Connective Tissue Formation in Wound Healing An experimental study

CONTENTS ORIGINAL PUBLICATIONS 5 ABBREVIATION 6 ABSTRACT 7 INTRODUCTION 9 REVIEW OF THE LITERATURE 11 WOUND HEALING 11 Blood clot formation 12 Inflammation 12 Epithelialization 13 Neovascularization 13 Formation of Granulation Tissue 15 Matrix Remodeling 16 Viscose cellulose sponges as a wound healing model 17 EXTRACELLULAR MATRIX 18 Collagens 18 Structure and biosynthesis of collagens 21 Fibrillar collagens 22 The gene structure of the fibril-forming collagens 23 Regulation of collagen genes 25 Type I collagen 28 Type III collagen 28 Type V collagen 29 Cell and ECM interaction 31 DEGRADATION OF EXTRACELLULAR MATRIX 33 Matrix metalloproteinases 33 Gelatinases 35 Other MMPs 35 Regulation of MMP activity 38 The role of MMPs and TIMPs in wound healing 40 GROWTH FACTORS IN WOUND HEALING 41 TGF-β 41 PDGF 46 CTGF 48 OUTLINE OF THE PRESENT STUDIES 53 MATERIALS AND METHODS 54 Animals 54 Experimental wound healing model 54 Histological staining for collagens 54 Hemoglobin 54 Determination of total tissue collagen and DNA 55 Collagen extraction, identification, and quantitation 55 Extraction of metalloproteinases and gelatinolytic activity assay 56 Primary antibodies 56 Immunohistochemistry 57 In Situ hybridization 57 Analysis of mRNA 58 Probes 59 Preparation of rat probes for type V collagen and MMP-2 and-9 60 Statistical analysis 61 RESULTS 62 Sponge implants [ I and II ] 62 Interstitial collagens in granulation tissue [ I and II ] 63 MMP-2 and MMP-9 in granulation tissue [ III ] 65 Growth factors in granulation tissue [ IV ] 67 DISCUSSION 70 Type I, III and V collagens in wound healing 71 MMP-2 and MMP-9 in wound healing 73 CTGF, TGF-β and PDGF in wound healing 75 CONCLUSIONS 78 ACKNOWLEDGEMENTS 80 REFERENCES 82 5 ORIGINAL PUBLICATIONS This thesis is based on the following original publications, which are referred to in the text by the Roman numerals I-IV. Cloning of cDNA for rat pro alpha1(V) collagen mRNA. Expression patterns of type I, type III and type V collagen genes in experimental granulation tissue. Expression and activity of matrix metalloproteinase-2 and-9 in experimental granulation tissue. Connective tissue growth factor and its correlation to other growth factors in experimental granulation tissue. 6 ABBREVIATION AP-1 activator protein-1 CCN Connective tissue growth factor/Cysteine-rich 61/Nephroblastoma over-expressed CTGF connective tissue growth factor ECM extracellular matrix EGF epidermal growth factor Egr-1 early growth response 1 FACIT fibril-associated collagens with interrupted triple helices FGF fibroblast growth factor GAPDH glyceraldehyde 3-phosphate dehydrogenase ICAM-1 intercellular adhesion …