Browsing by Subject "Tissue engineering"

Tissue-engineered skeletal muscle presents promising opportunities for developing high-fidelity in vitro models for investigating human muscle biology in the areas of regeneration and disease. In muscle regeneration, satellite cells (SCs) are essential for new muscle fiber formation; however, they lose their native quiescent state upon isolation, making in vitro studies of human SC function challenging. To optimally promote SC quiescence and enable exploration of SC dynamics in vitro, engineered muscle needs to recapitulate the native muscle microenvironment, which is comprised of muscle fibers, extracellular matrix, and other biochemical and mechanical cues. In disease modeling, mechanistic studies and therapeutic development are still extensively evaluated in animal models, which have limited translational relevance to patients. Specifically, Pompe disease is caused by a variety of mutations in the lysosomal enzyme acid alpha-glucosidase (GAA) that varyingly affect residual GAA activity and cannot be captured in the current GAA-/- mouse model. Therefore, human in vitro models are needed to enhance our mechanistic understanding of diseases and stimulate the development of effective therapies. To overcome these limitations, we set the dissertation goals to: 1) generate a pool of quiescent SCs and explore the mechanisms governing their formation and activation using an engineered skeletal muscle microenvironment and 2) develop a high-fidelity tissue-engineered skeletal muscle model for Pompe disease to investigate pathological mechanisms and test candidate therapies. To achieve these goals, we first compared methods for primary human myoblast expansion and found that p38 inhibition significantly increases the formation of Pax7+ cells in engineered 3D skeletal muscle tissues (“myobundles”). Gene expression analysis suggested that within the myobundle environment the Pax7+ cells adopt a quiescent phenotype (3D SCs), characterized by increased Pax7 expression, cell cycle exit, and Notch signaling activation relative to the original 2D expanded myoblasts. We then compared 3D SCs to previously described satellite-like cells that form alongside myotubes in 2D culture, termed reserve cells (RCs). Compared to RCs, 3D SCs showed an advanced quiescent phenotype characterized by a higher Pax7, Spry1, and Notch3 expression, as well as increased functional myogenesis demonstrated by formation of myobundles with higher contractile strength. To examine 3D SC activation, we tested several myobundle injury methods and identified treatment with a bee toxin, melittin, to robustly induce myofiber fragmentation, functional decline, and 3D SC proliferation. To further investigate the transcriptional processes describing how 2D myoblasts acquire 3D SC phenotype (i.e. deactivate) and how 3D SCs respond to injury (i.e. reactivate), we applied single cell RNA-sequencing (scRNA-seq) from which we discovered the existence of two subpools of 3D SCs—“quiescent” (qSC) and “activated” (aSC). The qSC subpool possessed greater expression of quiescence genes Pax7, Spry1, and Hey1, whereas the aSC subpool exhibited increased expression of inflammatory and differentiation markers. Furthermore, we performed trajectory inference along the deactivation process from 2D myoblasts to qSCs and identified deactivation-associated genes, included downregulated genes for proliferation, cytoskeletal reorganization, and myogenic differentiation. In response to tissue injury, we observed a decrease in the proportion of qSCs and an increase in the proportion of aSCs and committed myogenic progenitor cells suggestive of myogenic differentiation. In addition, we observed transcriptional changes within the aSC population reflective of SC activation in vivo, namely increased TNF- signaling, proliferation, and glycolytic and oxidative metabolism. These results strongly suggested that 3D SC heterogeneity and function recapitulate several aspects of native human SCs and could be applied to study human muscle regeneration and disease-associated SC dysfunction. To evaluate the myobundle system in the context of disease modeling, we developed the first 3D tissue-engineered skeletal muscle model of infantile onset Pompe disease (IOPD), the most severe form of Pompe disease. Diseased myobundles demonstrated characteristic GAA enzyme deficiency, accumulation of the GAA target glycogen, and lysosome enlargement. Despite exhibiting these key biochemical and structural hallmarks of disease, IOPD myobundles did not show deficits in contractile force generation or autophagic buildup. We therefore identified metabolic stress conditions that acutely targeted disease-associated abnormalities in the lysosomes and glycogen metabolism, which revealed impairments in contractile function and glycogen mobilization. To further elucidate the biological mechanisms underlying the phenotype of IOPD myobundles, we applied RNA sequencing (RNA-seq) and observed enrichment for terms consistent with Pompe disease phenotype including downregulation of gene sets involved in muscle contraction, increased endoplasmic reticulum stress, and reduced utilization of specific metabolic pathways. We then compared the transcriptomic profiles of GAA-/-¬ and wild-type mice to identify a Pompe disease signature and confirmed the presence of this signature in IOPD myobundles. Finally, treating IOPD myobundles with clinically used recombinant protein (rhGAA) therapy resulted in increased GAA activity, glycogen clearance, and a partial reversal of the disease signature, further confirming the utility of the myobundle system for studies of Pompe disease and therapy. In summary, this dissertation describes novel strategies for the formation and characterization of quiescent human SCs using the myobundle system. We present first-time application of scRNA-seq to engineered skeletal muscle, and uncover transcriptional descriptors of human myoblast deactivation and SC heterogeneity and activation. When utilizing human myobundles as a novel model of Pompe disease, we identified disease hallmarks and responses to therapy consistent with observations in Pompe patients. We anticipate that the findings and methods developed in this work will serve as a useful framework for the future engineering of regenerative human muscle for therapeutic and disease modeling applications.

Clinical studies have identified strong correlations between aging and the development of atherosclerosis. In particular, endothelial cell senescence is implicated in age-related changes in vasoreactivity. Oxidative stress is considered the primary source of endothelial cell (EC) senescence in vivo. EC senescence leads to abnormal proliferation of vascular smooth muscle cells, reduced vasoreactivity, enhanced vascular permeability, and greater adhesion of circulating monocytes and lipids. Endothelial senescence often occurs coincident with an inflammatory response within the endothelium. Recapitulating this mechanism of inducing EC senescence in vitro will facilitate a more precise understanding of how aging contributes to endothelial dysfunction and development of vascular diseases, particularly atherosclerosis. Additionally, evidence of vascular remodeling, particularly deposition of fibronectin and stiffening of the vessel wall matrix, is found in both older patients and atheroprone regions. The independent effects of these factors on the function of endothelial cells is poorly understood due to the inability to study them in isolation in vivo. The Truskey lab developed tissue-engineered blood vessels (TEBVs) which recapitulate the structure and function of an arteriole in vitro. These vessels can be fabricated rapidly, perfused immediately after fabrication, and reach functional maturity after a week. Measurements of endothelium-mediated vascular function confirm the presence of a healthy endothelium in the vessels for several weeks after initial fabrication. This in vitro system allows more precise control over the cellular and structural components of blood vessels than is possible with in vivo experiments. Ultimately, the development of a more robust in vitro model for atherosclerosis will contribute to an increased understanding of vascular disease progression and provide a platform for the evaluation of new drugs during preclinical trials. Specific Aim 1: Evaluate the functional effects of stress-induced senescence on TEBVs. Stress-induced senescence reduced endothelium-dependent vessel function and resulted in endothelial cell inflammation with minimal effects on the surrounding hNDFs. Stress-induced senescence was induced in vitro by treatment with hydrogen peroxide. 2-D cells and TEBVs were treated for 5 or 7 days with hydrogen peroxide. Cells in 2-D were stained for p21 to evaluate senescence, as well as key immune cell adhesion markers VCAM-1, ICAM-1, and E-Selectin. To characterize the effects on TEBVs, vasoreactivity in response to an endothelium-independent vasodilator (sodium nitroprusside) and vasoconstrictor (phenylephrine) were quantified, as well as endothelium-dependent vasoreactivity (acetylcholine). Immunostaining of p21 and VCAM-1 expression was also used to confirm that senescence and inflammation were induced in the TEBVs alongside the reduction in endothelium-dependent vasodilation. Specific Aim 2: Evaluate the capacity of stress-induced senescence to increase the monocyte adhesion and foam cell formation in the TEBVs Stress-induced senescence in TEBVs increased adhesion of circulating monocytes and foam cell formation in accumulated monocytes and medial hNDFs. Senescence was induced as in Aim 1, by treating vessels with hydrogen peroxide. The resulting increase in senescence, VCAM-1, and E-selectin increased adhesion of circulating monocytes to the vessel wall. To develop an atherogenesis model, low density lipoprotein was enzymatically modified into a more inflammatory state which is often identified within atherosclerotic lesions. Introducing enzyme-modified low-density lipoprotein (eLDL) alongside hydrogen peroxide treatment further increased endothelial cell activation. There was a significant increase in the percentage of ICAM-1 positive cells when eLDL was applied to endothelial cells alongside H2O2. hNDFs absorbed and retained eLDL, even without H2O2 in the growth media. When TEBVs were exposed to a combination of eLDL, H2O2, and cell-tracker red monocytes, endothelium-dependent vasoreactivity was significantly compromised. Lipid retention within the vessel wall was significant, as was adhesion of monocytes. Specific Aim 3: Evaluate the drug-responsiveness of the TEBV senescence model and the ability of geroprotective agents to reduce senescence-induced vascular dysfunction, monocyte adhesion, and foam cell formation. Development of a physiologically relevant model for vascular senescence can provide a valuable tool for evaluating the efficacy of drugs targeting atherosclerosis, particularly a new class of drugs in development called senolytics. Senolytics, and their sister drugs senomorphics, specifically target senescent cells and transiently disable the anti-apoptotic pathways that prolong their lives, reducing the burden of senescent cells within the tissue. Senomorphics target factors within the senescence-associated secretory pathway (SASP) to reduce cytokine production and inflammation. Dasatinib and quercetin, two senolytics, and tacrolimus, a senomorphic, were tested on CBECFCs growing in 2-D to see if they were effective at reducing the percentage of p21 positive (senescent) cells. Tacrolimus was found to be the best candidate and used in TEBV trials. TEBVs treated with tacrolimus for 48 hours after induction of senescence recovered significantly more endothelium-dependent vasoreactivity compared to vessels left to recover from H2O2 in normal growth media. Additionally, addition of tacrolimus for the duration of hydrogen peroxide treatment had an atheroprotective effect. Adhesion of monocytes and foam cell formation were significantly reduced compared to vessels without tacrolimus. In summary, the work presented here demonstrates that a TEBV model of vascular senescence can be generated in under two weeks using near-physiological levels of hydrogen peroxide. This model can be capitalized upon to model atherogenesis by adding only eLDL and monocytes. We were also able to effectively use the senomorphic tacrolimus to mitigate the effects of senescence on monocyte adhesion and lipid uptake. This system could be used to investigate other senolytics or test the efficacy and toxicity of novel drugs still in development.

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Continuous nanostructures, such as electrospun nanofibers, embedded with proteins may synergistically present the topographical and biochemical signals to cells for tissue engineering applications. In this dissertation, co-axial electrospinning is introduced as a mean to efficiently encapsulate and release protein and live entities while producing a tissue engineering scaffold with uniaxial topography. In the first specific aim, aligned poly (caprolactone) nanofibers encapsulated with BSA and growth factors were produced to demonstrate controlled release and bioactivity retention properties. Control over release kinetics is achieved by incorporation of poly(ethylene glycol) as a porogen in the shell of the fibers. PEG leaches out in a concentration and molecular weight dependent fashion, leading to BSA release half-lives that range from 1 -20 days. The second specific aim introduces the fabrication of virus and bacterial cell encapsulated electrospun fibers to achieve unique biological functionalization. Adenovirus encoding the gene for green fluorescent protein was efficiently encapsulated into the core of poly(caprolactone) fibers through co-axial electrospinning and subsequently released via the porogen-mediated process. Encapsulated bacterial cells were confined to fibers of varying core sizes, which provided an aqueous core environment for free mobility and allowed the bacterias to proliferate within the fibers.

In the third specific aim, the differentiation of skeletal myoblasts on aligned electrospun polyurethane fibers and in the presence of electromechanical stimulation were systematically studied. Skeletal myoblasts cultured on aligned polyurethane (PU) fibers showed more pronounced elongation, better alignment, upregulation of contractile proteins and higher percentage of striated myotubes compared to those cultured on random PU fibers and film. In the last specific aim, the controlled release aspect of co-axial electrospun fibers were combined with skeletal tissue engineering to serve as a therapeutic implant for the treatment of hemophilia. A non-viral, tissue engineering approach were taken to stimulate local lymphatic or vascular system in order to enhance transport near the FVIII-producing implants to provide effective and sustained treatment for hemophilia A. Stable FVIII-producing clones were engineered from isolated myoblasts and cultured on aligned, protein-releasing electrospun fibers to form skeletal myotubes. The implanted construct rapidly integrated with host tissue and selectively induced angiogenesis or lymphangiogenesis as a result of the encapsulated growth factors. Constructs inducing angiogenesis significantly enhanced the transport of produced FVIII and achieved hemophilia phenotypic correction over two months. The use of co-axial electrospun fibers to serve as controlled delivery and tissue engineering construct furthers the continued pursue of a more sophisticated and medically relevant implant scaffold design.

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary causes of death are stroke and cardiovascular disease at an average age of 14 years. It is known that loss or malfunction of smooth muscle cells (SMCs) in the vasculature leads to cardiovascular defects, however, the exact mechanisms are still not understood. The contribution of other vascular cell types, such as endothelial cells, is still not known due to the current limitations of studying such a rare disorder. Due to limitations of 2D cell culture, mouse models, and the limited HGPS patient pool, there is a need to develop improved models of HGPS to better understand the development of the disease and discover novel therapeutics.

To address these limitations, we produced a functional, three-dimensional tissue model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived cell sources from HGPS patients. To isolate the specific effects of HGPS SMCs, we initially used human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor and iPSC-derived SMCs (iSMCs). TEBVs fabricated from HGPS patient iSMCs and hCB-EPCs (HGPS iSMC TEBVs) showed disease attributes such as reduced vasoactivity, increased medial wall thickness, increased calcification, excessive extracellular matrix protein deposition, and cell apoptosis relative to TEBVs fabricated from primary mesenchymal stem cells (MSCs) and hCB-EPCs or normal patient iSMCs with hCB-EPCs. Treatment of HGPS iSMC TEBVs for one week with the rapamycin analog Everolimus (RAD001), increased HGPS iSMC TEBV vasoactivity and iSMC differentiation in TEBVs.

To improve the sensitivity of our HGPS TEBV model and study the effects of endothelial cells on the HGPS cardiovascular phenotype, we adopted a modified differentiation protocol to produce iPSC-derived vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) from normal and Progeria patient iPSC lines to create iPSC-derived vascular TEBVs (viTEBVs). Normal viSMCs and viECs showed structural and functional characteristics of vascular SMCs and ECs in 2D culture, while HGPS viSMCs and viECs showed various disease characteristics and reduced function compared to healthy controls. Normal viTEBVs had comparable structure and vasoactivity to MSC TEBVs, while HGPS viTEBVs showed reduced vasoactivity, increased vessel wall thickness, calcification, apoptosis and excess ECM deposition. In addition, HGPS viTEBVs showed markers of cardiovascular disease associated with the endothelium such as decreased response to acetylcholine, increased inflammation, and altered expression of flow-associated genes.

The treatment of viTEBVs with multiple Progeria therapeutics was evaluated to determine the potential of the HGPS viTEBV model to serve as a platform for drug efficacy and toxicity testing as well as to further elucidate the mechanisms behind each drugs mode of action. Treatment of viTEBVs with therapeutic levels of the farnesyl-transferase inhibitor (FTI), Lonafarnib, or Everolimus improved different aspects of HGPS viTEBV structure and function. Treatment with Everolimus alone increased response to phenylephrine, improved SMC differentiation and cleared progerin through autophagy. Lonafarnib improved acetylcholine response, decreased ECM deposition, decreased calcification and improved nitric oxide production. Most significantly, combined therapeutic treatment with both drugs showed an additive effect by improving overall vasoactivity, increasing cell density, increasing viSMC and viEC differentiation, and decreasing calcification and apoptosis in treated HGPS viTEBVs. On the other hand, toxic doses of both drugs combined resulted in significantly diminished HGPS viTEBV function through increased cell death. In summary, this work shows the ability of a tissue engineered vascular model to serve as an in vitro personalized medicine platform to study HGPS and potentially other rare diseases of the vasculature using iPSC-derived cell sources. It has also further identified a potential role of the endothelium in HGPS. Finally, this HGPS viTEBV model has proven effective as a drug testing platform to determine therapeutic and toxic doses of proposed therapeutics based on their specific therapeutic effects on HGPS viTEBV structure and function.

The purpose of this work is to determine if branched chain amino acids (BCAA) could have a causative role in the development of atherosclerosis. Atherosclerotic lesions occur in the vasculature and mediate the progression of cardiovascular disease (CVD). Advanced atherosclerotic lesions can lead to heart attacks or strokes. Conflicting evidence from previous studies has made it difficult to understand if BCAA help or hurt cardiovascular health, and it is not known if other pro-atherosclerotic factors cooperate with BCAA to accelerate atherosclerosis. While studies in human patients have shown that there is a correlation between BCAA levels in the blood and the development of diseases like metabolic syndrome and CVD, we cannot conclude that BCAA cause these diseases from association studies alone. Additionally, some studies in animals have shown that supplementation with BCAA supports cardiovascular health. Therefore, we need to determine if there is a mechanistic link between BCAA levels and atherosclerotic disease processes in human cells. This will also help us determine if BCAA could be a mechanistic link between metabolic syndrome and CVD.

In this work, we use a tissue engineered blood vessel (TEBV) model to determine the role of BCAA in the development of atherosclerosis. The TEBV model is an artificial blood vessel made of a collagen-based scaffold and populated with vascular cells, using similar tissue architecture and environmental stimuli of an artery in the human body. The TEBV system models the processes that occur in atherosclerosis, such as inflammation, loss of vasomotor tone, and interactions between white blood cells and vascular cells. Measuring these processes then allows us to predict what effect a novel risk factor, such as BCAA, would have on vascular health in the human body.

In chapter 2, we develop an “intermediate stage” lesion model in the TEBV system. Atherosclerotic lesions develop over many years, and since metabolic syndrome is often diagnosed in adults, many patients will have existing lesions. Therefore, it is important to expand upon existing models of early atherogenesis to include features that occur in later disease stages, such as remodeling of the vessel wall. We recapitulate the increase of a carbohydrate-based molecule called chondroitin sulfate (CS) that occurs in the atherosclerotic extracellular matrix in our TEBV model. While many studies have shown that CS enhances the development of atherosclerosis by affecting processes like lipid retention in the vessel wall and inflammation, there have not been many in vitro disease models that include the effects of CS-remodeling that occurs in the body In our work, we demonstrated that enriching the TEBV extracellular matrix with pathological levels of CS leads to an enhanced atherosclerotic response to treatment with modified low-density lipoprotein (LDL), including increased VCAM expression, a marker of inflammation in endothelial cells, and increased white blood cell adhesion to the vessel wall.

In chapter 3, we tested the effects of BCAA treatment on endothelial cell and TEBV health. We cultured cells and TEBVs in a low-BCAA medium that reflected the BCAA levels that occur in human serum. While a few other studies have looked at the effects of BCAA on human vascular cells, they did not use physiologically relevant levels of BCAA in their untreated controls, and used much higher levels of BCAA doses to test their effects. In our studies, we found that BCAA affect several key processes related to endothelial health, inducing oxidative stress in the mitochondria, inducing increased expression of redox-balancing enzymes, and slowing autophagy. This was consistent with results in TEBV experiments, where we saw that BCAA cooperate with another pro-atherosclerotic agent, oxidized LDL, to induce vasodilation dysfunction and increased white blood cell adhesion to the vessel wall.

In chapter 4, we evaluated the hypothesis that slowing BCAA catabolism is sufficient to induce buildup of BCAA in vascular cells, leading to an atherosclerotic phenotype. To slow BCAA catabolism, we used a dCas9-KRAB construct to repress the gene PPM1K. PPM1K plays a critical regulatory role in modulating BCAA levels in the cell by activating the rate-limiting enzyme in the BCAA metabolic pathway and stimulating BCAA breakdown. We found that repressing PPM1K effectively alters the active state of its target enzyme, BCKDH, and increases glutamine and serine levels in iPSC-derived endothelial cells. In TEBVs, we found that PPM1K repressed-endothelium induces a differential response to oxLDL treatment in causing vasodilation dysfunction, compared to the vehicle control. Thus, there may be a role for BCAA metabolism in enhancing an atherosclerotic phenotype induced by other pro-atherosclerotic factors.

In summary, we determined that BCAA can contribute to an atherosclerotic phenotype, specifically by affecting endothelial cell health. This conclusion is supported by our observations that BCAA affect several key molecular and functional markers of endothelial health, including mitochondrial oxidative stress, autophagy, vasodilation function, and white blood cell adhesion to the endothelium. Importantly, in TEBVs, the presence of other pro-inflammatory factors, such as oxLDL, enhanced these effects. Future research should aim to identify which of these processes may be a suitable target to interrupt the atherosclerotic risk of BCAA.

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

Articular cartilage is the connective tissue that lines the ends of long bones in diarthrodial joints, providing a low-friction load-bearing surface that can withstand a lifetime of loading cycles under normal conditions. Despite these unique and advantageous properties, the tissue possesses a limited capacity for self-repair due to its lack of vasculature and innervation. Total joint replacement is a well-established treatment for degenerative joint disease; however, the materials used in these procedures have a limited lifespan in vivo and will likely fail over time, requiring additional - and increasingly complicated - revision surgeries. For younger or more active patients, this risk is unacceptable. Unfortunately, alternative surgical options are not currently available, leaving pain management as the only viable treatment. In seeking to discover a new therapeutic strategy, the goal of this dissertation was to develop a functional tissue-engineered cartilage construct that may be used to resurface an entire diseased or damaged joint.

A three-dimensional (3-D) woven textile structure, produced on a custom-built miniature weaving loom, was utilized as the basis for producing novel composite scaffolds and cartilage tissue constructs that exhibited initial properties similar to those of native articular cartilage. Using polyglycolic acid (PGA) fibers combined with chondrocyte-loaded agarose or fibrin hydrogels, scaffolds were engineered with anisotropic, inhomogeneous, viscoelastic, and nonlinear characteristics prior to cultivation. However, PGA-based constructs showed a rapid loss of mechanical functionality over a 28 day culture period suggesting that the inclusion of other, less degradable, biomaterial fibers could provide more stable properties.

Retaining the original 3-D architecture and fiber/hydrogel composite construction, poly (epsilon-caprolactone) (PCL)-based scaffolds demonstrated initial biomechanical properties similar to those of PGA-based scaffolds. Long-term culture of 3-D PCL/fibrin scaffolds seeded with human adipose-derived stem cells (ASCs) showed that scaffolds maintained their baseline properties as new, collagen-rich tissue accumulated within the constructs.

In an attempt to improve the bioactivity of the PCL scaffold and further induce chondrogenic differentiation of seeded ASCs, we produced a hybrid scaffold system by embedding the 3-D woven structure within a porous matrix derived from native cartilage. We then demonstrated how this multifunctional scaffold could be molded, seeded, and cultured in order to produce an anatomically accurate tissue construct with potential for resurfacing the femoral head of a hip.

In summary, these findings provide valuable insight into a new approach for the functional tissue engineering of articular cartilage. The results of this work will hopefully lead to the discovery of new strategies for the long-term treatment of cartilage pathology.

To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype.

In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.

In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated.

Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.

The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain.

Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.

Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation.

Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.

In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

The aim of this dissertation was to develop new techniques for producing electrospun scaffolds for use in the tissue engineering of articular cartilage. We developed a novel method of imparting mechanical anisotropy to electrospun scaffolds that allowed the production of a single, cohesive scaffold with varying directions of anisotropy in different layers by employing insulating masks to control the electric field. We improved the quantification of fiber alignment, discovering that surface fibers in isotropic scaffolds show similar amounts of fiber alignment as some types of anisotropic scaffolds, and that cells align themselves in response to this subtle fiber alignment. We improved previous methods to improve cellular infiltration into tissue engineering scaffolds. Finally, we produced a new material with chondrogenic potential consisting of native unpurified cartilage which was electrospun as a composite with a synthetic polymer. This work provided advances in three major areas of tissue engineering: scaffold properties, cell-scaffold interaction, and novel materials.

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Nucleic acid delivery has applications ranging from tissue engineering to biologics development and manufacturing to vaccines and infectious disease. To improve delivery and extend viable expression over time, we turn to biomaterials as a method for sustained nucleic acid release and enhanced cell culture or tissue interaction. Here, we describe how cationic polymer and lipid condensed nucleic acids can be effectively loaded into injectable granular hydrogel scaffolds by stabilizing the condensed nucleic acid into a lyophilized powder, loading the powder into a bulk hydrogel, and then fragmenting the gel into hydrogel microparticles. The resulting microgels contain non-aggregated nucleic acid particles, can be annealed into an injectable microporous scaffold, and can effectively deliver nucleic acids to cells with a sustained rate of expression. We explore how this technology can improve the production of biologics, like antibodies and viruses, to overcome limitations of current batch processes. Our scaffolds allow for continuous biologics manufacturing, with sustained production upwards of 30 days. We also explore how our platform can improve tissue regeneration in disease models like dermal wounds by delivering nucleic acid drugs, namely DNA, mRNA, and therapeutic viruses. The loaded granular scaffolds allow cells to readily repopulate the missing tissue and drugs be locally released and taken up over time. Overall, our scaffold delivery approach is a customizable platform that can be tuned for many different applications.

High-throughput processing technologies hold critical position in biomedical research. These include screening of cellular response based on phenotypic difference and production of homogeneous chemicals and biologicals for therapeutic applications. The rapid development of microfluidics technology has provided an efficient, controllable, economical and automatable processing platform for various applications. In particular, emulsion droplet gains a lot of attention due to its uniformity and ease of isolation, but the application of water-in-oil (W/O) single emulsion is hampered by the presence of the oil phase which is incompatible with aqueous phase manipulation and the difficulty in modifying the droplet environment.

This thesis presents the development of a double emulsion (DE) droplet platform in microfluidics and two applications: (1) high-throughput screening of synthetic gene and (2) production of multicellular spheroids with adjustable microenvironment for controlling stem cell differentiation and liver tissue engineering. Monodisperse DE droplets with controllable size and selective permeability across the oil shell were generated via two microfluidics devices after optimization of device design and flow rates.

Next, bacterial cells bearing synthetic genes constructed from an inkjet oligonucleotide synthesizer were encapsulated as single cells in DE droplets. Enrichment of fluorescent signals (~100 times) from the cells allowed quantification and selection of functionally-correct genes before and after error correction scheme was employed. Permeation of Isopropyl β-D-1-thiogalactopyranoside (IPTG) molecules from the external phase triggered target gene expression of the pET vector. Fluorescent signals from at least ~100 bacteria per droplet generated clearly distinguishable fluorescent signals that enabled droplets sorting through fluorescence-activated cell sorting (FACS) technique.

In addition, DE droplets promoted rapid aggregation of mammalian cells into single spheroid in 150 min. Size-tunable human mesenchymal stem cells (hMSC) spheroids could be extracted from the droplets and exhibited better differentiation potential than cells cultured in monolayer. The droplet environment could be altered by loading matrix molecules in it to create spheroid-encapsulated microgel. As an example, hMSC spheroid was encapsulated in alginate or alginate-RGD microgel and enhanced osteogenic differentiation was found in the latter case.

Lastly, the capability of forming spheroids in DE droplet was applied in liver tissue engineering, where single or co-culture hepatocyte spheroids were efficiently produced and encapsulated in microgel. The use of alginate-collagen microgel significantly improved the long-term function of the spheroid, in a manner similar to forming co-culture spheroids of hepatocytes and endothelial progenitor cells at a 5 to 1 ratio. The hepatocyte spheroid encapsulated in microgel could be useful for developing bioartificial liver or drug testing platform or applied directly for hepatocyte transplantation.

Crystalline and amorphous materials have been extensively studied for their interesting properties, but they comprise a very small portion of the total materials space. The properties of most materials are a consequence of the interactions between their ordered and disordered components. This phenomenon is especially important in biology with materials such as silk and elastin owing their extraordinary attributes to the interactions of ordered and disordered domains at the inter- and intra- molecular levels. Recent insights in the emerging field of intrinsically disordered proteins have further highlighted the importance of order-disorder interactions as determinants of structural and chemical functions in multivalent proteins. While the significance of order-disorder interactions is well known and much work has been devoted to understanding their biological implications, little effort has been made to functionalize them for the development of new materials.

Recombinant protein polymers offer an interesting platform for determining how combinations of order and disorder lead to unique material properties as their molecular level control enables these components to be precisely mixed within a single polypeptide chain. This dissertation reports the successful design and application of a new class of recombinant materials inspired by the protein elastin, termed partially ordered polymers (POPs), to uncover the impact of single chain interactions between ordered domains and disordered regions on macroscopic material properties. These ‘smart’ protein materials: (1) are the first biopolymer system with temperature dependent phase behavior in which the aggregation and dissolution temperatures can be independently controlled, (2) are injectable as a solution that assembles under the stimulus of body heat into fractal-like, porous networks suitable for cell infiltration and remodeling, and (3) can be used to create microstructures with complex architectures and spatially segregated regions for applications in drug delivery and tissue engineering. This work expands the biomedical potential for protein-based materials as well as the available microarchitectures for biocompatible polymers, demonstrating that sequence level modulation of order and disorder is an untapped principle for the design of functional biomaterials.

With growing numbers of increasingly younger patients suffering debilitating arthropathies, the need for simple models that recapitulate the complex interplay between distinct joint tissues, and grafts that emulate these joint structures in their biological properties and their strength have become more urgent. The objective of this study is to engineer constructs of multiple tissue types by controlling the morphogenetic factors that direct stem cell differentiation and tissue formation either exogenously or via transduction of expression vectors. Our hypothesis is that sequential changes in exogenous growth factor delivery and also scaffold-mediated inducible regulation of morphogenetic gene expression and signaling in 3D-constructs of murine iPSCs will lead to the formation of both bone and cartilage tissue types, both as separate tissues, and as osteochondral constructs. In the first study, osteochondral organoids were grown in a scaffold-free system from a single iPSC cell source, creating tissues containing a distinct core with the genetic and extracellular matrix profile of articular cartilage surrounded by a shell with the genetic and extracellular matrix profile of bone. In the second study, chondrogenic, osteogenic, and osteochondral tissue grafts were grown by scaffold-mediated lentiviral delivery of differentiation factors expressed both constitutively and in a temporally inducible manner. These constructs will provide an excellent platform to study diseases of the osteochondral junction, to screen pharmacologic therapies affecting both cartilage and bone tissue, and as a next step toward making an implantable osteochondral graft for the direct treatment of joint defects.

The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro.

The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence.

Major results of the dissertation are as follows. Culture conditions of 100 μg/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.