Molecular characterisation of odontoblast during primary, secondary ...

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odontoblasts and pulp cells is evident and also in Hertwig’s Epithelial Root Sheath cells (B). Counterstained with haematoxylin. A: Bar=150µm, B,C,D: Bar= 60µm. ..... 106 Figure 36: Immunohistochemical analysis using anti-mouse beta-galactosidase antibody on first upper molars of 21 days post natal Msx2 +/- mice. Counterstained with haematoxylin. A: Bar=150µm, B,C,D: Bar= 60µm. ........................................... 106 Figure 37: Immunohistochemical analysis using anti-mouse beta-galactosidase antibody on first upper molar of 21 days post natal Msx2 -/- mice. Counterstained with haematoxylin. A: Bar=150µm, B,C,D: Bar= 60µm. ........................................... 107 Figure 38: Immunohistochemical analysis using anti-mouse DSP antibody on a first upper molar of 21 days post natal Msx2 -/- mouse. Counterstained with haematoxylin. A: Bar=150µm, B,C,D: Bar= 60µm, E: Bar=20µm. ............................................ 108 Figure 39: Coronal area of a first upper molar of a 21 days post natal Msx2 -/- mouse,. A: 7μm thickness, haematoxylin and eosin staining. B: Immunohistochemical analysis using anti-mouse DSP antibody. Counterstained with haematoxylin. Bar 70µm. DSP immunoreactivity is clearly visible for the embedded material in the dentine thickness. ....................................................................................................... 108 Figure 40: Immunohistochemical analysis using anti-mouse BrdU antibody on 14 days post natal Msx2 -/- animals, 12 hours after BrdU injection., no odontoblast cell division was noticeable. However, several cells mitoses were clearly highlighted in the bone area. A: Bar= 250µm, B,D: Bar= 100µm, C:Bar= 50µm. ..................................... 109 Figure 41: In Situ Hybridization of collagen Iα1 chain on first upper molar of a 14 days post natal Msx2 -/- mouse. Secretory activity of odontoblasts (even inverted polarity cells) was observed. A: bar=200µm, B: bar = 50µm C: bar= 20µm. ....................... 110 Figure 42: Quantitative RT-PCR analysis of Msx2 mRNA isolated from (A) incisor pulp mesenchymal cells of 3 months old Msx2 +/+, +/- and -/- mice, (B) MDPC23 cells (MDPC), MDPC 23 after transfection with Msx2 probe (+++) or lipofectamin ® vector only (MDPC vector), (C) OD21 (OD) and OD21 after transfection with Msx2(OD+++) probe or lipofectamin ® vector only (OD vector). (**: statistically significant). .................... 111 Figure 43: Quantitative RT-PCR analysis of DSPP mRNA isolated from (A) incisor pulp mesenchymal cells of 3 months old Msx2 +/+, +/- and -/- mice, (B) MDPC23 cells (MDPC), MDPC 23 after transfection with Msx2 probe (+++) or lipofectamin ® vector only (MDPC vector), (C) OD21 (OD) and OD21 after transfection with Msx2(OD+++) probe or lipofectamin ® vector only (OD vector). (**: statistically significant). .................... 112 Figure 44: Quantitative RT-PCR analysis of Osteocalcin (OC) mRNA isolated from (A) incisor pulp mesenchymal cells of 3 months old Msx2 +/+, +/- and -/- mice, (B) MDPC23 cells (MDPC), MDPC 23 after transfection with Msx2 probe (+++) or 12
lipofectamin ® vector only (MDPC vector), (C) OD21 (OD) and OD21 after transfection with Msx2(OD+++) probe or lipofectamin ® vector only (OD vector). (**: statistically significant). ........................................................................................ 112 Figure 45: (A) Histology of a mouse first upper molar capped with MTA, 11 weeks post operatively. 0.5µm thickness sections were stained with Methyl Blue/Azur II blue (50%:50%). The dentine bridge (db) was clearly visible, with inclusion of dentine chips. Dentine chips were probably impacted into the pulp tissue during pulp exposure. (B) Mouse first upper molar treated without MTA, 5weeks post operatively. No dentine bridge was visible. Histologically, the pulp tissue appears healthy with no inflammation evident. 7µm thickness cut sections stained with haematoxylin and eosin. ............................................................................................... 114 Figure 46: Histology at 2 weeks post-operatively. Haematoxylin and eosin (H&E) staining. The inflammation has already decreased, and few cells are present in contact with the material involved in tissue reorganisation. A specific line (→), with a high affinity for stain, demarcates the material and the pulp tissue. This line is in intimate contact with the material. At this stage no bacteria or inflammatory cells were visible. ....................................................................................................... 115 Figure 47: Mouse first upper molar, 5 weeks after pulp capping with MTA. (A) H&E staining. The dentine bridge (db) appears very clearly between material (mat) and the pulp; there is no visible gap between dentine bridge and dentine walls. In the thickness of the dentine bridge, a few tubules are evident; their course is not straight as in orthodentine (x 50). (B) Immunohistochemistry with the DSPP antibody of untreated mouse molar pulp (positive control). Staining of odontoblasts is evident, and DSPP is apparently also present in the extracellular matrix of the pulp (x50). (C) Negative control staining with omission of primary antibody (DSPP). (D) Immunohistochemistry with DSPP antibody of first upper molar tooth capped with MTA, 5 weeks post operatively (x50). Dentine bridge staining is very weak compared to orthodentine. (E) At higher magnification, DSPP expression is clearly visible in the cells in close contact with the dentine of the bridge (x100). .................................... 116 Figure 48: SEM observation of a mouse molar 5 weeks post operatively capped with MTA. Roots and pulp chamber floor were removed and the the pulp chamber roof was observed under different magnifications. (A) Magnification x60; dentine bridge appears clearly on the surface of the roof. (B) Observation of the dentine bridge at higher magnification (x200) (C) x4000 ultrastructure of the dentine bridge is different compared to orthodentine; it is more globular in appareance. (D) SEM observation at 13
Page 1 and 2: ECOLE DOCTORALE : Génétique, Cell
Page 3 and 4: ACKNOWLEDGEMENTS I wish to express
Page 5 and 6: Abstract : This research aimed to i
Page 7 and 8: CONTENTS List of Figures ………
Page 9 and 10: 4.5.2 MSX2 and pulp repair ........
Page 11 and 12: Figure 11 : Dentine bridge formatio
Page 13: minutes. (DMP = dentine matrix prot
Page 17 and 18: List of Tables: Table 1: Primer det
Page 19 and 20: 1.1 Towards a biological approach I
Page 21 and 22: Introduction odontoblast processes
Page 23 and 24: Introduction Secondary dentine: thi
Page 25 and 26: Introduction Understanding the dist
Page 27 and 28: Introduction terms of the communica
Page 29 and 30: Figure 5 : There is a differential
Page 31 and 32: Introduction Movement of the dentin
Page 33 and 34: 1.2.3.3 Consequences of new technol
Page 35 and 36: Introduction an apical nucleus, whe
Page 37 and 38: Introduction creates a physical bar
Page 39 and 40: Introduction polarisation and the d
Page 41 and 42: Introduction variations in pressure
Page 43 and 44: Introduction also express neurogeni
Page 45 and 46: 1.4.1 Reactionary dentinogenesis In
Page 47 and 48: Introduction responses, including a
Page 49 and 50: Introduction superficial cells move
Page 51 and 52: Introduction induce dentine bridge
Page 53 and 54: Introduction Because of its highly
Page 55 and 56: Introduction (Hayashi 1982). At 11
Page 57 and 58: Introduction To date, involvement o
Page 59 and 60: Introduction In deeper cavities, wh
Page 61 and 62: Introduction dental papilla cells.
Page 63 and 64: 2 Materials and Methods Material an
Page 65 and 66:
Material and methods containing; 12
Page 67 and 68:
2.1.2.3 Microarray analysis Materia
Page 69 and 70:
Material and methods 2.2 Part Two:
Page 71 and 72:
Material and methods a Modulyo free
Page 73 and 74:
Material and methods Figure 16: Pri
Page 75 and 76:
Material and methods To date, sever
Page 77 and 78:
Material and methods (pH=6.8) with
Page 79 and 80:
Material and methods (P), and calci
Page 81 and 82:
Material and methods (Sigma, la Ver
Page 83 and 84:
Material and methods visualized by
Page 85 and 86:
2.4.2.6 MSX2 over-expression in imm
Page 87 and 88:
Material and methods channel. Data
Page 89 and 90:
Results Amelogenin, IBSP, DCN, Alka
Page 91 and 92:
Results data previously reported fo
Page 93 and 94:
91 MAPK PATHWAY probe set GENE ID b
Page 95 and 96:
Results Alkaline phosphatase and am
Page 97 and 98:
Figure 24: Mouse upper incisor (A)
Page 99 and 100:
Figure 25: Phosphorylated p38 prote
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3.3 MSX2 proteins and dentinogenesi
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Figure 29: First upper molar in +/+
Page 105 and 106:
3.3.1.3 At 21days post natal : Resu
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Results expression, obtained using
Page 109 and 110:
Figure 37: Immunohistochemical anal
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3.3.4 BrdU IHC Results BrdU was inj
Page 113 and 114:
3.3.6 Quantitative RT-PCR (Q-RT-PCR
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Results 3.4 Design and use of a new
Page 117 and 118:
Results Figure 46: Histology at 2 w
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Results cells is also characteristi
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4 Discussion Discussion 4.1 Regulat
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Discussion odontoblast secretory be
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Discussion 4.2 Molecular characteri
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Discussion al. 2004; Magloire, Coub
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Discussion differences. Bone cells
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Discussion we can conclude that the
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Discussion pathway is complex and i
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Discussion odontoblast physiologica
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Discussion dentinogenesis. By perfo
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Discussion cellular and molecular s
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Discussion Notably, our results are
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Discussion contribute to reparative
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Discussion other species (Abedi, To
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Discussion dentinogenesis, some cel
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Discussion morphology. It was not p
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5 Conclusion Discussion Research on
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Discussion Figure 50: 14 months pos
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References during normal and in vit
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References Faraco, I. M., Jr. and R
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References Kidd, E. A., A. Banerjee
Page 161 and 162:
References antigen-expressing cells
Page 163 and 164:
References Smith, A. J. and H. Leso
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References Yamashiro, T., M. Tummer
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References Appendix 1: Full list of
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probe set GENE ID baseline mean (LS
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Appendix 3: Molecular characterizat
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Appendix 5: Evaluation of a new lab
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