ESTUDIO GMI. ESTUDIO PROTEÓMICO-COMPARACIÓN IMPLANTES DENTALES
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1 ESTUDIO GMI. ESTUDIO PROTEÓMICO-COMPARACIÓN IMPLANTES DENTALES
2 2 Hypothesis. Proteomic study of first protein layer Post-implantation, the biomaterial becomes in contact with the blood, resulting in protein adsorption on its surface (Andersson, 2005).
3 3 Proteomic study of first protein layer The physico-chemical characteristics influence the composition and conformation of the adsorbed proteins Cell Protein Biomaterial Superficial density Conformation Interaction strength ECM protein cell Substrate Chemical Topography Mechanical 3
4 4 Protein importance on biomaterial The first layer of adsorbed proteins will define the biological response to the material (Engberg, 2015). CELL Cell-Protein-Material interaction Regeneration
5 5 Protein importance on biomaterial Why not study the protein layer formed onto a biomaterial surface? Would it be possible to detect differences in the layer composition? Would it be possible to predict the biological response in vivo to the biomaterial using proteomics? CELL Cell-Protein-Material interaction Regeneration
6 6 PART 2. GMI proteomic study. Osteogenesis Immune response Bone tissue regeneration Fibrinolysis Bood coagulation
7 7 Proteomics: methodology 1 2 Incubation with human serum (3h 37ºC) Consecutive washes for the removal of non-adsorbed proteins H2O Mili-Q 100mM NaCl Tris-HCl ph 7.1 N=4 (4 implants)
8 8 Proteomics: methodology 1 Incubation with human serum (3h 37ºC) Biomaterial surface-attached protein elution 4% SDS, 100 mm DTT in 0.5M TEAB Final elution 2 Consecutive washes for the removal of non-adsorbed proteins H2O Mili-Q 100mM NaCl Tris-HCl ph 7.1 3
9 9 Proteomics: methodology 1 Biomaterial surface-attached protein elution 4% SDS, 100 mm DTT in 0.5M TEAB 2 Incubation with human serum (3h 37ºC) Final elution Consecutive washes for the removal of non-adsorbed proteins H2O Mili-Q 100mM NaCl Tris-HCl ph 7.1 Quantification and identification: Mass Spectrometry (MS/MS) 3
10 10 Proteomics: methodology 1 Biomaterial surface-attached protein elution 4% SDS, 100 mm DTT in 0.5M TEAB 2 Incubation with human serum (3h 37ºC) Final elution Consecutive washes for the removal of non-adsorbed proteins H2O Mili-Q 100mM NaCl Tris-HCl ph 7.1 Data analysis: PROGENESIS DAVID (Database for Annotation, Visualization and Integrated Discovery) Biological data base of protein families, useful to protein function identification and classification 4 Quantification and identification: Mass Spectrometry (MS/MS) 3
11 Proteomic comparative study ST GMI NR
12 Comparison GMI vs NR & ST Valor diferencial GMI Affinity GMI Accession Peptide count Unique peptides GMI/NR GMI/ST Description p value Ratio p value Ratio PROC_HUMAN 8 8 Vitamin K-dependent protein C 3,34E-06 17,56 1,17E-06 27,21 FA10_HUMAN 8 8 Coagulation factor X 2,40E-06 13,92 1,86E-04 17,21 FA7_HUMAN 3 3 Coagulation factor VII 1,80E-03 8,40 1,04E-04 16,27 TAGL2_HUMAN 2 2 Transgelin-2 3,20E-04 6,54 2,40E-05 11,54 SAA1_HUMAN 2 2 Serum amyloid A-1 protein 5,44E-04 4,33 1,46E-02 5,48 FA9_HUMAN 7 7 Coagulation factor IX 1,44E-02 2,82 2,44E-04 5,47 APOC1_HUMAN 3 3 Apolipoprotein C-I 3,51E-04 4,17 3,58E-04 5,05 FCN3_HUMAN 2 2 Ficolin-3 1,86E-03 3,22 5,42E-03 4,65 CAMP_HUMAN 2 2 Cathelicidin antimicrobial peptide 3,67E-02 2,59 3,93E-02 4,37 APOE_HUMAN Apolipoprotein E 1,32E-03 3,97 1,17E-03 3,61 APOC4_HUMAN 5 5 Apolipoprotein C-IV 7,95E-04 3,14 2,27E-05 3,60 APOA4_HUMAN Apolipoprotein A-IV 1,50E-05 3,81 1,83E-05 3,54 PROS_HUMAN Vitamin K-dependent protein S 6,52E-04 2,98 2,07E-03 2,87 C4BPB_HUMAN 2 2 C4b-binding protein beta chain 1,73E-02 1,96 4,79E-02 2,54 APOA1_HUMAN Apolipoprotein A-I 6,92E-06 3,64 2,38E-04 2,49 ANT3_HUMAN Antithrombin-III 3,58E-04 2,35 6,32E-04 2,33 THRB_HUMAN Prothrombin 2,09E-03 2,04 9,76E-04 2,31 C4BPA_HUMAN C4b-binding protein alpha chain 9,67E-04 2,76 9,04E-03 2,25 RAP1B_HUMAN 2 2 Ras-related protein Rap-1b 2,60E-02 2,22 2,17E-02 2,00 PON1_HUMAN Serum paraoxonase/arylesterase 1 5,56E-04 2,17 3,35E-03 1,75
13 Comparison GMI vs NR & ST Valor diferencial GMI Affinity GMI Accession Peptide count Unique peptides GMI/NR GMI/ST Description p value Ratio p value Ratio PROC_HUMAN 8 8 Vitamin K-dependent protein C 3,34E-06 17,56 1,17E-06 27,21 FA10_HUMAN 8 8 Coagulation factor X 2,40E-06 13,92 1,86E-04 17,21 FA7_HUMAN 3 3 Coagulation factor VII 1,80E-03 8,40 1,04E-04 16,27 TAGL2_HUMAN 2 2 Transgelin-2 3,20E-04 6,54 2,40E-05 11,54 SAA1_HUMAN 2 2 Serum amyloid A-1 protein 5,44E-04 4,33 1,46E-02 5,48 FA9_HUMAN 7 7 Coagulation factor IX 1,44E-02 2,82 2,44E-04 5,47 APOC1_HUMAN 3 3 Apolipoprotein C-I 3,51E-04 4,17 3,58E-04 5,05 FCN3_HUMAN 2 2 Ficolin-3 1,86E-03 3,22 5,42E-03 4,65 CAMP_HUMAN 2 2 Cathelicidin antimicrobial peptide 3,67E-02 2,59 3,93E-02 4,37 APOE_HUMAN Apolipoprotein E 1,32E-03 3,97 1,17E-03 3,61 APOC4_HUMAN 5 5 Apolipoprotein C-IV 7,95E-04 3,14 2,27E-05 3,60 APOA4_HUMAN Apolipoprotein A-IV 1,50E-05 3,81 1,83E-05 3,54 PROS_HUMAN Vitamin K-dependent protein S 6,52E-04 2,98 2,07E-03 2,87 C4BPB_HUMAN 2 2 C4b-binding protein beta chain 1,73E-02 1,96 4,79E-02 2,54 APOA1_HUMAN Apolipoprotein A-I 6,92E-06 3,64 2,38E-04 2,49 ANT3_HUMAN Antithrombin-III 3,58E-04 2,35 6,32E-04 2,33 THRB_HUMAN Prothrombin 2,09E-03 2,04 9,76E-04 2,31 C4BPA_HUMAN C4b-binding protein alpha chain 9,67E-04 2,76 9,04E-03 2,25 RAP1B_HUMAN 2 2 Ras-related protein Rap-1b 2,60E-02 2,22 2,17E-02 2,00 PON1_HUMAN Serum paraoxonase/arylesterase 1 5,56E-04 2,17 3,35E-03 1,75
14 Comparison GMI vs NR & ST Affinity GMI p value GMI/NR CO8A_HUMAN 6 6 Complement component C8 alpha chain 1,86E-02 0,48 2,48E-02 0,51 CO5_HUMAN Complement C5 2,05E-02 0,57 4,78E-02 0,49 FETUA_HUMAN 7 7 Alpha-2-HS-glycoprotein 9,65E-03 0,50 1,08E-03 0,40 KNG1_HUMAN Kininogen-1 4,16E-05 0,18 6,98E-05 0,33 CFAB_HUMAN Complement factor B 1,59E-03 0,36 2,06E-02 0,33 FA11_HUMAN Coagulation factor XI 3,64E-03 0,18 6,27E-03 0,33 AMBP_HUMAN 8 8 Protein AMBP 1,75E-02 0,36 5,48E-03 0,32 IGHA1_HUMAN 11 4 Ig alpha-1 chain C region 3,58E-02 0,40 1,34E-03 0,28 FCN1_HUMAN 2 2 Ficolin-1 9,97E-04 0,34 7,85E-03 0,26 IGHA2_HUMAN 8 1 Ig alpha-2 chain C region 4,70E-02 0,33 5,53E-03 0,24 HPT_HUMAN Haptoglobin 4,57E-03 0,24 4,07E-03 0,22 CERU_HUMAN Ceruloplasmin 4,97E-03 0,24 3,71E-04 0,22 CBG_HUMAN 2 2 Corticosteroid-binding globulin 6,75E-04 0,27 5,97E-03 0,22 ANGT_HUMAN 6 6 Angiotensinogen 2,81E-03 0,32 2,07E-03 0,21 AACT_HUMAN Alpha-1-antichymotrypsin 2,49E-02 0,28 4,84E-04 0,20 ITIH1_HUMAN 10 9 Inter-alpha-trypsin inhibitor heavy chain H1 6,01E-03 0,30 2,50E-03 0,19 ALBU_HUMAN Serum albumin 4,64E-02 0,28 1,13E-02 0,18 A1AT_HUMAN Alpha-1-antitrypsin 9,39E-03 0,24 1,05E-03 0,17 TRFE_HUMAN Serotransferrin 1,33E-02 0,18 5,42E-03 0,15 A1AG2_HUMAN 7 5 Alpha-1-acid glycoprotein 2 1,29E-02 0,13 2,30E-02 0,12 A1AG1_HUMAN 8 6 Alpha-1-acid glycoprotein 1 6,34E-03 0,13 3,51E-03 0,12 ZA2G_HUMAN Zinc-alpha-2-glycoprotein 9,72E-04 0,17 7,13E-04 0,12 AFAM_HUMAN Afamin 6,15E-03 0,16 1,22E-03 0,12 A2MG_HUMAN Alpha-2-macroglobulin 9,66E-03 0,15 3,84E-03 0,11 HEMO_HUMAN Hemopexin 4,43E-02 0,27 4,91E-03 0,10 A1BG_HUMAN 9 9 Alpha-1B-glycoprotein 4,38E-03 0,15 2,28E-03 0,09 A2GL_HUMAN 4 4 Leucine-rich alpha-2-glycoprotein 1,77E-02 0,09 6,02E-03 0,08 Ratio p value GMI/ST Ratio
15 15 Coagulation cascade This protein display a key role in blood clotting Osteogenesis Immune response Bone tissue regeneration Bood coagulation Fibrinolysis However, blood coagulation is a high regulated system. ANT3, PROC, PROS could ensure the correct process development in order to avoid an acute inflammation and thrombosis
16 16 Immune response Complement system cascade Desproportionated adsoption of complement system proteins was not detected. Osteogenesis Immune response Bone tissue regeneration Fibrinolysis Bood coagulation
17 17 Osteogenesis Osteogenesis Immune response Bone tissue regeneration Fibrinolysis Valor diferencial GMI Bood coagulation
18 18 Key protein? Proteómica Sr Proteómica Ca SAE-Ti vs Ti
19 19 Papel de la vitamina K y la ApoE: importancia en el metabolismo óseo Transporte de Vitamina K a través de Apo E Rodrigues, A. et al. Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures. Bone 51, (2012)
20 20 Osteogenic potential ApoE Increase osteoblastic differentiation Decrease osteoclastic differentiation
21 21 Antiinflammatory potential ApoE Macrophage polarization M1 Mф M2 M1 M2 Formation of new bone Early phase Bone formation Bone remodellation Formation fibrous capsule M1 Acute inflammation Chronic inflammation Fibrous encapsulation M2
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