MECHANOBIOLOGY OF COMPLEX LOADING IN FUNCTIONAL SPINAL UNITS by Robert Allen ...

391 Pages·2014·4.72 MB·English

MECHANOBIOLOGY OF COMPLEX LOADING IN FUNCTIONAL SPINAL UNITS

by
Robert Allen Hartman
Bachelor of Science, University of Pittsburgh, 2008
Master of Science, University of Pittsburgh, 2010

Submitted to the Graduate Faculty of
The Swanson School of Engineering in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy

University of Pittsburgh
2014
UNIVERSITY OF PITTSBURGH
SWANSON SCHOOL OF ENGINEERING

This dissertation was presented

by

Robert Allen Hartman

It was defended on
July 21, 2014
and approved by
James D. Kang, MD, Department of Orthopaedic Surgery
Richard E. Debski, PhD, Department of Bioengineering
Bryan N. Brown, PhD, Department of Bioengineering
Dissertation Director: Gwendolyn A. Sowa, MD, PhD, Department of Physical Medicine &
Rehabilitation, Department of Bioengineering, and Department of Orthopaedic Surgery

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Copyright © by Robert Hartman
2014
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MECHANOBIOLOGY OF COMPLEX LOADING IN FUNCTIONAL SPINAL UNITS

Robert Allen Hartman, Ph.D.
University of Pittsburgh, 2014

A majority of back pain, a costly condition and leading cause of disability, is mechanical in origin
involving the intervertebral disc, facet joints, or ligamentum flavum. Mechanical loading may be
beneficial or detrimental to spinal tissues depending on loading mode, magnitude, frequency, and
duration. Ex vivo mechanobiology systems have been used to explore how axial loading
parameters influence intervertebral disc biology, but flexion/extension (F/E) and combined
rotations, loading modes relevant to back pain, have not been investigated. Moreover, biological
responses in facet cartilage (FC) and ligamentum flavum (LF) have not been studied. A novel
experimental platform was developed to assess simultaneous biological responses to six degrees-
of-freedom (DOF) loading of intact functional spinal units (FSUs) in annulus fibrosus (AF),
nucleus pulposus (NP), FC and LF. A bioreactor previously validated for assessment of axially
compressed FSUs was attached to a robotic testing system and validated for rigid fixation and
unrestricted movement in F/E and axial torsion (AT). At first, neutral F/E of varying range-of-
motion and cycle number was applied. F/E loading elicited a predominantly catabolic response
from spinal tissues with significant up-regulation of catabolic gene expression in AF and FC.
Range-of-motion modulated aggrecan fragmentation in AF. AT was then added to F/E in small
and large magnitudes to simulate mild and severe axial asymmetries treated clinically. F/E with
coupled AT was pro-inflammatory in all spinal tissues and was pro-catabolic in AF and LF. In
FC, which is gapped by torsion on one side and compressed on the other, pro-inflammatory
changes were higher in gapped joints, and catabolic loss of matrix was higher in compressed joints.
These findings point to a role for altered segmental mechanics in driving pro-inflammatory,
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catabolic processes in spinal tissues that may play a role in spinal disorders involved in back pain.
Finally, multiple regression analysis was performed to assess how well mechanical responses
predicted changes in gene expression. Mechanical predictors accounted for more variation in gene
expression in FC and LF than AF and NP. The development of this system provides spine and
orthopaedic research with a novel experimental platform that can evaluate complex loading and
simulated in vivo motions.

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TABLE OF CONTENTS
PREFACE ............................................................................................................................... XVII
INTRODUCTION......................................................................................................................... 1
1.0 BACKGROUND ............................................................................................................... 4
1.1 BACK PAIN ......................................................................................................... 4
1.2 SPINAL FUNCTION .......................................................................................... 6
1.2.1 Mechanotransduction .................................................................................... 10
1.3 DEGENERATION: MECHANICAL CONSEQUENCES ........................... 13
1.3.1 Compositional & Mechanical Changes ........................................................ 14
1.3.2 Cellular Changes and Mechanotransduction.............................................. 17
1.4 MECHANICS IN TREATMENT .................................................................... 18
1.5 MECHANOBIOLOGY RESEARCH .............................................................. 21
1.5.1 In vitro Studies ............................................................................................... 22
1.5.2 In vivo Studies ................................................................................................ 24
1.5.3 Ex vivo Systems .............................................................................................. 28
2.0 GOAL AND SPECIFIC AIMS ...................................................................................... 33
2.1 SYSTEM DEVELOPMENT............................................................................. 33
2.2 SPECIFIC AIM 1 – FLEXION/EXTENSION................................................ 35
2.3 SPECIFIC AIM 2 – COMPLEX LOADING .................................................. 36
2.4 REGRESSION MODELING ........................................................................... 37
3.0 SYSTEM DEVELOPMENT .......................................................................................... 38
3.1 SYSTEM REQUIREMENTS ........................................................................... 38
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3.2 KINEMATIC AND KINETIC PRECISION OF THE TESTING SYSTEM ..
............................................................................................................................. 40
3.2.1 Introduction ................................................................................................... 40
3.2.2 Methods .......................................................................................................... 41
3.2.2.1 Robot Testing System ......................................................................... 41
3.2.3 Results ............................................................................................................. 46
3.2.3.1 Robot Testing System ......................................................................... 46
3.2.4 Conclusions..................................................................................................... 50
3.2.5 Acknowledgements ........................................................................................ 52
3.3 RIGIDITY OF THE FIXATION SYSTEM .................................................... 53
3.3.1 Introduction ................................................................................................... 53
3.3.2 Methods .......................................................................................................... 53
3.3.2.1 Statistics ............................................................................................... 58
3.3.3 Results ............................................................................................................. 59
3.3.3.1 Interface Motion .................................................................................. 59
3.3.3.2 Stiffness ................................................................................................ 62
3.3.4 Conclusions..................................................................................................... 63
3.3.5 Acknowledgements ........................................................................................ 65
3.4 ATTACHMENT TO ROBOT TESTING SYSTEM ...................................... 66

3.4.1 Repeatable attachment .................................................................................. 67
3.4.2 COR position .................................................................................................. 69
3.4.3 Orientation about COR................................................................................. 71
3.4.4 Conclusion ...................................................................................................... 72
3.5 MEMBRANE EFFECTS .................................................................................. 73
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4.0 MECHANICAL LOADING PROTOCOL DEVELOPMENT .................................. 77
4.1 INTRODUCTION ............................................................................................. 77
4.2 PARAMETER DETERMINATION ............................................................... 80
4.2.1 Loading duration ........................................................................................... 80
4.2.2 Flexion/extension moment magnitudes ........................................................ 81
4.2.3 Axial torsion moment magnitudes ............................................................... 86
4.2.4 Loading rate ................................................................................................... 87
5.0 FLEXION/EXTENSION: RANGE-OF-MOTION & CYCLES ................................ 89
5.1 INTRODUCTION ............................................................................................. 89
5.2 METHODS ......................................................................................................... 91
5.2.1 Specimen Preparation ................................................................................... 91
5.2.2 Ex vivo Flexion/Extension ............................................................................. 91
5.2.3 Biological Assessments .................................................................................. 95
5.2.4 Statistical Analysis ......................................................................................... 97
5.3 RESULTS ........................................................................................................... 98
5.3.1 Ex vivo Flexion/Extension- Mechanical Characterization ......................... 98
5.3.2 Biological Assessment of Flexion/Extension: Range-of-Motion ................ 99
5.3.3 Biological Assessment of Flexion/Extension: Cycles and Duration ........ 102
5.4 DISCUSSION ................................................................................................... 104
6.0 COMPLEX LOADING: FLEXION/EXTENSION AND AXIAL TORSION ........ 110
6.1 INTRODUCTION ........................................................................................... 110
6.2 METHODS ....................................................................................................... 112
6.2.1 Specimen Preparation ................................................................................. 112
6.2.2 Ex vivo Combined Loading: Axial Torsion + Flexion/Extension ........... 113
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6.2.2.1 Mechanical Assessments ................................................................... 113
6.2.2.2 Biological Assessments ...................................................................... 114
6.2.3 Statistical Analysis ....................................................................................... 116
6.3 RESULTS ......................................................................................................... 117
6.3.1 Mechanical Response: Axial Torsion + Flexion/Extension ...................... 117
6.3.2 Biological Response: Relative Gene Expression ...................................... 120
6.3.3 Biological Response: Western Blotting ..................................................... 123
6.4 DISCUSSION ................................................................................................... 126
7.0 MECHANICAL CONTRIBUTION OF FSU COMPONENTS ............................... 132
7.1 INTRODUCTION ........................................................................................... 132
7.2 METHODS ....................................................................................................... 133
7.3 RESULTS ......................................................................................................... 135
7.4 DISCUSSION ................................................................................................... 142
7.4.1 Neutral F/E ................................................................................................... 142
7.4.2 Combined loading: AT+F/E ....................................................................... 144
7.4.3 Limitations ................................................................................................... 146
7.4.4 Conclusions................................................................................................... 147
7.4.5 Acknowledgements ...................................................................................... 148
8.0 REGRESSION ANALYSIS: CORRELATING MECHANICAL AND
BIOLOGICAL RESPONSES ...................................................................................... 149

8.1 INTRODUCTION ........................................................................................... 149
8.2 METHODS ....................................................................................................... 152
8.2.1 Mechanical Factor Reduction..................................................................... 155
8.2.2 Principal Component Analysis ................................................................... 155
8.2.3 Multiple Regression-Part I: Hierarchical Entry Rationale .................... 156
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8.2.4 Multiple Regression-Part II: Final Regression ........................................ 159
8.3 RESULTS ......................................................................................................... 161
8.3.1 Data Reduction: Autocorrelation .............................................................. 161
8.3.2 Principal Component Analysis ................................................................... 163
8.3.3 Multiple Regression—Part I: Important Predictors Identified .............. 164
8.3.4 Multiple Regression—Part II: Summary in All Tissues ......................... 165
8.3.5 Tissue-Specific Regression Analysis........................................................... 168
8.3.6 Annulus Fibrosus (AF) ................................................................................ 168
8.3.6.1 MMP-1 ............................................................................................... 170
8.3.6.2 MMP-3 ............................................................................................... 171
8.3.6.3 ADAMTS-5 ........................................................................................ 172
8.3.6.4 ACAN ................................................................................................. 173
8.3.7 Facet Cartilage (FC) .................................................................................... 175
8.3.7.1 ADAMTS-5 ........................................................................................ 176
8.3.7.2 COX-2 ................................................................................................ 177
8.3.7.3 ACAN ................................................................................................. 179
8.3.8 Nucleus Pulposus (NP) ................................................................................ 180
8.3.8.1 ACAN ................................................................................................. 181
8.3.9 Ligamentum Flavum (LF) .......................................................................... 183
8.3.9.1 MMP-1 ............................................................................................... 184
8.3.9.2 ADAMTS-5 ........................................................................................ 185
8.3.9.3 COX-2 ................................................................................................ 187
8.3.10 Comparisons Across Tissues ..................................................................... 188
8.4 DISCUSSION ................................................................................................... 189
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