EGF-Iike repeat domains) (ITGBLl), mRNA Consensus includes Homo sapiens 303090 protein phosphatase 1, regulatory

3655,3 2,04 (inhibitor) subunit 5 Homo sapiens Kallmann syndrome 1 sequence (KALI), mRNA (enhanced in

3569,8 2,06 metastasizing esophageal cancer) Homo sapiens latent transforming growth factor beta binding protein 1 (LTBP1),

3433,9 2,04 mRNA

3310,9 4,14 Human ovarian beta-A inhibin mRNA, complete cds. Cluster includes AL049370: Homo sapiens mRNA; cDNA

3105,8 2,10 DKFZp586D0918/cds=UNKNO N/ug=Homo sapiens 13350 Homo sapiens, adipose differentiation-related protein, clone MGC: 10598, mRNA,

3092, 1 2, 18 complete cds. Homo sapiens IL-1 receptor antagonist IL-lRa (IL-1RN) gene, alternatively spliced

2916,9 2,06 forms, complete cds.

2789,7 2,18 Human extracellular matrix protein 1 (ECM1) mRNA, complete cds.

2772,4 2,04 Consensus includes Homo sapiens 80552 dermatopontin Homo sapiens bone gamma-carboxyglutamate (gla) protein (osteocalcin) (BGLAP),

2742,6 6, 18 mRNA

2675,9 2,04 Consensus includes Homo sapiens 21858 trinucleotide repeat containing 3

2491,0 2,04 Human mRNA for beta-l,4-galactosyltransferase, complete cds.

2410,2 2,20 Homo sapiens metallothionein 1H (MT1H), mRNA

2383,6 2,04 Consensus includes DPT gene for Dermatopontin, ESTs, an STS and GSSs Homo sapiens cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1

2346,3 2,04 (glaucoma 3, primary infantile) (CYP1B1), mRNA Cluster Incl. AI806793;wfl5d05.xl UNKNOWN /ug=Homo sapiens 239450

2277,0 2,04 /len=555 Homo sapiens fibroblast growth factor receptor 3 (achondroplasia, thanatophoric

2268,0 2,04 dwarfϊsm) (FGFR3), transcript variant 1, mRNA.

2226,6 2,25 Homo sapiens growth arrest and DNA-damage-inducible, beta (GADD45B), mRNA

2220,6 2,04 Homo sapiens neurogenic extracellular slit protein Slit2 mRNA, complete cds.

2189,8 2,04 Homo sapiens lipoprotein lipase (LPL), mRNA

2168,3 2,04 Homo sapiens eukaryotic translation initiation factor 5A (EIF5A), mRNA

2159,8 2,06 Homo sapiens fatty acid binding protein 4, adipocyte (FABP4), mRNA

2159,6 2,22 Consensus includes Homo sapiens 94070 osteomodulin

2069,4 2,04 Consensus includes Homo sapiens mRNA full length insert integrin beta-like 1 y* F* Description

1964,7 2,04 Homo sapiens dermatopontin (DPT), mRNA

1899,7 2,04 Homo sapiens follistatin (FST), transcript variant FST344, mRNA

1811,8 2,04 Consensus includes Homo sapiens 249239 collagen, type VIII, alpha 2 Consensus includes Homo sapiens 245188 tissue inhibitor of metalloproteinase 3

1810,9 2,04 (Sorsby fundus dystrophy, pseudoinflammatory)

1799,9 2,04 Homo sapiens prostaglandin 12 (prostacyclin) synthase (PTGIS), mRNA

1788,3 2,04 Homo sapiens ectonucleotide pyrophosphatasephosphodiesterase 1 (ENPP 1 ), mRNA

1785,2 2,04 Consensus includes Homo sapiens 139033 paternally expressed 3

1764,1 2,04 Homo sapiens proline arginine-rich end leucine-rieh repeat protein (PRELP), mRNA

1725,3 4, 14 Human mRNA for fructose- 1,6-bisphosphatase, complete cds.

1705,2 2,06 Homo sapiens actin, alpha, cardiac muscle (ACTC), mRNA Homo sapiens, prostaglandin D2 synthase (21kD, brain), clone MGC;14559, mRNA,

1647,7 2,18 complete cds

1529,7 2,04 Homo sapiens complement component 3 (C3), mRNA

1521,5 2,06 Consensus includes Homo sapiens 245188 tissue inhibitor of metalloproteinase 3

1505,6 2,04 Homo sapiens osteomodulin (OMD), mRNA Homo sapiens cartilage oligomeric matrix protein (pseudoachondroplasia, epiphyseal

1375,8 2,10 dysplasia 1, multiple) (COMP), mRNA Human bcl-1 mRNA, complete cds. -Homo sapiens 82932 cyclin Dl (PRAD1 :

1370,7 2,04 parathyroid adenomatosis 1) Homo sapiens serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen

1365,0 2,04 activator inhibitor type 1 ) mRNA

1360,4 2,20 Homo sapiens tropo yosin 1 (alpha) (TPM1 ), mRNA

1360,0 2,04 Homo sapiens MAB21 2 protein (MAB21 L2) mRNA, complete cds.

1297,0 2,04 Homo sapiens similar to S68401 (cattle) glucose induced gene (HSl 1 19D91), mRNA

1289,5 2,04 Consensus includes Homo sapiens 180878 lipoprotein lipase Homo sapiens SRY (sex determining region Y)-box 9 (campomelic dysplasia,

1269,0 2,04 autosomal sex-reversal) (SOX9), mRNA

1239,3 2,04 Homo sapiens putative transmembrane protein (NMA), mRNA

1157,9 2,04 Homo sapiens heptacellular carcinoma novel gene-3 protein (LOC51339), mRNA Consensus includes Homo sapiens 13350 Homo sapiens mRNA; cDNA

1144,3 2, 14 DKFZp586D0918 (from clone DKFZp586D0918)

1138,4 2,17 Homo sapiens heat shock 70kD protein IB (HSPA1B), mRNA Consensus includes sapiens 154654 cytochrome P450, subfamily I (dioxin-inducible),

1098,6 2,04 polypeptide 1 Consensus includes Homo sapiens 85112 insulin-like growth factor 1 (somatomedin

1046,3 2,04 C) Consensus includes Homo sapiens 241257 latent transforming growth factor beta

1038,4 2,04 binding protein 1 Homo sapiens large conductance calcium- and voltage-dependent potassium channel

1016,0 2,04 alpha subunit (MaxiK) mRNA Homo sapiens major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1),

1005,4 2,06 mRNA.

1000,8 2,08 Homo sapiens glypican 1 (GPC1), mRNA

994,8 2,04 Homo sapiens complement-cl q tumor necrosis factor-related protein mRNA

960,5 2,04 Homo sapiens aldehyde dehydrogenase 1 family, member A3 (ALDH1A3), mRNA

943,9 2,04 Homo sapiens mRNA for KIAA1199 protein, partial cds.

942,1 2,06 Homo sapiens sarcolipin (SLN), mRNA

939,9 2,04 Homo sapiens interleukin 1 receptor, type II (IL1R2), mRNA

933,9 2,04 Homo sapiens kinesin family member 13B (KIF13B), mRNA

926,6 2,04 Consensus includes Homo sapiens 137168 OLF-1EBF associated zinc finger gene Homo sapiens sema domain, immunoglobulin domain (Ig), secreted, (semaphorin) 3C

913,4 2,04 (SEMA3C), mRNA

912,2 2,04 Homo sapiens vitamin D (1,25- dihydroxyvitamin D3) receptor (VDR), mRNA

910,7 2,04 Consensus includes Homo sapiens 81134 interleukin 1 receptor antagonist y* F* Description

908,9 2,04 HSU84487 Human CX3C chemokine precursor, mRNA, alternatively spliced, complete cds.

896,8 2,04 Homo sapiens syndecan 4 (amphiglycan, ryudocan) (SDC4), mRNA

879,1 2,04 Homo sapiens hemoglobin, delta (HBD), mRNA

875,5 2,06 Homo sapiens activating transcription factor 3 (ATF3), mRNA

837,6 2,04 Homo sapiens putative lymphocyte G0G1 switch gene (G0S2), mRNA

787,3 2,18 Human c-erb-B-2 mRNA. (neuroglioblastoma derived oncogene homolog) Homo sapiens small inducible cytokine A3 (homologous to mouse Mip-la) (SCYA3).

770,2 2,04 mRNA

765,9 2,04 Homo sapiens troponin C2, fast (TNNC2), mRNA

745,1 2,06 Homo sapiens IgG Fc binding protein (FC(GAMMA)BP) mRNA Consensus includes Homo sapiens 74520 spinocerebellar ataxia I

738,5 2,04 (olivopontocerebellar ataxia 1, autosomal dominant, ataxin 1)

735,8 2,04 Consensus includes Homo sapiens 198427 hexokinase 2 Homo sapiens hypothetical protein FLJ11808 (FLJ11808), mRNA.

714,5 2,04 PROD=hypothetical protein FLJ11808

702,4 2,08 Homo sapiens cystatin SN (CST1), mRNA Homo sapiens hypothetical protein MGC2827 (MGC2827), mRNA.

690,9 2,04 PROD=hypothetical protein MGC2827

688,5 2,04 Homo sapiens deiodinase, iodothyronine, type II (DI02), transcript variant 1, mRNA

688,5 2,04 Consensus includes Homo sapiens 286049 phosphoserine aminotransferase

679,0 2,12 Homo sapiens actin, gamma 2, smooth muscle, enteric (ACTG2), mRNA

661,1 2,04 Consensus includes Homo sapiens 81134 interleukin 1 receptor antagonist Homo sapiens mRNA; cDNA DKFZp434A0530 (from clone DKFZp434A0530);

653,9 2,04 complete cds.

652,0 4,08 Homo sapiens aquaporin 9 (AQP9), mRNA.

650,7 2,04 Homo sapiens BCL2-related protein Al (BCL2A1), Mrna Consensus includes Homo sapiens 85112 insulin-like growth factor 1 (somatomedin

632,6 2,04 C) Homo sapiens C-type (calcium dependent, carbohydrate-recognition domain) lectin,

632,1 2,04 superfamily member 5 (CLECSF5), mRNA Homo sapiens mRNA for KIAA0611 protein, partial cds. Homo sapiens 70604

624,0 2,04 ATPase, Class II, type 9A

618,7 2,18 Homo sapiens glypican 4 (GPC4), mRNA

616,8 2,04 Homo sapiens meningioma (disrupted in balanced translocation) 1 (M 1), mRNA

608,1 2,06 Consensus includes Homo sapiens 139033 paternally expressed 3

607,6 8,22 Homo sapiens thrombospondin 4 (THBS4), mRNA

597,3 2,04 Homo sapiens early growth response 3 (EGR3), mRNA

572,4 2,04 Homo sapiens vascular endothelial growth factor mRNA, complete cds Consensus includes Homo sapiens 80261 enhancer of filamentation 1 (cas-like

569,7 2,14 docking; Crk-associated substrate related) Consensus includes Homo sapiens 22907 mRNA; cDNA DKFZp586F071 (from

552,4 2,04 clone DKFZp586F071) Homo sapiens hypothetical protein FLJ1 1082 (FLJ1 1082), mRNA. Homo sapiens

549,3 2,04 31792 hypothetical protein FLJ11082

547,9 4,16 Homo sapiens pentaxin-related gene, rapidly induced by IL-1 beta (PTX3), mRNA

547,5 2,04 Homo sapiens myosin, heavy polypeptide 2, skeletal muscle, adult (MYH2), mRNA

543,7 2,04 Homo sapiens scrapie responsive protein 1 (SCRG1), mRNA

533,6 2,04 Homo sapiens carbonyl reductase 3 (CBR3), mRNA. gb;NM_001236.2 Consensus includes Homo sapiens 144630 nuclear receptor subfamily 2, group F,

524,6 2,04 member 1

524,0 2,04 Homo sapiens serum constituent protein (MSE55), mRNA Homo sapiens potassium intermediate small conductance calcium-activated channel,

520,2 2,06 subfamily N, member 4 (KCNN4), mRNA.

518,2 2,06 Consensus includes Homo sapiens 290 phospholipase A2, group V y* F* Description 517,3 2,06 Homo sapiens sodium calcium exchanger (NCKX3), mRNA 515,5 2,06 Homo sapiens actin, alpha 1, skeletal muscle (ACTAl), mRNA

Table la: Preferred selection of Table 1 # v* F* Description Function Context Homo sapiens small inducible cytokine B-cell and monocyte chemokine, overexpressed in 1 8942,3 2,04 subfamily B (Cys-X-Cys), member 14 growing epithelial cells and in infiltrating (BRAK) (SCYB14), mRNA inflammatory cells Induction of MMP-7, -9 and -12 from epithelial , ~4 Homo sapiens matrix metalloproteinase 7 cells serves as a good prognostic for invasiveness 8169,0 (matrilysin, uterine) (MMP7), mRNA of oral cancer. Increased in infiltrating inflammatory cells. Belongs to family of CCN proteins; activates stimulation of cell proliferation, migration, and adhesion, as well as angiogenesis and tumorigenesis. Significant correlation exists ,. „ . Human connective tissue growth factor, 7517,6 between CTGF mRNA levels versus tumor grade, ' complete cds gender, and pathology of gliomas. Activates fϊbrogenic tissue in co-stimulatory approach with epithelial growth activation. Correlates with better survival in gliomas. Co-expression with CTGF; angiogenic inducer; significant associations were found between Homo sapiens CYR61 mRNA, complete CYR61 expression versus tumor grade, pathology, 7171,8 2,04 cds. gender, and age at diagnosis. Combined activation of fibrotic tissue in combination with epihelial growth responses. Induction and activation of polymorphonuclear 5733,9 2,06 Homo sapiens interleukin 8 (IL8), mRNA leucocytes MAGP-2 is a small glycoprotein that is specifically associated with fϊbrillin-containing microfibrils. Human microfibril-associated glycoprotein- 5364,2 4,12 MAGP-2 promotes the attachment and spreading of 2 MAGP-2 mRNA, complete cds. fibroblasts. Human fibroblasts and osteoblasts adhere strongly to MAGP-2 via integrin O_vd₃. The expression of nerve growth factor (NGF) is increased after injury, in part mediated by effects on astrocytes of pro-inflammatory mediators and cytokines produced by immune cells. Conversely, Human nerve growth factor (HBNF-1) 5142,8 2,04 cells of the immune system express NGF receptors, mRNA and NGF signaling modulates immune function. Increased production of NGF can suppress inflammation by switching the immune response to an anti-inflammatory, suppressive mode. Homo sapiens platelet derived growth Highly expressed at sites of epithelial openings and 8 4461,2 2,04 factor C (PDGFC), mRNA=secretory damage growth factor-like protein fallotein Homo sapiens integrin, beta-like 1 (with TGF-D 1 -associated integrin response in increased 9 3727, 1 4, 18 EGF-Iike repeat domains) (ITGBLl), mesenchymal growth mRNA Beta-A inhibin (Activin A) is a member of the Human ovarian beta-A inhibin mRNA, TGF-D family, augments the mesenchymal 10 3310,9 4, 14 complete cds. response of TGF-0 and is found in developing liver fibrosis Extracellular matrix protein 1 (ECM1) is a secreted 1 1 _?7_SO 7 1 _i S Hu an extracellular matrix protein glycoprotein involved in bone formation and ' ' ' ' (ECM1) mRNA, complete cds. angiogenesis. ECM1 regulates endochondral bone formation, stimulates proliferation of endothelial cells, and induces angiogenesis. Homo sapiens bone gamma- 12 Osteocalcin levels from peri-implantitis sites are 2742,6 6,18 carboxyglutamate (gla) protein (osteocalcin) (BGLAP), mRNA significantly higher than in healthy implants Homo sapiens Kallmann syndrome 1 Kal-1 gene disrupt epidermal morphogenesis, 13 3569,8 2,06 sequence (KALI), mRNA (enhanced in resulting in closure defects metastasizing esophageal cancer) 14 ι ι _Q Dermatopontin interacts with transforming growth y_ήo₄4, ₇/ - z₇, ru_i44 H _mo_RNo_A sapiens dermatopontin (DPT), factor beta and enhances its biological activity. Angiogenetic effect: Activation of endothelial cells 15 17_QQ O _? 04. Ho o sapiens prostaglandin 12 via integrin D_VD₃ cause angioneogenesis by '^V ' (prostacyclin) synthase (PTGIS), mRNA prostacyclin production. Homo sapiens, prostaglandin D2 synthase ^• 16 1647,7 2,18 (2 lkD, brain), clone MGC: 14559, mRNA, Direct inflammatory effect complete cds . , ,_ „ . ₀. 17 ' ' of

Small Ieucine rich proteoglycan facilitating early _i sn< _f. i _fli Homo sapiens osteomodulin (OMD), nu:>,o z,U4 „„_>, » pulp development, identifying initial steps of dental mRNA bone formation. 1 607 ₍ R 9? ^Homo sapiens thrombospondin 4 (THBS4), Induced in fibrogenesis and epithelial- ' ' mRNA mesenchymal transition (EMT) ™ --- _r, -, , -. Homo sapiens actin, gamma 2, smooth „ . ~ _c, . . . 20 679,0 2,12 , ^H ₄ . , . P ~_{> T}Λ_{I Λ} Specific fibroblast protein muscle, enteric (ACTG2), mRNA ^{r v} Homo sapiens major histocompatibility 21 1005,4 2,06 complex, class II, DQ alpha 1 (HLA- Activation of cellular immunity DQA1), mRNA. -..-i _{m Λ} „ _Λ„ Homo sapiens vascular endothelial growth . ,. ,. „ 22 572,4 2,04 „ , _n . . . . , ^s Activation of angiogenesis factor mRNA, complete cds " ° Homo sapiens pentaxin-related gene, 23 547,9 4,16 rapidly induced by IL-1 beta (PTX3), Activation of angiogenesis mRNA

Table 2: Repressed genes caused by a titanium implant (polished titanium according Branemark) in comparison to a polyethylene implant F* = factor against reference implant (polyethylene) V* = Expression rate V* F* Description -11329,6 -2,28 Homo sapiens cathepsin K (pycnodysostosis) (CTSK), mRNA. -10653,4 -2,72 Human saposin proteins A-D mRNA, complete cds. -9776,6 -2,20 Homo sapiens interferon, gamma-inducible protein 30 (IFI30), mRNA -9593,3 -2,32 Human MHC class II HLA-DR-alpha mR A, complete cds. ,... , _ , , Consensus includes PROD=M130 antigen cytoplasmic variant 1, =Homo sapiens 74076 CD163 -/o-.o,l -z,lo .. antigen -6875,9 -5,20 Homo sapiens heme oxygenase (decycling) 1 (HMOX1), mRNA -6795,9 -2,96 Homo sapiens cystatin C (amyloid angiopathy and cerebral hemorrhage) (CST3), mRNA -6718,0 -3,04 Homo sapiens alpha-2-macroglobulin (A2M), mRNA -5796,3 -2,52 Homo sapiens CD 163 antigen (CD 163), mRNA -4856,9 -2,36 Homo sapiens ribosomal protein S26 (RPS26), mRNA V* F* Description

-4785 9 -2 36 Consensus includes Homo sapiens 914 Human mRNA for SB classll histocompatibility antigen ' ' alpha-chain

-4456,8 -2,80 Consensus includes Homo sapiens 105700 secreted frizzled-related protein 4

-4137,2 -3,56 Consensus includes Homo sapiens 119129 collagen, type IV, alpha 1 /FL=gb:NM_001845.1

-3996,9 -2,52 Homo sapiens prolylcarboxypeptidase (angiotensinase C) (PRCP), mRNA

-3764,5 -4,48 Consensus Homo sapiens 78146 plateletendothelial cell adhesion molecule (CD31 antigen) - .„, „ , - , Homo sapiens phosphodiesterase 4C, cAMP-specific (dunce (Drosophila)-homo log ' ' phosphodiesterase El) (PDE4C), mRNA

-3408,1 -2,68 Consensus includes Homo sapiens 108885 collagen, type VI, alpha 1

-3350,8 -3,04 Homo sapiens granulin (GRN), mRNA

-3265,8 -2,24 Homo sapiens Kreisler (mouse) maf-related leucine zipper homolog (KRML), mRNA

-3243,9 -2, 16 Homo sapiens glutaminyl-tRNA synthetase (QARS), mRNA

-31 0,9 -2,80 Consensus includes Homo sapiens 75617 collagen, type IV, alpha 2

-3137,0 -2,36 Consensus includes Homo sapiens 283330 hypothetical protein PRO 1843

-2838,7 -6,56 Homo sapiens ribonuclease, RNase A family, 1 (pancreatic) (RNASE1), mRNA

„_-„ » . _ . Homo sapiens, Similar to eukaryotic translation initiation factor 3, subunit 8 (1 lOkD), clone ' ' MGC:8693, mRNA, complete cds

-2660,8 -3,28 Homo sapiens fenestrated-endothelial linked structure protein (FELS) mRNA, complete cds

-2644,2 -5,20 Human uncoupling protein homolog (UCPH) mRNA, complete cds

-2608,6 -4,56 Homo sapiens collagen, type XV, alpha 1 (COL15A1), mRNA

-2566,9 -3,16 Consensus includes Homo sapiens 180577 granulin

-2476,4 -2,12 Consensus includes Homo sapiens 77522 major histocompatibility complex, class II, DM alpha

-2367,2 -3,52 Homo sapiens cathepsin C (CTSC), mRNA τi_\ * o i_f. Homo sapiens, non-POU-domain-containing, octamer-binding, clone MGC:3380, mRNA, complete cds

-2263,5 -2,60 Homo sapiens caveolin 1, caveolae protein, 22kD (CAV1), mRNA _??4 s _{7 (} Homo sapiens colony stimulating factor 1 receptor, formerly McDonough feline sarcoma viral (v- fms) oncogene homolog (CSF1R), mRNA

-2218,6 -2,68 Homo sapiens CD20-like precusor (LOC64166), mRNA

_{?7fi8 1 ? 74.} Consensus includes Homo sapiens 100293 O-linked N-acetylglucosamine (GlcNAc) transferase ' ^" ' (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyI transferase)

-2198,2 -3,44 Homo sapiens growth arrest-specific 1 (GAS1), mRNA

-2007,2 -3,04 Consensus includes Homo sapiens 247984 Human glutamine synthetase pseudogene

- 1990,8 -2,04 Homo sapiens cellular repressor of E 1 A-stimulated genes (CREG), mRNA.1

-1 84,7 -2,28 Homo sapiens protective protein for beta-galactosidase (galactosialidosis) (PPGB), mRNA

-1974,0 -3,52 Consensus includes Homo sapiens 8136 endothelial PAS domain protein 1

-1948,0 -4,20 Consensus includes Homo sapiens 74602 aquaporin 1 (channel-forming integral protein, 28kD)

-1934,5 -6,76 Homo sapiens SPARC-like 1 (mast9, hevin) (SPARCL1), mRNA

-1930,4 -4,64 Homo sapiens spondin 2, extracellular matrix protein (SPON2), mRNA

-1928,4 -2,28 Homo sapiens spermidinespermine Nl-acetyltransferase (SAT), mRNA _{1 i?}^s o _o no Consensus includes Homo sapiens 296398 Homo sapiens mRNA; cDNA DKFZp586El 124 (from ' ' clone DKFZp586EU24); complete cds

-1851,3 -2,20 Homo sapiens endoglin (Osler-Rendu-Weber syndrome 1) (ENG), mRNA , „ ,, , „ _nn Consensus includes Homo sapiens 8850 a isintegrin and metalloproteinase domain 12 (meltrin alpha)

-1823,8 -2,20 Consensus includes Homo sapiens 155218 ElB-55kDa-associated protein 5

- 1820,0 -2,64 Homo sapiens nidogen 2 (NID2), mR A V* F* Description

-1815,0 -2,08 Homo sapiens hypothetical protein FLJ20700 (FLJ20700), mRNA

-1799,8 -6,80 Homo sapiens von Willebrand factor (VWF), mRNA

- 1797,8 -2,56 Homo sapiens cell surface glycoprotein P 1 H 12 precursor, mRNA, complete cds

-1781,3 -2,12 Homo sapiens cat eye syndrome chromosome region, candidate 1 (CECRl), mRNA

-1734,3 -3,60 Homo sapiens macrophage myristoylated alanine-rich C kinase substrate (MACMARCKS), mRNA

-1711 8 -3 04 Consensus includes Homo sapiens 164036 Homo sapiens AKAP350C mRNA sequence, ' ' alternatively spliced

-1692,9 -2,24 Consensus includes Homo sapiens 752 FK506-binding protein lA (12kD)

-1644,6 -3,84 Human intercrine-alpha (hlRH) mRNA, complete cds.

-1629,7 -8,48 Homo sapiens acid phosphatase 5, tartrate resistant (ACP5), mRNA 1626 6 -2 12 Human mRNA for Id- 1 H, complete cds. Homo sapiens 75424 inhibitor of DNA binding 1 , ' ' dominant negative helix-loop-helix protein

-1613,4 -3,92 Homo sapiens eukaryotic translation initiation factor 3, subunit 8 (1 lOkD) (EIF3S8), mRNA. 1604 4 -2 04 Homo sapiens, eukaryotic translation initiation factor 3, subunit 4 (delta, 44kD), clone MGC:2053, mRNA, complete cds. ϊ 597 0 2 32 Consensus includes gb: Homo sapiens 8025, Homo sapiens clone 23767 and 23782 mRNA ' ' sequences

-1593,0 -2,32 Homo sapiens hypothetical protein FLJ11029 (FLJ11029), mRNA.

-1577,4 -2,52 Homo sapiens T-cell, immune regulator 1 (TCIRG1), mRNA. , . „ ,, „ Homo sapiens, Similar to malic enzyme 2, D(+)-dependent, mitochondrial, clone MGC:5187, mRNA, complete cds. _l s _{1 7} e, _i R Consensus includes gb: Homo sapiens 148598 Homo sapiens cDNA FLJ1 1452 fis, clone

-i3 i /,o -2,88 _{HEMBA 1 00}]4₃₅

- 1488,7 -2,88 Homo sapiens adaptor-related protein complex 1 , sigma 2 subunit (AP 1 S2), mRNA. ₁47_{0 Q} 6 Consensus includes gb: Homo sapiens 3337 transmembrane 4 superfamily member 1

-i4/y,y -i, /^b _/FL--_{g :M9}o657.1

-1478,6 -6,28 Consensus includes gb: Homo sapiens 9305 angiotensin receptor-like 1 /FL=gb:NM_005161.1

-1478,6 -4,28 Homo sapiens glutamate-ammonia ligase (glutamine synthase) (GLUL), mRNA. , .,, . „ _ΛO Consensus includes gb: Homo sapiens 82685 CD47 antigen (Rh-related antigen, integrin-associated

-1420,4 -Z,Uo . , . signal transducer)

-1422,1 -6,20 Homo sapiens matrix metalloproteinase 13 (collagenase 3) (MMP13), mRNA.

-1389,0 -5,52 Human leukocyte surface protein (CD31) mRNA, complete cds

-1387,6 -2,16 Consensus includes gb: Homo sapiens 52184 hypothetical protein FLJ20618

-1374,0 -5,00 Homo sapiens EGF-like-domain, multiple 6 (EGFL6), mRNA. Homo sapiens eukaryotic translation initiation factor 2, subunit 3 (gamma, 52kD) (EIF2S3),

-1367,0 -2,08 mRNA.

-1339,8 -2,68 Homo sapiens peptidylprolyl isomerase C (cyclophilin C) (PPIC), mRNA. _ι . . _n . , _m Consensus includes gb: Homo sapiens 4835 eukaryotic translation initiation factor 3, subunit 8

-1330,3 -3,92 _{(] 1()kD)}

-1312,9 -5,84 Homo sapiens stromal cell-derived factor 1 (SDF1), mRNA.

-1279,6 -2,88 Consensus includes gb: Homo sapiens 205353 ectonucleoside triphosphate diphosphohydrolase 1

-1273,5 -2,44 Consensus includes gb: Homo sapiens 198282 phospholipid scramblase 1

-1268,1 -8,80 Human MHC class II DQ-beta associated with DRw6, DQwl protein, complete cds.

-1265,9 -3,40 Consensus includes gb: Homo sapiens 303157 T cell receptor beta locus , ,,, . „ _n . Consensus includes gb: Homo sapiens 198253 major histocompatibility complex, class II, DQ alpha

-1/03,1 -y,U4 . 1761 1 08 Consensus includes gb: Homo sapiens 206882 Homo sapiens mRNA for FLJ00032 protein, partial ' ' cds

-1248,4 -3,36 Homo sapiens golgi membrane protein GP73 (LOC51280), mRNA.

V* F* Description

-931,7 -2,08 Homo sapiens arginyl aminopeptidase (aminopeptidase B) (RNPEP), mRNA.

-918 8 2 56 Homo sapiens mRNA for CC chemokine 16530 small inducible cytokine subfamily A (Cys-Cys), ' ' member 18, pulmonary and activation-regulated

-910,9 -2,32 Homo sapiens protein tyrosine phosphatase, receptor type, C (PTPRC), mRNA.

-910,7 -2,16 Consensus includes Homo sapiens 179526 upregulated by 1,25-dihydroxyvitamin D-3

-905,3 -3,00 Homo sapiens NAD(P)H menadione oxidoreductase 2, dioxin-inducible (NMOR2), mRNA.

-901,9 -5,52 Homo sapiens aquaporin 1 (channel-forming integral protein, 28kD) (AQP1), mRNA.

-897,6 -3,28 Consensus includes Homo sapiens 75416 DAZ associated protein 2 _o„ , . „ „, Consensus includes Homo sapiens 160483 erythrocyte membrane protein band 7.2 (stomatin)

-^δyo,ι -z, ^/o /_FL=g_b:M81635 i _gb:NM_004099.1

-893,3 -3,20 Homo sapiens hypothetical protein MGC5363 (MGC5363), mRNA.

-892,7 -2,56 Homo sapiens frizzled (Drosophila) homolog 4 (FZD4), mRNA.

-883,0 -2,64 Homo sapiens, Similar to cytochrome c-like antigen, clone MGC:2960, mRNA, complete cds.

-882,4 -4,20 Human CD34 mRNA, complete cds.

-873,9 -2,96 Homo sapiens KIAA0570 gene product (KIAA0570), mRNA.

-867,1 -6,04 Homo sapiens integral membrane protein 2A (ITM2 A), mRNA.

-864,7 -2,08 Homo sapiens, quinone oxidoreductase homolog, clone MGC:8642, mRNA, complete cds.

-863,0 -2,24 Homo sapiens cysteine and glycine-rich protein 2 (CSRP2), mRNA.

-862,9 -2,20 Homo sapiens hemopoietic cell kinase (HCK), mRNA.

-862,9 -2,20 Human apolipoprotein Al regulatory protein (ARP-1) mRNA, complete cds.

-858,9 -2,04 Homo sapiens cytochrome c-1 (CYC 1), mRNA.

-855,0 -8,20 Homo sapiens LR8 protein (LR8), mRNA.

-852,0 -4,32 Consensus includes Homo sapiens 23262 ribonuclease, RNase A family, k6

-851,4 -2,68 Homo sapiens frizzled (Drosophila) homolog 1 (FZD1), mRNA.

-845,6 -2,96 Consensus includes Homo sapiens cDNA FLJ12112 fis, clone MAMMA1000043

-838,9 -2,68 Homo sapiens 172207 non-POU-domain-containing, octamer-binding

-784,3 -2,28 Homo sapiens microsomal glutathione S-transferase 2 (MGST2), mRNA.

-782,7 -4,92 Homo sapiens downregulated in ovarian cancer 1 (DOC1), mRNA.

-778,0 -6,88 Consensus includes Homo sapiens 198253 major histocompatibility complex, class II, DQ alpha 1

-770,3 -2,08 Consensus includes Homo sapiens 106070 cyclin-dependent kinase inhibitor 1C (p57, Kip2)

-770,0 -2,64 Homo sapiens, clone MGC:4419, mRNA, complete cds. Homo sapiens 185055 BENE protein , _sn Consensus includes Homo sapiens 8265 transglutaminase 2 (C polypeptide, protein-glutamine- gamma-glutamyltransferase)

-767,1 -4,48 Homo sapiens 57958 EGF-TM7-latrophilin-related protein

-767,1 -3,08 Consensus includes Homo sapiens 177781 Homo sapiens, clone MGC:5618, mRNA, complete cds

-764,2 -2,04 Homo sapiens complement component 2 (C2), mRNA.

-761,2 -7,44 Consensus includes Homo sapiens 73931 major histocompatibility complex, class II, DQ beta 1

-756,2 -3,16 Human T-cell receptor rearranged beta-chain V-region (V-D-J) mRNA, complete cds.

-752,6 -2,24 Consensus includes Homo sapiens 79307 RacCdc42 guanine exchange factor (GEF) 6

-743,9 -2,08 Consensus includes Homo sapiens 166982 phosphatidylinositol glycan, class F

-743,6 -5,24 Homo sapiens cadherin 5, type 2, VE-cadherin (vascular epithelium) (CDH5), mRNA.

- ., „ . ₀₀ Homo sapiens, Similar to immunoglobulin kappa constant, clone MGC: 12418, mRNA, complete

-/4₃, -4,oo ■ cds.

-741,5 -2,56 Homo sapiens FXYD domain-containing ion transport regulator 6 (FXYD6), mRNA. V* F* Description

-736,5 -2,20 Homo sapiens WD repeat domain 6 (WDR6), mRNA.

-732,0 -2,84 Cluster Inch Homo sapiens Jagged2 (JAG2) mRNA, complete cds

-725,9 -3, 12 Homo sapiens tetranectin (plasminogen-binding protein) (TNA), mRNA.

-720,9 -4,44 Homo sapiens solute carrier family 21 (prostaglandin transporter), member 2 (SLC21 A2), mRNA.

-717 2 2 08 Consensus includes Homo sapiens 250773 signal sequence receptor, alpha (translocon-associated protein alpha) 708 9 3 00 Human Lyn B protein mRNA, complete cds. =Homo sapiens 80887 v-yes-1 Yamaguchi sarcoma ' ' viral related oncogene homolog 706 3 7 97 Homo sapiens special AT-rich sequence binding protein 1 (binds to nuclear matrixscaffold- ' ' associating DNAs) (SATBl), mRNA.

-703,8 -2,04 Consensus includes Homo sapiens mRNA for KIAA0328 protein, partial cds.

-695,8 -2,40 Homo sapiens 80426 brain and reproductive organ-expressed (TNFRSF1 A modulator)

-691,1 -3,28 Homo sapiens bone morphogenetic protein 1 (BMP1), transcript variant BMP1-4, mRNA. 688 3 2 32 Consensus includes Homo sapiens 168383 intercellular adhesion molecule 1 (CD54), human ' ' rhinovirus receptor

-686,6 -2,80 Homo sapiens opsin 3 (encephalopsin) (OPN3), mRNA.

-672,5 -5,16 Homo sapiens putative integral membrane transporter (LC27), mRNA.

-671,2 -2,88 Consensus includes Homo sapiens 237825 signal recognition particle 72kD

-670,6 -2,84 Homo sapiens calcitonin receptor-like (CALCRL), mRNA.

-669, 1 -4,48 Homo sapiens MSTP032 protein (MSTP032), mR A.

-667,8 -2,60 Consensus includes Homo sapiens 239489 TIAl cytotoxic granule-associated RNA-binding protein

-666,3 -3,72 Consensus includes Homo sapiens 75709 mannose-6-phosphate receptor (cation dependent)

-663,1 -2,52 Homo sapiens testin (DKFZP586B2022), mRNA.

-655,0 -3,56 Homo sapiens paraoxonase (PON2) mRNA, with alternatively spliced exon 3, complete cds.

-653,2 -2,52 Homo sapiens Homer, neuronal immediate early gene, 3 (HOMER-3), mRNA.

-652,4 -2,36 Homo sapiens protocadherin 12 (PCDH12), mRNA.

-645,9 -2,84 Homo sapiens intercellular adhesion molecule 2 (ICAM2), mRNA.

-644,4 -2,80 Homo sapiens KIA A 1036 protein (KIAA 1036), mRNA.

-636,2 -2,12 Homo sapiens formyi peptide receptor 1 (FPR1), mR A.

-627,3 -2,32 Homo sapiens catalase (CAT) mRNA, complete cds.

-625,5 -2,08 Consensus includes Homo sapiens 155976 cullin 4B

-619,7 -4,00 Homo sapiens six transmembrane epithelial antigen of the prostate (STEAP), mRNA.

-617,4 -3,80 Homo sapiens regulator of G-protein signalling 5 (RGS5) mRNA, complete cds.

-615,3 -2,24 Homo sapiens hypothetical protein FLJ20093 (FLJ20093), mRNA.

-610,1 -2,36 Homo sapiens hypothetical protein FLJ20006 (FLJ20006), mRNA.

-607,3 -3,24 Homo sapiens protein kinase (cAMP-dependent, catalytic) inhibitor gamma (PKIG), mRNA.

-603,8 -4,16 Homo sapiens vascular endothelial junction-associated molecule (VE-JAM), mRNA.

-602,4 -2,00 Homo sapiens guanylate binding protein 2, interferon-inducible (GBP2), mRNA.

-601,9 -2,28 Consensus includes Homo sapiens 100293 O-linked N-acetylglucosamine (GlcNAc) transferase

-599,2 -2,52 Homo sapiens hypothetical protein FLJ20401 (FLJ20401), mRNA.

-597,3 -4,40 Homo sapiens hypothetical protein FKSG63 (FKSG63), mRNA. ,_q. , „ „„ Homo sapiens lymphocyte cytosolic protein 2 (SH2 domain-containing leukocyte protein of 76kD) ^lfi -Z,³Z _(LCP2)_; mRNA.

-589,2 -3,68 Consensus includes Homo sapiens 89643 transketolase (Wernicke-Korsakoff syndrome)

-577,9 -2,56 Cluster Inch Human semaphorin III family homolog mRNA, complete cds V* F* Description -570,0 -4,20 Homo sapiens ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), mRNA. -569,0 -2,64 Homo sapiens, HBS1 (S. cerevisiae)-like, clone MGC:1869, mRNA, complete cds. -557,6 -2,44 Homo sapiens CGI-27 protein (LOC51072), mRNA. -554,9 -3,08 Consensus includes Homo sapiens 78746 phosphodiesterase 8A -554,8 -3,00 Homo sapiens latrophilin (KIAA0786), mRNA. -552,4 -3,40 Consensus includes Homo sapiens 79283 selectin P ligand -551 ,5 -2,72 Homo sapiens EphB4 (EPHB4) mRNA. 546 8 -2 60 Human cbl-b mRNA, complete cds. =Homo sapiens 3144 Cas-Br-M (murine) ectropic retroviral transforming sequence b -542,6 -4,28 Consensus includes Homo sapiens 97199 complement component C 1 q receptor -542,0 -3,28 Homo sapiens hypothetical protein FLJ 13465 (FLJ 13465), mRNA. -535,8 -2,76 Consensus includes Homo sapiens 13453 splicing factor 3b, subunit 1, 155kD „ . „ ,„ Homo sapiens 301724 Homo sapiens mRNA; cDNA DKFZp434E248 (from clone -^3/4,^/ -z,ou _DKFZp434_E248); complete cds -b523,5 -2,24 Homo sapiens glioma pathogenesis-related protein (RTVP1), mRNA. -522,2 -3,08 Homo sapiens transmembrane 4 superfamily member 2 (TM4SF2), mRNA. .-,. . - „„ Homo sapiens claudin 5 (transmembrane protein deleted in velocardiofacial syndrome) (CLDN5), mRNA. -519,4 -3,16 Homo sapiens Rab27 isoform mRNA, complete cds. -515,4 -2,60 Human mitochondrial N AD(P)+ dependent malic enzyme mRNA, complete cds.

Table la is a preferred selection of the polynucleotide library of Table 1 and includes 23 of the most relevant genes and gene expression products (including mRNA).

In principle, it could be shown that at least 15 members of the genes and gene expression products as specified in at least one of the Tables 1 and 2 play an important role in osseointegration of dental implants. It could be shown that preferably 15 - 50, more preferably 20 - 30 members of any of Tables 1 and 2 are, as a rule, sufficient to cover the most relevant genes, gene transcripts and gene translation products participated in up- and / or downregulation in osseointegration of dental implants allowing to obtain a discriminating gene pattern, namely to distinguish between normal individuals and patients suffering from failing or suboptimum osseointegration caused by dental implants made from plastics and metals, including surface-modified titanium, titanium alloys, titanium oxides, titanium coatings with bone cements, silicon, organic polymers, or polypeptides influencing adhesion and growth factors. The invention relates also to a polynucleotide library useful to differentiate a normal cell from a cell of patients with regular and failing osseointegration wherein the pool of polynucleotide sequences or fragments thereof correspond substantially to any combination of at least one polynucleotide sequence selected among those included in each one of predefined polynucleotide sequences sets indicated above, useful in differentiating a normal cell from a cell from patients suffering from failing osseointegration. Differences in gene expression are accepted as different when the signals from patient material are at least two fold lower or higher than those from comparative material, be it from healthy volunteers material or from other diseased material. This threshold is commonly accepted for the evaluation of expression arrays. The invention additionally provides a method for identifying gene expression or genomic DNA of infective agents including bacteria, yeasts, fungi, or viruses as cellular parasites in cells from patients with failing osseointegration.

In summary the invention relates to the following items:

• A polynucleotide array essentially consisting of a solid support and a specific polynucleotide library representing the genetic situation under testing or reference conditions in the environment of dental implants in the human jaw bone, the library being composed of a cluster of different probes of cDNA, RNA or mRNA, wherein said probes are immobilized on said solid support and derive from samples of human tissue obtained from the direct environment of an artificial dental prosthesis which has been implanted into the jaw of a human individual, and said cluster comprises a number of members of cDNA, RNA, mRNA or gene translation products related thereto, as specified in Table 1, Table 2, wherein said number comprises at least fifteen members of these Tables.

• A corresponding polynucleotide array, wherein said gene cluster comprises the members of cDNA, RNA, or mRNA, or translation products related thereto as specified in Table la.

• A corresponding polynucleotide array, wherein the artificial dental prosthesis causes a pathological condition in the tissue surrounding it compared with the natural bone, or a reference implant causing essentially no pathological condition under the same conditions.

• A corresponding polynucleotide array, wherein the pathological condition is inflammation due to fibroproliferative processes during disintegrated wound healing, or an exaggerated immune response to the artificial implant. • A corresponding polynucleotide array, wherein the genes of the gene cluster related to said artificial dental prosthesis are upregulated and / or overexpressed compared to the corresponding genes related to the natural bone or said reference implant. • A corresponding polynucleotide array, wherein the pathological conditition is due to misdirected or failing vascularization of bone tissue or failing production of growth factors in context with the implantation of said artificial dental prosthesis.

• A corresponding polynucleotide array, wherein the genes of the gene cluster related to said artificial dental prosthesis are downregulated and / or underexpressed compared to the corresponding genes related to the natural bone or said reference implant.

• A corresponding polynucleotide array, wherein said reference implant essentially consists of polished titanium.

• A corresponding polynucleotide array, wherein said artificial dental prosthesis essentially causes no pathological condition in the tissue surrounding it compared with the natural bone under the same conditions.

• A corresponding polynucleotide array, wherein said artificial dental prosthesis essentially consists of polished titanium

• The use of a polynucleotide array as defined above, for ex-vivo testing of an artificial dental implant or a modification thereof in context with its biological acceptance in view of misdirected or failed osseointegration and / or fibrotic wound healing.

• The corresponding use, wherein said osseointegration and wound healing is further influenced by the individual's condition or disease.

• The corresponding use, wherein said osseointegration and wound healing is further influenced by a pharmaceutically effective drug.

• The corresponding use, wherein said individual's condition or disease is further influenced by a pharmaceutically effective drug.

• The corresponding use, wherein said pharmaceutically effective drug is an agonist or an antagonist of one ore more of the genes, gene transcripts or gene translation products as depicted in Table 1, Table la and Table 2.

• A method for generating a specific polynucleotide library representing the genetic situation under testing conditions or reference conditions in the environment of dental implants in the jaw bone of a human individual, and comprising the genes, gene transcripts or gene translation products as depicted in Table 1, Table la and Table 2, the method comprising the steps: (i) isolating RNA, mRNA or cDNA from a reference sample deriving from the tissue which was taken from the direct environment of a reference dental prosthesis implanted in the jaw bone of a human individual, wherein said reference dental prosthesis causes an essentially regular and non-pathological osseointegration under reference conditions; (ii) isolating RNA, mRNA or cDNA from a testing sample deriving from the tissue which was taken from the direct environment of a testing dental prosthesis implanted in the jaw bone of a human individual, wherein said testing dental prosthesis may cause a misdirected or pathological osseointegration under testing conditions; (iii) hybridizing each of said polynucleotide probes of the reference and testing sample obtained in steps (i) and (ii) with polynucleotide probes of the human genome immobilized on the support of a human genome array. (iv) separating and identifying the hybridization products of the reference and testing sample obtained in step (iii) by standard separating and detecting means, (v) comparing the identified hybridization products of the testing sample to that of the reference sample, and (vi) selecting those identified polynucleotides which are overxepressed or underexpressed in the testing sample and reference sample, respectively, compared to the corresponding reference sample and testing sample, respectively, or vice versa, thus forming the respective specific polynucleotide library

• A corresponding method, wherein the misdirected or pathological osseointegration is due to inflammation and fibroproliferative processes during disintegrated wound healing.

• A corresponding method, wherein a reference dental prosthesis is used which is made of polished titanium.

• A corresponding method, wherein the tissues of the reference and testing sample derive from the bone or root canal or chamber or the vicinity thereof.

• A corresponding method, wherein the tissue of the testing and / or reference sample is taken from an individual in a specific condition or suffering from a specific disease. • A corresponding method, wherein the individual's condition or disease is further influenced by a pharmaceutically effective drug.

• A method for preparing a polynucleotide array as specified above by immobilizing the polynucleotide probes of the polynucleotide library obtained by a method as specified above, onto a solid support of an array or chip matrix. • A method of testing an artificial dental prosthesis with respect to its biological acceptance under specific conditions after implantation into the jaw bone of a human individual by means of generating genetic profiles, in comparison to a reference implant with a known respective genetic profile, the method comprises the steps: (i) isolating RNA, mRNA or cDNA deriving from tissue taken from the direct environment of said artificial dental prosthesis,; (ii) hybridizing each of said polynucleotide probes obtained in steps (i) with the polynucleotide probes of a reference polynucleotide array representing the genetic profile of the reference implant, wherein said reference array is an array as specified above, and (iii) identifying quality and quantity of the hybridization products and specifying thereof the genes which are overexpressed or underexpressed in comparison to said reference implant.

EXAMPLE Functional clusters of gene activation may be defined by different approaches: a. the search for single gene mutations, b. by knocking out gene functions that have been attributed to specific cellular functions by use of in vitro cell systems, and c. by defining the coherent functions of gene transcription within a context that is clinically defined. The first opportunity is hampered by the fact that mutations are scarcely the only reason for complex functional pathologies, the second by the fact that cell lines in most instances will not represent the biology of a given tissue, and the last one by the complexity and the sheer number of genes involved in a complex regulation. On the other hand, this approach provides the unique possibility of avoiding the dangerous bias of "picking" target gene functions solely in accordance to one's own perception. To be effective, this opportunity requires the construction of several distinct clinical 'filter' systems which allow for the sorting of gene functions that are intimately involved within the regulative process in question. Using the approach of transcriptional screening, it is possible to generate several effective filter systems for fibrotic wound healing by use of different fibrosing diseases including fibrotic osseointegration, by use of in vitro culture of fibro(myo)blasts from fibrotic tissue and by developing the first therapeutic approach directed against fibrotiv wound healing itself. This approach has also been used for sorting out gene functions that are directly involved in the functional steering process of misdirected wound healing in fibrosis. In the case of osseointegration of dental implants, it is necessary to define four functional states: A. Effective osseointegration defined by material B. Effective osseointegration defined by material and bone condition C. Fibrotic osseointegration defined by material D. Fibrotic osseointegration defined by material and bone condition Thus, the first approach used was to define the conditions B., C. and (partly) D.: by applying two implants representing different material conditions (fibrosing vs. non-fibrosing) into one bone condition (healthy), the first subtractive transcriptional screen was generated. It represents gene functions that are helpful in osseointegration (up-regulated gene functions in the case of regular osseointegration by titanium implants) and must be avoided to hinder fibrotic osseointegration (down-regulated gene functions by titanium versus plastic implants). This extremely valuable set of data provides the basis for defining condition A, especially in the case of patients with critical bone (conditions A., B. and D.). Each step of the process will reduce the number of gene functions critical for effective wound healing in osseointegration and by that "pick" the functional ones. This effect has already been observed in the process of influencing fibrotic wound healing in the lung leading to and at the same time defining the first therapeutical approach for fibrotic wound healing, which was the initial strategy used according to this invention. While doing so, another set of data directly related to the process of fibrotic wound healing representing a different organ background has been generated that will serve as the positive control for the "entity" of misdirected wound healing itself. The strategy outlined above will not only generate a unique test system for final definition of the process of effective osseointegration, but also the means to generate and control future implant surfaces. Every patient obtains two dental implants. The first one is an implant based on polyethylene which causes fibrosis during wound healing with a high probability. The second one is based on pure titanium which is known for regular wound healing without inflammation in most of the cases. Patients are investigated in two course scheme groups (5 patients of each group). Group A: Explantation of the implants after two weeks of implantation; Group B: Explantation of the implants after four weeks of implantation. From each patient two bone biopsies are taken before implantation (TO). After two weeks (Tl) tissue samples from the jaw bone around the implant of Group I patients are taken, as well as after four weeks (T2) the corresponding samples from Group II patients. This procedure allows the determination of differences regarding the efficacy of the implants used as well as the sequential analysis of the possible occurrence of pathological conditions such as inflammation and tissue fibrosis. All samples obtained by these biopsies are investigated by histological and molecular biotechnological means. For this reason the tissue in the direct vicinity of the lower region of the implant was removed for isolation of cytoplasm RNA. Molecular analysis of the bone material obtained as described is carried out by means of real - 5 time RT-PCR, and gene transcription is investigated for genes which are characteristic and responsible for the following physiological processes: - Th2-dominat chronic cellular inflammatory and fibrosing processes (IFN-gamma, IL-13), - growth factor production (TGF-beta, CTGF), - activation of osteoblasts during regular bone cell growth (BMP-2, Runx2, osteocalcin), I o - regulation of apoptosis. Furthermore, the treatment is controlled by x-ray analysis and computer tomography.

Claims

PATENT CLAIMS

1. A polynucleotide array essentially consisting of a solid support and a specific polynucleotide library representing the genetic situation under testing or reference conditions in the environment of dental implants in the human jaw bone, the library being composed of a cluster of different probes of cDNA, RNA or mRNA, wherein said probes are immobilized on said solid support and derive from samples of human tissue obtained from the direct environment of an artificial dental prosthesis which has been implanted into the jaw of a human individual, and said cluster comprises a number of members of cDNA, RNA, mRNA or gene translation products related thereto, as specified in Table 1 , Table 2, wherein said number comprises at least fifteen members of these Tables.

2. A polynucleotide array according claim 1, wherein said gene cluster comprises the members of cDNA, RNA, or mRNA, or translation products related thereto as specified in Table la.

3. A polynucleotide array according to claim 1 or 2, wherein the artificial dental prosthesis causes a pathological condition in the tissue surrounding it compared with the natural bone, or a reference implant causing essentially no pathological condition under the same conditions.

4. A polynucleotide array according to claim 3, wherein the pathological condition is inflammation due to fibroproliferative processes during disintegrated wound healing, or an exaggerated immune response to the artificial implant.

5. A polynucleotide array according to claim 4, wherein the genes of the gene cluster related to said artificial dental prosthesis are upregulated and / or overexpressed compared to the corresponding genes related to the natural bone or said reference implant.

6. A polynucleotide array according to claim 3, wherein the pathological conditition is due to misdirected or failing vascularization of bone tissue or failing production of growth factors in context with the implantation of said artificial dental prosthesis.

7. . A polynucleotide array according to claim 5, wherein the genes of the gene cluster related to said artificial dental prosthesis are downregulated and / or underexpressed compared to the corresponding genes related to the natural bone or said reference implant.

8. A polynucleotide array according to any of the claims 3 - 7, wherein said reference implant essentially consists of polished titanium.

9. A polynucleotide array according to claim 3, wherein said artificial dental prosthesis essentially causes no pathological condition in the tissue surrounding it compared with the natural bone under the same conditions.

10. A polynucleotide array according to claim 9, wherein said artificial dental prosthesis essentially consists of polished titanium

11. Use of a polynucleotide array according to any of the claims 1 - 10, for ex-vivo testing of an artificial dental implant or a modification thereof in context with its biological acceptance in view of misdirected or failed osseointegration and / or fibrotic wound healing.

12. Use of claim 11, wherein said osseointegration and wound healing is further influenced by the individual's condition or disease.

13. Use of claim 11, wherein said osseointegration and wound healing is further influenced by a pharmaceutically effective drug.

14. Use according to claim 12, wherein said individual's condition or disease is further influenced by a pharmaceutically effective drug.

15. Use according to claim 13 or 14, wherein said pharmaceutically effective drug is an agonist or an antagonist of one ore more of the genes, gene transcripts or gene translation products as depicted in Table 1, Table la and Table 2.

16. A method for generating a specific polynucleotide library representing the genetic situation under testing conditions or reference conditions in the environment of dental implants in the jaw bone of a human individual, and comprising the genes, gene transcripts or gene translation products as depicted in Table 1, Table la and Table 2, the method comprising the steps: (i) isolating RNA, mRNA or cDNA from a reference sample deriving from the tissue which was taken from the direct environment of a reference dental prosthesis implanted in the jaw bone of a human individual, wherein said reference dental prosthesis causes an essentially regular and non-pathological osseointegration under reference conditions; (ii) isolating RNA, mRNA or cDNA from a testing sample deriving from the tissue which was taken from the direct environment of a testing dental prosthesis implanted in the jaw bone of a human individual, wherein said testing dental prosthesis may cause a misdirected or pathological osseointegration under testing conditions; (iii) hybridizing each of said polynucleotide probes of the reference and testing sample obtained in steps (i) and (ii) with polynucleotide probes of the human genome immobilized on the support of a human genome array. (iv) separating and identifying the hybridization products of the reference and testing sample obtained in step (iii) by standard separating and detecting means, (v) comparing the identified hybridization products of the testing sample to that of the reference sample, and (vi) selecting those identified polynucleotides which are overxepressed or underexpressed in the testing sample and reference sample, respectively, compared to the corresponding reference sample and testing sample, respectively, or vice versa, thus forming the respective specific polynucleotide library

17. A method according to claim 16, wherein the misdirected or pathological osseointegration is due to inflammation and fibroproliferative processes during disintegrated wound healing.

18. A method of claim 16 or 17, wherein a reference dental prosthesis is used which is made of polished titanium.

19. A method according to any of the claims 16 - 18, wherein the tissues of the reference and testing sample derive from the bone or root canal or chamber or the vicinity thereof.

20. A method according to any of the claims 16 - 19, wherein the tissue of the testing and / or reference sample is taken from an individual in a specific condition or suffering from a specific disease.

21. A method of claim 20, wherein the individual's condition or disease is further influenced by a pharmaceutically effective drug.

22. A method for preparing a polynucleotide array as specified in any of the claims 1 - 10 by immobilizing the polynucleotide probes of the polynucleotide library obtained by a method of any of the claims 14 - 19, onto a solid support of an array or chip matrix.

23. A method of testing an artificial dental prosthesis with respect to its biological acceptance under specific conditions after implantation into the jaw bone of a human individual by means of generating genetic profiles, in comparison to a reference implant with a known respective genetic profile, the method comprises the steps: (i) isolating RNA, mRNA or cDNA deriving from tissue taken from the direct enviromnent of said artificial dental prosthesis,; (ii) hybridizing each of said polynucleotide probes obtained in steps (i) with the polynucleotide probes of a reference polynucleotide array representing the genetic profile of the reference implant, wherein said reference array is the array of claim 9 or the array of any of the claims 3 - 8, and (iii) identifying quality and quantity of the hybridization products and specifying thereof the genes which are overexpressed or underexpressed in comparison to said reference implant.