Mechanical and mechanobiological influences on bone fracture repair - identifying important cellular characteristics A catalogue record is available from the Eindhoven University of Technology Library ISBN 978-90-386-1146-4 Copyright © 2007 by H. Isaksson All rights reserved. No part of this book may be reproduced, stored in a database or retrieval system, or published, in any form or in any way, electronically, mechanically, by print, photoprint, microfilm or any other means without prior written permission of the author. Cover design: Jorrit van Rijt, Oranje Vormgevers Printed by Universiteitsdrukkerij TU Eindhoven, Eindhoven, The Netherlands. Financial support from the AO Foundation, Switzerland is gratefully acknowledged. AO Foundation Research Mechanical and mechanobiological influences on bone fracture repair - identifying important cellular characteristics PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Technische Universiteit Eindhoven, op gezag van de Rector Magnificus, prof.dr.ir. C.J. van Duijn, voor een commissie aangewezen door het College voor Promoties in het openbaar te verdedigen op maandag 26 november 2007 om 16.00 uur door Hanna Elisabet Isaksson geboren te Linköping, Zweden Dit proefschrift is goedgekeurd door de promotoren: prof.dr.ir. H.W.J. Huiskes en prof.dr.ir. K. Ito Copromotor: dr. C.C. van Donkelaar To my family and friends for all their support through these years vii Contents Contents vii Summary ix List of original publications xi 1 Introduction 1 2 Bone fracture healing and computational modeling of bone mechanobiology 7 3 Comparison of biophysical stimuli for mechano-regulation of tissue differentiation during fracture healing 27 4 Corroboration of mechano-regulatory algorithms: Comparison with in vivo results 41 5 Bone regeneration during distraction osteogenesis: Mechano-regulation by shear strain and fluid velocity 55 6 A mechano-regulatory bone-healing model based on cell phenotype specific activity71 7 Determining the most important cellular characteristics for fracture healing, using design of experiments methods 91 8 Remodeling of fracture callus in mice can be explained by mechanical loading 107 9 Discussion and conclusions 123 Appendix A: Theoretical development of finite element formulation for modeling cellular activity………… 135 Appendix B: Taguchi orthogonal arrays and design of experiments methods 141 References 147 Samenvatting 163 Acknowledgement 165 Curriculum Vitae 167 ix Mechanical and mechanobiological influences on bone fracture repair - identifying important cellular characteristics Summary Fracture repair is a complex and multifactorial process, which involves a well-programmed series of cellular and molecular events that result in a combination of intramembranous and endochondral bone formation. The vast majority of fractures is treated successfully. They heal through ‘secondary healing’, a sequence of tissue differentiation processes, from initial haematoma, to connective tissues, and via cartilage to bone. However, the process can fail and this results in delayed healing or non-union, which occur in 5-10% of all cases. A better understanding of this process would enable the development of more accurate and rational strategies for fracture treatment and accelerating healing. Impaired healing has been associated with a variety of factors, related to the biological and mechanical environments. The local mechanical environment can induce fracture healing or alter its biological pathway by directing the cell and tissue differentiation pathways. The mechanical environment is usually described by global mechanical factors, such as gap size and interfragmentary movement. The relationship between global mechanical factors and the local stresses and strains that influence cell differentiation can be calculated using computational models. In this thesis, mechano-regulation algorithms are used to predict the influence of mechanical stimuli on tissue differentiation during bone healing. These models used can assist in unraveling the basic principles of cell and tissue differentiation, optimization of implant design, and investigation of treatments for non-union and other pathologies. However, this can only be accomplished after the models have been suitably validated. The aim of this thesis is to corroborate mechanoregulatory models, by comparing existing models with well characterized experimental data, identify shortcomings and develop new computational models of bone healing. The underlying hypothesis throughout this work is that the cells act as sensors of mechanical stimuli during bone healing. This directs their differentiation accordingly. Moreover, the cells respond to mechanical loading by proliferation, differentiation or apoptosis, as well as by synthesis or removal of extracellular matrix. In the first part of this work, both well-established and new potential mechano-regulation algorithms were implemented into the same computational model and their capacities to predict the general tissue distributions in normal fracture healing under cyclic axial load were compared. Several algorithms, based on different biophysical stimuli, were equally well able to predict normal fracture healing processes (Chapter 3). To corroborate the algorithms, they were compared with extensive in vivo experimental bone healing data. Healing under two distinctly different mechanical conditions was compared: axial compression or torsional rotation. None of the established algorithms properly predicted the spatial and temporal tissue distributions observed experimentally, for both loading modes and time points. Specific Summary x inadequacies with each model were identified. One algorithm, based on deviatoric strain and fluid flow, predicted the experimental results the best (Chapter 4). This algorithm was then employed in further studies of bone regeneration. By including volumetric growth of individual tissue types, it was shown to correctly predict experimentally observed spatial and temporal tissue distributions during distraction osteogenesis, as well as known perturbations due to changes in distraction rate and frequency (Chapter 5). In the second part of this work, a novel ‘mechanistic model’ of cellular activity in bone healing was developed, in which the limitations of previous models were addressed. The formulation included mechanical modulation of cell phenotype and skeletal tissue-type specific activities and rates. This model was shown to correctly predict the normal fracture healing processes, as well as delayed and non-union due to excessive loading, and also the effects of some specific biological perturbations and pathological situations. For example, alterations due to periosteal stripping or impaired cartilage remodeling (endochondral ossification) compared well with experimental observations (Chapter 6). The model requires extensive parametric data as input, which was gathered, as far as possible, from literature. Since many of the parameter magnitudes are not well established, a factorial analysis was conducted using ‘design of experiments’ methods and Taguchi orthogonal arrays. A few cellular parameters were thereby identified as key factors in the process of bone healing. These were related to bone formation, and cartilage production and degradation, which corresponded to those processes that have been suggested to be crucial biological steps in bone healing. Bone healing was found to be sensitive to parameters related to fibrous tissue and cartilage formation. These parameters had optimum values, indicating that some amounts of soft tissue production are beneficial, but too little or too much may be detrimental to the healing process (Chapter 7). The final part of this work focused on the remodeling phase of bone healing. Long bone post- fracture remodeling in mice femora was characterized, including a new phenomenon described as ‘dual cortex formation’. The effect of mechanical loading modes on fracture- callus remodeling was evaluated using a bone remodeling algorithm, and it was shown that the distinct remodeling behavior observed in mice, compared to larger mammals, could be explained by a difference in major mechanical loading mode (Chapter 8). In summary, this work has further established the potential of mechanobiological computational models in developing our knowledge of cell and tissue differentiation processes during bone healing in general, and fracture healing and distraction osteogenesis in particular. The studies presented in this thesis have led to the development of more mechanistic models of cell and tissue differentiation and validation approaches have been described. These models can further assist in screening for potential treatment protocols of pathophysiological bone healing. [...]... endochondral bone formation strongly resembles the embryonic development of long bones (Ferguson et al., 1999) Angiogenesis occurs in parallel with endochondral ossification, eventually leading to erosion of mineralized cartilage and deposition of bone (Mark et al., 2004) 12 Bone fracture healing and computational modeling of bone mechanobiology Remodeling Once bony bridging of the callus has occurred and. .. osteocytes and bone- lining cells differentiate from mesenchymal stem cells, and osteoclasts from hemopoietic stem cells (Owen, 1970) 8 Bone fracture healing and computational modeling of bone mechanobiology 2.1.2 Bone formation and growth Bone forms, grows and resorbs continuously, by remodeling processes The formation of bone occurs by two methods, intramembranous and endochondral ossification These... Hermey, 1996) 2.1.1 Bone structure and composition Morphologically, bones are classified as cortical or trabecular (cancellous) bone Cortical bone forms the outer shell of every bone It is compact, stiff and strong and has a high resistance to all loads: bending, axial and torsion, which are especially important in the shafts of long bones (Buckwalter et al., 1996a) In contrast, trabecular bone is a less... proliferation and differentiation of mesenchymal precursor cells into osteoblasts, osteoclasts and chondrocytes (Linkhart et al., 1996) They also appear to stimulate both endochondral and intramembranous 14 Bone fracture healing and computational modeling of bone mechanobiology bone formation BMP signaling leads to activation of genes for proliferation and differentiation along the chondrogenic and osteogenic... Woven bone is laid down rapidly and has randomly oriented collagen fibers, and low strength In adults it is observed mainly at sites of repair, at tendon or ligament attachments and in pathological conditions In contrast, the collagen fibers in lamellar bone are aligned and are much stronger Woven bone is mostly replaced by lamellar bone during growth or repair (Buckwalter et al., 1996b) Bone consists... Occasionally a fibro-cartilaginous pseudoarthrosis or ‘false’ joint forms (Figure 2-5 ) Some types of non-unions, such as pseudoarthrosis and hypertrophic non-unions are usually treated mechanically Other types of non-unions related to infection, aseptic or septic tissues are treated biologically Treatments can also include the use of traditional bone grafts Figure 2-5 : Non-union represented by a mid-diaphyseal... osteogenesis bone forms just as rapidly as during fracture healing, and as long as distraction force is applied, bone regeneration can be sustained almost indefinitely (Einhorn, 1998a) Hence, it is a suitable model for studying the potential mechanisms that stimulate bone formation and examination of the role of mechanical forces 16 Bone fracture healing and computational modeling of bone mechanobiology... around chondrocytes, which swell osmotically, and are suited to support hydrostatic pressure only Hence, he identified strain and pressure, as 18 Bone fracture healing and computational modeling of bone mechanobiology two distinct stimuli, stimulating or allowing fibrous tissue and cartilage, respectively Primary bone formation requires a stable, low-strain mechanical environment and endochondral bone formation... responsible for bone adaptation to the mechanical environment (Wolf, 1892), resulting in bone thickening in regions of increased stress and bone thinning in regions of decreased stress Figure 2-1 : Schematic diagram of haverisan remodeling (Reprinted from Rüedi et al (2007), Copyright by AO Publishing, Davos, Switzerland) 2.1.3 Bone fracture Bone fractures when its strain limit is exceeded A fracture disrupts... mineral component resists compression and the collagen fibers resist tension and shear (van der and Garrone, 1991; Marks and Hermey, 1996) The remainder of the skeleton consists of cells and blood vessels There are four different cell types in human bones: osteoblasts, osteoclasts, bone lining cells, and osteocytes Osteoblasts are bone forming cells They line the surfaces of the bones and produce osteoid . Mechanical and mechanobiological influences on bone fracture repair - identifying important cellular characteristics Summary Fracture repair is a complex and. differentiation are reviewed and theories and algorithms described. 2 2 Chapter 2 8 2.1 Bone and bone fracture The adult human skeleton consists of 206 bones.