R-3 Repository :: Browsing by Author "Mikos, Antonios G"

Browsing by Author "Mikos, Antonios G"

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3D Printing of Biomaterials for Cranial Bone Tissue Engineering
(2022-05-23) Koons, Gerry L.; Mikos, Antonios G
Successful materials design for bone tissue engineering requires an understanding of the composition and structure of native bone tissue, as well as appropriate selection of biomimetic natural or tunable synthetic biomaterials, such as polymers, bioceramics, and composites. Scalable fabrication technologies that enable unparalleled control over construct architecture, such as 3D printing, can then be employed to process these biomaterials into suitable forms for bone tissue engineering. In this dissertation, an overview of materials design considerations for bone tissue engineering applications is provided. Additionally, demonstrated approaches for addressing the requirements of 3D printing with growth factors, including direct inclusion of growth factors with the biomaterial during printing or intermediary encapsulation of growth factors in delivery vehicles such as microparticles or nanoparticles, are described. Specifically, the 3D printing temperature is correlated to the bioactivity of osteogenic growth factor released from polymeric constructs. The effects of particulate delivery vehicle loading on 3D printing accuracy and scaffold degradation are also characterized. Subsequently, scaffolds composed of different ratios of β-tricalcium phosphate to hydroxyapatite were characterized for calcium and phosphate ion release under aqueous conditions, and for autologous bone-forming capacity in vivo in a rat bone augmentation model involving implantation of 3D printed bioceramic scaffolds against the calvarial periosteum. Finally, unmet needs and current challenges in the development of ideal materials for bone tissue regeneration are discussed, and emerging strategies in the field of bone tissue engineering are highlighted.
Adeno-Associated Virus-Based Gene Delivery Approaches for Tissue Engineering
(2018-06-04) Lee, Esther Joy; Mikos, Antonios G
Tissue engineering constitutes non-trivial processes that can be promoted through combinations of cells, materials and bioactive factors. The formation of tissues and organs demands spatiotemporal precision that cannot, however, be achieved through sole reliance on microenvironment-based strategies. Gene delivery therefore offers a valuable avenue, whereby direct modifications can be made to dynamic systems such as cell populations. The work presented in this thesis explores several strategies for improving control with the intricacies of tissue engineering applications in mind and additionally leverages elements from synthetic biology and synthetic virology. Optogenetic tools based on light-sensitive proteins offer an exquisite option for fine-tuning expression levels with minimal interference and at low cost. Adeno-associated virus (AAV) is employed as the candidate gene delivery vehicle due to its ability to infect many cell types and good safety profile in humans, which bode well for clinical applications. The body of work is grouped into three parts. First, the design and construction of a consolidated red/far red light activatable gene regulatory circuit was pursued with the intent to tune levels of bone morphogenetic protein-2 (BMP-2) expression. The assembly of a large construct had its share of obstacles that eventually led to a downsized strategy, and a number of lessons were accrued from the duration of this endeavor. Second, an all-in-one AAV platform based activatable by blue light was developed and characterized in a series of light induction experiments. Finally, reverse transduction of three AAV serotypes ranging in degree of infectivity in HeLa cells was explored to determine if this strategy could lead to improved transduction efficiency. Parallel comparisons of standard and reverse infection approaches revealed that transduction efficiency could be improved for less amenable serotypes with the latter protocol. We then investigated whether increased transduction efficiency could be explained by higher cellular uptake of AAV. However, internalization assay results could not provide a full explanation for this phenomenon.
Advances in 3D bioprinting for regenerative medicine applications
(Oxford University Press, 2024) Loukelis, Konstantinos; Koutsomarkos, Nikos; Mikos, Antonios G; Chatzinikolaidou, Maria
Biofabrication techniques allow for the construction of biocompatible and biofunctional structures composed from biomaterials, cells and biomolecules. Bioprinting is an emerging 3D printing method which utilizes biomaterial-based mixtures with cells and other biological constituents into printable suspensions known as bioinks. Coupled with automated design protocols and based on different modes for droplet deposition, 3D bioprinters are able to fabricate hydrogel-based objects with specific architecture and geometrical properties, providing the necessary environment that promotes cell growth and directs cell differentiation towards application-related lineages. For the preparation of such bioinks, various water-soluble biomaterials have been employed, including natural and synthetic biopolymers, and inorganic materials. Bioprinted constructs are considered to be one of the most promising avenues in regenerative medicine due to their native organ biomimicry. For a successful application, the bioprinted constructs should meet particular criteria such as optimal biological response, mechanical properties similar to the target tissue, high levels of reproducibility and printing fidelity, but also increased upscaling capability. In this review, we highlight the most recent advances in bioprinting, focusing on the regeneration of various tissues including bone, cartilage, cardiovascular, neural, skin and other organs such as liver, kidney, pancreas and lungs. We discuss the rapidly developing co-culture bioprinting systems used to resemble the complexity of tissues and organs and the crosstalk between various cell populations towards regeneration. Moreover, we report on the basic physical principles governing 3D bioprinting, and the ideal bioink properties based on the biomaterials’ regenerative potential. We examine and critically discuss the present status of 3D bioprinting regarding its applicability and current limitations that need to be overcome to establish it at the forefront of artificial organ production and transplantation.
Biodegradable Hydrogel Composites for Growth Factor and Stem Cell Delivery in Osteochondral Tissue Engineering
(2016-02-24) Lu, Steven; Mikos, Antonios G
Cartilage has a limited endogenous ability for self-repair and current clinical treatments for damaged or diseased cartilage tissue are insufficient. Additionally, there is a biological and mechanical interplay between cartilage and the underlying subchondral bone, linking the pathogenesis/regeneration of both tissues. Thus, this thesis seeks to develop hydrogel composites as growth factor and cell delivery vehicles to study the regeneration of osteochondral tissue. First, we investigated the release of growth factors from acellular hydrogel composites containing gelatin microparticles (GMPs) to stimulate the repair of cartilage tissue in an in vivo osteochondral defect model. Transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) were delivered from the chondral layer of bilayered hydrogel composites while the subchondral layer remained growth factor-free. Results demonstrated that dual delivery of TGF-β3 and IGF-1 did not synergistically enhance cartilage repair, regardless of release kinetics, and the delivery of IGF-1 alone positively stimulated osteochondral tissue repair. Subsequently, we focused on improving the repair of the subchondral bone. The second part of this thesis investigated the delivery of IGF-1 and bone morphogenetic protein-2 (BMP-2) from the chondral and subchondral layers, respectively, of bilayered scaffolds in vivo. Results showed that BMP-2 enhanced subchondral bone repair, and that while the dual delivery of both growth factors did not improve cartilage repair, they synergistically enhanced subchondral bone formation over the delivery of IGF-1 alone. Using the results from this study, we also investigated relationships between specific cartilage and bone repair metrics to provide a fuller understanding of the osteochondral repair process. Correlation analysis revealed an intrinsic association between the degree of subchondral bone formation and cartilage surface regularity. Lastly, the third part of this thesis investigated the hydrogel composites as stem cell delivery vehicles. Degradable GMPs were used as temporary adherent substrates for anchorage-dependent mesenchymal stem cells (MSCs). MSCs were seeded onto GMPs and subsequently encapsulated in hydrogels to investigate their role on influencing MSC differentiation and aggregation. Non-seeded MSCs co-encapsulated with GMPs in the hydrogels were used as a control for comparison. Results revealed that MSC-seeded GMPs exhibited more cell-cell contacts, greater chondrogenic potential, and a down-regulation of osteogenic markers compared to the controls. Overall, these hydrogel composites demonstrate potential as growth factor and cell delivery vehicles for the stimulation and study of osteochondral tissue regeneration.
Carbon-Based Nanomaterials and their Medical Applications
(2015-02-06) Samuel, Errol Loïc Graeme; Tour, James M.; Billups, Wilbur E; Mikos, Antonios G
The Tour lab has previously demonstrated that antibody-targeted drug-loaded carbon nanoparticles, called PEGylated hydrophilic carbon clusters (PEG-HCCs), can be utilized for cancer-specific drug delivery both in vitro and in vivo. In this work, we append a range of receptor-binding peptides to PEG-HCCs, and show that these peptidyl-PEG-HCCs have enhanced utility in killing a number of different cancers in vitro and in vivo. Moreover, we can potentiate cancer-specific toxicity by using peptide-targeted PEG-HCCs to deliver xenobiotic drug pump inhibitors to cancer cells simultaneously with chemotherapy. With the plethora of drug delivery vehicles currently under study, we have diverted our attention to capitalizing on the intrinsic properties of our PEG-HCCs; most notably, their antioxidant activity. PEG-HCCs show high capacity to annihilate reactive oxygen species (ROS) such as superoxide and hydroxyl radicals, show no reactivity toward nitric oxide, and can be functionalized with targeting moieties without loss of activity. PEG-HCCs therefore offer an exciting new area of study for treatment of numerous ROS-induced human pathologies. Furthermore, a new class of carbon particles developed in the Tour lab, graphene quantum dots derived from coal, possess the same properties and are even more promising, given their inexpensive starting material and simple preparation. In many neurodegenerative diseases, inflammation is associated with an increase in ROS. We have found that PEG-HCCs are immunomodulatory, reducing inflammation by suppression of the effector memory T cell response. Further, PEG-HCCs can be modified in order to visualize them both in vitro and in vivo using magnetic resonance imaging (MRI). Our data, along with previous work, demonstrates potential T cell tracking utility of our nanoparticles and their ability to modulate inflammation. Finally, a different nanostructured material, graphene nanoscaffolds prepared form graphene oxide, was explored as a bioscaffold for neuronal regeneration after spinal cord injury (SCI). The graphene nanoscaffolds adhered well to the spinal cord tissue and there was no area of pseudocyst around the scaffolds suggestive of cytotoxicity. Instead, histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells around the graphene nanoscaffolds. Thus, it may provide a scaffold for the ingrowth of regenerating axons after SCI.
Customizable Bone Constructs and Tunable Scaffolds for Craniofacial Tissue Engineering
(2020-07-28) Watson, Emma; Mikos, Antonios G
The repair of large craniofacial defects remains a challenge, and repair with the clinical gold standard of a fibular flap can lead to donor site morbidity. In cases of tumor removal, growth factors may be contraindicated, and in trauma an infection may prevent immediate definitive reconstruction. A two-stage approach to craniofacial tissue engineering involves the implantation of a space maintainer within the defect site to optimize the site for receiving a customized bone graft grown elsewhere within the body adjacent to periosteum. The space maintainers can be loaded with antibiotics to clear infections and fabricated from novel polymers with varied mechanical properties to create a robust tissue pocket for transfer. In this dissertation, we sought to develop tissue engineering and biomaterials-based strategies for the reconstruction of large, complicated craniofacial defects. In the first specific aim, the two-stage strategy for mandibular repair is further challenged with two complex ovine models. We tested the ability of an antibiotic-loaded space maintainer to treat a mandibular infection, while simultaneously determining the effects of an untreated mandibular infection on the bone growth within the bioreactors. We showed that the antibiotic-loaded space maintainer was capable of clearing or preventing a Staphyloccus aureus mandibular infection, and that an untreated mandibular infection led to increased bone growth of more mature bone within the bioreactor chambers. We also showed that autograft-filled bioreactors led to increased new bone formation with more robust mechanical properties than commercially available cancellous bone chips. In another sheep model, we increased our defect size to the entire height of the mandible, creating a defect that was more exposed to mechanical forces than previously tested. Using fixation considered standard for a human with a similar defect, several animals experienced dehiscence and hardware failure. Radiographic analysis of the bioreactor tissue from implantation to transfer to integration within the mandible showed remodeling over time, but the tissue did not reach the same radiographic values as the unoperated contralateral side. Taken together, these two studies demonstrated that a space maintainer and bioreactor two-stage strategy is promising if fixation of the mandible is adequate to prevent hardware failure, micromotion, and mucosal dehiscence. For the second aim, we worked to commercialize our porous space maintainer through submissions to and interactions with the Food and Drug Administration (FDA). Our device was to proceed along the 510(k) pathway for a significant risk device, requiring an early feasibility study (EFS) and investigational device exemption (IDE) approval. To acquire the appropriate approvals, we developed protocols and created specimens for a battery of biocompatibility testing, proving that our device was cytocompatible, non-mutagenic, non-sensitizing, non-irritating, and non-toxic. We further worked with clinical collaborators to draft a clinical protocol for approval from the FDA to begin our EFS. Finally, the third specific aim involved the investigation of a class of novel polymers, synthesized from potentially antimicrobial monomers. These polymers were created from differing lengths of diols reacted with diacids to create polymers with tunable mechanical properties. We performed a main effects analysis on molecular weight, thermal characteristics, and mechanical properties to determine how they were impacted by the feed ratios of the polymers. We further expanded the tested ratios of the diacids and demonstrated that the mechanical properties could be varied predictably. However, the cytotoxicity also varied across groups, with high succinic acid incorporation leading to high levels of cell death. Although this platform was tunable, the effects of mechanical properties of the substrate and release of cytotoxic compounds on cell fate would be difficult to dissect. The overall goal of this thesis was to expand on the two-stage strategy of utilizing a space maintainer and bioreactor to repair craniofacial defects and to develop and investigate the applicability of a novel polymer system as a tunable tissue engineering platform. We demonstrated that antibiotic-loaded space maintainers are efficacious at clearing mandibular infection and that the two-stage approach to bone tissue engineering is promising even in larger, load-bearing defects. We worked to commercialize this technology and to create a polymer system with tunable mechanical properties. The translation of tissue engineering strategies has great potential to assist in patient treatment.
Development and Characterization of Modular Biomaterial Platforms for Cartilage Tissue Engineering Applications
(2022-06-17) Pearce, Hannah Abigail; Mikos, Antonios G
Biomaterials are a powerful tool in tissue engineering. Biomaterials impart cues to the body, can be encapsulated with cells, promote cellular infiltration, and direct differentiation. Beneath these functions of biomaterials are the physicochemical properties that drive much of these phenomena. This thesis outlines the systematic characterization and demonstration of how material physicochemical properties dictate biomaterial behavior and ultimately determine the success of achieving tissue remodeling in a cartilage explant model. Firstly, a thiolated gelatin microparticle (GMP) platform was developed and characterized. For this work it was hypothesized that by thiolating gelatin before microparticle formation, a versatile platform would be created that preserves the cytocompatibility of gelatin, while enabling post-fabrication modification. The thiolated GMPs were demonstrated to be a biocompatible platform for mesenchymal stem cell attachment. Additionally, the thiolated particles were able to be covalently modified with a peptide, demonstrating their promise as a platform for drug delivery applications. Focusing next on an injectable carrier, a poly(N-isopropylacrylamide) (p(NiPAAm)) based hydrogel was developed to determine how covalent peptide conjugation influences hydrogel behavior in vitro. Using a modular poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) crosslinker, peptides were synthesized and covalently clicked to PdBT via azide/alkyne click chemistry. The peptides did not significantly impact the mass sol fraction while significantly increasing the equilibrium swelling of the hydrogels. This hydrophilicity of the network was demonstrated to be the most important factor in dictating hydrogel behavior over time in vitro. Focusing next on how these complex physiochemical properties can influence cell behavior in a biologically relevant ex vivo system, a study was designed in which the charge and thermogelation behavior of p(NiPAAm)-based hydrogels was investigated. A positively, neutrally, or negatively charged peptide was conjugated to the PdBT crosslinker and cellular infiltration and tissue integration were assessed in the explant. Negatively charged hydrogels whose thermogelation behavior changed over time were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation. This thesis demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future tissue engineering strategies.
Development of a High-Throughput 3D Tumor Model for Bone Sarcomas
(2016-10-21) Santoro, Marco; Mikos, Antonios G; Ludwig, Joseph A
Preclinical drug testing commonly relies on the use of two-dimensional (2D) cultures, which allow for rapid drug screening in vitro. However, 2D cultures are unable to capture the complexity of the native three-dimensional (3D) tumor microenvironment, resulting in a lack of correspondence between preclinical data and clinical trial outcomes. The establishment of high-throughput 3D models able to describe distinctive aspects of the tumor niche would advance our understanding of tumor biology and would allow for drug testing in a physiologically relevant setup. Along this rationale, this thesis focuses on the development of a high-throughput 3D tumor model of bone sarcoma based on tissue-engineered polymeric scaffolds in combination with a flow perfusion bioreactor. First, we investigated the effects of flow perfusion on a 3D culture of Ewing sarcoma (ES) cells. We found that increasing levels of flow-derived shear stress promoted the secretion of insulin-like growth factor-1 (IGF-1) which, in turn, resulted in shear stress-dependent cell sensitivity to the IGF-1 receptor (IGF-1R) blockade, a central player in ES progression. We then leveraged these findings to culture ES cells on 3D-printed scaffolds under flow perfusion conditions. By designing 3D scaffolds with a defined porosity gradient, ES cells were exposed to a shear stress gradient that resulted in a gradient in cell response. In this way we sought to model variable levels of shear stress present within ES tumors due to intratumoral heterogeneity. In the final part of this thesis we investigated how the simultaneous presence of mesenchymal stem cells (MSCs) and flow perfusion affected drug sensitivity and phenotype of ES cells. We showed that the presence of MSCs within the coculture induces a progressive inhibition of cell growth and resistance to the IGF-1R blockade, highlighting the role of mechanically-sensitive mesodermal stroma on ES drug resistance. Overall, in this thesis we present a tissue-engineered tumor model that reliably mimics key features of the bone microenvironment, specifically the effects of biomechanical stimulation and of tumor-stroma interactions. The model hereby developed is amenable to further mechanistic studies on tumor biology and allows for a more accurate high-throughput screening of novel drug candidates.
Development of Extracellular Matrix-Based Biomaterials for Musculoskeletal Tissue Engineering
(2023-08-08) Hogan, Katie JoAnna; Mikos, Antonios G
Extracellular matrix (ECM)-based materials, which provide tissue-specific biochemical cues for cell recruitment, proliferation, and differentiation, have been the subject of significant research for cartilage, bone, and muscle tissue engineering. The use of advanced fabrication techniques such as 3D-printing (3DP) and electrospinning enable the fabrication of scaffolds with macro- and microarchitecture that further aids in these applications. In the initial aims of this thesis, decellularized cartilage ECM (cdECM) and demineralized bone matrix (DBM) were adapted into composite colloidal 3DP inks for the fabrication of 3DP constructs with tunable tissue-specific ECM content and photocrosslinking for cartilage and bone regeneration. First, photo-reactive cdECM was combined with photo-reactive gelatin nanoparticles (GNPs) in composite hydrogel-colloidal composite inks. Increased GNP content increased cdECM-GNP ink printability, and increased photocrosslinking was found to decrease cdECM-GNP scaffold swelling and degradation rates and increase biomolecule retention, demonstrating control over scaffold physicochemical properties. Next, photo-reactive DBM nanoparticles (DBM-NPs) were synthesized and combined with photo-reactive GNPs to create colloidal composite 3DP inks and scaffolds. The addition of DBM-NPs into composite colloidal inks did not impact ink printability, and photocrosslinking was demonstrated to decrease scaffold swelling and degradation kinetics, showing the tunability of these properties. An in vitro assessment of mesenchymal stem cell osteogenesis showed the osteoconductivity of DBM-NP-incorporating 3DP constructs. In the final component of this thesis, electrospun aligned decellularized skeletal muscle ECM (mdECM) microfiber meshes with variable crosslinking densities were implanted in an in vivo rat model of volumetric muscle loss, and increased crosslinking was associated with increased expression of markers for angiogenesis and myogenesis. This difference was thought to be related to more prolonged release of biochemical cues over time, emphasizing the importance of crosslinking in controlling presentation of mechanical and biochemical cues. Together, these studies present a variety of strategies for adapting ECM-based materials for high-throughput, precise fabrication methods suitable for the tissues of interest. The completion of this thesis and development of these techniques has resulted in fabrication platforms for ECM-derived biomaterial scaffolds with crosslinking systems for tunable physicochemical properties and biomolecule presentation which have broad applications across the field of tissue engineering.
Development of Injectable, Dual Thermally and Chemically Gelling Hydrogels for Craniofacial Bone Tissue Engineering
(2016-04-07) Vo, Tiffany N; Mikos, Antonios G
The objective of this work was to design a novel injectable hydrogel system capable of delivering osteoprogenitor cell populations for the minimally invasive regeneration of craniofacial bone. To this end, injectable and biodegradable hydrogels comprising thermogelling macromers and diamine-functionalized crosslinkers were developed that undergo dual thermogelation at physiological temperature and concomitant chemical crosslinking. The thermogelling macromers and crosslinker were each successfully synthesized and their physicochemical properties such as swelling behavior, mechanical properties, and degradation as a function of polymer content, crosslinking density, crosslinker length, and degree of hydrogel hydrophilicity were established. Each of these factors was found to have no significant effects on hydrogel cytocompatibility when tested with cells in vitro, except in the highest concentrations in a solution osmolality-dependent manner. Biocompatibility of the acellular hydrogels was established in a critical size rat cranial defect through analysis of the tissue response and fibrous capsule. The hydrogels also demonstrated an ability to undergo a hydrophobicity-dependent mineralization and partial bony bridging in vivo despite the absence of cells, bioactive factors, or initial mineral content. To evaluate the hydrogel system as a cell delivery vehicle, hydrogel composites were created through incorporation of gelatin microparticles and rat mesenchymal stem cells were encapsulated for a period of 28 days. Cell viability and mineralization were enhanced, whereas markers of osteogenic differentiation were modulated with gelatin microparticle loading. Altering the cell encapsulation density and osteogenic predifferentiation resulted in only in short-term effects on in vitro osteogenesis, suggesting optimization of cells is required. Investigation of the stem cell-laden composite hydrogels in vivo demonstrated significant mineralization, bony bridging, and most promising, direct bone-implant contact and tissue infiltration that are not commonly observed with hydrogels in this orthotopic model. The results suggest that these injectable, dual-gelling hydrogels show great potential for stem cell delivery in craniofacial bone tissue engineering applications.
Development of Novel Bioinks for Studying Multi-Material Architecture within Osteochondral Tissue Engineering
(2022-08-09) Bedell, Matthew Linden; Mikos, Antonios G
Symptomatic osteoarthritis and other forms of cartilage disease are a burden for millions of adults worldwide. Due to the low vascularity and the intrinsic lack of the ability to regenerate, articular cartilage tissue is a challenge to palliate once damaged, and surgical approaches fall short of creating long-term solutions for cartilage repair. Also, the ability of implanted cartilage or biomaterials to laterally integrate into the diseased cartilage site as well as the role of the subchondral bone region are still not fully understood. Tissue engineering, which combines materials with cells and bioactive factors to address questions within bioengineering and medicine, has been investigated for the study of osteochondral defects. The most recent studies of osteochondral regeneration have introduced 3D printing techniques, which can increase the degree of control over a printed implant’s cellular and architectural complexity. Among these novel approaches is the bioprinting of continuous composition gradients, which mimic osteochondral physiology and have not been investigated for their mechanical effects nor their biological implications on osteochondral tissue integration. The study of these emergent properties arising from the interactions facilitated by the continuous interface and transition between two phenotypically-distinct, cell-encapsulating bioink phases is the fundamental question of this thesis. While selected previous works have examined similar questions in vitro with injectable or castable cell culture environments, the adaptation of those promising hydrogels into printable bioinks is still a challenge preventing them from being studied for further questions about more biomimetic architectural complexity and 3D structure – questions that a reliably printable system can address. But despite decades of work in these specific sub-fields of biomaterial 3D printing and bioprinting, standardized methods for how to evaluate the printability and subsequent biological effects of bioinks are still being studied. The body of work described herein meets the needs of the greater bioprinting and tissue engineering communities by both studying novel bioinks to answer new questions within osteochondral tissue engineering and by documenting the methods, assays, and standards by which the inks were translated from mere hydrogels into a robust set of bioinks that encapsulate living cells. Specifically, we put forward a high-throughput method that evaluates hydrogel polymer systems as candidates for the base material of future bioinks with a given cell type based on viability, proliferation, and immunostaining in 3D culture, using less than 1 million cells and 1 mL per ink to observe and compare biological outcomes. Then, we studied biological additives within a gelatin-based ink that induced bone and cartilage extracellular matrix (ECM) production in encapsulated stem cells, and we also compiled a diverse array of methods to study printability additives, resulting in several novel osteochondral bioinks for extrusion, inkjet, and digital light processing 3D bioprinting. Select bioinks were then used to answer questions about architecture within an osteochondral explant model, and here, we observed that architecture did indeed make a difference in the cells’ ECM deposition and integration into the natural tissue. Altogether, this body of work demonstrates how important 3D factors are when considering tissue-engineered constructs for regenerative medicine, and it also chronicles a vast array of protocols and methods to serve as a reference for broader scientific forums interested in studying those interactions in a 3D environment.
Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering
(Oxford University Press, 2023) Hogan, Katie J; Öztatlı, Hayriye; Perez, Marissa R; Si, Sophia; Umurhan, Reyhan; Jui, Elysa; Wang, Ziwen; Jiang, Emily Y; Han, Sa R; Diba, Mani; Jane Grande-Allen, K; Garipcan, Bora; Mikos, Antonios G
Demineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles (DBM-NPs, ∼400 nm) were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks (100:0, 95:5 and 75:25 GNP-MA:DBM-NP-MA) did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an ∼40% greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline (PBS). Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, ∼45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.
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Dissecting the Effects of Targeted Radiation on the Bone Microenvironment
(2024-04-17) Barrios, Sergio; Mikos, Antonios G; Dondossola, Eleonora
Radium-223 (223Ra) is a bone-targeting, alpha particle-emitting radionuclide approved for the treatment of patients with metastatic prostate cancer that is currently being tested in a variety of clinical trials for a diverse set of bone-related disease. 223Ra has been shown to accumulate on mineralized bone tissue due to its calcium-mimetic properties and is highly enriched in areas with high bone turnover like the growth plates of long bones. Recent clinical studies have shown a significant fracture rate increase associated with the use of 223Ra, predominantly in tumor-free bones (68% of fractures). However, the biological mechanisms underlying this bone fragility are still unclear and generate a great concern in the clinical setting. Therefore, preclinical studies addressing the role of 223Ra-mediated modulation of the bone stromal components and the consequences on bone mechanical properties are much needed. In this thesis, we combined mechanical testing, micro-CT, and conventional endpoint analysis with fluorescent reporter mice for ex vivo 3D spatial biology microscopy analysis to clarify the effects of 223Ra on bone stromal components including osteoblasts, osteoclasts, adipocytes, and blood vessels. This approach assumes relevance when studying the response to 223Ra, which is spatially confined within 100 μm of the bone interface, due to the short penetration of alpha particles. Additionally, a novel tool for bone tissue image analysis was developed and validated. Overall, this thesis advances our understanding in bone biology and the unexplored impact of alpha-particle radiation on the bone microenvironment.
Engineering Three Dimensional In Vitro Models of Bone Tumors for Drug Testing and Mechanistic Studies
(2015-04-15) Fong, Li Shan Eliza; Mikos, Antonios G; Farach-Carson, Mary C; Navone, Nora M; Miller, Jordan S; Kasper , Fred K
Development of anti-cancer therapeutics has been traditionally reliant on two-dimensional (2D) systems and animal models, both of which have major limitations that contribute to the poor clinical translation of preclinical findings. The goal of this thesis work was to develop three-dimensional (3D) in vitro models of bone malignancies for accurate drug testing and mechanistic studies. To this end, I investigated the use of different 3D scaffolds to recreate the distinct in vivo bone niches relevant for these bone cancers in vitro. First, I evaluated the use of electrospun poly(ε-caprolactone) scaffolds to provide 3D architectural cues for the culture of Ewing sarcoma (EWS) cells. 3D-cultured EWS cells were remarkably different from the same cells cultured in 2D, and more similar to those grown in vivo with respect to morphology, growth kinetics, and protein expression. This work underscored the importance of providing a 3D context for tumor growth in vitro. The second part of this thesis investigated the use of 3D hyaluronan (HA) hydrogels to support the culture of bone metastatic prostate cancer (PCa) cells. Due to their high fidelity to the tumor of origin, there is an emerging interest in the use of patient-derived xenograft (PDX) models to overcome the limitations of cancer cell lines. However, existing PDX culture systems are few and limited. Hence, I sought to develop an in vitro PCa PDX model by first establishing a method to enrich for PCa PDX tumor cells, then evaluated the ability of 3D hyaluronan (HA) hydrogels to maintain the viability, morphology, growth and phenotype of the encapsulated tumor cells. This work demonstrated the feasibility of using a 3D scaffold-based approach to culture PDX tumor cells in vitro. Lastly, I incorporated integrin-binding and matrix metalloproteinase-degradable peptides to HA hydrogels to support osteoblast culture with PCa PDX cells in 3D. Through this 3D co-culture system, the in vivo structural organization, phenotype, as well as biochemical crosstalk between PCa and osteoblasts in bone was recapitulated. In this work, I demonstrate for the first time, the feasibility of co-culturing PDX tumor cells with stromal cells in vitro using a tunable 3D system for controlled mechanistic investigations.
Injectable Biomimetic Hydrogel Platforms for Cartilage Regeneration
(2021-03-04) Kim, Yu Seon; Mikos, Antonios G
Regeneration of articular cartilage has been a clinical challenge as the tissue lacks the ability to regenerate itself due to its low cell density and avascularity. Surgical methods that are currently being implemented for cartilage repair frequently result in the formation of mechanically inferior fibrous cartilage, leaving a large room for improvement in terms of their clinical outcomes. The field of tissue engineering and regenerative medicine has therefore been exploring innovative solutions for delivering cells and bioactive factors that will, in conjunction, guide the regeneration of healthy cartilage. In this thesis, we sought to develop an injectable therapeutic platform that mimics the cartilage microenvironment and could be used as a cell/biomolecule delivery system for cartilage regeneration applications. To this effect, an injectable hydrogel was developed which consisted of a poly(N-isopropylacrylamide)-based thermogelling macromer (TGM) and chondroitin sulfate (CS)-based network-forming macromers. In this design, the two CS macromers – modified with adipic acid dihydrazide and N-hydroxysuccinimide, respectively – covalently interacted with each other to form a CS network, with which the TGM could further crosslink and form an injectable hydrogel system. The addition of CS, an anionic polysaccharide, greatly increased the degree of swelling of the hydrogel. The molar content of CS affected the rate of degradation, compressive modulus, as well as cytotoxicity of the hydrogel where hydrogels with higher CS content degraded faster, demonstrated higher compressive strength, but were also more cytotoxic. To establish this system into a cell and biomolecule delivery vehicle, we further reinforced the three-component hydrogel system described in the first study with poly(amidoamine), a crosslinker developed specifically for crosslinking TGM. The resulting TGM-CS dual-network hydrogel was used to investigate the effect of poly(L-lysine) (PLL), a novel bioactive factor shown to affect the skeletal development pathway, on the chondrogenesis of cocultures of mesenchymal stem cells (MSCs) and chondrocytes. PLL did not affect the swelling nor degradation properties of the hydrogel and demonstrated good retention within the bulk hydrogel over 28 days in vitro. In addition, PLL with different molecular weights and concentrations did not affect the viability of encapsulated MSCs. Further studies with different coculture ratios of MSCs and chondrocytes revealed that, while PLL seemed to affect the expression of chondrogenic and hypertrophic genes during early stages of the in vitro culture, the long-term chondrogenesis was mostly governed by the fraction of chondrocytes in cocultures. Histological analysis revealed that the study groups with higher fraction of chondrocytes showed denser secretion of cartilage-like matrix. In the last part of the thesis, we developed an ex vivo study using cartilage explants isolated from articular cartilage of pigs to further investigate the effect of the presence of PLL and chondrocytes on the mechanical properties of the hydrogel. By injecting the hydrogel within the defects generated in the center of the cartilage explants, we were able to analyze both the hydrogel surface stiffness and the degree of integration with the surrounding cartilage.
Injectable, Click Functionalized Hydrogels for Osteochondral Tissue Engineering
(2020-12-03) Guo, Jason L; Mikos, Antonios G
Articular cartilage injury affects millions around the world and results in hundreds of thousands of procedures being performed in the US alone. Tissue engineering has emerged as a promising strategy for the generation of functional cartilage tissue. Since the repair of articular cartilage and its function depends on the regeneration of multiple tissues within the osteochondral unit – including cartilage and underlying subchondral bone – osteochondral tissue engineering (OTE) is of great interest to scientists and clinicians. The use of injectable hydrogels and three-dimensionally (3D) printed scaffolds represent two approaches in which biomaterial constructs – often encapsulated with cells – are delivered directly to the site of defect, where they will support tissue regeneration. However, these hydrogels and scaffolds, which are often composed of synthetic polymers, are typically bioinert and thus produce a suboptimal biological response from encapsulated and native cells. The conjugation or delivery of tissue-specific biomolecules for cartilage and bone is thus a critical prerequisite for induction of osteochondral tissue regeneration. This doctoral thesis addresses the need for biologically relevant constructs by developing a modular, click functionalized hydrogel system that utilizes tissue-specific biomolecular cues for bone or cartilage regeneration (Figure 1). To achieve this goal, we developed a novel polymeric crosslinker, poly(glycolic acid)–poly(ethylene glycol)–poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) that can click conjugate biomolecules of diverse size and chemical character via mild, aqueous alkyne-azide cycloaddition. PdBT can be functionalized with bone- and cartilage-specific biomolecules and then used as a crosslinker for the thermoresponsive polymer poly(N-isopropylacrylamide-co-glycidyl methacrylate) (P(NIPAAM-co-GMA)), generating rapidly crosslinked, cytocompatible, and highly swollen hydrogels. We characterized the bioactivity of bone- and cartilage-specific hydrogels in vitro, evaluating their capacity to promote the chondrogenesis and osteogenesis of encapsulated mesenchymal stem cells (MSCs). Additionally, we fabricated bilayered hydrogels with bone- and cartilage-specific components for in vivo implantation in a rabbit model. To further explore the utility of our click conjugation scheme, we applied this chemistry to the tissue-specific functionalization of 3D printable polyesters, an emerging area of interest within the field of tissue engineering. Our first objective was to synthesize PdBT and click conjugate it with osteogenic biomolecules – such as osteogenic bone marrow homing peptide 1 (BMHP1) and glycine-histidine-lysine peptide (GHK) – and chondrogenic biomolecules – such as chondroitin sulfate (CS) and N-cadherin peptide (NC). Injectable hydrogels were developed and characterized by mixing biofunctionalized PdBT crosslinkers and P(NIPAAM-co-GMA). Our second objective was to encapsulate MSCs within these hydrogels and characterize the effects of biomolecule identity and concentration on chondrogenesis and osteogenesis in vitro. Our third objective was to implant bilayered hydrogels for the repair of bone and cartilage in a rabbit femoral condyle defect model to study in vivo osteochondral tissue repair. Our last object was to apply this click bioconjugation scheme to a new application in the biological functionalization of 3D printable synthetic polyesters. Ultimately, the bioconjugation toolset developed in this thesis provides a platform for the tissue-specific functionalization of synthetic polymers for tissue engineering applications.
Leveraging Biomaterials for the Treatment of Large Infected Tissue Defects
(2017-04-25) Tatara, Alexander M; Mikos, Antonios G
While the body has an incredible ability to heal, host and external factors may overwhelm its innate regenerative capacity. In these instances, a tissue defect may occur. Tissue defects (regions of either necrotic tissue or void space) are highly susceptible to microbial invasion. Given their proximity to the native micro- and mycobiome, craniofacial and cutaneous defects are at particularly high risk for chronic contamination and infection. The combination of tissue loss and infection creates a negative feedback loop: 1) lack of vascularized healthy tissue leads to a locally immunocompromised area; 2) pathogens are able to colonize the tissue defect and eventually invade the margins of healthy tissue; 3) the resulting pathogenic attack and inflammatory response results in tissue injury and necrosis at the defect border; and 4) the tissue defect expands. Two possible mechanisms to break this cycle are to restore vascularized tissue to the defect site or clear the infection to restore the body’s ability to regenerate tissue. In this dissertation, we seek to develop technologies to treat large tissue defects susceptible to infection through biomaterials-based strategies. First, we investigated and optimized the in vivo bioreactor platform for generating autologous free tissue flaps for mandibular reconstruction. As the engineered tissues grown in these bioreactors are vascularized, their use in mandibular repair restores circulation to the defect site. In a large animal model of disease, we demonstrated that these bioreactors did not require harvested donor tissue, exogenous stem cells, or growth factors in order to generate bony flaps suitable for reconstruction. When transferred to a large mandibular defect in a physiologically-relevant ovine model, these engineered tissues were capable of integrating with the native host tissue for functional craniofacial repair. In the second aim of this work, space maintenance was explored to facilitate the repair of large tissue defects by stimulating the growth of a healthy soft tissue envelope around the defect space as well as functioning as a depot for local delivery of antimicrobial agents to prevent and/or treat infection of the vulnerable large tissue defect. Porous space maintainers were fabricated per good manufacturing practice, subjected to electron beam irradiation, and evaluated for suitability of subsequent mechanical properties. These porous space maintainer devices were then implanted in a superior marginal defect adjacent to the oral mucosa in the mandibular diastema of an ovine model of disease. After nine weeks, space maintainers were removed and the defect was reconstructed with tissue-engineered vascularized flaps generated in 3D-printed bioreactors. Even in a challenging defect environment with proximity to the oral flora and under mechanical load, the space maintainers were able to prevent collapse of tissue into the defect site and maintain a healthy soft tissue envelope for repair. Infection was associated with the single failed case during the use of this strategy. Given the high risk of infection in the setting of large craniofacial defects, econazole-eluting porous space maintainers were developed for the local delivery of an antimicrobial therapeutic during space maintenance. Compared to traditional solid space maintainers, porous space maintainers were able to better inhibit the in vitro growth of common fungal and bacterial pathogens and may be of value in the treatment of tissue defects with infection. Finally, the last aim of this thesis specifically examined novel antifungal approaches for the treatment of infected tissue defects. As there is currently a dearth of animal models of fungal infection relevant to tissue engineering, a murine model with a large cutaneous defect infected with Aspergillus fumigatus was established. A new class of diol-based aliphatic polyesters was synthesized and characterized as polymers whose degradation products have inherent antifungal properties. These diol-based polymers were then fabricated into microparticles and loaded with traditional antifungal therapeutics. After demonstrating extended release of therapeutics in vitro, the microparticles were used to locally treat fungal infection in a large cutaneous defect in an immunocompromised murine model of disease. In sum, this body of work explores biomaterials-based approaches to treat large infected defects, through restoration of tissue and/or by mitigating infection. From leveraging the body’s own innate healing capacity to create vascularized tissue suitable for defect reconstruction (in vivo bioreactors), to utilizing traditional biomaterials in innovative ways (antifungal-eluting bone cement-based space maintainers), to developing new biomaterials with specific antimicrobial applications in mind, we have created a number of strategies to aid in the regeneration of large tissue defects.
Tailored Release of Bioactive Factors from Composite Multidomain Peptide Hydrogels
(2016-03-28) Wickremasinghe, Navindee Charya; Hartgerink, Jeffrey D; Marti, Angel A; Mikos, Antonios G
Multidomain peptides (MDP) self-assemble to form nanofibrous scaffolds well suited to tissue engineering and regeneration strategies. MDPs can present bioactive cues that promote vital biological responses. Orthogonal self-assembly of MDP and growth factor-loaded liposomes generate supramolecular composite hydrogels. This thesis demonstrates the ability to create a unique hydrogel, developed by stepwise self-assembly of multidomain peptide fibers and liposomes, and presents its potential for in vivo applications. Chapter One of the thesis presents an introduction to the above work with background spanning from the role of self-assembling peptides and hydrogels in tissue engineering, to current strategies for therapeutic angiogenesis and wound healing. Chapter Two addresses the design and characterization of a composite hydrogel containing MDP and liposomes. Results showed that structural and mechanical integrity of the peptide nanofibers, lipid vesicles and the composite gel are retained. The two-component gel allows for controlled release of bioactive factors at multiple time points and indicates bimodal release of two growth factors from the same system. These MDP-Liposome Composites (MLCs) were injected in vivo for targeted, localized delivery of growth factors, and Chapter Three details how they functioned in vivo. Placental growth factor-1 (PlGF-1) was shown to temporally stimulate VEGF-receptor activation in vitro in endothelial cells, and robust vessel formation in vivo. MLCs provide a novel method for the time controlled delivery of growth factors from within highly biocompatible and injectable hydrogels. Time controlled release guided by MLCs induces an unprecedented level of growth factor-mediated neovascular maturity. Use of cytokine-loaded MDP hydrogels to accelerate diabetic wound healing is another in vivo application explored in Chapter Four of this thesis. Delivery of a pro-healing cytokine IL-4 via MDP hydrogels have resulted in enhanced healing of full-thickness dermal wounds on the backs of genetically diabetic mice. Compared to controls, wounds treated with IL-4-MDP composite gels showed higher wound closure, M2 macrophage polarization, re-epithelialization, granulation tissue formation and angiogenesis. The conclusion chapter, Chapter Five, discusses how the above in vivo success of composite MDP hydrogels speaks to their potential to function as a unique protein delivery platform for tissue regeneration.
Tissue-Engineered Microenvironments to Model Mechanical Cues and Tumor-Associated Macrophages in Osteosarcoma
(2022-10-07) Chim, Letitia Kai-Ling; Mikos, Antonios G
Current in vitro models employed to study cancer biology at the preclinical phase rely on hard, flat surfaces that lack the complex array of physical and biochemical signaling cues provided by the architecture, mechanical properties, and heterotypic cell interactions within the tumor microenvironment. As such, drug candidates that appear promising during in vitro testing frequently fail in preclinical animal testing or worse, in clinical trials with patients. Osteosarcoma is the most common primary tumor of the bone and is characterized by a high degree of inter- and intra-tumor heterogeneity. This heterogeneity and the absence of physiologically relevant preclinical models has meant that developing new treatments for osteosarcoma has been especially slow. In this thesis, techniques from tissue engineering—using combinations of cells, scaffolds, and biological factors to create tissue constructs—were employed to model the osteosarcoma tumor microenvironment. First, mechanically tunable scaffolds were developed by coaxially electrospinning poly(ε-caprolactone)/gelatin core-shell fibers and were used to investigate the effects of three-dimensional architecture and substrate stiffness on osteosarcoma cell phenotype and response to treatment. Nuclear localization of YAP and TAZ increased as the substrate stiffness decreased, and the three-dimensional environment in part downregulated the IGF-1R/mTOR signaling cascade and altered the efficacy of combination therapy with doxorubicin and agents targeting IGF-1R/mTOR compared to monolayer controls. Then, having demonstrated that the architecture and mechanical properties alter the phenotype of osteosarcoma cells, tumor-associated macrophages, the most prevalent infiltrating immune cells in the tumor microenvironment, were introduced to the engineered model. Co-culturing osteosarcoma cells with macrophages within the scaffolds for 24 hours resulted in the microenvironment becoming highly inflamed, as indicated by elevated levels of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6, and the effect was most pronounced upon moderately stiff scaffolds. Increased levels of TNFα and IL-6 correlated with resistance to doxorubicin treatment, and inhibition of STAT3 diminished inflammation-related resistance but did not improve the efficacy of doxorubicin. This work highlights the advantages that tunable and physiologically relevant models can bring to the study of cancer biology and the development of novel therapeutics.
Tissue-Engineered Tumor Microenvironments for Bone Sarcoma
(2019-07-30) Molina, Eric Rodolfo; Mikos, Antonios G
Preclinical methods interrogation of cancer biology and of evaluation of potential therapeutics rely heavily on the use of monolayer, adherent culture on tissue culture treated polystyrene dishes. However, these techniques ignore characteristics of the tumor microenvironment that have been found responsible for essential elements of pathogenesis in tumors. While a variety of in vitro tumor models exist and mouse models with patient derived xenograft (PDX) tissue remain the gold standard in preclinical testing, there is more investigation needed into how various physical and biochemical elements of the microenvironment contribute to cancer pathogenesis. Osteosarcoma (OS) and Ewing’s sarcoma (ES) are the two most common primary tumor of bone. Although advancements in combination therapy and surgical resection have improved outcomes, identification and targeting of essential pathogenic signaling has been difficult as evidenced by myriad failed clinical trials and the absence of biologically targeted therapeutics for these diseases. In this thesis, we employ the techniques of tissue engineering to develop bone tumor microenvironments suitable for the interrogation of elements in sarcoma tumor niche. We sought to determine how these elements contribute to changes in cell phenotype, proliferation, activation of critical pathogenic pathways, and response to therapy. The first objective of this thesis was to engineer a 3D electrospun poly(ε-caprolactone) (PCL) microenvironment to determine the effects of bone-like extracellular matrix (ECM) and mineral components on ES cells. We achieved this by culturing mesenchymal stem cells in osteogenic medium on electrospun scaffolds PCL scaffolds and decellularizing the scaffold after 12 days to generate PCL-ECM constructs. In our first specific aim we show that 3D microenvironments contribute to decreased proliferation and the downregulation of the insulin-like growth factor 1(IGF-1R)/mechanistic target of rapamycin signaling (mTOR) cascade compared to monolayer controls. Further we determine that cells in 3D environments also became resistant to combination therapy with doxorubicin and IGF-1R/mTOR targeted therapy. While the ECM and mineral components in 3D scaffolds increased growth and recapitulated some morphological aspects characteristic of ES tumors in patients, there did not appear to be any difference in IGF-1R/mTOR activation or therapeutic response between ES cultured in either electrospun PCL scaffolds or PCL-ECM constructs. Our second objective was to engineer a mechanically tunable 3D electrospun environment that could be used to interrogate the effects of architecture and stiffness on osteosarcoma. In our second aim, we develop a system of 3D mechanically tunable scaffolds utilizing coaxial electrospinning of PCL:gelatin (core:shell) fibers. We determine by quantitative confocal image analysis that as stiffness is decreased in 3D microenvironments, OS cells increase the nuclear to cytoplasmic ratio (N:C ratio) of the mechanoresponsive proteins, yes-associated protein 1 (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). We further determine that 3D microenvironments contribute to a downregulation of the IGF-1R/mTOR cascade and resistance to combination therapy with chemotherapy and IGF-1R/mTOR targeted therapy compared to monolayer controls in OS. Our third objective was to correlate YAP N:C ratio, TAZ N:C ratio, nuclear IGF-1R, and nuclear phosphorylated IGF-1R (pIGF-1R) in tumor sections from 37 osteosarcoma patients with clinical aspects of disease, histological characterization of tumors, and overall survival. We determined that strong correlations exist between YAP and TAZ N:C ratio and IGF-1R nuclear staining intensity and pIGF-1R staining intensity. Interestingly we also found that pIGF-1R nuclear staining strongly correlated with YAP and TAZ N:C ratio. We determine that nuclear pIGF-1R and YAP N:C ratio were higher in tumors with a chondroblastic histotype and that Nuclear pIGF-1R, YAP N:C ratio, and TAZ N:C ratio were lower in high grade bone osteosarcoma subtypes compared to all other subtypes. Univariate and multivariate analysis of outcomes indicated that high pIGF-1R and possibly low YAP N:C ratio may be negative prognostic indicators for overall survival. The overall goal of this work was to examine how the microenvironment can be engineered to modulate and study essential pathogenic signaling and the generation of therapy resistant phenotypes in sarcoma. Through this thesis, we have demonstrated that architectural elements and the stiffness of tumor environments contribute to phenotypic changes, herald increased therapy resistance in vitro, and affect the expression and localization of potentially prognostic indicators in sarcoma. We highlight the importance of incorporating controllable microenvironmental elements in in vitro cancer biology which can be engineered to study myriad aspects of the tumor microenvironment and recapitulate more accurately the phenotypes of cells found in tumors from patients.

Browsing by Author "Mikos, Antonios G"

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