Tissue engineering is an emerging area with an aim to create artificial tissues or organs by employing methods of biology, engineering and material science. In tissue engineering, scaffolds are three-dimensional (3D) structure made from biomaterials with highly interconnected pore networks or microstructure, and are used to provide the mechanical and biological cues to guide cell differentiation in order to form desired three-dimensional tissues or functional organs. Hence, tissue scaffold plays a critical role in tissue engineering. However, fabrication of such scaffolds has proven to be a challenge task. One important barrier is the inability to fabricate scaffolds with designed pore size and porosity to mimic the microstructure of native tissue. Another issue is the prediction of process-induced cell damage in the cell-involved scaffold fabrication processes. By addressing these key issues involved in the scaffold fabrication, this research work is aimed at developing methods and models to represent the dispensing-based solid free form scaffold fabrication process with and without the presence of living cells.<p> The microstructure of scaffolds, featured by the pore size and porosity, has shown to significantly affect the biological and mechanical properties of formed tissues. As such, during fabrication process the ability to predict and determine scaffold pore size and porosity is of great importance. In the first part of this research, the flow behaviours of the scaffold materials were investigated and a model of the flow rate of material dispensed during the scaffold fabrication was developed. On this basis, the pore size and porosity of the scaffolds fabricated were represented by developing a mathematical model. Scaffold fabrication experiments using colloidal gels with different hydroxylapatite volume fractions were carried out and the results obtained agreed with those from model simulations, indicating the effectiveness of the models developed. The availability of these models makes it possible to control the scaffold fabrication process rigorously, instead of relying upon a trial and error process as previously reported.<p> In the scaffold fabrication process with the presence of living cells, cells are continuously subjected to mechanical forces. If the forces exceed certain level and/or the forces are applied beyond certain time periods, cell damage may result. In the second part of this research, a method to quantify the cell damage in the bio-dispensing process is developed. This method consists of two steps: one step is to establish cell damage models or laws to relate cell damage to the hydrostatic pressure / shear stress that is applied on cells; and the second step is to represent the process-induced forces that cells experience during the bio-dispensing process and then apply the established cell damage law to model the percent cell damage in the process. Based on the developed method, the cell damage percents in the scaffold fabrication processes that employ two types of dispensing needles, i.e., tapered and cylindrical needles, respectively, were investigated and compared. Also, the difference in cell damage under the high and low shear stress conditions was investigated, and a method was developed to establish the cell damage law directly from the bio-dispensing process. To validate the aforementioned methods and models, experiments of fabricating scaffolds incorporating Schwann cells or 3T3 fibroblasts were carried out and the percent cell damage were measured and compared with the simulation results. The validated models allow one to determine of the influence of process parameters, such as the air pressure applied to the process and the needle geometry, on cell damage and then optimize these values to preserve cell viability and/or achieve the desired cell distribution within the scaffolds.

Current treatments to intervertebral disc degeneration alter spine biomechanics and have complications. Tissue engineering offers an approach to regenerate a biological disc that provides flexibility and stability to, and integrates with the spine. To date, a scaffold that mimics the extracellular matrix composition and mechanical strength of a native disc is lacked. In this project, a biphasic scaffold was fabricated using glycosaminoglycan (GAG) and collagen, the prevalent ma-trix components in a native disc. It also adapted the structure of the disc, with la-mellae of collagen surrounding a collagen-GAG (CG) core. The first part of this project studied chemical modification of CG and evaluated the physiochemical and biological properties of modified CGs. As only loosely bound by GAG under physiological environment, collagen was modified by deamination, methylation and amination, and yielded Deaminated, Methylated and Aminated CGs upon co-precipitation with GAG. While GAG was mostly lost within 1 day in Untreated and Deaminated CGs, 20% and 40% GAG was retained after 6 days in Methylated and Aminated CGs respectively. In cell-seeded Aminated CG, over 60% GAG was retained after 8 days. Aminated CG, having the highest GAG/HYP of 4.5, best simulated the GAG-rich nucleus pulposus tissue. In ultrastructural analysis, Aminated CG consisted of abundant granular sub-stances that resembled the nucleus pulposus. Despite the differential initial number adhered to the CG scaffolds, human mesenchymal stem cells (hMSCs) had over 90% viability at all time points. Cell morphology was distinct, being round in Untreated and Methylated CGs but elongated in Deaminated and Aminated ones. The adhesion of hMSCs via collagen receptor, integrin alpha2beta1, was observed in all CG scaffolds, while adhesion via general matrix receptor, integrin alphaV, was extensive in all but Aminated CG. Based on improved GAG incor-poration and retention, which approximate the matrix composition of nucleus pulposus, Aminated CG was chosen as the core of the biphasic scaffold. The second part of this project studied lamination in biphasic disc scaffold and evaluated its mechanical properties in creep, recovery and dynamic loadings. A process was optimized to encapsulate a CG under physiological condition whilst producing an intact collagen gel, which allowed the CG to retain more GAGs and to be confined by the annulus structurally as was in the disc. This encasing approach was repeated for multiple lamellae, one lamella per day. Scaffolds with more lamellae had increased viscous compliance in creep and recovery, which was explained by the less laminated scaffolds being overloaded. Another lamination approach replaced most encasing lamellae with coiling ones. Despite low sample size, it was shown that this combined approach produced scaffolds with lower elastic and viscous compliances and longer equilibrating time in both creep and recovery, and higher complex modulus under dynamic loading. Full recovery was not achieved by any scaffold. This study demonstrated that a biphasic disc scaffold, made of GAG and collagen, contained similar matrix components to native disc, was almost mechanically comparable to the disc, and was cyto-compatible. It paved way towards tissue engineering of intervertebral disc and the intervertebral disc motion segment. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Bone graft substitute is a continuously developing field in orthopedics. When compared to tradition biomaterial in the field such as PLA or PCL, elastomer like polyurethane offers advantages in its high elasticity and flexibility, which establish an intimate contact with surrounding bones. This tight contact can provide a stable bone-material interface for cell proliferation and ingrowth of bone. The aim of this study is to evaluate the osteogenesis capabilities of a porous polyurethane scaffold in a critical size bone defect. In this study, a porous scaffold synthesized from segmented polyurethane is put under in vitro and in vivo tests to evaluate its potential in acting as a bone graft substitute for critical size bone defects. In vitro results indicate osteoblast-like cells are proliferating on the polyurethane scaffold during the 21-days experiment. Cells express their normal morphology when seeded on polyurethane under fluorescent staining. Although cells show a relatively lower cell activity then that seeded on culture plate, they share a similar alkaline phosphatase activity profile with the controls during the experiment period. In the in vivo animal model, reconstructed images from micro CT scanning indicates there are bone ingrowth inside the scaffold. Histology also indicates a tight interface has formed between bone and polyurethane, with osteogenic cells proliferating on the surface. The result has indicates polyurethane is a potential material for orthopedics in acting as a bone graft substitute. / published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

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Thesis (M.S.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Cells are complex active materials that display fascinating phenomena in response to changes in their physical environments. It is well established that the physical environment dictates cell fate and function; nevertheless, the standard method of culturing and studying cells is on stiff 2- dimensional Petri dishes and glass cover slips. The difference in the magnitude of the stiffness of the substrate in addition to the 2-dimensional character, leads to an incomplete and perhaps misleading picture of the cellular process under scrutiny. As such, an entire field has been dedicated to developing materials that more closely match the characteristics of the natural cellular milieu: biomaterials. Despite significant progress in the field, we are still far from fully recapturing the native environment. Importantly, many of the current strategies for engineering 3-dimensional biomaterials have specific applications yet lack flexibility to be adapted to a wide variety of functions. Our approach is to repurpose existing complex, readily available materials to create a platform for biomaterial production; our biomaterials are derived from plant tissue. Plants have evolved over millions of years to attain structures with intricate geometries for specialized functions. Due to the wide variety of plant structures, one can easily select a plant-based material with analogous features to the tissue of interest. A series of investigations are presented on these novel biomaterials to demonstrate this approach, quantify the mechanical properties, and study the cellular responses. First, we developed a method of processing plant materials to yield decellularized, cellulose-based, biocompatible scaffolds that can be repopulated with mammalian cells. We then created composite materials by casting hydrogels around the cellulose-based scaffolds, which allowed us to incorporate distinct temporal and spatial cues to the local cell populations. Spatial organization of tissues and tissue interfaces remains a primary challenge in biomedical engineering, as tissue interfaces mark complex transitional zones between distinct cell populations. Replicating and repairing this intricate delineation of cell types and mechanical profiles has proven to be a major concern in regenerative medicine. As such, we sought to develop a platform for engineered tissue interfaces, wherein components are combined in a modular fashion into a functional unit. The mechanical cues of the microenvironment affect a plethora of cellular processes, namely cell migration, proliferation, and differentiation. Consequently, the rheological properties of our decellularized, plant-based scaffolds were thoroughly investigated. An in-depth knowledge of the mechanics of the underlying substrate is required to guide future applications and refinements of this technology. The potential applications of these 3-dimensional constructs, as demonstrated through our findings, include designing in vitro models of tissue interactions, new biomaterials for in vivo applications, and studies on fundamental cellular processes. We highlight the significance of our results in a collection of scientific articles, which are presented in the body of this thesis (Chapters 2-5). This work is focused on the use of plant- derived cellulose materials, which forms a subsection of the cellulose biomaterial field. A review article centered on the use of cellulose materials for tissue engineering serves as an introductory chapter.

OBJECTIVE: In today’s world, diabetes has become an ever-growing crisis, with no definitive cure yet found. In a report conducted by the American Diabetes Association in March of 2018, it was noted that 1.5 million Americans are diagnosed with diabetes each year (ADA, 2018). Insulin is both a limited and expensive source, with prices of Lispro, a rapid-acting insulin, costing upwards of $306 per 1000 units, to Glargine, a basal-analog insulin, costing $298 per 1000 units (McEwen et. al, 2017). Because of this, many diabetics are left with no alternatives to properly treat their blood sugars and maintain a healthy HbA1c level, a laboratory measure of glucose bound to hemoglobin that indicates a diabetic’s blood sugar over a two to three-month period (Mayo Clinic, 2018). Even with insulin treatment, diabetics can suffer from microvascular complications ranging from nephropathy, retinopathy, or even death thereafter if not properly cared for (Klein et. al, 2005). In turn, many researchers have delved into analyzing and perfecting a potential treatment procedure known as islet transplantation that can serve to eliminate the necessity of insulin injections and pump devices, and replace the beta cells destroyed by complications from Type I diabetes. Islet transplantation is the process of extracting healthy islets from the pancreas of an organ donor, purifying the islets in cell culture media that works to recover islet cells, known as islet isolation, and injecting the isolated islets into diabetic recipients whose beta cells are nonfunctional (Alejandro et. al, 2018). The goal of this procedure is to restore proper function of endogenous islets in the body, which contain beta cells that work to secrete insulin and better regulate the body’s glucose metabolism. While pancreatic islet transplantation can reverse diabetes, the process is inefficient, with many islets lost to hypoxemia before the islets become vascularized (Kumatzu et. al, 2018). We hypothesize that by prevascularizing islets ex-vivo, and using a gelatinous scaffold seeded with endothelial cells, one can avoid ischemic induced loss. This will ensure that islets are delivered the necessary oxygen and nutrients they need in order to restore endogenous function. By inserting this prevascularized device into the subcutaneous space of C57 BL/6 mice, islets can be surrounded by a vast blood network, allowing them to function similarly to when they are in the pancreas. If completed properly, this could ease the difficulty of diabetics continuously having to self-regulate their blood sugar levels by multiple injections of exogenous insulin each day. METHODS: Prior to implanting a prevascularized device into the mouse model, we isolated and purified healthy islets from the pancreases of C57 B/L 6 mouse donors, using the steps outlined in the Edmonton Protocol (Shapiro et. al, 2006). Each device could house approximately 300-400 islets, so about two to three mouse donors were used per vascularized graft implanted. In conjunction with IVIVA Medical, the functionalized, three-dimensional islet graft was created and contained a perfusable vascular bed to better ensure islet survival and improve integration immediately after implantation. Mice were monitored daily to ensure the graft was stable inside the subcutaneous space and to ensure the mice were not experiencing any adverse reactions from the implant. On specified days post-implantation, the graft was explanted from the mouse, along with the surrounding tissue, to analyze the foreign body reaction experienced from the implantation and whether a vascular network formed. The tissue sample was then sent to the Histopathology Department for further processing and analysis. RESULTS: In all three groups in the study, foreign body reactions were expressed by the recruitment and presence of multiple cell lines, including macrophages, dendritic cells, and B and T lymphocytes. While immune cells proliferated, there were limited endothelial cells and islets present post-implantation, indicating the presence of hypoxemia, poor vascular formation, and a potent inflammatory response, ultimately leading to islet dysfunction. CONCLUSION: While prevascularizing the scaffolds helped them better perfuse while in the subcutaneous space, we found that the inflammatory reaction, coupled with improper islet seeding, did not initially lead to islet graft survival. With modifications, we plan to create a stronger vascular network to surround the islet cells that would ensure their durability and survival in the long-term. In utilizing this data, future research can work to better stabilize islet cells, with the end goal of translating this work into human models in the near future.

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