Fabrication of Tissue Engineering Scaffolds Using Stereolithography Benita M. Comeau 226 Pages Directed by Dr. Clifford L. Henderson New methods and materials for the fabrication of hierarchically structured, 3D tissue scaffolds using stereolithography (SL) are presented. The ability to chemically modify selected areas on a scaffold is one way to direct cell growth in deliberate patterns; which is necessary for the engineering of complex, functioning tissues. SL will allow for the building of complex 3D structures with well defined geometries, and a second level of order is created by subsequent modification of chemical groups via catalyzing a de-protection event through exposure to another wavelength of light. The investigated system utilizes an acid-catalyzed de-protection event to change the surface chemistry of an SL-made polymer, analogous to conventional chemically amplified photoresists. The chemical modification alters the surface energy, affecting how proteins interact with the material. This allows selective areas to be more favorable towards cell adhesion. The results of this work include the identification of cytocompatible photo-acid generators that are necessary for the acid-catalyzed de-protection, the demonstration that traditional photolithographic materials may be used for cell patterning, quartz crystal microbalance studies which illuminate why these patterning methods work, the design and performance of a mirror array based stereolithographic apparatus capable of multi-wavelength exposures, and the synthesis and formulation of a novel stereolithographic resin for use in this system. The findings suggest that this system has great potential for use in cell and tissue studies, and possibilities for future use and research are discussed.

In this dissertation, small and large NaCl particle-derived surfaces (Ra > 40 microns) were generated on 2D Zn materials, and the surfaces were carefully studied concerning topography, corrosion behavior, and bone cell compatibility. Increases in surface roughness accelerated the corrosion rate, and cell viability was maintained. This method was then extended to 3D porous scaffolds prepared by a hybrid AM/casting technique. The scaffolds displayed a near-net shape, an interconnected pore structure, increasing porosity paralleled to an increased corrosion rate, an ability to support cell growth, and powerful antibacterial properties. Lastly, nano/micro (Rz 0.02–1 microns) topographies were generated on 2D Zn materials, and the materials were comprehensively studied with special attention devoted to corrosion behavior, biocompatibility, osteogenic differentiation, immune cell response, hemocompatibility, and antibacterial performance. For the first time, the textured nonhemolytic surfaces on Zn were shown to direct cell fate, and the micro-textures promoted bone cell differentiation and directed immune cells away from an inflammatory phenotype.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

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Engineering therapeutic approaches to wound healing can be divided into two major categories of fibrous and non-fibrous approaches. There has been significant progress in designing artificial skin products to replace autografting. For patients with non-healing/hard-to-heal wounds, there is an unmet clinical need for inexpensive skin substitutes to be transplanted. In skin regeneration area of research, electrospinning is a very commonly used method of production of grafts for wound healing applications, owing its popularity to the fibrous nature of the resultant product, which mimics the extracellular matrix of the native skin. Despite the high degree of porosity in conventional electrospun scaffolds, the small pore size effectively limits the penetration of cells into the scaffold. Transplantation of such scaffolds with poor cell infiltration abilities may lead to a range of negative consequences, from prolongation of the first/destructive phase of inflammation to rejection of the scaffolds. Several experimental approaches have been developed to generate interfibrillar space in the electrospun scaffolds, including but not limited to modifications of the electrospinning set-up and inclusion of sacrificial components. It has been reported that scaffolds with larger pore diameters in the range of ~ 40-100 μm can modulate, moderate and reduce acute inflammatory responses of the body, by influencing macrophages biological behavior, and direct the course of the wound healing process to the tissue remodeling phase. Macrophages are the major cell component of innate immune system and play critical roles in clearance of pathogens, resolution of inflammation and wound healing following an injury. Macrophages are characterized by their diversity and plasticity. In response to environmental stimuli, they acquire different functional phenotypes of pro-inflammatory (M1) or anti-inflammatory (M2). In this thesis, we developed a novel unique gradient porous structure from a plant-based “green” soy protein isolate (SPI) with improved pore size for macrophages to infiltrate. We further showed the ability of the scaffold to modulate phenotype switch in macrophages in vitro and in vivo. The proposed scaffold, moreover, appeared to support transition of the inflammation process from the destructive to the constructive phase in vivo. Based on the promising results of this thesis, we propose our newly developed scaffold has the ability to be used as a new therapeutic modality for treatment of non-healing chronic wounds. / Bioengineering

In the field of tissue engineering, a major area of study is developing bone scaffolds that will provide support for osteoblasts. Despite many advances in recent years there is still a significant need for new bio-based 3-D porous scaffolds that possess sufficient initial mechanical properties to prevent immediate failure upon implantation. Ovalbumin (OVA), a glycoprotein from chicken egg whites, has been use to fabricate biodegradable, porous hydrogel bone scaffolds that promote osteoblast attachment and proliferation. Although ovalbumin scaffolds encourage bioactivity and are naturally resorbed into the body after bone regeneration, they are also very fragile. Extremely stiff cellulose nanocrystals (CNCs), derived from wood pulp, can be utilized to reinforce these scaffolds while improving biocompatibility. When chemically modified to incorporate surface amine groups, cellulose nanocrystals become capable of covalently crosslinking with the OVA matrix for improved mechanical resilience. Three concentrations (2, 5, 10 wt. %) of CNCs were incorporated and crosslinked to form nanocomposite scaffolds then were compared to pure OVA scaffolds. After fabrication, pore size morphology was compared between each CNC loading using SEM. The images revealed that the 10 wt. % CNC concentration doubled the pore compared to pure OVA scaffolds. Under high magnification, the CNCs were incorporated into the pore walls, providing a contoured surface. AFM was applied to analyze the topography of OVA with CNCs present. The surfaces laden with CNCs had a higher mean surface roughness, but was insufficient to impact cell behavior. Compression testing was carried out on both Instron and DMA machines to demonstrate any reinforcing effect provided by the CNCs. While the compressive modulus remained constant, the elastic limit and strain increased with CNC loading, indicating a change in the resilience of the reinforced scaffolds. With a MTT Assay, it was shown that MC3T3-E1 preosteoblasts significantly increase in metabolic activity on 2 wt. % films and scaffolds, an indication of proliferation. All scaffolds had a net increase in metabolic activity suggesting overall biocompatibility for OVA scaffolds and those incorporating CNCs. Overall, the 5 wt. % scaffolds had the highest mechanical strength and had a positive cell response. / Master of Science

After skeletal muscle damage, satellite cells move towards the injured area to assist in regeneration. However, these cells are rare as their numbers depend on the age and composition of the injured muscle. This regeneration method often results in scar tissue formation along with loss of function. Although several treatment methods have been investigated, no muscle replacement treatment currently exists. Tissue engineering attempts to create, repair, and/or replace damaged tissue by combining cells, biomaterials, and tissue-inducing substances such as growth factors. Electrospinning produces a non-woven scaffold out of biomaterials with fiber diameters ranging from nanometers to microns to create an extracellular-like matrix on which cells attach and proliferate. Our focus is on synthetic polymers, specifically poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), and poly(ε-caprolactone) (PCL). Skeletal muscle cells grown on electrospun scaffolds tend to elongate and fuse together thus, mimicking natural tissue. Electrical stimulation has been shown to increase the number of cells fused in culture and decreased the time needed in culture for cells to contract. Therefore, a conductive element was added to each scaffold, specifically polyaniline (PANi), gold nanoparticles (Au Nps), and multi-walled carbon nanotubes (MWCNT). Our project goal is to create a polymeric, conductive, and biocompatible scaffold for skeletal muscle regeneration. PANi and PDLA were mixed to form the following solutions 24% (83% PDLA/17% PANi), 24% (80% PDLA/20% PANi), 22% (75%PDLA/25% PANi), 29% (83% PDLA/17% PANi), and 29% (80% PDLA/20% PANi). Only the 75/25 electrospun scaffold was conductive and had a calculated conductivity of 0.0437 S/cm. Scaffolds with larger amounts of PANi were unable to be electrospun. PDLA/PANi scaffolds were biocompatible as primary rat skeletal muscle cells cultured in vitro did attach. However, the scaffolds shrunk, degraded easily, and became brittle. Although PDLA/PANi scaffolds were easily manufactured, our results indicate that this polymer mixture is not appropriate for skeletal muscle scaffolds. PLLA and Au Nps were electrospun together to form three composite scaffolds: 7% Au-PLLA, 13% Au-PLLA, and 21% Au-PLLA. These were compared to PLLA electrospun scaffolds. Measured scaffold conductivities were 0.008 ± 0.015 S/cm for PLLA, 0.053 ± 0.015 S/cm for 7% Au-PLLA, 0.076 ± 0.004 S/cm for 13% Au-PLLA, and 0.094 ± 0.037 S/cm for 21% Au-PLLA. It was determined via SEM with a Bruker energy dispersive x-ray spectrometer (EDS) that the Au Nps were not evenly distributed within the scaffolds as they had agglomerated. Rat primary muscle cells cultured on the three Au-PLLA scaffolds displayed low cellular activity. A second cell study was conducted to determine Au NPs toxicity. The results show that the Au Nps were not toxic to the cells and the low cellular activity may be a marker for myotube fusion. Elastic modulus and yield stress values for the three Au-PLLA scaffolds measured on days 0, 7, 14, 21, and 28 were much larger than skeletal muscle tissue. Due to the larger mechanical properties and Au Nps agglomeration, a third polymer and conductive element scaffold was investigated. PCL was chosen as the new synthetic polymer as it had a lower elastic modulus and high elongation. MWCNT were chosen as the conductive element as they disperse well within PCL when acid functionalized. A third component was added to the scaffold to help it move similar to skeletal muscle. Ionic polymer gels (IPG) are hydrogels that respond to an external stimulus such as temperature, pH, light, and electric field. A poly(acrylic acid)/poly(vinyl alcohol) (PAA/PVA) mixture is one type of IGP that responds to an electric field. The scaffolds were coaxially electrospun so that each fiber had a PCL-MWCNT interior with a PAA/PVA sheath. These scaffolds were compared to electrospun PCL and PCL-MWCNT ones. The addition of MWCNT to the PCL did increase scaffold conductivity. Actuation of the PCL-MWCNT-PAA/PVA scaffold occurred when 15V and 20V were applied. All three scaffolds had rat primary skeletal muscle cells attached but, more multinucleated cells with actin interaction were seen on PCL-MWCNT-PAA/PVA scaffolds. Once again the mechanical properties were greater than muscle, but because of its ability to actuate we believe the PCL-MWCNT-PAA/PVA scaffold has potential as a bioartificial muscle. Further characterization of the PCL-MWCNT-PAA/PVA included varying the ratios of PAA/PVA, smaller crosslinking times, and lower amounts of MWCNT. Four ratios, 83/17, 60/40, 50/50, and 40/60, were successfully coaxially electrospun with PCL and MWCNT. Overall, very few differences were seen between the four ratios in conductivity, cellular biocompatibility, actuation angular speed, and mechanical properties. The 83/17 and 40/60 ratios were chosen for additional investigation into mechanical properties and actuation. As the mechanical properties of the two types of scaffolds did not change significantly through degradation, lower PVA crosslinking times were tested. No significant effects were found and it was hypothesized that the evaporation of the solution played a role in the crosslinking process. The smaller MWCNT amount scaffolds also did not significantly affect the mechanical properties or the actuation angular speeds. More work into lowering the scaffold mechanical properties while increasing the actuation angular speed is necessary. Though the mechanical properties for the 83/17 and 40/60 scaffolds remained high compared to skeletal muscle, we also looked for differences in in vivo biocompatibility. Both scaffolds were implanted into the right vastus lateralis muscle of Sprague-Dawley rats. The left vastus lateralis muscle served as either the PBS injected sham surgery or an unoperated control. Biocompatibility was evaluated using enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH), levels, fibrosis formation, inflammation, scaffold cellular infiltration, and neovascularization on days 7, 14, 21, and 28 post-implantation. Fibrotic tissue formation, inflammation, and elevated CK and LDH levels were observed initially but responses decreased during the four week study. Cells infiltrated the scaffolds and histological staining showed more fibroblasts than myogenic cells initially but over time, the fibroblasts decreased and myogenic cells increased. Neovascularization of both scaffolds was also recorded. PCL-MWCNT-PAA/PVA scaffolds were determined to be biocompatible, but some differences between the two types were noted. The 83/17 scaffolds caused less of a response from the body compared to the 40/60 scaffolds and had more myogenic cells attached. However, the 40/60 scaffolds had a larger number of blood vessels running through the scaffold. In conclusion, we have successfully fabricated a polymeric, conductive, and biocompatible scaffold that can actuate for skeletal muscle tissue engineering. Although our results are promising, more work is necessary to continue developing and refining the scaffold. / Ph. D.

Bio-based materials are a viable alternative to synthetic materials for tissue engineering. Although many bio-based materials have been used, Ovalbumin (OA) has not yet been researched to create 3D structures that promote cellular responses. Micro-porous scaffolds are a promising construct for bone tissue regeneration; therefore OA crosslinked with three different concentrations (10%, 15% and 20%) of glutaraldehyde (GA) was used in this research. After fabrication, a porous morphology was observed using SEM. Average pore sizes were found to be comparable to scaffolds previously shown to promote cellular response. A TNBS assay determined percent crosslinking in the scaffolds, however there was no significant difference in percent crosslinking despite differing GA concentrations used. Possible explanations include an excess of GA was used. Using DSC, a glass transition temperature (Tg) was found for control indicating the scaffolds are amorphous. Average dry and wet compressive strengths were also found. As expected, differing GA concentrations had no significant effect on Tg and average compressive strengths due to an excess used. Scaffolds were mechanically tested at 37°C with no significant difference found; therefore these scaffolds can be used in the body. It was shown through cell studies that MC3T3-E1 pre-osteoblast cells significantly increased in number on the 10% and 15% scaffolds, therefore cell proliferation occurred. Because of a positive cellular response, 10% GA scaffolds were used for differentiation studies that showed an increase in osteocalcin at 21 days and alkaline phosphatase levels for scaffolds cultured for 14 days. Overall OA scaffolds have shown to be a promising 3D construct for bone tissue regeneration. / Master of Science

No / This title provides a comprehensive and authoritative review on recent advancements in the application and use of composite scaffolds in tissue engineering. Chapters focus on specific tissue/organ (mostly on the structure and anatomy), the materials used for treatment, natural composite scaffolds, synthetic composite scaffolds, fabrication techniques, innovative materials and approaches for scaffolds preparation, host response to the scaffolds, challenges and future perspectives, and more. Bringing all the information together in one major reference, the authors systematically review and summarise recent research findings, thus providing an in-depth understanding of scaffold use in different body systems.

No / This title provides a comprehensive and authoritative review on recent advancements in the application and use of composite scaffolds in tissue engineering. Chapters focus on specific tissue/organ (mostly on the structure and anatomy), the materials used for treatment, natural composite scaffolds, synthetic composite scaffolds, fabrication techniques, innovative materials and approaches for scaffolds preparation, host response to the scaffolds, challenges and future perspectives, and more. Bringing all the information together in one major reference, the authors systematically review and summarise recent research findings, thus providing an in-depth understanding of scaffold use in different body systems.