Mechano-Biological Computer Model of Scaffold-Supported Bone Regeneration: Effect of Bone Graft and Scaffold Structure on Large Bone Defect Tissue Patterning

Large segmental bone defects represent a clinical challenge for which current treatment procedures have many drawbacks. 3D-printed scaffolds may help to support healing, but their design process relies mainly on trial and error due to a lack of understanding of which scaffold features support bone regeneration. The aim of this study was to investigate whether existing mechano-biological rules of bone regeneration can also explain scaffold-supported bone defect healing. In addition, we examined the distinct roles of bone grafting and scaffold structure on the regeneration process. To that end, scaffold-surface guided migration and tissue deposition as well as bone graft stimulatory effects were included in an in silico model and predictions were compared to in vivo data. We found graft osteoconductive properties and scaffold-surface guided extracellular matrix deposition to be essential features driving bone defect filling in a 3D-printed honeycomb titanium structure. This knowledge paves the way for the design of more effective 3D scaffold structures and their pre-clinical optimization, prior to their application in scaffold-based bone defect regeneration

Titanium Lattice Structures Produced via Additive Manufacturing for a Bone Scaffold: A Review

The progress in additive manufacturing has remarkably increased the application of lattice materials in the biomedical field for the fabrication of scaffolds used as bone substitutes. Ti6Al4V alloy is widely adopted for bone implant application as it combines both biological and mechanical properties. Recent breakthroughs in biomaterials and tissue engineering have allowed the regeneration of massive bone defects, which require external intervention to be bridged. However, the repair of such critical bone defects remains a challenge. The present review collected the most significant findings in the literature of the last ten years on Ti6Al4V porous scaffolds to provide a comprehensive summary of the mechanical and morphological requirements for the osteointegration process. Particular attention was given on the effects of pore size, surface roughness and the elastic modulus on bone scaffold performances. The application of the Gibson-Ashby model allowed for a comparison of the mechanical performance of the lattice materials with that of human bone. This allows for an evaluation of the suitability of different lattice materials for biomedical applications

In silico assessment of the bone regeneration potential of complex porous scaffolds

Mechanical environment plays a crucial role in regulating bone regeneration in bone defects. Assessing the mechanobiological behavior of patient-specific orthopedic scaffolds in-silico could help guide optimal scaffold designs, as well as intra- and post-operative strategies to enhance bone regeneration and improve implant longevity. Additively manufactured porous scaffolds, and specifically triply periodic minimal surfaces (TPMS), have shown promising structural properties to act as bone substitutes, yet their ability to induce mechanobiologially-driven bone regeneration has not been elucidated. The aim of this study is to i) explore the bone regeneration potential of TPMS scaffolds made of different stiffness biocompatible materials, to ii) analyze the influence of pre-seeding the scaffolds and increasing the post-operative resting period, and to iii) assess the influence of patient-specific parameters, such as age and mechanosensitivity, on outcomes. To perform this study, an in silico model of a goat tibia is used. The bone ingrowth within the scaffold pores was simulated with a mechano-driven model of bone regeneration. Results showed that the scaffold's architectural properties affect cellular diffusion and strain distribution, resulting in variations in the regenerated bone volume and distribution. The softer material improved the bone ingrowth. An initial resting period improved the bone ingrowth but not enough to reach the scaffold's core. However, this was achieved with the implantation of a pre-seeded scaffold. Physiological parameters like age and health of the patient also influence the bone regeneration outcome, though to a lesser extent than the scaffold design. This analysis demonstrates the importance of the scaffold's geometry and its material, and highlights the potential of using mechanobiological patient-specific models in the design process for bone substitutes

Mechanobiologische Optimierung von Gerüsten für große Knochendefekte

Bone has the remarkable capability to regenerate without forming any scar tissue upon a given injury. However, this intrinsic healing capacity can be impaired in some critical cases, such as large bone defects where a large piece of bone is missing. The current gold standard treatment for such defects is autologous bone grafting, where bone material is taken from another location in the patient and put into the defect to support it mechanically and biologically. Nevertheless, this treatment has several drawbacks, in particular the need for an additional surgery associated with donor site morbidities and a limited availability of bone material. An alternative treatment strategy is the use of 3D-printed bone scaffolds. Such implants are produced synthetically before the chirurgical intervention, can be made of metal, polymer or ceramic, and implanted into various defects to support their regeneration. However, how such scaffolds interact with the bone regeneration process has remained poorly understood so far, preventing scaffolds to become clinical routine practice in large bone defect treatment. In addition, scaffold design has mainly relied on trial-and-error approaches, requiring long, costly and ethically questionable experiments to assess the scaffold healing potential. This critical clinical issue was the starting point of this project. The aim of the thesis was therefore to propose an in silico methodology to design scaffolds for large bone defect regeneration that would be optimised for their ability to support the endogenous bone healing process. To do so, specific mechanisms of scaffold-supported large bone defect regeneration were investigated by means of a predictive computer model for bone regeneration. Next, the effect of individual scaffold design parameters were studied. In addition, the dynamics of the bone healing process was investigated by comparing healing outcome predictions at different healing time points. Lastly, a computational framework for bone scaffold optimisation was developed with the objective of maximising the amount of regenerated bone. The in silico investigation of scaffold-supported large bone defect regeneration used a coupled finite element analysis and agent-based model to simulate various experimental studies. The comparison between in silico predictions and experimental results revealed specific features of the regeneration process: (1) the lack of biological stimulation in large bone defects, that can be compensated by specific scaffold material or the addition of bone graft and (2) guidance provided by the scaffold surfaces on the cellular migration and tissue deposition processes. Specific scaffold designs were varied by changing the pore size, strut size or material properties. Overall, porous stiff scaffolds or more bulky softer scaffolds were found to be most beneficial for bone regeneration. However, precise pore or strut size recommendations depended largely on the underlying scaffold architecture, suggesting that a "one-fits-them-all" approach cannot be used to design optimal scaffolds. In addition, the modelling of different time horizons (initial mechanics and bone regeneration simulation) highlighted the dynamics of the bone regeneration process. Conclusions based on different time horizons contradicted each other: several scaffold designs seemed very favourable initially but were actually not predicted to support bone regeneration well. These observations emphasised the need for a dynamic optimisation process that would take the regeneration process into account. Lastly, a computational framework for bone scaffold design optimisation was developed. Its objective was defined as to maximise regenerated bone volume according to the mechanobiological bone regeneration model; scaffold designs were parametrised with a few variables (e.g. pore sizes or material properties). A surrogate optimisation approach was chosen to deal with the computationally demanding regeneration simulations: a simplified input-output relationship was derived from a set of initial simulations and used for the optimisation process to avoid too many demanding simulation runs. The results presented in the thesis proved the feasibility of the approach on a simplified cubic scaffold and a more realistic cylindrical scaffold. These results suggested that the initial design and its parametrisation should be carefully chosen to improve the optimisation outcome. Future work should further validate the computer model for scaffold-supported bone regeneration. A particular emphasis should be put on improving the computational efficiency of such models for their use in optimisation purposes. Next, more realistic, clinically-based scaffold designs should be employed in the optimisation framework, in combination with experiments. In that way, knowledge on scaffold healing potential and informed scaffold design for experiments could be improved iteratively.Knochen hat die bemerkenswerte Fähigkeit, sich nach einer Verletzung ohne Narbenbildung zu regenerieren. Allerdings kann diese inhärente Fähigkeit in bestimmten kritischen Fällen beeinträchtigt werden, zum Beispiel im Fall von großen Knochendefekten, bei denen ein großes Knochenstück fehlt. Die derzeitige Standardbehandlung für solche Defekte beinhaltet den Einsatz von autologem Knochenmaterial, wobei Knochenmaterial des Patienten aus einer defektfernen Stelle entnommen und in den Defekt implementiert wird, um ihn mechanisch und biologisch zu unterstützen. Diese Behandlungsmethode hat jedoch mehrere wesentliche Nachteile wie beispielsweise den zusätzlich erforderlichen chirurgischen Eingriff, der mit Morbidität an der Entnahmestelle verbunden ist, und die begrenzte Verfügbarkeit des Knochenmaterials. Eine alternative Behandlungsstrategie besteht darin, 3D-gedruckte Knochengerüste zu benutzen. Solche Implantate, bestehend aus Metall, Polymer oder Keramik, werden synthetisch vor dem chirurgischen Eingriff hergestellt und finden Anwendung in unterschiedlichen Defekten, um deren Regeneration zu unterstützen. Bislang bleibt unklar, wie genau solche Gerüste den Knochenregenerationsprozess beeinflussen, was deren alltägliche klinische Anwendung für die Behandlung von großen Knochendefekten unterbunden hat. Zur Bewertung des klinischen Potenzials eines Gerüstes wurde vor allem die Versuch-und-Irrtum-Methode herangezogen, welche langwierige, teure und mitunter ethisch fragwürdige Experimente erfordert. Dieses wichtige klinische Problem galt als Ausgangspunkt dieses Projekts. Ziel der Doktorarbeit war es also, eine in silico Methode zu entwickeln, um Gerüste für die Regeneration großer Knochendefekte zu konzipieren, die den endogenen Knochenheilungsprozess optimal unterstützen. Dazu wurden spezifische Mechanismen der gerüstgestützten Regeneration großer Knochendefekte anhand eines prädiktiven Computermodells für Knochenregeneration untersucht. Anschließend wurde der Effekt individueller Gerüstparameter studiert. Hierbei wurde die Dynamik des Knochenregenerationsprozesses erforscht, indem Heilungsvorhersagen nach bestimmten Heilungszeitpunkten miteinander verglichen wurden. Letztens wurde ein Computer-Framework für die Optimierung der Knochengerüste mit dem Ziel entwickelt, das Volumen regenerierten Knochens zu maximieren. Die in silico Untersuchung der gerüstgestützten Regeneration großer Knochendefekte baute auf einer gekoppelten Finite-Elemente-Analyse und agentenbasierte Modellierung auf, um verschiedene Experimente zu simulieren. Der Vergleich zwischen den in silico Vorhersagen und den experimentellen Ergebnissen lieferte mechanistische Einblicke in den Regenerationsprozess: (1) der Mangel an biologischer Stimulation in großen Knochendefekten, der durch den Einsatz von Gerüstmaterial oder Knochenmaterial kompensiert werden kann, und (2) der Einfluss der Gerüstflächen auf die Prozesse von Zellmigration und Gewebeanlagerung. Unterschiedliche Gerüste wurden simuliert, indem die Porengröße, die Stützengröße oder die Materialeigenschaften verändert wurden. Generell zeigte sich, dass poröse steife Gerüste oder voluminösere weichere Gerüste die Knochenregeneration am vorteilhaftesten unterstützen. Allerdings hingen die optimalen Poren- oder Stützengrößen größtenteils von der zugrunde liegenden Gerüstarchitektur ab, was darauf hinweist, dass eine allgemeingültige Empfehlung nicht angewendet werden kann, um optimale Gerüste zu konzipieren. Darüber hinaus zeigte die Modellierung von verschiedenen Zeitintervallen (initiale Mechanik und Simulation der Knochenregeneration) die Dynamik des Knochenregenerationsprozesses. Ergebnisse aus verschiedenen Zeitintervallen standen nämlich im Widerspruch zueinander: mehrere Gerüste schienen initial sehr vorteilhaft aber der Modellvorhersagen zufolge wurde die Knochenregeneration dann nicht gut unterstützt. Diese Feststellung bestätigte den Bedarf eines dynamischen Optimierungsprozesses, der auch den Regenerationsprozess berücksichtigen würde. Letztens wurde ein Computer-Framework für die Optimierung von Knochengerüsten entwickelt. Als Ziel wurde die Maximierung des Volumens des regenerierten Knochens nach dem mechanobiologischen Knochenregenerationsmodell gesetzt. Gerüste wurden anhand einiger Variablen (etwa Porengröße oder Materialeigenschaften) parametrisiert. Eine Surrogat-Optimierungsvorgehensweise wurde wegen der rechenintensiven Regenerationssimulationen gewählt: ein vereinfachtes Input-Output-Verhältnis wurde von einer Gruppe von initialen Simulationsläufen abgeleitet und für den Optimierungsprozess benutzt, um zu viele rechenintensive Simulationsläufe zu vermeiden. Die Ergebnisse dieser Doktorarbeit beweisen das Anwendungspotenzial dieser Methode für ein vereinfachtes kubisches Gerüst und ein realistischeres zylindrisches Gerüst. Diese Ergebnisse zeigen, dass das initiale Design des Gerüsts und dessen Parametrierung bedacht ausgesucht werden sollten, um den Optimierungsausgang zu verbessern. Zukünftige Forschungsarbeit sollte die Validierung des Computermodells für die gerüstgestützte Regeneration großer Knochendefekte fortsetzen. Insbesondere ist darauf zu achten, die rechnerische Effizienz dieser Modelle für deren Anwendung für Optimierungszwecke zu verbessern. Zusätzlich sollten realistischere, klinisch-basierte Gerüste für das Optimierungsverfahren mit komplementären Experimenten benutzt werden. Auf dieser Weise kann ein grundlegendes Verständnis für das Heilungspotenzial des Gerüstes geschaffen und ein fundiertes Design von experimentellen Gerüsten iterativ verbessert werden

In silico assessment of the bone regeneration potential of complex porous scaffolds.

Mechanical environment plays a crucial role in regulating bone regeneration in bone defects. Assessing the mechanobiological behavior of patient-specific orthopedic scaffolds in-silico could help guide optimal scaffold designs, as well as intra- and post-operative strategies to enhance bone regeneration and improve implant longevity. Additively manufactured porous scaffolds, and specifically triply periodic minimal surfaces (TPMS), have shown promising structural properties to act as bone substitutes, yet their ability to induce mechanobiologially-driven bone regeneration has not been elucidated. The aim of this study is to i) explore the bone regeneration potential of TPMS scaffolds made of different stiffness biocompatible materials, to ii) analyze the influence of pre-seeding the scaffolds and increasing the post-operative resting period, and to iii) assess the influence of patient-specific parameters, such as age and mechanosensitivity, on outcomes. To perform this study, an in silico model of a goat tibia is used. The bone ingrowth within the scaffold pores was simulated with a mechano-driven model of bone regeneration. Results showed that the scaffold's architectural properties affect cellular diffusion and strain distribution, resulting in variations in the regenerated bone volume and distribution. The softer material improved the bone ingrowth. An initial resting period improved the bone ingrowth but not enough to reach the scaffold's core. However, this was achieved with the implantation of a pre-seeded scaffold. Physiological parameters like age and health of the patient also influence the bone regeneration outcome, though to a lesser extent than the scaffold design. This analysis demonstrates the importance of the scaffold's geometry and its material, and highlights the potential of using mechanobiological patient-specific models in the design process for bone substitutes

Bone regeneration in patient-specific scaffolds from microfluidics to computational simulation

Los trastornos musculoesqueléticos y sus correspondientes enfermedades óseas son una de las principales causas de dolor y discapacidad, así como una carga social y económica para nuestra sociedad. Cuando la función articular se ve afectada o los defectos óseos son demasiado grandes para los injertos óseos, los implantes protésicos son el método estándar para tratar los trastornos musculoesqueléticos graves, aunque existe la necesidad clínica de que los implantes permanezcan activos durante un período de tiempo más largo y reduzcan las tasas de revisión. Para abordar la mayor durabilidad de los implantes ortopédicos, recientemente han surgido implantes impresos en tres dimensiones (3D) para fabricar superficies porosas específicas del paciente en la superficie del hueso-implante, mejorando así la fijación biológica del implante. La traslación de los principios de la medicina regenerativa a la ortopedia permitiría definir una nueva generación de implantes que completen la transición de materiales inertes a andamios bioactivos que guíen el proceso de regeneración ósea. A corto plazo, es probable que los andamios ortopédicos regenerativos impresos en 3D aumenten la vida útil del implante, mientras que a largo plazo puedan degradarse una vez que el tejido huésped esté completamente reparado. El objetivo global de esta tesis es evaluar el potencial regenerativo asociado a los andamiajes óseos impresos en 3D para aplicaciones ortopédicas específicas del paciente.Para ello, el primer estudio tuvo como objetivo determinar el papel del entorno mecánico del huésped en el proceso de regeneración ósea guiado por andamios óseos impresos en 3D en aplicaciones de carga. Se desarrolló un modelo computacional de regeneración ósea impulsada por un mecanismo en andamios porosos y se basó en la especificidad del sujeto, el sitio de implantación y la sensibilidad al entorno mecánico. A continuación, se simuló el crecimiento óseo en el interior de andamiajes porosos de titanio implantados en el fémur distal y la tibia proximal de tres cabras y se comparó con los resultados experimentales. Los resultados mostraron que el crecimiento óseo en el interior cambió de un patrón de distribución homogéneo, cuando los andamios estaban en contacto con el hueso trabecular, a un crecimiento óseo localizado cuando los andamios se implantaron en una ubicación diafisaria. En general, la dependencia de la respuesta osteogénica de la biomecánica del huésped sugirió que, desde una perspectiva mecánica, el potencial regenerativo dependía tanto del andamio como del entorno del huésped.El segundo estudio de esta tesis tuvo como objetivo evaluar la actividad osteogénica específica del paciente en un entorno controlado in vitro donde las células óseas humanas, aisladas de sujetos individuales, imitan los rasgos esenciales del proceso de formación ósea. Los sistemas in vitro tradicionales ya permitieron demostrar que los osteoblastos humanos primarios embebidos en una matriz fibrada de colágeno se diferencian en osteocitos en condiciones específicas. Por lo tanto, se planteó la hipótesis de que la traslación de este entorno a la escala de órgano en un chip crea una unidad funcional mínima para recapitular la maduración de los osteoblastos hacia los osteocitos y la mineralización de la matriz. Con este propósito, se sembraron osteoblastos humanos primarios en un hidrogel de colágeno de tipo I, para conocer mejor el papel de la densidad de siembra de células en su diferenciación a osteocitos. Los resultados muestran que las células cultivadas a mayor densidad aumentan la longitud de la dendrita con el tiempo, dejan de proliferar, exhiben morfología dendrítica, regulan positivamente la actividad de la fosfatasa alcalina y expresan marcadores de osteocitos. Este estudio reveló que los sistemas de microfluídica son una estrategia funcional que permite crear un modelo de tejido óseo específico del paciente e investigar el potencial osteogénico individual de las células óseas del paciente.En conjunto, los resultados de esta tesis enfatizan la importancia de utilizar un sistema de modelado múltiple al investigar el proceso de regeneración in vivo guiado por armazones óseos específicos adecuados al paciente. Ambos actores de una estrategia regenerativa libre de células in situ, a saber, el andamio y el paciente, tienen un efecto significativo en el resultado regenerativo final y necesitan ser modelados. Las técnicas avanzadas de in vitro e in silico, combinadas con datos de in vivo, evalúan aspectos distintivos del proceso de regeneración ósea para aplicaciones específicas del paciente. Las futuras estrategias personalizadas de ingeniería de tejidos podrían depender de la integración de esos modelos para mitigar en última instancia la variabilidad en el proceso de regeneración ósea guiado por un andamio específico para el paciente.<br /

Optimisation and validation of a custom-designed perfusion bioreactor for bone tissue engineering: Flow assessment and optimal culture environmental conditions

Various perfusion bioreactor systems have been designed to improve cell culture with three-dimensional porous scaffolds, and there is some evidence that fluid force improves the osteogenic commitment of the progenitors. However, because of the unique design concept and operational configuration, the experimental setups of perfusion bioreactor systems are not always compatible. To reconcile results from different systems, the thorough optimisation and validation of the experimental configuration are required in each system. In this study, optimal experimental conditions for a perfusion bioreactor were explored in 3 steps. First, an in silico modelling was performed using a scaffold geometry obtained by microCT and an expedient geometry parameterised with porosity and permeability to assess the accuracy of calculated fluid shear stress and computational time. Then, environmental factors for cell culture were optimised, including the volume of the medium, bubble suppression, and medium evaporation. Further, by combining the findings, it was possible to determine the optimal flow rate at which cell growth was supported but osteogenic differentiation was triggered. Here, we demonstrated that fluid shear stress, ranging from nearly 0 to 15 mPa, was sufficient to induce osteogenesis, but cell growth was severely impacted by the volume of perfused medium, the presence of air bubbles, and medium evaporation, all of which are common concerns in perfusion bioreactor systems. This study emphasises the necessity of optimisation of experimental variables, which may often be underreported or overlooked, and indicates steps which can be taken to address issues common to perfusion bioreactors for bone tissue engineering.publishedVersio

Multiscale modeling of bone tissue Mechanobiology

Mechanical environment has a crucial role in our organism at the different levels, ranging from cells to tissues and our own organs. This regulatory role is especially relevant for bones, given their importance as load-transmitting elements that allow the movement of our body as well as the protection of vital organs from load impacts. Therefore bone, as living tissue, is continuously adapting its properties, shape and repairing itself, being the mechanical loads one of the main regulatory stimuli that modulate this adaptive behavior. Here we review some key results of bone mechanobiology from computational models, describing the effect that changes associated to the mechanical environment induce in bone response, implant design and scaffold-driven bone regeneration