||The role of burn blister fluid in burn wound neovascularization
||Institute of Clinical Medicine
Burn blister fluids
Burn wound healing is a complex and dynamic process, which involves an interaction between different cells and mediators. This process can be divided into inﬂammatory phase, proliferative phase, and remodeling phase. Neovascularization, beginning during the inﬂammatory phase of wound healing, is an imperative stage of wound healing. The whole process consists of not only angiogenesis but also adult vasculogenesis.
Superﬁcial partial thickness burn (SPTB) heals within 2 weeks without scarring. Deep partial thickness burn (DPTB), on the other hand, heals beyond 2 weeks and requires aggressive treatment to prevent hypertrophic scarring. Burn blisters on the skin are a hallmark of not only SPTB but also DPTB. Previous study reported that blisters contained several cytokines with beneﬁcial effects on keratinocyte proliferation. However, the effect of burn blister ﬂuids on the neovascularization has not been fully explored. Burn hypertrophy scar usually exhibits in a hypervascular status. DPTB wounds often result in hypertrophy scar, we assume that the neovascularization between SPTB and DPTB wounds may be different.
To verify this hypothesis, neovasculogenic effect of two different kinds of burn blister ﬂuids was tested and compared in vitro and in vivo. The results show DPTB blister ﬂuids more predominantly support neovascularization than do SPTB wounds in the early stage of wound healing.
Following the above study, we are particularly interested in investigating the factors modulating the differential angiogenic activity of these fluids. An expression analysis of angiogenic factors in burn fluids was conducted. Upon finding a significant difference of angiogenin between the two different burn fluids, the role of angiogenin was assessed in vitro, in vivo and human study. We find that angiogenin clearly involves in the neovascularization of burn wound healing.
Rapid and accurate assessment of burn depth is the most important determinant of burn wound management in the early stage of burn injury. However, assessment of second-degree burn wounds with intact blisters has always been difficult, even for experienced surgeons. Although our finding demonstrates a significantly higher angiogenin expression in DPTB blister fluids than formed in SPTB fluids, screening the angiogenin in burn blister fluids for determination of burn depth should be further studied. Nevertheless, application of burn fluid contents to observe cell-mediators interaction in burn wounds is of considerable interest to those investigating the processes of burn wound healing.
CONTENT LIST PAGE
Chinese abstract 3
English abstract 5
Chapter 1 Burn Blister in Burn Wound Healing
1.1 Phases of Normal Wound Healing 15
1.1.1 Inflammation phase 15
1.1.2 Proliferative phase 16
22.214.171.124 Angiogenesis 17
126.96.36.199 Vasculogenesis 18
188.8.131.52.1 Characterization of circulating EPC 19
184.108.40.206.2 Role of EPCs in Neovascularization 20
220.127.116.11.3 Mechanisms by which EPCs promote 21
18.104.22.168.4 Mobilization of EPCs 22
22.214.171.124.5 Chemotaxis of EPCs 22
126.96.36.199.6 Homing and differentiation of EPCs 23
188.8.131.52.7 Vasculogenesis in wound healing 24
1.1.3 Remodeling phase 26
1.2 The Burn Wound 27
1.2.1 Anatomy of normal skin 27
1.2.2 Thermal effect on skin 28
1.2.3 Depth of the Burn 29
1.2.4 First-degree burn 29
1.2.5 Second-degree burn 29
1.2.6 Third-degree burn 30
1.2.7 Fourth-degree burn 30
1.2.8 Assessment of burn wound depth 30
184.108.40.206 Clinical evaluation 31
220.127.116.11 Biopsy and histology 32
18.104.22.168 Thermography 33
22.214.171.124 Vital dyes 34
126.96.36.199 Indocyanine green video angiography 35
188.8.131.52 Laser Doppler flowmetry 35
184.108.40.206 Nuclear imaging 37
220.127.116.11 Noncontact and high-frequency 38
1.3 Burn blister 39
1.3.1 Occurrence of blister 39
1.3.2 Advantages of blisters intact 40
1.3.3 Advantages of blister removal 41
1.4 Conclusion 43
1.5 Goal of Ph.D project 44
Chapter 2 The Effect of Superficial And Deep Partial Thickness Burn Blister Fluid in Burn Wound Neovascularization
2.1 Introduction 46
2.2 Materials and Methods 46
2.2.1. Patient samples 46
2.2.2 Immunohistochemistry 47
2.2.3 Cell proliferation assay 48
2.2.4 Isolation and differentiation of circulating 48
2.2.5 Isolation of CD34+ and CD14+ cells 49
2.2.6 Transwell migration assay 49
2.2.7 Real-time polymerase chain reaction (PCR) 50
2.2.8 Characterization of endothelial cells 50
2.2.9 Matrigel plug assay 51
2.2.10 Statistical analysis 51
2.3 Results 51
2.3.1 Microvascular density in different burn 51
2.3.2 Proliferative ability of endothelial cells in 52
burn blister ﬂuids
2.3.3 Migration ability of CACs in burn blister 52
2.3.4 Endothelial gene expression in burn blister 52
2.3.5 Production of functional endothelial cells in 53
burn blister fluids
2.3.6 In-vivo animal study of burn blister fluids 54
2.3.7 Purity of CD34+ and CD14+ cells 54
2.3.8 Migration ability of CD34+ and CD14+ cells 54
in burn blister fluids
2.3.9 The endothelial gene expression of CD34+ 55
and CD14+ cells in burn blister fluids
2.3.10 Endothelial characteristics of CD34+ and 55
CD14+ cells in burn blister fluids
2.4 Discussion 55
2.5 Conclusion 58
Chapter 3 Factors modulating the angiogenic activity of burn blister fluids
3.1 Introduction 60
3.2 Materials and Methods 60
3.2.1. Patient samples 60
3.2.2 Cytokine antibody assay 61
3.2.3 Enzyme-linked immunosorbent assay (ELISA) 61
3.2.4 Cell proliferation assay 62
3.2.5 CAC isolation and differentiation 62
3.2.6 Endothelial cell characterization 62
3.2.7 Matrigel plug assay 63
3.2.8 Immunohistochemistry 63
3.2.9 Statistical analysis 64
3.3 Results 64
3.3.1 Differential angiogenesis-related cytokine 64
expression in burn blister fluids
3.3.2 Effect of endothelial cell proliferation by 65
3.3.3 Correlation of angiogenin expression with 66
3.3.4 CACs differentiation by angiogenin inhibition 66
3.3.5 Angiogenin neutralization attenuated 67
neovascularization in vivo
3.3.6 Angiogenin expression in human burn wound 67
3.4 Discussion 68
3.5 Conclusion 72
Chapter 4 Conclusion 73
Chapter 5 Future Work 75
Reference List 76
List of thesis related publications 122
Curriculum Vitae 123
LIST of TABLES
Table 1 The number of CAC differentiation in patients 95
according to burn severity
Table 2 ELISA analysis of cytokines in blister fluids 96
Table 3 Angiogenin staining intensity in human burn 97
LIST of FIGURES
Figure 1. Common methods of “EPC” culture 98
Figure 2. Origin and differentiation of endothelial 99
Figure 3. Immunohistochemical staining of CD31 in 100
SPTB and DPTB wounds
Figure 4. The effect of burn ﬂuids on endothelial 101
Figure 5. The migration ability of peripheral blood 102
mononuclear cells induced by SPTB or DPTB
Figure 6. The effect of burn blister ﬂuids on the 103
endothelial mRNA expression
Figure 7A. The effects of burn blister fluids on CACs 104
Figure 7B. Flow cytometry analysis of endothelial 105
differentiation of CACs
Figure 8. In vivo angiogenic activity of burn 106
Figure 9. Purity of CD34+ and CD14+ cells 107
Figure 10. Migration ability of CD34+ and CD14+ 108
cells in DPTB blister fluid
Figure 11. The vWF mRNA gene expression of cultured 109
CD34+ and CD14+ cells in DPTB blister
Figure 12A.Flow cytometry analysis of CACs 110
differentiation from cultured CD34+,
CD34-, CD14+ and CD14- cells.
Figure 12B.Differentiation of cultured CD34+ and 111
CD14+ cells in DPTB blister fluids
Figure 13. Differential expression of cytokines in 112
SPTB and DPTB fluids
Figure 14. Time course of cytokines expression in 113
burn blister fluid following thermal
Figure 15. The effect of anti-angiogenin antibodies 114
on endothelial cell proliferation
Figure 16. The effect of different levels of 115
anti-angiogenin antibody on endothelial
Figure 17. The effect of angiogenin on CAC 116
Figure 18. The effect of neutralizing antibody to 117
angiogenin on CAC differentiation induced
by DPTB fluid
Figure 19. The effect of VEGF-A neutralization on 118
DPTB fluid-induced CAC differentiation
Figure 20. Angiogenin neutralization in DPTB fluids 119
reduced neovascularization in Matrigel plugs
Figure 21. The relationship between angiogenin and 120
neovascularization in human burn wounds
Figure 22. Summary of differential neovascularization 121
between superficial and deep partial
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