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.

yes / Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Im-munohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrody-namic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of a-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

Yes / Fabrication of extracellular matrix (ECM)-like scaffolds (in terms of structural-functional) is the main challenge in skin tissue engineering. Herein, inspired by macromolecular components of ECM, a novel hybrid scaffold suggested which includes silk/hyaluronan (SF/HA) bio-complex modified by PCP: [polyethylene glycol/chitosan/poly(ɛ-caprolactone)] copolymer containing collagen to differentiate human-adipose-derived stem cells into keratinocytes. In followed by, different weight ratios (wt%) of SF/HA (S1:100/0, S2:80/20, S3:50/50) were applied to study the role of SF/HA in the improvement of physicochemical and biological functions of scaffolds. Notably, the combination of electrospinning-like and freeze-drying methods was also utilized as a new method to create a coherent 3D-network. The results indicated this novel technique was led to ~8% improvement of the scaffold's ductility and ~17% decrease in mean pore diameter, compared to the freeze-drying method. Moreover, the increase of HA (>20wt%) increased porosity to 99%, however, higher tensile strength, modulus, and water absorption% were related to S2 (38.1, 0.32 MPa, 75.3%). More expression of keratinocytes along with growth pattern similar to skin was also observed on S2. This study showed control of HA content creates a microporous-environment with proper modulus and swelling%, although, the role of collagen/PCP as base biocomposite and fabrication technique was undeniable on the inductive signaling of cells. Such a scaffold can mimic skin properties and act as the growth factor through inducing keratinocytes differentiation.

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.

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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