Developing a 3D full-thickness skin model based on thiol-norbornene chemistry

Abstract

[eng] Collagen-based gels are widely used as the standard for in vitro skin tissue models due to their biocompatibility and structural similarity to natural skin. However, they have notable drawbacks, such as batch-to-batch variability and gel contraction, which can affect the consistency and reliability of experimental results. To address these issues, various alternatives have been proposed. However, developing a 3D model capable of mimicking the morphology and physiology of the in vivo tissue still represents a challenge. In this thesis, we focused on developing a 3D full-thickness skin model using photocrosslinking gels based on thiol-norbornene chemistry to overcome these limitations. Our initial approach involved creating RGD-functionalized norbornene-pullulan-based gels. The incorporation of the RGD peptide, which is known for enhancing cell adhesion, was aimed at improving cell-matrix interactions to support the development of a functional epithelial layer. The results were promising, as the epithelized-dermal skin models formed successfully, indicating that the norbornene-pullulan gels provided an adequate substrate for skin cell attachment and proliferation. This success laid the groundwork for developing more complex and physiologically relevant skin models. To further refine our model, we investigated single (SN) and interpenetrating network (IPN) systems, based on norbornene dextran (+/- agarose) resulting in robust full-thickness skin models. The norbornene dextran-based gels provided a stable and supportive dermal compartment, where a well-differentiated epidermis on top was observed. A key advantage of our norbornene-based gel systems is their ability to be finely tuned and customized through photocrosslinking. This method allows for precise control over gel properties such as stiffness, which is critical for replicating the mechanical environment of skin tissue. Additionally, the thiol-norbornene chemistry used in our gels is known for its rapid and efficient crosslinking, minimizing the potential for cytotoxicity and preserving cell viability during the gelation process. One of the most significant outcomes of our research was demonstrating the potential to vascularize these gel systems. Vascularization is crucial for in vitro skin models as it facilitates nutrient and oxygen delivery to the tissue, promoting cell survival and function. By incorporating endothelial cells, we successfully induced the formation of capillary-like structures within the gel matrix. This advancement not only enhances the physiological relevance of our skin model but also opens new possibilities for studying skin-related diseases and drug testing in a more realistic in vitro environment. Finally, we also developed a method for the creation of hydrogels with gradients of stiffness, for strategically favoring the incorporation of different cell types depending on the matrix’s stiffness. In conclusion, this thesis presents a significant advancement in developing in vitro skin tissue models. By using photocrosslinking gels based on thiol-norbornene chemistry, we addressed key limitations associated with traditional collagen-based gels. The RGD-functionalized norbornene-pullulan gels supported the formation of epithelized-dermal skin models, while the norbornene dextran-based IPN systems provided robust and more reliable full-thickness skin models with a well-differentiated epidermis. Furthermore, the ability to vascularize these gels enhances their potential for in vitro applications, making our model a competitive alternative for studying skin biology, disease mechanisms, and drug testing. Our findings underscore the importance of developing customizable and valid gel systems for tissue engineering applications. The versatility and tunability of thiol-norbornene-based gels make them a promising platform for creating various tissue models beyond the skin, potentially advancing the field of regenerative medicine and in vitro testing. Future research should focus on further optimizing these gels and exploring their applications in other tissue types, paving the way for more accurate and functional in vitro models.

[spa] Los geles basados en colágeno se usan ampliamente como estándar para modelos de tejido cutáneo in vitro debido a su biocompatibilidad y similitud estructural con la piel natural. Sin embargo, tienen desventajas como la variabilidad entre lotes y la contracción del gel. Se han propuesto varias alternativas, pero desarrollar un modelo 3D que imite la morfología y fisiología del tejido in vivo sigue siendo un desafío. En esta tesis, desarrollamos un modelo tridimensional de piel de grosor completo utilizando geles de entrecruzamiento por luz basados en la química tiol-norborneno. Inicialmente, creamos geles de norborneno-pululano funcionalizados con RGD, un péptido que mejora la adhesión celular, las interacciones célula-matriz y apoyar el desarrollo de una capa epitelial funcional. Los resultados fueron prometedores, logrando formar modelos de piel dermoepitelizados con éxito. Para refinar nuestro modelo, investigamos sistemas de red única y interpenetrante (IPN), basados en dextrano de norborneno (+/- agarosa), y creamos modelos de piel robustos. Estos geles proporcionaron un compartimento dérmico estable y de apoyo, observándose una epidermis bien diferenciada en la parte superior. Una ventaja clave de nuestros geles es su capacidad para ser ajustados mediante entrecruzamiento por luz, permitiendo un control preciso sobre propiedades como la rigidez, crucial para replicar el entorno mecánico del tejido cutáneo. Además, la química utilizada minimiza la citotoxicidad y preserva la viabilidad celular. Un resultado significativo fue demostrar el potencial para vascularizar estos sistemas. La vascularización es crucial para facilitar la entrega de nutrientes y oxígeno al tejido. Al incorporar células endoteliales, logramos formar estructuras capilares dentro de la matriz del gel, mejorando la relevancia fisiológica y abriendo nuevas posibilidades para estudiar enfermedades, y pruebas de medicamentos. También desarrollamos hidrogeles con gradientes de rigidez, favoreciendo la incorporación de diferentes tipos de células según la rigidez de la matriz. Esta tesis presenta un avance significativo en el desarrollo de modelos de tejido cutáneo in vitro. Los geles basados en tiol-norborneno abordan las limitaciones de los geles de colágeno tradicionales, proporcionando modelos de piel dermoepitelizados y de grosor completo más robustos y confiables. La capacidad de vascularizar estos geles mejora su potencial para aplicaciones in vitro, siendo una alternativa competitiva.