This study analyzed the feasibility of using gas foaming to create Poly Lactic-co-Glycolic Acid (PLGA) scaffolds for use as a substrate in bone tissue engineering and set out to determine whether the presence of osteoblasts on these scaffolds enhanced their material stiffness. The process of bone formation involves osteoblasts depositing extracellular matrix and calcifying this matrix with calcium phosphate crystals (Hasegawa et al., 2017) and pits between 30-40μm in diameter on tissue engineering scaffold surfaces have been shown to best promote osteogenic activity in the presence of bone-forming cells (Halai et al., 2014).The scaffolds were determined to contain pits within this 30-40μm range and the ability of osteoblasts to lay down and calcify extracellular matrix on gas foamed PLGA scaffolds was confirmed by the image analysis of inverted optical microscope images of Alizarin Red S-stained scaffold cryosectionsThe presence of osteogenic activity combined with the desired scaffold porosity led us to conclude that gas foaming PLGA scaffolds are a feasible method of scaffold fabrication for bone tissue engineering and allowed us to optimize the gas foaming apparatus as an instrument to be used in further bone tissue engineering experiments at California Polytechnic State University, San Luis Obispo.However, this study failed to determine whether the presence of osteoblasts improved the material stiffness of the PLGA scaffolds due to a lack of statistical significance in compression testing results.

Yes / The use of biological macromolecules like quince seed mucilage (QSM), as the common curative practice has a long history in traditional folk medicine to cure wounds and burns. However, this gel cannot be applied on exudative wounds because of the high water content and non-absorption of infection of open wounds. It also limits cell-to-cell interactions and leads to the slow wound healing process. In this study to overcome these problems, a novel QSM-based hybrid scaffold modified by PCL/PEG copolymer was designed and characterized. The properties of this scaffold (PCL/QSM/PEG) were also compared with four scaffolds of PCL/PEG, PCL/Chitosan/PEG, chitosan, and QSM, to assess the role of QSM and the combined effect of polymers in improving the function of skin tissue-engineered scaffolds. It was found, the physicochemical properties play a crucial role in regulating cell behaviors so that, PCL/QSM/PEG as a smart/stimuli-responsive bio-matrix promotes not only human-adipose stem cells (h-ASCs) adhesion but also supports fibroblasts growth, via providing a porous-network. PCL/QSM/PEG could also induce keratinocytes at a desirable level for wound healing, by increasing the mechanobiological signals. Immunocytochemistry analysis confirmed keratinocytes differentiation pattern and their normal phenotype on PCL/QSM/PEG. Our study demonstrates, QSM as a differentiation/growth-promoting biological factor can be a proper candidate for design of wound dressings and skin tissue-engineered substrates containing cell.

The tumor microenvironment plays an important role in regulating cancer cell behavior. The tumor microenvironment describes the cancer cells, and the surrounding endothelial cells, fibroblasts, and mesenchymal stem cells, along with the extracellular matrix (ECM). The tumor microenvironment stiffens as cancer undergoes malignant progression, providing biophysical cues that promote invasive, metastatic cellular behaviors. This project investigated the influence of three dimensional (3D) chitosan-alginate (CA) scaffold stiffness on the morphology, growth, and migration of green fluorescent protein (GFP) – transfected MDA-MB-231 (231-GFP) breast cancer (BCa) cells. The CA scaffolds were produced by the freeze casting method at three concentrations, 2 wt%, 4 wt%, and 6 wt% to provide different stiffness culture substrates. The CA scaffold material properties were characterized using scanning electron microscopy imaging for pore structure and compression testing for Young's Modulus. The BCa cell cultures were characterized at day 1, 3, and 7 timepoints using Alamar Blue assay for cell number, fluorescence imaging for cell morphology, and single-cell tracking for cell migration. Pore size calculations using SEM imaging yielded pore sizes of 253.29 ± 52.45 µm, 209.55 ± 21.46 µm, and 216.83 ± 32.63 µm for 2 wt%, 4 wt%, and 6 wt%, respectively. Compression testing of the CA scaffolds yielded Young's Modulus values of 0.064 ± 0.008 kPa, 2.365 ± 0.32 kPa and 3.30 ± 0.415 kPa for 2 wt%, 4 wt%, and 6 wt% CA scaffolds, respectively. The results showed no significant difference in cell number among the 3D CA scaffold groups. However, the 231-GFP cells cultured in 2 wt% CA scaffolds possessed greater cellular size, area, perimeter, and lower cellular circularity compared to those in 4 wt% and 6 wt% CA scaffolds, suggesting a more prominent presence of cell clusters in softer substrates compared to stiffer substrates. The results also showed cells in 6 wt% CA having a higher average cell migration speed compared to those in 2 wt% and 4 wt% CA scaffolds, indicating a positive relationship between substrate stiffness and cell migration velocity. Findings from this experiment may contribute to the development of enhanced in vitro 3D breast tumor models for basic cancer research using 3D porous biomaterial scaffolds.

No description available.

No description available.

No description available.

El objetivo principal de esta tesis doctoral es el diseño de un andamio polimérico bicapa macroporoso para la regeneración del complejo osteocondral. El material empleado para la fabricación del constructo ha sido el ácido poli(L-láctico), un polímero biodegradable de la familia de los poliésteres. Una de las capas del andamio ha sido diseñada para asistir la regeneración del cartílago articular. La otra capa sirve de anclaje al hueso subcondral, y se diferencia de la anterior en sus propiedades mecánicas y bioactividad. Este comportamiento ha sido logrado por combinación del ácido poli(L-láctico) con nanopartículas inorgánicas. Ambas capas están unidas entre sí por una fina capa de material no poroso que evita el flujo de células de una parte a otra del constructo. Para lograr este objetivo se realizó un primer estudio de diseño variando la morfología de los andamios hasta obtener aquella arquitectura más adecuada para la regeneración de ambos tejidos. Se varió parámetros de síntesis tales como la concentración de polímero y el ratio entre polímero y porógeno. Los andamios fueron evaluados mecánica y fisicoquímicamente y se seleccionó los parámetros de síntesis del ácido poli(L-láctico) que dieron mejores resultados. En la regeneración del tejido es esencial conocer cómo variarán las propiedades del material una vez sea implantado y comience su degradación. Por lo tanto, fue considerado oportuno realizar un estudio de degradación del material in vitro en diversas condiciones. El estudio de la degradación fue realizado en condiciones estáticas durante 6, 12, 18, 24 semanas y 1 año y en condiciones dinámicas durante 1, 2, 4 y 6 semanas. Se evaluó tanto las características mecánicas como las fisicoquímicas tras los diversos tiempos de la degradación. Posteriormente, y para aumentar las características mecánicas y la bioactividad del anclaje óseo, se incorporó distintas cantidades de nanopartículas inorgánicas de hidroxiapatita y sílice a los andamios. / Deplaine ., H. (2012). Scaffold design and characterisation for osteochondral tissue regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/14638

[EN] Abstract The main purpose of this thesis is the design and characterization of an experimental articular cartilage model. The in vitro model is composed of a macro and micro- porous Polycaprolactone scaffold with a Poly(Vinyl Alcohol) filling. The scaffold/hydrogel construct has been subjected to repeating number of freezing and thawing cycles in order to crosslink the hydrogel inside the scaffold's pores. The Poly(Vinyl Alcohol) resembles the growing cartilaginous tissue inside the scaffolds pores, as it gets denser and stiffer for each cycle of freezing and thawing. The in vitro model allows studying a variety of characteristics of the scaffold and hydrogel, revealing interesting features. The importance of water flow on the mechanical properties is studied, so as the influence of micro-porosity. It can be seen that the mechanical properties of the porous scaffolds are influenced in distinct ways by the hydrogel density and micro-porosity of the scaffold. The permeability of the scaffolds is studied and is seen independent of crosslinking density of the hydrogel inside the porous scaffolds. The experimental cartilage model has also been applied on a macro porous acrylic scaffold. The results show that the water has different effect on the mechanical properties, for macro, or macro and micro-porous scaffolds. The in vitro cartilage model has elastic modulus, aggregate modulus and permeability values in the same order as human articular cartilage. The model is useful to predict the mechanical behavior of porous scaffolds in vivo. A scaffold implant device for animal studies has been designed based on a previous patent of the research group, and implanted in two different in vivo trials in sheep. The results show that the fixation and anchoring to the subchondral bone improve the tissue repair and diminish alterations in the subchondral bone. ¿ / [ES] Resumen El objetivo principal de esta tesis doctoral es el diseño y caracterización de un modelo de cartílago articular experimental. El modelo in vitro se compone de un scaffold micro- y macroporoso de Policaprolactona con un relleno de Poli(Vinil Alcohol). El constructo scaffold/hidrogel ha sido sometido a ciclos consecutivos de congelación y descongelación con objeto de entrecruzar el hidrogel dentro de los poros del scaffold. El Poli(Vinil Alcohol) mimetiza al tejido de cartílago que se regenerará en los poros, ya que en cada ciclo de congelación y descongelación se vuelve más denso y duro. El modelo in vitro permite estudiar una gran variedad de características del scaffold e hidrogel, revelando fenómenos interesantes para la ingeniería tisular. Se ha estudiado la importancia del flujo de agua a través del scaffold en las propiedades mecánicas, así como la influencia de la microporosidad. Se ha podido constatar que la densidad del hidrogel y la microporosidad influyen de distinta forma en las propiedades mecánicas de los scaffolds porosos. Se ha estudiado la permeabilidad de los scaffolds, que ha resultado ser independiente de la densidad de entrecruzamiento del hidrogel dentro de sus poros. El modelo experimental de cartílago se ha aplicado también a un scaffold macroporoso acrílico. Los resultados muestran que el agua tiene un efecto distinto en las propiedades mecánicas de los scaffolds macroporosos y en los micro- macroporosos. El modelo de cartílago in vitro tiene valores del modulo elástico, módulo agregado y permeabilidad que son del mismo orden de magnitud que los del cartílago articular humano. El modelo permite predecir el comportamiento mecánico in vivo de scaffolds porosos. Se ha diseñado un dispositivo de implante de scaffold para experimentos en animales basado en una patente del grupo de investigación, que ha sido implantado en dos ensayos in vivo diferentes en ovejas. Los resultados muestran que la fijación y anclaje al hueso subcondral tiene un gran papel en la reparación del tejido. / [CA] Resum L'objectiu principal d'aquesta tesi doctoral és el disseny i caracterització d'un model de cartílag articular experimental. El model in vitro es compon d'un scaffold micro- i macroporós de Policaprolactona amb un farciment de Poli(Vinil Alcohol). El constructe scaffold/hidrogel ha estat sotmès a cicles consecutius de congelació i descongelació amb l'objectiu d'entrecreuar l'hidrogel dins del porus del scaffold. El Poli(Vinil Alcohol) mimetitza al teixit de cartílag que es regenerarà en el porus, ja que en cada cicle de congelació i descongelació es torna més dens i dur. El model in vitro permet estudiar una gran varietat de característiques del scaffold i hidrogel, posant de manifest fenòmens interessants per a l'enginyeria tissular. S'ha estudiat la importància del flux d'aigua a través del scaffold en les propietats mecàniques, així com la influència de la microporositat. S'ha pogut constatar que la densitat de l'hidrogel i la microporositat influeixen de distinta manera en les propietats mecàniques dels scaffolds porosos. S'ha estudiat la permeabilitat dels scaffolds, que ha resultat ser independent de la densitat d'entrecreuament de l'hidrogel dins dels seus porus. El model experimental de cartílag s'ha aplicat també a un scaffold macroporós acrílic. Els resultats mostren que l'aigua té un efecte distint en les propietats mecàniques dels scaffolds macroporosos i en els micro- macroporosos. El model de cartílag in vitro té valors del mòdul elàstic, mòdul agregat i permeabilitat que són del mateix ordre de magnitud que els del cartílag articular humà. El model permet predir el comportament mecànic in vivo de scaffolds porosos. S'ha dissenyat un dispositiu d'implant de scaffold per a experiments en animals basat en una patent del grup d'investigació, que ha segut implantat en dos assaigs in vivo diferents en ovelles. Els resultats mostren que la fixació i ancoratge a l'os subcondral té un gran paper en la reparació del teixit. / Vikingsson, LKA. (2015). An experimental model to mimic the mechanical behavior of a scaffold in a cartilage defect [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/53912

Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro-and nano- architectures, which provides signaling cues and influences cell behavior. Mimicking this three dimensional environment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique with controlled fiber diameter (microns to less than 100 nm), orientation and spacing in single and multiple layers are demonstrated as biological scaffolds. Hierarchical nanofiber assemblies were developed to control single cell shape (shape index from 0.15 to 0.57), nuclei shape (shape index 0.75 to 0.99) and focal adhesion cluster length (8-15 micrometer). To further investigate single cell-ECM biophysical interactions, nanofiber nets fused in crisscross patterns were manufactured to measure the "inside out" contractile forces of single mesenchymal stem cells (MSCs). The contractile forces (18-320 nano Newton) were found to scale with fiber structural stiffness (2 -100 nano Newton/micrometer). Cells were observed to shed debris on fibers, which were found to exert forces (15-20 nano Newton). Upon CO? deprivation, cells were observed to monotonically reduce cell spread area and contractile forces. During the apoptotic process, cells exerted both expansive and contractile forces. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology. / Master of Science

The goal of this work was to design and build a mask projection microstereolithography (MPμSL) 3D printing system to characterize, process, and quantify the performance of novel photopolymers. MPμSL is an Additive Manufacturing process that uses DLP technology to digitally pattern UV light and selectively cure entire layers of photopolymer resin and fabricate a three dimensional part. For the MPμSL system designed in this body of work, a process was defined to introduce novel photopolymers and characterize their performance. The characterization process first determines the curing characteristics of the photopolymer, namely the Critical Exposure (Ec) and Depth of Penetration (Dp). Performance of the photopolymer is identified via the fabrication of a benchmark test part, designed to determine the minimum feature size, XY plane accuracy, Z-axis minimum feature size, and Z-axis accuracy of each photopolymer with the system. The first characterized photopolymer was poly (propylene glycol) diacrylate, which was used to benchmark the designed MPμSL system. This included the achievable XY resolution (212 micrometers), minimum layer thickness (20 micrometers), vertical build rate (360 layers/hr), and maximum build volume (6x8x36mm3). This system benchmarking process revealed two areas of underperformance when compared to systems of similar design, which lead to the development of the first two research questions: (i) 'How does minimum feature size vary with exposure energy?' and (ii) 'How does Z-axis accuracy vary with increasing Tinuvin 400 concentration in the prepolymer?' The experiment for research question (i) revealed that achievable feature size decreases by 67% with a 420% increase in exposure energy. Introducing 0.25wt% of the photo-inhibitor Tinuvin 400 demonstrated depth of penetration reduction from 398.5 micrometers to 119.7 micrometers. This corresponds to a decrease in Z-axis error from 119% (no Tinuvin 400) to 9% Z-axis error (0.25% Tinuvin 400). Two novel photopolymers were introduced to the system and characterized. Research question (iii) asks 'What are the curing characteristics of Pluronic L-31 how does it perform in the MPμSL system?' while Research Question 4 similarly queries 'What are the curing characteristics of Phosphonium Ionic Liquid and how does it perform in the MPμSL system?' The Pluronic L-31 with 2wt% photo-initiator had an Ec of 17.2 mJ/cm2 and a Dp of 288.8 micrometers, with a minimum feature size of 57.3 ± 5.7 micrometers, with XY plane error of 6% and a Z-axis error of 83%. Phosphonium Ionic Liquid was mixed in various concentrations into two base polymers, Butyl Diacrylate (0% PIL and 10% PIL) and Poly Ethylene Dimethacrylate (5% PIL, 15% PIL, 25% PIL). Introducing PIL into either base polymer caused the Ec to increase in all samples, while there is no significant trend between increasing concentrations of IL in either PEGDMA or BDA and depth of penetration. Any trends previously identified between penetration depth and Z accuracy do not seem to extend from one resin to another. This means that overall, among all resins, depth of penetration is not an accurate way to predict the Z axis accuracy of a part. Furthermore, increasing concentrations of PIL caused increasing % error in both XY plane and Z-axis accuracy . / Master of Science