Bone and cartilage constructs are often plagued with mechanical failure, poor nutrient transport, poor tissue ingrowth, and necrosis of embedded cells. However, advances in computer aided design (CAD) and computational modeling enable the design of scaffolds with complex internal michroarchitectures and the a priori prediction of their transport and mechanical properties, such that the design of constructs satisfying the needs of the tissue environment can be optimized. The goal of this research is to investigate the capability of additive manufacturing technologies to create designed microarchitectured tissue engineering scaffolds for bone and cartilage regeneration. This goal will be achieved by pursuing the following two objectives: (1) the manufacture of bioresorbable thermoplastic scaffolds by selective laser sintering (SLS) (2) and the manufacture of hydrogel scaffolds by large area maskless photopolymerization (LAMP). SLS is a laser based additive manufacturing method in which an object is built layer-by-layer by fusing powdered material using a computer-controlled scanning laser. LAMP is a massively parallel ultraviolet curing-based process that can be used to create hydrogels from a photomonomer on a large-scale (558x558mm) while maintaining extremely high feature resolution (20µm). In this research, SLS is used to process polycaprolactone (PCL) and composites of PCL with hydroxyapatite (HA) for bone tissue engineering applications while LAMP is used to process polyethylene glycol diacrylate (PEGDA) which can be used for hard and soft tissue applications.

This study explored hydrostatic pressure as a mechanobiological parameter to control in vitro endothelial cell tubulogenesis in 3-D hydrogels as a model microvascular tissue engineering approach. For this purpose, the present investigation used an endothelial spheroid model, which we believe is an adaptable microvascularization strategy for many tissue engineering construct designs. We also aimed to identify the operating magnitudes and exposure times for hydrostatic pressure-sensitive sprout formation as well as verify the involvement of VEGFR-3 signaling. For this purpose, we used a custom-designed pressure system and a 3-D endothelial cell spheroid model of sprouting tubulogenesis. We report that an exposure time of 3 days is the minimum duration required to increase endothelial sprout formation in response to 20 mmHg. Notably, exposure to 5 mmHg for 3 days was inhibitory for endothelial spheroid lengths without affecting sprout numbers. Moreover, endothelial spheroids exposed to 40 mmHg also inhibited sprouting activity by reducing sprout numbers without affecting sprout lengths. Finally, blockade of VEGFR-3 signaling abolished the effects of the 20-mmHg stimuli on sprout formation. Based on these results, VEGFR-3 dependent endothelial sprouting appears to exhibit a complex pressure dependence that one may exploit to control microvessel formation.

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Cardiovascular disease (CVD) affects more than 70 million Americans and is the number one cause of mortality in the United States. Because the regenerative capacity of adult tissues such as the heart is limited, human embryonic stem cells (hESC) have emerged as a source for potential cardiac therapies. However, despite the use of a variety of biochemical differentiation protocols, current yields of hESC-derived cardiomyocytes (CM) have been low. In the case of hESC-CM, which are inherently electromechanically active, additional forms of inducing a mature cardiac fate have not been fully explored. In order to non-invasively visualize and quantify biochemical, electrical, and mechanical stimulation on hESC-CM differentiation in future studies, a bioreactor imaging system has been developed and is described in this report. / text

Creating effective cellular interfaces that can provide specific cellular signals is important for a number of fields ranging from tissue engineering to biosensors. Electroconducting polymers, especially polypyrrole (PPy), have attracted much attention for use in numerous biomedical applications since they provide a potential platform for local delivery of electrical stimuli to target tissues. To effectively modulate cellular functions at neural interfaces, it is essential to incorporate a range of extracellular cues into conducting polymers according to specific applications, such as nerve guidance conduits and implantable neural probes. For nerve regeneration scaffolds, three dimensional forms are desired for control of critical properties, such as porosity, mechanical strength, and topography. However, most researchers have worked on conventional two-dimensional PPy films, which cannot mimic a native three-dimensional architecture. Thus, a portion of my work has focused on introducing three-dimensional nanofibrous features into PPy. I have investigated various coating conditions to obtain uniform and conductive nanofibers. Effectiveness of electrical stimulation through the conducting nanofibers was confirmed by in vitro PC12 cell culture. The effects of different conducting nanofiber topographies (random and aligned) on cell adhesion and neurite outgrowth were examined in conjunction with electrical stimulation. The benefits of immobilized-NGF could be combined with electrical stimuli, which could be an ideal platform for neural tissue engineering scaffolds. Thus, I have modified conducting polymers to display neurotrophic activity. Nerve growth factor (NGF) was chemically immobilized on two dimensional and three dimensional PPy substrates. Specific chemical conjugation was achieved and characterized using diverse techniques. Immobilized NGF was as effective as exogenous NGF in medium in inducing neurite development and extension. NGF immobilized on functionalized PPy substrates was stable in a physiological solution and under electrical stimulation, indicating effective prolonged activity. I also investigated another important application of conducting polymer-based materials for neural interfacing - passivating electrodes with a biocompatible polysaccharide, hyaluronic acid (HA). I synthesized electrically polymerizable HA by chemically conjugating amine-functionalized pyrrole derivatives with HA. This coating was stable under physiological conditions for three months and resistant to enzymatic degradation. In vitro studies have shown the minimal adhesion and migration of astrocytes on the HA-coated electrodes. Implantation of HA-coated commercial probes into rat cortices for three weeks revealed attenuated reactive astrocyte responses from the coated wires, and the importance of glial interaction with non-conducting sites was demonstrated. / text

This thesis presents a melt electrospinning technique to fabricate highly porous and controllable poly (ε-caprolactone) (PCL) microfibers for tissue engineering applications and rehabilitation applications. Electrospinning without solvents via melt methods may be an attractive approach to tissue engineering of cell constructs where solvent accumulation or toxicity is an issue. This method is also able to produce microfibers with controllable parameters. However, the fiber diameters resulting from melt electrospinning processes are relatively large when compared to the fibers from solution electrospinning. The typical microfiber diameter from melt electrospinning was reported to be approximately 0.1mm. In order to further develop the melt electrospinning technique, we focused on the design of a melt electrospinning setup based on numerical analysis using the Solidworks 2013 simulation package and practically established a melt electrospinning setup and thermal control system for accurate experiments. One of main purposes of this thesis is the build-up of mathematical modeling to control and predict the electrospun microfiber via a more intricate understanding of their parameters such as the nozzle diameter, applied voltage, distance between the nozzle and counter electrode, temperature, flow rate, linear transitional speed, among others. The model is composed of three parts: 1) melt electrospinning process modeling, 2) fibrous helix movement modeling, and 3) build-up of microfibers modeling. The melt electrospinning process model describes an electric field, the shape of jet’s continuously changing shape, and how the polymer melt is stretched into a Taylor cone and a straight jet. The fibrous helix movement model describes movement of electrospun microfibers influenced by Lorentz force, which moves along the helix pattern. Lastly, the build-up microfiber modeling describes the accumulation of the extruded microfibers on both flat and round counter electrodes based on the physical forces involved. These models are verified by experimental data from our own customized melt electrospinning setup. Moreover, the fabricated scaffolds are tested by seeding neural progenitors derived from murine R1 embryonic stem cell lines and it demonstrates the potential of scaffolds for tissue engineering applications. To increase cell attachment and proliferation, highly porous microfibers are fabricated by combination of melt electrospinning and particulate leaching technique. Finally, auxetic stretchable PCL force sensors are fabricated by melt electrospinning for hand rehabilitation. These stretchable sensors can be used to measure applied external loads or displacement and are also attachable to various substrates. We have attempted to apply the sensors to real human hand in order to prove their functionality. / Graduate / jko@me.uvic.ca

Implanted biomaterials often induce inflammation that frequently leads to the foreign body response, fibrosis, and the failure of the implant. Thus, it is important to evaluate how cells interact with materials to promote a more regenerative response. It is critical to determine how to modulate the response of tissue resident innate immune cells, as they are among the first cells to interact with implanted materials. Among tissue resident innate immune cells are mast cells, which are inflammatory sentinels that degranulate and orchestrate the fate of other cell populations, such as monocytes/macrophages and lymphocytes. Mast cells have also been reported to play a vital role in the foreign body response of implanted biomaterials as well as angiogenesis. The goal of this study was to determine how to modulate mast cell responses to electrospun scaffolds by altering scaffold architecture and composition to promote anti-inflammatory and regenerative cell-scaffold interactions. Scaffold architecture was manipulated by changing either fiber diameter or pore diameter and mast cell responses were mediated by endogenous and exogenous DAMPs (i.e. IL-33 and LPS, respectively). Particularly in response to IL-33, scaffolds with increased fiber and pore diameter promoted less inflammatory cytokine and chemokine release while increasing angiogenic cytokine release. Additionally, taking scaffolds that promoted increased inflammatory cytokine expression and increasing the pore diameter alone dampened inflammatory cytokine expression. The next question we wanted to answer was how might the degradative byproducts of scaffolds alter mast cell inflammatory responses. Given the widespread use of polylactic acid, we decided to investigate this question using lactic acid as a degradative byproduct. In the presence of physiologically relevant levels of lactic acid, IL-33- and IgE-mediated inflammatory cytokines and chemokines are suppressed, while angiogenic cytokines are enhanced. This response was shown to be pH- and MCT1-dependent and was recapitulated in primary human skin mast cells as well as in vivo. In summary, scaffold architecture and the presence of select polymer degradative byproducts have the potential of selectively suppressing inflammatory cytokines and enhancing angiogenic cytokines.

<p>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.</p><p> 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. </p><p> 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. </p><p> 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.</p> / Dissertation

<p>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.</p><p>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. </p><p>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.</p><p>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.</p> / Dissertation

<p>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.</p><p> 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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p> / Dissertation