Designing Hydrogel-Based Bone-On-Chips for Personalized Medicine

The recent development of bone-on-chips (BOCs) holds the main advantage of requiring a low quantity of cells and material, compared to traditional In Vitro models. By incorporating hydrogels within BOCs, the culture system moved to a three dimensional culture environment for cells which is more representative of bone tissue matrix and function. The fundamental components of hydrogel-based BOCs, namely the cellular sources, the hydrogel and the culture chamber, have been tuned to mimic the hematopoietic niche in the bone aspirate marrow, cancer bone metastasis and osteo/chondrogenic differentiation. In this review, we examine the entire process of developing hydrogel-based BOCs to model In Vitro a patient specific situation. First, we provide bone biological understanding for BOCs design and then how hydrogel structural and mechanical properties can be tuned to meet those requirements. This is followed by a review on hydrogel-based BOCs, developed in the last 10 years, in terms of culture chamber design, hydrogel and cell source used. Finally, we provide guidelines for the definition of personalized pathological and physiological bone microenvironments. This review covers the information on bone, hydrogel and BOC that are required to develop personalized therapies for bone disease, by recreating clinically relevant scenarii in miniaturized device

Estudio de diferenciación y mineralización in vitro de osteoblastos en dispositivos microfluídicos

La ausencia de producción de matriz ósea en scaffolds e implantes, supone una limitación en el campo de la ingeniería de tejidos para la remodelación ósea. Los sustitutos óseos no consiguen replicar la capacidad de remodelación que posee este tejido. Por ello, es necesario conocer qué estímulos ayudan a favorecer este proceso a nivel celular. El desarrollo de modelos óseos in vitro pretende ayudar a entender qué mecanismos promoverían la formación de hueso, así como el estudio aislado de determinadas patologías o terapias potenciales. Para llevar esto a cabo, se han utilizado dispositivos microfluídicos, dentro de los cuales se siembras las células óseas embebidas en una matriz colagenosa. Lo que se ha hecho en este trabajo ha sido una siembra de osteoblastos humanos en una matriz densa de colágeno. El principal objetivo es conseguir el modelo óseo mediante un cultivo de osteoblastos primarios humanos a largo plazo. Además, se quiere medir el comportamiento ostegénico de las células y estudiar si existe una completa diferenciación a osteocitos. Se ha conseguido ver que, en las condiciones estudiadas, existe comportamiento osteogénico del cultivo: demostrado por la expresión de la enzyma ALP y el calcio liberado a la matriz. Sin embargo, todavía no ha sido posible obtener una completa diferenciación ya que no hay expresión de la proteína DMP1 en la matriz y la actividad enzimática no se reduce con el tiempo. Con lo que concluído a lo largo de estos experimentos y comparando con la información obtenida de la literatura, se deduce que la principal necesidad es aumentar la densidad celular dentro del gel y, si es posible, extender los tiempos de la experimentación

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 /

Towards in silico Models of the Inflammatory Response in Bone Fracture Healing.

peer reviewedIn silico modeling is a powerful strategy to investigate the biological events occurring at tissue, cellular and subcellular level during bone fracture healing. However, most current models do not consider the impact of the inflammatory response on the later stages of bone repair. Indeed, as initiator of the healing process, this early phase can alter the regenerative outcome: if the inflammatory response is too strongly down- or upregulated, the fracture can result in a non-union. This review covers the fundamental information on fracture healing, in silico modeling and experimental validation. It starts with a description of the biology of fracture healing, paying particular attention to the inflammatory phase and its cellular and subcellular components. We then discuss the current state-of-the-art regarding in silico models of the immune response in different tissues as well as the bone regeneration process at the later stages of fracture healing. Combining the aforementioned biological and computational state-of-the-art, continuous, discrete and hybrid modeling technologies are discussed in light of their suitability to capture adequately the multiscale course of the inflammatory phase and its overall role in the healing outcome. Both in the establishment of models as in their validation step, experimental data is required. Hence, this review provides an overview of the different in vitro and in vivo set-ups that can be used to quantify cell- and tissue-scale properties and provide necessary input for model credibility assessment. In conclusion, this review aims to provide hands-on guidance for scientists interested in building in silico models as an additional tool to investigate the critical role of the inflammatory phase in bone regeneration

Primary Human Osteoblasts Cultured in a 3D Microenvironment Create a Unique Representative Model of Their Differentiation Into Osteocytes

{Microengineered systems provide an in vitro strategy to explore the variability of individual patient response to tissue engineering products, since they prefer the use of primary cell sources representing the phenotype variability. Traditional in vitro systems already showed that primary human osteoblasts embedded in a 3D fibrous collagen matrix differentiate into osteocytes under specific conditions. Here, we hypothesized that translating this environment to the organ-on-a-chip scale creates a minimal functional unit to recapitulate osteoblast maturation toward osteocytes and matrix mineralization. Primary human osteoblasts were seeded in a type I collagen hydrogel, to establish the role of lower (2.5 × 105 cells/ml) and higher (1 × 106 cells/ml) cell density on their differentiation into osteocytes. A custom semi-automatic image analysis software was used to extract quantitative data on cellular morphology from brightfield images. The results are showing that cells cultured at a high density increase dendrite length over time, stop proliferating, exhibit dendritic morphology, upregulate alkaline phosphatase (ALP) activity, and express the osteocyte marker dental matrix protein 1 (DMP1). On the contrary, cells cultured at lower density proliferate over time, do not upregulate ALP and express the osteoblast marker bone sialoprotein 2 (BSP2) at all timepoints. Our work reveals that microengineered systems create unique conditions to capture the major aspects of osteoblast differentiation into osteocytes with a limited number of cells. We propose that the microengineered approach is a functional strategy to create a patient-specific bone tissue model and investigate the individual osteogenic potential of the patient bone cells

COMMBINI:an experimentally-informed COmputational Model of Macrophage dynamics in the Bone INjury Immunoresponse

Bone fracture healing is a well-orchestrated but complex process that involves numerous regulations at different scales. This complexity becomes particularly evident during the inflammatory stage, as immune cells invade the healing region and trigger a cascade of signals to promote a favorable regenerative environment. Thus, the emergence of criticalities during this stage might hinder the rest of the process. Therefore, the investigation of the many interactions that regulate the inflammation has a primary importance on the exploration of the overall healing progression. In this context, an in silico model named COMMBINI (COmputational Model of Macrophage dynamics in the Bone INjury Immunoresponse) has been developed to investigate the mechano-biological interactions during the early inflammatory stage at the tissue, cellular and molecular levels. An agent-based model is employed to simulate the behavior of immune cells, inflammatory cytokines and fracture debris as well as their reciprocal multiscale biological interactions during the development of the early inflammation (up to 5 days post-injury). The strength of the computational approach is the capacity of the in silico model to simulate the overall healing process by taking into account the numerous hidden events that contribute to its success. To calibrate the model, we present an in silico immunofluorescence method that enables a direct comparison at the cellular level between the model output and experimental immunofluorescent images. The combination of sensitivity analysis and a Genetic Algorithm allows dynamic cooperation between these techniques, enabling faster identification of the most accurate parameter values, reducing the disparity between computer simulation and histological data. The sensitivity analysis showed a higher sensibility of the computer model to the macrophage recruitment ratio during the early inflammation and to proliferation in the late stage. Furthermore, the Genetic Algorithm highlighted an underestimation of macrophage proliferation by in vitro experiments. Further experiments were conducted using another externally fixated murine model, providing an independent validation dataset. The validated COMMBINI platform serves as a novel tool to deepen the understanding of the intricacies of the early bone regeneration phases. COMMBINI aims to contribute to designing novel treatment strategies in both the biological and mechanical domains.</p

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

Engineering in-plane mechanics of electrospun polyurethane scaffolds for cardiovascular tissue applications

Effective cardiovascular tissue surrogates require high control of scaffold structural and mechanical features to match native tissue properties, which are dependent on tissue-specific mechanics, function heterogenicity, and morphology. Bridging scaffold processing variables with native tissue properties is recognized as a priority for advancing the biomechanical performance of biomedical materials and when translated to clinical practice, their efficacy. Accordingly, this study selected electrospinning on a rotating cylindrical target as an apparatus of broad application and mapped the relationship between key processing variables and scaffold mechanics and structure. This information was combined with mechanical anisotropy ranges of interest for the three main categories of tissue surrogated in cardiovascular tissue engineering: heart valve leaflets, ventricle wall, and large diameter blood vessels. Specifically, three processing variables have been considered: the rotational velocity and the rastering velocity of the mandrel and the dry (single nozzle – polymer only) vs wet (double nozzle – polymer plus phosphate buffer saline solution) fabrication configuration. While the dry configuration is generally utilized to obtain micro-fiber based polymeric mats, the wet fabrication is representative of processing conditions utilized to incorporate cells, growth factors, or micro-particles within the fibrous scaffold matrix. Dry and wet-processed electrospun mats were fabricated with tangential and rastering velocities within the 0.3-9.0 m/s and 0.16-8 cm/s range respectively. Biaxial mechanics, fiber network, and pore microarchitectures were measured for each combination of velocities and for each fabrication modality (dry and wet). Results allowed identification of the precise combination of rotational and rastering velocities, for both dry and wet conditions, that is able to recapitulate the native cardiovascular tissue anisotropy ratio. By adopting a simple and broadly utilized electrospinning layout, this study is meant to provide a repeatable and easy to access methodology to improve biomimicry of the in plane-mechanics of heart valve leaflets, ventricular wall, and large diameter blood vessels

Mechano-driven regeneration predicts response variations in large animal model based on scaffold implantation site and individual mechano-sensitivity

It is well founded that the mechanical environment may regulate bone regeneration in orthopedic applications. The purpose of this study is to investigate the mechanical contributions of the scaffold and the host to bone regeneration, in terms of subject specificity, implantation site and sensitivity to the mechanical environment. Using a computational approach to model mechano-driven regeneration, bone ingrowth in porous titanium scaffolds was simulated in the distal femur and proximal tibia of three goats and compared to experimental results. The results showed that bone ingrowth shifted from a homogeneous distribution pattern, when scaffolds were in contact with trabecular bone (max local ingrowth 12.47%), to a localized bone ingrowth when scaffolds were implanted in a diaphyseal location (max local ingrowth 20.64%). The bone formation dynamics revealed an apposition rate of 0.37±0.28%/day in the first three weeks after implantation, followed by limited increase in bone ingrowth until the end of the experiment (12 weeks). According to in vivo data, we identified one animal whose sensitivity to mechanical stimulation was higher than the other two. Moreover, we found that the stimulus initiating bone formation was consistently higher in the femur than in the tibia for all the individuals. Overall, the dependence of the osteogenic response on the host biomechanics means that, from a mechanical perspective, the regenerative potential depends on both the scaffold and the host environment. Therefore, this work provides insights on how the mechanical conditions of both the recipient and the scaffold contribute to meet patient and location-specific characteristics

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