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

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

\u3cp\u3eWhile 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.\u3c/p\u3

The relationship between proteoglycan loss, overloading-induced collagen damage, and cyclic loading in articular cartilage

Objective The interaction between proteoglycan loss and collagen damage in articular cartilage and the effect of mechanical loading on this interaction remain unknown. The aim of this study was to answer the following questions: (1) Is proteoglycan loss dependent on the amount of collagen damage and does it depend on whether this collagen damage is superficial or internal? (2) Does repeated loading further increase the already enhanced proteoglycan loss in cartilage with collagen damage? Design Fifty-six bovine osteochondral plugs were equilibrated in phosphate-buffered saline for 24 hours, mechanically tested in compression for 8 hours, and kept in phosphate-buffered saline for another 48 hours. The mechanical tests included an overloading step to induce collagen damage, creep steps to determine tissue stiffness, and cyclic loading to induce convection. Proteoglycan release was measured before and after mechanical loading, as well as 48 hours post-loading. Collagen damage was scored histologically. Results Histology revealed different collagen damage grades after the application of mechanical overloading. After 48 hours in phosphate-buffered saline postloading, proteoglycan loss increased linearly with the amount of total collagen damage and was dependent on the presence but not the amount of internal collagen damage. In samples without collagen damage, repeated loading also resulted in increased proteoglycan loss. However, repeated loading did not further enhance the proteoglycan loss induced by damaged collagen. Conclusion Proteoglycan loss is enhanced by collagen damage and it depends on the presence of internal collagen damage. Cyclic loading stimulates proteoglycan loss in healthy cartilage but does not lead to additional loss in cartilage with damaged collagen

Towards a load bearing hydrogel: A proof of principle in the use of osmotic pressure for biomimetic cartilage constructs

Cartilage defects occur frequently and can lead to osteoarthritis. Hydrogels are a promising regenerative strategy for treating such defects, using their ability of mimicking the native extracellular matrix. However, commonly used hydrogels for tissue regeneration are too soft to resist load-bearing in the joint. To overcome this, an implant is being developed in which the mechanical loadbearing function originates from the osmotic pressure generated by the swelling potential of a charged hydrogel, which is restricted from swelling by a textile spacer fabric. This study aims to quantify the relationship between the swelling potential of the hydrogel and the compressive stiffness of the implant

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

Lithography-based additive manufacturing of customized bioceramic parts for medical applications

In the current study, materials and systems for the fabrication of customized bioceramic parts by using lithography-based additive manufacturing techniques (AMT) are presented. By using this modified system based on digital mirror devices, which relies on a selectively polymerization of a photosensitive ceramic filled resin, structures with a resolution of 40 µm can be generated. By modifying the working DLP-system (Digital Light Processing) a resolution of 25 µm could be reached. The building volume ranges from 77 x 43 x 115 mm to 115 x 65 x 160 mm, depending on the used optics. Photocurable ceramic suspensions with a high solid loading of ceramic powders can be processed. Depending on the ceramic powder and the field of application, delicate bioceramic parts with coordinated properties made of alumina, tricalcium phosphate (TCP) or bioactive glass were fabricated and characterized.status: publishe

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

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Cartilage contact pressure in the knee in the presence of a focal defect

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A new Nanoindentation Protocol for identifying the elasticity of undamaged extracellular bone tissue

\u3cp\u3eWhile the quest for understanding and even mimicking biological tissue has propelled, over the last decades, more and more experimental activities at the micro and nanoscales, the appropriate evaluation and interpretation of respective test results has remained a formidable challenge. As a contribution to tackling this challenge, we here describe a new method for identifying, from nanoindentation, the elasticity of the undamaged extracellular bone matrix. The underlying premise is that the tested bovine bone sample is either initially damaged (i.e. exhibits micro-cracks a priori) or that such micro-cracks are actually induced by the nanoindentation process itself, or both. Then, (very many) indentations may relate to either an intact material phase (which is located sufficiently far away from micro-cracks), or to differently strongly damaged material phases. Corresponding elastic phase properties are identified from the statistical evaluation of the measured indentation moduli, through representation of their histogram as a weighted sum of Gaussian distribution functions. The resulting undamaged elastic modulus of bovine femoral extracellular bone matrix amounts to 31 GPa, a value agreeing strikingly well both with direct quasi-static modulus tests performed on SEM-FIB-produced micro-pillars (Luczynski et al., 2015), and with the predictions of a widely validated micromechanics model (Morin and Hellmich, 2014). Further confidence is gained through observing typical indentation imprints under Scanning Electron Microscopy (SEM), which actually confirms the existence of the two types of micro-cracks as described above.\u3c/p\u3