Ciencias Exactas y Ciencias de la Salud
Permanent URI for this collectionhttps://hdl.handle.net/11285/551039
Pertenecen a esta colección Tesis y Trabajos de grado de las Maestrías correspondientes a las Escuelas de Ingeniería y Ciencias así como a Medicina y Ciencias de la Salud.
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- Biofabrication of anisotropic constructs using extrusive chaotic printing(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2023-12-01) Hernandez Medina, David Hyram; Trujillo de Santiago, Grissel; mtyahinojosa, emipsanchez; Bolívar Monsalve, Edna Johana; Castillo, Jimmy; Esquivel Alfaro, Marianelly; Escuela de Ingeniería y Ciencias; Campus Monterrey; Álvarez, Mario MoisésAligned tissue constitutes a considerable percentage of the body mass, and it is this anisotropic characteristic which confers certain mechanical and functional properties to the tissue. For creating tissue-like structures that resembles the body, one relevant challenge lies on using biomaterials and shaping them to create aligned structures. These constructs serve as scaffolding materials that promote cell proliferation and differentiation that can eventually become a working tissue. In this study, continuous chaotic printing was used to fabricate highly oriented printed scaffolds and bioprinted cell-laden constructs. First, we assessed the effectiveness of a chaotic extrusion printhead, containing a Kenics Static Mixer (KSM), as a tool to align fibrillar inks. In short, soft fibrillar materials (i.e., alginate-cellulose and collagen) were chaotically printed into 1 mm thick filaments and scaffold anisotropy was assessed in terms of printed microstructures orientation. Filaments showed orientation up to 68% in a -15° to 15° region where the main axis (i.e., aligned fiber) correspond to 0°. Moreover, we assessed the capability of chaotic bioprinting to produce aligned and pre-vascularized skeletal-muscle-like tissues. Briefly, fibers were bioprinted using three inks: a hydrogel loaded with myoblasts (C2C12 cells), a non-crosslinkable hydrogel to create inner vessels inside the fiber, and a high viscosity hydrogel loaded with mesoporous bioactive glass (MBG) to provide mechanical robustness to the fiber. A comparison was made between homogeneous (pre mixed) fibers and pre-vascularized fibers with a layered inner structure. The constructs were cultured up to 21 days and demonstrated high viability (>85%) and a significant relation in the orientation trend of the F-actin filaments with the stratification. Overall, we demonstrated that chaotic printing is a practical tool for fabricating anisotropic constructs with both, fibrillar inks and cell-laden constructs.
- Development of a GelMA-based bioink enhanced with minimally-processed tissue for the fabrication of skeletal muscle(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2020-06-12) Tavares Negrete, Jorge Alfonso; TAVARES NEGRETE, JORGE ALFONSO; 744164; Trujillo de Santiago, Grissel; ilquio/puemscuervo; Pérez Carrillo, Esther; Mertgen, Anne-Sophie; Santiago Miramontes, Ma. de los Ángeles de; Escuela de Ingeniería y Ciencias; Campus Monterrey; Álvarez, Mario MoisésBioprinting, an emerging technology that uses living cells and biomaterials for fabricating engineered tissues with complex three-dimensional (3D) architecture, has become popular as an alternative to conventional 3D-culture techniques. In typical extrusion bioprinting, a bioink (i.e., generally a suspension of cells in a hydrogel) is extruded through a printing head to build an artificial 3D tissue in a layer-by-layer-fashion. Bioinks are key components of a bioprinting process. Bioinks have to meet appropriate biological and rheological characteristics to be printable and to provide a proper microenvironment to cells. Here, we present a simple strategy to develop cost-effective bioinks based on gelatin methacryloyl (GelMA) enhanced with fetal minimally processed muscle tissue (MPT). This strategy is intended to supplement bioinks with growth factors, glycosaminoglycans and proteins from fetal tissue, due to its biochemical composition rich in growth factors and peptides. As a first demonstrative model, we supplemented GelMA hydrogels with 0.5, 1, 2% dried and powdered MPT derived from a goat or calf fetus. The biochemical characterization of MPT showed that our minimally processed technique preserves more than 65% of GAG content compared to traditional methods (i.e. decellularization). The rheological profile of our hydrogels was analyzed to determine a suitable working-window of printing parameters, all inks have shear thinning behavior. Cell-culture experiments showed that the incorporation of MPT in the hydrogels influences the metabolic activity of the myoblast cells (C2C12) and confers structural stability to the hydrogels for cultures of up to 28 days in comparison to pristine GelMA hydrogels. The cell orientation of the GelMA-MPT bioprinted constructs (measured with image analysis) was up to 60% resembling the tissue coherence and architecture of native tissue. Our results demonstrate that our materials can be used as cost-effective bioinks for the bioprinting of skeletal muscle models.
- Fabrication of highly perfusable gelatin-methacryloyl (GelMA) constructs using flow-based strategies(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2020-06-05) Pedroza González, Sara Cristina; TRUJILLO DE SANTIAGO, GRISSEL; 256730; Trujillo de Santiago, Grissel; RR, emipsanchez; González Gamboa, Ivonne; Mertgen, Anne-Sophie; De Santiago Miramontes, Ma. de los Ángeles; Escuela de Ingeniería y Ciencias; Campus Monterrey; Álvarez, Mario MoisésOne of the most important challenges when engineering tissues in vitro is the creation of viable thick constructs. The diffusion of gas and nutrients severely limits the size of engineered constructs. Therefore, the incorporation of perfusable lumen structures within thick engineered tissues is needed for enabling gas exchange, perfusion of nutrients, and waste removal down to the depth of the tissue. Current biofabrication techniques used to create perfusable networks in thick 3D constructs are limited in resolution and control, and they require sophisticated or expensive tools. In this work, we propose a simple technique to develop perfusable hydrogel constructs based on the use of a 3D flow-based biofabrication technique, namely the mini Journal Bearing (mJB), and by employing sacrificial inks. Through the action of regular flows induced in a mJB and the flow-advection of two different hydrogels, we created constructs with an internal sacrificial structure. We used gelatin methacryloyl (GelMA) as a permanent hydrogel matrix, and a drop (100 µL) of gelatin as a fugitive ink/bioink. Here we present a thorough characterization of the microarchitecture and porosity of these constructs. Especially, we demonstrated how permeability increased within these constructs. Additionally, aiming to mimic the architectural complexity of natural tissues, we added nanotopographical cues to our constructs by the incorporation of elongated flexuous plant viruses, namely Turnip Mosaic Virus (TuMV). We conducted our in vitro experiments using myoblasts cells as a biological model and characterized their biological response through time. We fabricated three different types of cell-laden-constructs: GelMA with suspended cells, GelMA with a gelatin ink loaded with cells, and GelMA with a gelatin ink loaded with cells and TuMV. Cells were able to grow faster and for longer in GelMA/gelatin constructs than in pristine-GelMA constructs. While an intricate network of cells was developed after 28 days of culture within permeabilized GelMA/gelatin constructs, only surface proliferation was observed in dense constructs made exclusively with GelMA. The use of GelMA/gelatin-TuMV had an evident morphological effect on cell attachment and proliferation. TuMV 3D meshes providing additional scaffolding within the lumina. While myoblast alignment was strongly evident in GelMA/gelatin where cells adhered mainly to the lamellae walls, in GelMA/gelatin-TuMV constructs, cells alignment was attenuated by interaction with the 3D micromesh of TuMV.
- Biofabrication of nanoenhanced hydrogel fibers for muscle tissue engineering using surface chaotic flows: Chaotic 2D-printing(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2020) Frías Sánchez, Ada Itzel; TRUJILLO DE SANTIAGO, GRISSEL; 256730; FRIAS SANCHEZ, ADA ITZEL; 887018; ALVAREZ, MARIO MOISES; 26048; Trujillo de Santiago, Grissel; RR; Tamayol, Ali; Ponz Ascaso, Fernando; Samandari, Mohamadmahdi; School of Engineering and Sciences; Campus Monterrey; Alvarez, Mario MoisésMultiple human tissues exhibit a fibrous nature. Therefore, the fabrication of hydrogel filaments for biomedical engineering applications is a trending topic. Current tissue models are made of materials that often require further enhancement for appropriate cell attachment, proliferation and differentiation. Here we present a simple strategy, based on the use of mathematically modelable surface chaotic flows, to fabricate continuous, long and thin filaments of gelatin methacryloyl (GelMA) added with Turnip mosaic virus (TuMV) for enhanced muscle tissue engineering. The fabrication of these filaments was achieved by chaotic advection in a finely controlled and miniaturized version of the journal bearing (JB) system. A drop of a pre-gel solution of GelMA was injected on a higher-density viscous fluid (glycerin) and a chaotic flow was applied through an iterative process. The hydrogel drop exponentially deformed and elongated to generate a fiber, which was then photocrosslinked under exposure to UV light. Computational fluid dynamics (CFD) simulations were conducted for the design and prediction of our results. GelMA fibers were then used as scaffolds for C2C12 myoblast cells, and the effect of adding plant-based viral nanoparticles (VNP) to the hydrogel fibers as nano-scaffolds for cellular growth was evaluated. Chaotic 2D-printing was proven to be a viable method for the fabrication of hydrogel fibers. CFD simulations accurately predicted the lengths of the printed fibers, and a correlation coefficient of R2=0.9289 was determined from the experimental and simulated data of the first two cycles. The hydrogel fibers were effective scaffolds for muscle cells and show potential to be used as cost-effective models for muscle tissue engineering purposes. TuMV significantly increased the metabolic activity of the cell-seeded fibers (p<0.05), strengthened cell attachment throughout the first 28 days, improved cell alignment to ~50%, and promoted the generation of structures that resemble natural mammal muscle tissue.
- Desarrollo y caracterización de adhesivos tisulares basados en zeína(Instituto Tecnológico y de Estudios Superiores de Monterrey) Cuéllar Monterrubio, Aimé Alexandra; 856068; 856068; 856068; Trujillo de Santiago, Grissel; Lara Mayorga, Itzel; Escuela de Ingeniería y Ciencias; Escuela de Ingeniería y Ciencias; Campus Monterrey; Álvarez, Mario MoisésEn la práctica médica, las cirugías son procedimientos rutinarios que requieren de suturas para cerrar las incisiones. Las suturas son efectivas uniendo los tejidos, pero generalmente son asociadas con procesos inflamatorios, irritación e infecciones. Los adhesivos tisulares son materiales prometedores para sustituir las suturas, pero hoy en día las opciones de adhesivos tisulares no son efectivas cerrando heridas (v.gr., selladores de fibrina utilizados como complementos de suturas) y presentan citotoxicidad (v.gr., cianoacrilatos). La presente tesis presenta el desarrollo y caracterización de un adhesivo tisular sintonizable basado en zeína. La zeína es una proteína obtenida del maíz que pertenece a la familia de las prolaminas. Esta proteína es una materia prima ideal para los adhesivos quirúrgicos, ya que es naturalmente adhesiva, estable en condiciones húmedas, biocompatible, biodegradable, de origen vegetal (el cual reduce los riesgos de transmisión viral), bajo costo y fácil de procesar. El adhesivo tisular consiste en una base de zeína disuelta en etanol. Se evaluó el desempeño mecánico y biológico de los adhesivos analizando el efecto del ácido acético como aditivo cosolvente y PEG 400 como aditivo plastificante. El adhesivo se preparó mediante la adición de 5 g de zeína en polvo a 15 mL de etanol a una temperatura de 65ºC y en agitación constante de 300 rpm por 45 minutos. Las propiedades adhesivas, fisicoquímicas y biológicas de los adhesivos se caracterizaron mediante pruebas mecánicas, microscopía, FTIR, índice de hinchazón y respuesta celular in vitro. Posteriormente, los adhesivos se formularon con nanopartículas de plata (AgNPs; por sus siglas en inglés “Argentum nanoparticles”), a concentraciones de 1.5 mg/mL y 3 mg/mL para conferir propiedades antimicrobianas frente a Staphylococcus aureus. Adicionalmente, los adhesivos tisulares se analizaron mecánicamente (ensayos de esfuerzo cortante por cizallamiento) siguiendo los lineamientos reportados en la norma ASTM F2255-05 donde se comparó con la matriz de zeína. La caracterización mecánica mostró que los aditivos afectaron el desempeño adhesivo de las formulaciones. Por ejemplo, se observa una tendencia a incrementar el Módulo de Young y la tensión máxima al agregar aditivos siendo mayor en presencia de ambos (PEG 400 y ácido acético). La microestructura del adhesivo y de su interfaz al ser adherido a piel porcina fue observada mediante Microscopía Electrónica de Barrido (SEM, por sus siglas en inglés). La formulación que contenía PEG 400 y ácido acético muestra una microestructura que sugiere una interacción adhesiva entre la piel porcina y nuestro adhesivo tisular. También el adhesivo fue sometido a pruebas de cito compatibilidad in vitro donde se cultivaron fibroblastos BJ sobre el adhesivo de zeína y la matriz y se observó el crecimiento y la proliferación celular donde se muestra un incremento poblacional en cada una de las muestras. Finalmente la actividad anti- bacterial fue evaluada incorporando AgNPs en polvo a nuestros adhesivos. Los adhesivos basados en zeína a diferentes concentraciones de AgNPs fueron expuestos a S. aureus donde observamos inhibición bacteriana en las formulaciones con altas concentraciones de nanopartículas. Nuestros resultados presentados en esta tesis sugieren que la zeína es una matriz polimérica con potencial para emplearse como adhesivo tisular para heridas superficiales, coadyuvante a suturas quirúrgicas y como adhesivo con agente anti – bacteriano. Sus características se pueden ajustar fácilmente para coincidir con las propiedades mecánicas de diferentes tejidos para una amplia variedad de aplicaciones.
- Hybrid GelMA-Turnip mosaic virus(TuMV) scaffolds for enhanced fibroblast proliferation: Effect of EGF conjugation on the TuMV surface(Instituto Tecnológico y de Estudios Superiores de Monterrey) Lobo-Zegers, Matías José; 861337; Trujillo de Santiago, Grissel; González-Gamboa, Ivonne; Menchaca-Arredondo, Jorge Luis; School of Engineering and Sciences; School of Engineering and Sciences; Campus Monterrey; Álvarez, Mario MoisesTissue Engineering promises to deliver real solutions in several relevant fronts of modern medicine including regenerative medicine, pharmacological screening, and fundamental biomedical research. Modern tissue engineering techniques rely heavily in the use of hydrogels to support cell proliferation in 2D and 3D cultures. Often, these need functionalization with signaling molecules or biological factors to promote cell proliferation, differentiation and viability. Growth factors (GFs) are naturally occurring proteins that stimulate cellular growth, proliferation and differentiation. However, GFs translation into clinical applications is limited due to their short effective half-life, low stability, and rapid inactivation by enzymes under physiological conditions. Protein immobilization techniques combined with nanomaterial carriers have shown promise in augmenting the delivery, stability and effectiveness of GFs. Viral nanoparticles have uniform and well-defined nano-structures and can be produced in large quantities. Several protein based systems such as plant viral nanoparticles have been tested in biomedical applications due to their biosafety, biocompatibility, and surface modification availability. In this work, we propose the development of smart-tailored hydrogels: Gelatin methacryloyl (GelMA) with a nanoscaffold of Turnip mosaic virus(TuMV) functionalized with Epidermal Growth Factor (EGF) for sustained release of the growth factor to cells in 2D and 3D cultures. EGF-conjugated nanostructured hydrogel samples promoted at least a 30\% increase in cell proliferation, viability and attachment compared to unbound EGF, at equivalent concentrations in 2D and 3D cultures. This validates TuMV as an immobilization platform for multimeric conjugation of the growth factor. Combined with GelMA, they provide a two-way immobilization system for EGF, augmenting its stability and exposition to the cells.
- On the improvement of the process of synthesis of gelatin methacryloyl (GelMA) hydrogels and development of a hybrid nanoparticle-GelMA-based bioink for tissue engineering(Instituto Tecnológico y de Estudios Superiores de Monterrey) Sánchez Rodríguez, Víctor Hugo; 852663; 852663; 852663; Trujillo de Santiago, Grissel; González Gamboa, Ivonne; School of Engineering and Sciences; School of Engineering and Sciences; Campus Monterrey; Álvarez Hernández, Mario MoisésGelatin methacryloyl (GelMA) is a semisynthetic biomaterial that conserves some relevant advantages of native collagen such as the presence of cell-binding domains with protease-cleavage sites. GelMA-based hydrogels are currently used as biomaterials to develop cell-laden systems for several biomedical applications. GelMA provides the necessary physiological microenvironment and biocompatibility for tissue engineering studies. However, currently used methods to produce GelMA do not consider a strict control of the key parameters of the reaction process (i.e., mixing, location and rate of addition of methacrylic anhydride, and pH) which leads to batch to batch inconsistencies and low yields. In this work, a semi-automated process to synthesize and purify GelMA is presented. Briefly, the method considered (a) the use of a custom-made jacketed reactor with temperature, agitation, and pH control, (b) the addition of methacrylic anhydride (MA), the key reagent of the synthesis, by a controllable syringe pump, and (c) a continuous dialysis stage, carried through a set of peristaltic pumps, follows the procedure shortening effectively the time needed for methacrylic acid removal. Automated equipment and pH control reduce synthesis and purification times and enhance reactivity, leading to a higher degree of substitution. Using this synthesis and purification strategy, we conducted multiple sets of GelMA production and correlated reaction conditions to final yield and quality of the product. As a result of this process intensification, the required time of synthesis and purification was significantly reduced, and the amount of water required for the removal of cytotoxic residues (i.e., methacrylic acid) was minimized. While conventional protocols require a 2 weeks preparation time and result in an inconsistent quality of the final product, the methodologies presented here yield consistent quality GelMA in 5 days. Moreover, this work presents results on the rates of methacryloyl functionalization during the reaction stage and the rate of methacrylic acid removal during the purification stage. The degree of methacrylation (DoM), the exposure to UV light (intensity and time) during the photo-crosslinking time of GelMA hydrogels, as well as the gelatin and photoinitiator type and concentration have a strong and direct influence on the mechanical, physicochemical, properties of this biomaterial, which at the same time influence cellular behavior. Finally, we developed a proof-of-principle strategy to use our hydrogel as a nano-composite bio-ink for 3D printing. To fulfill the goal of proper rheology for GelMA extrusion, a variety of bioinks were prepared by mixing GelMA, gelatin, and halloysite-nanoparticles (HNT). The concentration of GelMA, gelatin, and nanoparticles was varied to create an executable 3D ink at room temperature taking advantage of a two-step thermal- / photo-crosslinking strategy. This approach demonstrated to obtain reproducible cell-laden scaffolds by using extrusion as a tool for 3D printing.
