Mechanobiologische Herkunft der Knochen-Porosität und -Elastizität: ein experimenteller und computergestützter Mehrskalen-Ansatz

Zusammenfassung in deutscher SpracheBone mechanobiology comprises all the processes by which bones 'sense' and 'react on' mechanical loading, through the corresponding activity of biological cells and biochemical factors. In this context, the transfer of mechanical loads from the macroscopic scale down to the cellular level is governed by the hierarchical interaction of bone, as well as its mechanical properties; thereby, elasticity and porosity play a particularly eminent role. The latter two quantities, shortly reviewed in Chapter 1, as well as the interdependencies of these properties and their relationship with bone mechanobiology are investigated in the present thesis, by means of experiments and computer simulations. Notably, both approaches are guided by the concept of multiscale continuum (poro)micromechanics, an essential theoretical framework when dealing with a multiscale, hierarchically structured material such as bone. In Chapter 2, a multiscale mathematical model for simulation of bone remodeling is presented, describing the porosity-specific processes and relationships between bone cells, biochemical factors, and mechanical loads occuring at the level of the vascular and lacunar pores. Particularly, the mechanical stimuli acting on the bone cells involved in bone remodeling are quantified in terms of hydrostatic pore pressures, estimated from the macroscopic loading by means of a continuum (poro)micromechanics representation of bone. The model is then validated quantitatively and qualitatively with experimental data from literature, showing the infuence of different mechanical loading conditions on bone adaptation for various animal species. Chapters 3 and 4 deal with determination of the elastic modulus of bone by means of a new method which, based on the concept of statistical nanoindentation and an evolutionary algorithm, can distinguish between damaged and undamaged material phases - or, more generally, between indents where the elastic half space theory applies, or not (e.g., due to the presence or initiation of microcracks). More precisely, in Chapter 3, the elastic modulus of undamaged, cortical bone, at the scale of the extracellular matrix, is determined throughout different plane sections through the midshaft of a human femur, and the differences in stiffness between endosteal and periosteal regions, as well as between loaded and not loaded areas are investigated. In Chapter 4, Young-s modulus of intact bovine extracellular femur bone is investigated. In both chapters, the hypothesis that nanoindentation testing may also deliver elasticity values related to damaged material is checked, by imaging microcracks with a Scanning Electron Microscope (SEM). Finally, in Chapters 5 and 6, experimental methods are employed for determination of the mechanical properties of ceramic materials for bone tissue engineering scaffold production, namely baghdadite (Ca3ZrSi2O9) and Bioglass®. Ideally, such scaffolds should reproduce the properties of bone as closely as possible. In the case of baghdadite, scaffolds seeded with bone cells have shown good biological properties in vivo, but research on their mechanical properties are scarce. In Chapter 5, by means of statistical nanoindentation combined with ultrasonic tests, the elasticity of porous baghdadite is characterized across a wide range of material porosities. In the case of Bioglass® scaffolds, mechanical properties have been measured before, and require improvement in order to come close to those of trabecular bone. The study in Chapter 6 investigates, by means of multiscale ultrasound-nanoindentation measurements, the possibilities of enhancing the stiffness of these scaffolds by coating them with various types of polymers.Knochen-Mechanobiologie umfasst alle Prozesse, bei denen Knochen durch die Aktivität von Zellen oder biochemischen Faktoren eine mechanische Belastung erfahren. In diesem Sinne ist die Lastübertragung von makroskopischer zu zellulärer Ebene von hierarchischen Knochen-Interaktionen, sowie von mechanischen Eigenschaften geleitet; dabei spielen die Elastizität und die Porosität eine wesentliche Rolle. In dieser Arbeit werden diese beiden Eigenschaften (kurz erläutert in Kapitel 1), ihre gegenseitige Abhängigkeit, sowie ihre Zusammenhänge mit Knochen-Mechanobiologie durch Experimente und computergestützte Simulationen untersucht. Beide Vorgehen basieren auf der Theorie der mehrskaligen Kontinuums-(Poro)Mikromechanik, einem wesentlichen Ansatz bei der Untersuchung von mehrskaligen, hierarchisch strukturierten Materialien wie Knochen. In Kapitel 2 wird ein mehrskaliges mathematisches Modell für die Simulation von Knochenremodellierung vorgestellt, das die Porosität-spezifischen Prozesse und Zusammenhänge zwischen Zellen, biochemischen Faktoren und mechanischen Belastungen beschreibt, die auf Ebene der vaskularen Poren und Lakunen stattfinden. Die mechanischen Stimuli, die auf die in Knochen-Remodellierung involvierten Zellen wirken, werden als hydrostatische Porendrücke quantifiziert, und von der makroskopischen Belastung mit Hilfe einer kontinuums-(poro)mikromechanischen Darstellung von Knochen berechnet. Das Modell wird anschließend quantitativ und qualitativ mit auf Literatur basierenden experimentellen Werten validiert, die den Einfluss von unterschiedlichen mechanischen Belastungsbedingungen auf Knochenadaptierung für verschiedenartige Tierspezies belegen. Die Kapitel 3 und 4 behandeln die Bestimmung des Elastizitätsmoduls von Knochengewebe mittels einer neuen Methode, die - basierend auf dem Konzept der statistischen Nanoindentation und einem evolutionären Algorithmus - zwischen beschädigten und unbeschädigten Materialphasen unterscheiden kann - oder, im Allgemeinen, zwischen Indents, bei denen die Theorie des elastisches Halbraums gültig oder ungültig (z.B. aufgrund existierender oder neugebildeter Mikrorisse) ist. In Kapitel 3 wird der Elastizitätsmodul von unbeschädigtem menschlichem Femur auf extrazellulärer Ebene bestimmt und an verschiedenen anatomischen Positionen und Belastungsrichtungen verglichen. Die Unterschiede in Steifigkeit zwischen Endost und Periost, sowie zwischen belasteten und unbelasteten Regionen werden untersucht. In Kapitel 4 wird der Elastizitätsmodul von intaktem Rinder-Femur auf extrazellulärer Ebene ermittelt. In beiden Kapiteln wird die Hypothese, dass Nanoindentations Tests auch Elastizitätswerte von beschädigtem Material liefern, durch Abbildung von Mikrorissen mit einem Scanning Electron Microscope (SEM) untersucht. Schlussendlich werden in den Kapiteln 5 and 6 experimentelle Methoden zur Bestimmung der mechanischen Eigenschaften von keramischen Materialien für die Produktion von Tissue Engineering Knochengerüsten, Baghdadite (Ca3ZrSi2O9) und Bioglass®, angewandt. Idealerweise sollten diese Gerüste die Eigenschaften von Knochen so genau wie möglich reproduzieren. Für Baghdadite zeigten mit Knochenzellen besetzte Gerüste in vivo gute biologische Eigenschaften, jedoch sind ihre mechanischen Eigenschaften kaum erforscht. In Kapitel 5 wird die Elastizität von Baghdadite unterschiedlicher Porosität mit statistischer Nanoindentation und Ultraschall- Tests charakterisiert. Für Bioglass® Gerüste wurden bereits mechanische Eigenschaften gemessen; diese müssen jedoch verbessert werden, um den Eigenschaften von trabekulärem Knochen möglichst genau zu entsprechen. Die Studie in Kapitel 6 untersucht, wie die Steifigkeit dieser Gerüste durch verschiedene Polymer- Beschichtungen mittels mehrskaliger Ultraschall-Nanoindentations-Messungen verbessert werden kann.23

A mathematical multiscale model of bone remodeling, accounting for pore space-specific mechanosensation

While bone tissue is a hierarchically organized material, mathematical formulations of bone remodeling are often defined on the level of a millimeter-sized representative volume element (RVE), “smeared” over all types of bone microstructures seen at lower observation scales. Thus, there is no explicit consideration of the fact that the biological cells and biochemical factors driving bone remodeling are actually located in differently sized pore spaces: active osteoblasts and osteoclasts can be found in the vascular pores, whereas the lacunar pores host osteocytes – bone cells originating from former osteoblasts which were then “buried” in newly deposited extracellular bone matrix. We here propose a mathematical description which considers size and shape of the pore spaces where the biological and biochemical events take place. In particular, a previously published systems biology formulation, accounting for biochemical regulatory mechanisms such as the RANK-RANKL-OPG pathway, is cast into a multiscale framework coupled to a poromicromechanical model. The latter gives access to the vascular and lacunar pore pressures arising from macroscopic loading. Extensive experimental data on the biological consequences of this loading strongly suggest that the aforementioned pore pressures, together with the loading frequency, are essential drivers of bone remodeling. The novel approach presented here allows for satisfactory simulation of the evolution of bone tissue under various loading conditions, and for different species; including scenarios such as mechanical dis- and overuse of murine and human bone, or in osteocyte-free bone. © 2017 Elsevier Inc

Modal analysis of nanoindentation data, confirming that reduced bone turnover may cause increased tissue mineralization/elasticity

It is widely believed that the activities of bone cells at the tissue scale not only govern the size of the vascular pore spaces (and hence, the amount of bone tissue available for actually carrying the loads), but also the characteristics of the extracellular bone matrix itself. In this context, increased mechanical stimulation (in mediolateral regions of human femora, as compared to anteroposterior regions) may lead to increased bone turnover, lower bone matrix mineralization, and therefore lower tissue modulus. On the other hand, resorption-only processes (in endosteal versus periosteal regions) may have the opposite effect. A modal analysis of nanoindentation data obtained on femurs from the Melbourne Femur Research Collection (MFRC) indeed confirms that bone is stiffer in endosteal regions compared to periosteal regions (E̅endost = 29.34 ± 0.75 GPa > E̅periost = 24.67 ± 1.63 GPa), most likely due to the aging-related increase in resorption modeling on endosteal surfaces resulting in trabecularization of cortical bone. The results also show that bone is stiffer along the anteroposterior direction compared the mediolateral direction (E̅anteropost = 28.89 ± 1.08 GPa > E̅mediolat = 26.03 ± 2.31 GPa), the former being aligned with the neutral bending axis of the femur and, thus, undergoing more resorption modeling and consequently being more mineralized. © 2018 Elsevier Lt

Comparison of DENSE-FISP and DENSE-FID for assessment of cartilage strain computation under compressive loading.

status: publishe

Micro-elasticity of porous ceramic baghdadite : A combined acoustic-nanoindentation approach supported by homogenization theory

Bone tissue engineering aims at repairing damaged bone and restoring its functions with the help of biocompatible materials cultivated with cells and corresponding growth factors [1]. Besides being osteoconductive and osteoinductive, the bone substitute or scaffold should exhibit sufficient porosity for good vascular and tissue ingrowth, while not overly compromising the overall mechanical properties of the implant, i.e. its stiffness and strength. The design process of such scaffolds requires a multitude of in vitro and in vivo experiments and has proven to be a challenging task, thus giving rise to the wish for rational, computer-aided design of biomaterials, regarding not only biological and cell transport aspects, but also mechanics. Highly porous baghdadite (Ca3ZrSi2O9) scaffolds have shown promising biological responses when used for the repair of critical size defects in rabbit radial bones [2]. However, the mechanical properties of these scaffolds require further investigation. Therefore, by using structure-property relations derived from ultrasound and nanoindentation experiments, and on the basis of theoretical and applied micromechanics, the current research aims at applying the state-of-the-art methods in computational biomechanics and biomaterials to this new material to investigate its elastic properties

Micro-poro-elasticity of baghdadite-based bone tissue engineering scaffolds: A unifying approach based on ultrasonics, nanoindentation, and homogenization theory

Microstructure-elasticity relations for bone tissue engineering scaffolds are key to rational biomaterial design. As a contribution thereto, we here report comprehensive length measuring, weighing, and ultrasonic tests at 0.1 MHz frequency, on porous baghdadite (Ca3ZrSi2O9) scaffolds. The resulting porosity-stiffness relations further confirm a formerly detected, micromechanically explained, general relationship for a great variety of different polycrystals, which also allows for estimating the zero-porosity case, i.e. Young modulus and Poisson ratio of pure (dense) baghdadite. These estimates were impressively confirmed by a physically and statistically independent nanoindentation campaign. comprising some 1750 indents. Consequently, we can present a remarkably complete picture of porous baghdadite elasticity across a wide range of porosities, and, thanks to the micromechanical understanding, reaching out beyond classical elasticity, towards poroelastic properties, quantifying the effect of pore pressure on the material system behavior. (C) 2014 The Authors. Published by Elsevier B.V

Non-invasive assessment of loading-mediated mechanics of healthy and diseased cartilage models using high-field DENSE MR-Imaging

status: Published onlin

Influence of the choice of material law coefficients on the predictive accuracy of Finite Element modelling of strain distribution in cartilage.

status: accepte

Combined enzymatic degradation of proteoglycans and collagen significantly alters intratissue strains in articular cartilage during cyclic compression

As degenerative joint diseases such as osteoarthritis (OA) progress, the matrix constituents, particularly collagen fibrils and proteoglycans, become damaged, therefore deteriorating the tissue's mechanical properties. This study aims to further the understanding of the effect of degradation of the different cartilage constituents on the mechanical loading environment in early stage OA. To this end, intact, collagen- and proteoglycan-depleted cartilage plugs were cyclically loaded in axial compression using an experimental model simulating in vivo cartilage-on-cartilage contact conditions in a micro-MRI scanner. Depletion of collagen and proteoglycans was achieved through enzymatic degradation with collagenase and chondroitinase ABC, respectively. Using a displacement-encoded imaging sequence (DENSE), strains were computed and compared in intact and degraded samples. The results revealed that, while degradation with one or the other enzyme had little effect on the contact strains, degradation with a combination of both enzymes caused an increase in the means and variance of the transverse, axial and shear strains, particularly in the superficial zone of the cartilage. This effect indicates that the balance between cartilage matrix constituents plays an essential role in maintaining the mechanical properties of the tissue, and a disturbance in this balance leads to a decrease of the load bearing capacity associated with degenerative joint diseases such as OA.status: publishe

Combined enzymatic degradation of proteoglycans and collagen significantly alters intratissue strains in articular cartilage during cyclic compression

As degenerative joint diseases such as osteoarthritis (OA) progress, the matrix constituents, particularly collagen fibrils and proteoglycans, become damaged, therefore deteriorating the tissue's mechanical properties. This study aims to further the understanding of the effect of degradation of the different cartilage constituents on the mechanical loading environment in early stage OA. To this end, intact, collagen- and proteoglycan-depleted cartilage plugs were cyclically loaded in axial compression using an experimental model simulating in vivo cartilage-on-cartilage contact conditions in a micro-MRI scanner. Depletion of collagen and proteoglycans was achieved through enzymatic degradation with collagenase and chondroitinase ABC, respectively. Using a displacement-encoded imaging sequence (DENSE), strains were computed and compared in intact and degraded samples. The results revealed that, while degradation with one or the other enzyme had little effect on the contact strains, degradation with a combination of both enzymes caused an increase in the means and variance of the transverse, axial and shear strains, particularly in the superficial zone of the cartilage. This effect indicates that the balance between cartilage matrix constituents plays an essential role in maintaining the mechanical properties of the tissue, and a disturbance in this balance leads to a decrease of the load bearing capacity associated with degenerative joint diseases such as OA