UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1103 | ISSN 0346-6612 | ISBN 978-91-7264-319-2 Innervation patterns and locally produced signal substances in the human patellar tendon – of importance when understanding the processes of tendinosis Patrik Danielson Umeå 2007 From the Department of Integrative Medical Biology, Anatomy, and the Department of Surgical and Perioperative Sciences, Sports Medicine, Umeå University, Umeå, Sweden Department of Integrative Medical Biology Section for Anatomy Umeå University SE-901 87 Umeå, Sweden Copyright © Patrik Danielson, 2007 ISSN: 0346-6612 ISBN: 978-91-7264-319-2 Printed in Sweden at Print and Media, Umeå University, Umeå 2007 Figures 1, 3 and 4: Illustrations by Patrik Danielson Figure 2: Image by Gustav Andersson, redrawn from Józsa and Kannus, 1997 Figure 5: Ultrasonographic image provided by Håkan Alfredson All previously published papers were reproduced with permission from the publisher. Patrik Danielson, 2007 CONTENTS ABBREVIATIONS................................................................................................... 6 ABSTRACT.............................................................................................................. 7 LIST OF ORIGINAL PAPERS ................................................................................ 8 1. INTRODUCTION ...................................................................................... 9 1.1 The human patellar tendon.................................................................... 9 1.1.1 Anatomy .................................................................................................... 9 1.1.2 General tendon histology.......................................................................... 10 1.1.3 Blood supply ............................................................................................ 12 1.1.4 Innervation............................................................................................... 13 1.1.5 Tendon cells; metabolism, properties, and possible stem cell-like features. 14 1.2 Patellar tendinosis (Jumper’s knee)....................................................... 15 1.2.1 Definition of tendinosis, and terminology................................................. 15 1.2.2 Location, clinical symptoms, and diagnostics ............................................ 15 1.2.3 Epidemiology ........................................................................................... 17 1.2.4 Etiology, pathogenesis, and pathology....................................................... 18 1.2.5 Histopathological tissue changes ............................................................... 21 1.2.6 Treatment methods .................................................................................. 22 1.3 Production in non-neuronal cells of signal substances traditionally associated with neurons........................................................................ 25 2. AIMS........................................................................................................... 27 3. MATERIAL AND METHODS.................................................................. 28 3.1 Patient material.................................................................................... 28 3.1.1 Tendinosis patients................................................................................... 28 3.1.2 Normal controls ....................................................................................... 28 3.1.3 Ethics ....................................................................................................... 28 3.2 Sampling ............................................................................................. 30 3.2.1 Surgery (tendinosis tendons)..................................................................... 30 3.2.2 Biopsies from normal tendons................................................................... 30 3.3 Processing of tissue............................................................................... 30 3.4 Sectioning............................................................................................ 31 3.5 Immunohistochemistry........................................................................ 31 3.5.1 Immunofluorescence (TRITC and FITC)................................................. 31 3.5.2 Peroxidase-antiperoxidase (PAP) immunostaining..................................... 32 3.5.3 Immunostaining using EnVision® detection ............................................ 33 3.5.4 Primary antibodies.................................................................................... 33 3.5.5 Control stainings, including preabsorbtions .............................................. 35 3.6 Histochemical staining......................................................................... 36 3.7 In situ hybridization (ISH)................................................................... 36 4 Innervation patterns and locally produced signal substances in the human patellar tendon 3.8 Microscopic examination, and evaluation............................................. 37 4. RESULTS.................................................................................................... 39 4.1 General histology................................................................................. 39 4.1.1 Tissue morphology ................................................................................... 39 4.1.2 Tenocytes; morphology and frequency...................................................... 40 4.2 Innervation patterns............................................................................. 40 4.2.1 General innervation.................................................................................. 40 4.2.2 Sensory innervation, and presence of NK-1 receptors................................ 41 4.2.3 Cholinergic innervation, and presence of M2 receptors in nerve structures and blood vessel walls ............................................................................... 42 4.2.4 Sympathetic innervation, and presence of adrenergic receptors in nerve structures and blood vessel walls................................................................ 43 4.3 Presence in tenocytes of synthesizing enzymes and receptors for signal substances traditionally associated with neurons ................................... 44 4.3.1 Cholinergic system ................................................................................... 44 4.3.2 Catecholaminergic system......................................................................... 46 4.4 Innervation patterns in relation to local production of cholinergic/catecholaminergic signal substances; summary of results..... 47 5. DISCUSSION............................................................................................. 48 5.1 Morphological aspects.......................................................................... 48 5.1.1 Paratenon and endotenon ......................................................................... 48 5.1.2 Tenocytes and/or tenoblasts?..................................................................... 49 5.2 Methodological aspects ........................................................................ 49 5.3 Innervation of the patellar tendon........................................................ 50 5.3.1 Nerve patterns .......................................................................................... 50 5.3.2 Receptors.................................................................................................. 51 5.3.3 Possible implications with regard to blood vessel regulation in tendinosis .. 51 5.4 Evidence of a local production of signal substances traditionally associated with neurons, and of a presence of receptors for these, in tenocytes ...... 52 5.4.1 Possible implications in relation to tissue changes in tendinosis (proliferation, degeneration, apoptosis) ........................................................................... 52 5.4.2 Stem cell-like characteristics of tenocytes/tenoblasts? ................................. 53 5.4.3 Possible implications regarding chronic tendon pain ................................. 54 6. CONCLUSIONS........................................................................................ 56 FUNDING............................................................................................................. 57 ACKNOWLEDGEMENTS................................................................................... 58 REFERENCES ....................................................................................................... 60 PERMISSION FROM PUBLISHERS ................................................................... 66 5 Patrik Danielson, 2007 ABBREVIATIONS ACh acetylcholine AChE acetylcholine esterase α1-AR α1-adrenoreceptor (adrenergic receptor subtype α1) α2A-AR α2A-adrenoreceptor (adrenergic receptor subtype α2A) β1-AR β1-adrenoreceptor (adrenergic receptor subtype β1) BSA bovine serum albumin CGRP calcitonin gene-related peptide ChAT choline acetyltransferase FITC fluorescein isothiocyanate htx hematoxylin ISH in situ hybridization -LI -like immunoreactions mAChR muscarinic acetylcholine receptor M2R M2 receptor (mAChR subtype M2) MRI magnetic resonance imaging NK-1 R neurokinin-1 receptor NPY neuropeptide Y PAP peroxidase-antiperoxidase PBS phosphate-buffered saline PGP 9.5 protein gene product 9.5 SP substance P TH tyrosine hydroxylase TRITC tetramethylrhodamine isothiocyanate