Mathematical modelling of tissue-engineering angiogenesis

We present a mathematical model for the vascularisation of a porous scaffold following implantation in vivo. The model is given as a set of coupled non-linear ordinary differential equations (ODEs) which describe the evolution in time of the amounts of the different tissue constituents inside the scaffold. Bifurcation analyses reveal how the extent of scaffold vascularisation changes as a function of the parameter values. For example, it is shown how the loss of seeded cells arising from slow infiltration of vascular tissue can be overcome using a prevascularisation strategy consisting of seeding the scaffold with vascular cells. Using certain assumptions it is shown how the system can be simplified to one which is partially tractable and for which some analysis is given. Limited comparison is also given of the model solutions with experimental data from the chick chorioallantoic membrane (CAM) assay

Modeling of mass transfer and fluid flow in perfusion bioreactors

Tissue engineering is an emerging field with the aim to produce artificial organs and tissues for transplant treatments. Cultivating cells on scaffolds by means of bioreactors is a critical step to forming the organ or tissue substitutes prior to transplantation. Among various bioreactors, the perfusion bioreactor is known for its enhanced convection through the cell-scaffold constructs. The enhanced convection will significantly increase the mass transport and at the same time, will increase the shear stress acting on the cells and scaffolds. To manipulate the scaffold-based cell culture process, knowledge of the mass transport and fluid flow (featured by flow velocity and shear stress) in bioreactors is required. Due to the complicated microstructure and multiphase flow involved in this process, the development of models for capturing the aforementioned knowledge has proven to be a challenging task. In this research, the mass transport and fluid flow in scaffolds cultivated in perfusion bioreactors was studied using numerical methods. In the first stream of this research, a novel mathematical model was developed to represent the nutrient transport and cell growth within three-dimensional scaffolds. Based on the developed model, the effect of such factors as the scaffold porosity, the culture time, and the flow rate were investigated. In the second stream, the flow field within the scaffold was studied with an emphasis on representing the shear stress distribution over the scaffold surface. The commercial computational fluid dynamics software ANSYS-CFX was used to simulate and represent the effect of factors, such as the diameter of the scaffold strand, the horizontal span between two strands, and the flow rate, on the shear stress distribution. Results showed that the nutrient concentration and cell volume fraction are time dependent and sensitive to the porosity and flow rate. The diameters of the strands, the horizontal span and the flow rate affect the magnitude of the shear stress. The knowledge obtained in this study provides new insight into the scaffold-based cell culture process in perfusion bioreactors and allows for potential optimization of the cell culture process by regulating the process parameters as well as the scaffold structure during its fabrication

In silico mechano-chemical model of bone healing for the regeneration of critical defects: The effect of BMP-2

The healing of bone defects is a challenge for both tissue engineering and modern ortho- paedics. This problem has been addressed through the study of scaffold constructs com- bined with mechanoregulatory theories, disregarding the influence of chemical factors and their respective delivery devices. Of the chemical factors involved in the bone healing pro- cess, bone morphogenetic protein-2 (BMP-2) has been identified as one of the most power- ful osteoinductive proteins. The aim of this work is to develop and validate a mechano- chemical regulatory model to study the effect of BMP-2 on the healing of large bone defects in silico. We first collected a range of quantitative experimental data from the literature con- cerning the effects of BMP-2 on cellular activity, specifically proliferation, migration, differen- tiation, maturation and extracellular matrix production. These data were then used to define a model governed by mechano-chemical stimuli to simulate the healing of large bone de- fects under the following conditions: natural healing, an empty hydrogel implanted in the de- fect and a hydrogel soaked with BMP-2 implanted in the defect. For the latter condition, successful defect healing was predicted, in agreement with previous in vivo experiments. Further in vivo comparisons showed the potential of the model, which accurately predicted bone tissue formation during healing, bone tissue distribution across the defect and the quantity of bone inside the defect. The proposed mechano-chemical model also estimated the effect of BMP-2 on cells and the evolution of healing in large bone defects. This novel in silico tool provides valuable insight for bone tissue regeneration strategies

IV.3. Bioreactors in tissue engineering.

IV.3. Bioreactors in tissue engineering

Microfluidic-based 3d fibroblast migration studies in biomimetic microenvironments

Cell migration in 3D is a fundamental process in many physiological and pathological phenomena. Indeed, migration through interstitial tissue is a multi-step process that turns out from the cell-ECM interaction. It is a dynamic and complex mechanism that depends on the physic-chemical balance between the cell and its surrounding. Early stage of deep dermal wound healing process is a relevant migratory example, in which the fibroblast is the epicenter: the recruitment of the fibroblasts -by chemotaxis of PDGF-BB- to the clotted wound occurs. Likewise, this work focuses on studying the major underlying mechanisms of 3D fibroblast migration and the main microenvironmental cues involved within. To do so, we have confined two physiologically relevant hydrogels, made of collagen and fibrin, within microfluidic platforms. Firstly, an integral comparative study of biophysical and biomechanical properties of both gels is presented. In these results, we have overcome the wide diversity of the existing data and special stress has been done in order to compare the microstructural arrangement, resistance to flow and elasticity. On the other hand, controlled chemical gradients have been generated and characterized within the microfluidic devices. Since biomolecules interact as purely diffusive factors or bound to the matrix proteins, in this work, distribution of PDGF-BB and TGF-ß1 across collagen and fibrin gels has been quantified. Finally, by taking advantage of the biophysico-chemical definition, we have characterized the migratory responses of human fibroblasts within the microsystems in the presence of a chemoattractant (PDGF-BB). Our results demonstrate that the local microarchitecture of the hydrogels determines the migratory properties of human fibroblasts in response to controlled chemotactic and haptotactic gradients, in a myosin II-dependent manner.La migración celular en 3D es fundamental en muchos fenómenos fisiológicos y patológicos. La migración, la cual resulta de la interacción célula-matriz, es un mecanismo dinámico y complejo que depende del equilibrio entre la célula y su entorno físico-químico. Concretamente, la etapa temprana del proceso de cicatrización de heridas profundas es un proceso migratorio ejemplar, en el cual el fibroblasto es el epicentro: se produce el reclutamiento de los fibroblastos -por quimiotaxis de PDGF-BB- del tejido circundante al coágulo. Este trabajo se centra en el estudio de los principales mecanismos subyacentes de la migración de fibroblastos en 3D y las principales señales microambientales involucradas en ella. Para ello, se han empleado modelos in vitro haciendo uso de plataformas microfluídicas para confinar dos hidrogeles fisiológicamente relevantes, compuestos por colágeno y fibrina. En primer lugar, se presenta un estudio comparativo integral de las propiedades biofísicas y biomecánicas de los hidrogeles. En estos resultados, se ha hecho especial hincapié en comparar la conformación microestructural, la resistencia al flujo de fluido y la elasticidad. Por otro lado, se han generado y caracterizado gradientes químicos dentro de los dispositivos. Puesto que las biomoléculas interactúan como factores puramente difusivos o adheridos a las proteínas de la matriz, en este trabajo se ha cuantificado la distribución de PDGF-BB y TGF-β1, en colágeno y fibrina. Finalmente, mediante esta definición físico-química, se ha caracterizado la respuesta migratoria de fibroblastos humanos dentro de los microdispositivos en presencia de un factor químico (PDGF-BB). Los resultados aquí mostrados demuestran que la microarquitectura local de los hidrogeles determina las propiedades migratorias de fibroblastos humanos en respuesta a gradientes quimiotácticos y haptotácticos, de manera dependiente de la miosina II

Development and characterisation of MSC-seeded decellularised airway scaffolds for regenerative bioengineering

Tracheal tissue engineering (TE) is a potential solution for long tracheal lesions and recent clinical experience yielded promising results but challenges remain with respect to measurable criteria for acceptance of decellularised scaffolds, optimisation of cell seeding and understanding the biology of the seeded cells post attachment. Confirming previous data from our group, I showed cellular clearance of DC scaffolds and significant reduction in total DNA levels but observed retention of residual nuclear materials within hyaline cartilage and submucosa. Evaluation of extracellular matrix components demonstrated retention of collagen and glycosaminoglycan and disrupted basement membrane components. The novel use of dynamic mechanical analysis (DMA) to measure the viscoelastic properties of tracheal cartilage in addition to tensile testing, provided the first demonstration of preservation of native viscoelastic mechanical properties after decellularisation. To overcome the limitations of passive cell seeding, I conceived partial surface dehydration (PSD) conditioning of scaffolds which significantly improved cell seeding/attachment efficiency to (96.46% 1.710) and I confirmed survival of MSCs on the scaffold in vitro. Multiphoton imaging showed limited scaffold infiltration but revealed two, distinct cell morphologies dependent on the presence or absence of adventitia. These showed different RNA transcriptomic profiles and differential gene expression. Seeded MSCs upregulated transcripts of bioactive paracrine factors associated with tissue repair, including ECM remodelling, pro-angiogenesis, antifibrosis, chemoattraction and immunomodulatory properties. Cells seeded into the adventitial layer upregulated more bioactive factors and showed lower cellular stress, suggesting a favourable effect of maintaining adventitial layer. The data presented herein form a coherent series of experiments providing novel data to the field of tracheal tissue engineering which address important GMP issues such as in-process acceptance criteria for scaffolds and data to support the rationale of autologous MSC seeding prior to implantation. These results allowed us to manufacture an improved clinical product for a compassionate case

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 /