Dynamic nanostructured scaffolds as advanced biomaterials

Growing replacement tissues and organs in the laboratory will revolutionise healthcare; however, the maturation of cells into functional tissue constructs requires the controlled presentation of biochemical factors within a mechanically suitable scaffold. In nature, the presentation of such signals is provided through factors and structures existent within the nanoarchitecture of the extracellular matrix (ECM); therefore, in tissue engineering there is significant need to develop dynamic advanced artificial tissue constructs capable of mimicking the complexities of the native ECM. The requirement for bioactive, innervated constructs that contain biologically relevant signals delivered through tuneable mechanisms has yet to be achieved. One approach to address this key-challenge is offered through bioprinting, which allows for the controlled spatial distribution of bioinks containing cells, structures and signals within a single printed construct. However, currently bioprinting applications are severely limited by bioink function - with the majority of bioinks either lacking sufficient mechanical properties or biochemical signalling. Therefore, there is a key need to develop bioinks which adequately mimic the native ECM on a nanostructured, chemical level - particularly in establishing effective control over cell fate and tissue innervation. Tissue composition and extracellular signalling varies substantially between tissue-types, and therefore, advanced approaches that allow for ease of mechanical and biological tuneability through modular mechanisms would provide a practical avenue for bioink development. Self-assembling peptides (SAPs) are a unique class of biomaterials capable of spontaneously forming simple biomimetic structures which entangle to form highly hydrated, bioactive networks with favourable conditions for cell maturation. These biomaterials are easily tuned through modification of amino acid sequence, enabling tailored control over biochemical signalling between cells and scaffold. This provides the ability to artificially replicate natural signalling in a controlled manner - bringing about desired cell behaviour. Using these peptides, a variety of synergistic ECM-protein analogues have been developed, including Fmoc-FRGDF containing fibronectin's attachment motif RGD, and Fmoc-DIKAV, containing laminin's attachment motif IKVAV. Fmoc-SAPs possess the ability to be further functionalised through macromolecule addition, allowing for the presentation of charged, developmentally or structurally-important macromolecules on the surface of peptide fibrils. These macromolecules can integrate with the peptide networks, facilitating additional signalling and allowing for mechanical tunability. Here, we take advantage of these properties to develop an advanced and dynamic bioink for bioprinting applications. Initially, material enhancement is investigated through development of multi-sequence scaffolds. Specifically, Fmoc-FRGDF is combined with a synergistic cell attachment motif PHSRN, either through sequence engineering (Fmoc-FRGSFPHSRN) or through control over assembly properties (Fmoc-FRGDF/Fmoc-PHSRN coassembly). Here, the coassembled (Fmoc-FRGDF/Fmoc-PHSRN) system forms a synergistic network which promotes the attachment, proliferation and migration of muscle cells in vitro. The potential of Fmoc-SAP multi-sequence scaffolds is further investigated through the development of an artificial tumour microenvironment for cancer-cell studies. Here, Fmoc-FRGDF is combined with Fmoc-DIKVAV and used as a spheroid (LLC, NOR-10, LLC + NOR-10) micro-environment. The coassembled Fmoc-FRGDF/Fmoc-DIKVAV microenvironment enhances cancer-cell growth and progression compared to 2D cultures, non-encapsulate spheroids, and spheroids encapsulated in agarose. Agarose was selected as a control owing to the similar physical properties yet lack of biofunctionalisation. Results from this study reinforce the potential of Fmoc-SAPs as advanced microenvironments, and further support the ease of biological functionalisation inherent with this material. Further scaffold functionalisation is investigated through macromolecule addition. Here, one of two macromolecules are coassembled into a Fmoc-FRGDF network. The first macromolecule is fucoidan, a seaweed-derived polysaccharide with known anti-inflammatory properties, while the second is versican, a developmentally important proteoglycan which plays a variety of roles in muscle development. Versican was selected owing to its charge similarity to fucoidan, yet vastly different biological function. Fucoidan addition was found to increase fibre bundling and alter hydrogel mechanical properties, while versican addition had no substantial effect on hydrogel mechanics when compared to an Fmoc-FRGDF empty-vector control. Cell morphology was substantially altered by macromolecule addition, with fucoidan samples resulting in smaller, rounder cells with fewer multinucleated syncytia compared to an Fmoc-FRGDF control, while versican hydrogels showed an initial decrease in cell-size and multinucleation after 24h and a comparable cell-size and multinucleation following 72h. Here, it is possible that macromolecule addition perturbs cells attachment, and therefore, macromolecule selection is a key consideration. Interestingly, the regain of cell morphological characteristics in versican-containing hydrogels following 72h indicates the ability of cells to break-down versican, while the maintenance of small, round cells in the fucoidan hydrogels shows an inability for cells to break down fucoidan. The ability of Fmoc-SAPs to form components in bioinks is investigated through assembly with gelatin methacryloyl (GelMA) macromolecules. Initially, GelMA nanostructure and mechanical properties are investigated in response to increased degree of methacrylation or increased control. Here, structure-function relationships are drawn, and 18% methacryloyl Gelma (LM-GelMA) is selected for further bioink development owing to favourable thermoresponsive viscoelastic properties and improved strain tolerance. LM-GelMA assembly with coassembled Fmoc-FRGDF/Fmoc-PHSRN is investigated as a potential avenue to develop biologically and mechanically tuneable hydrogels. The incorporation of Fmoc-SAPs allows for control over sequence selection, while control over mechanical properties is offered through GelMA inclusion. LM-GelMA/Fmoc-FRGDF/Fmoc-PHSRN (FPG-Hybrid) bioinks demonstrate enhanced printability and are shown to support primary myoblast differentiation. The potential of Fmoc-SAP/GelMA bioinks to act as a modular bioink toolkit is further investigated through Fmoc-FRGDF/Fmoc-PHSRN substitution with Fmoc-DIKVAV, to develop a neural-suitable bioink (DIKVAV-Hybrid). This DIKVAV-Hybrid bioink demonstrated unique mechanical morphological properties and is shown to support rat cortical neurosphere viability. Throughout this project, the networks have been vigorously characterised through various analytical techniques, including micro/nanoimaging (Transmission electron microscopy, Atomic force microscopy, Cryo-scanning electron microscopy), Small-angle X-ray scattering, Small-angle neutron scattering, rheology, and spectroscopy; while the overall effectiveness of these systems have been analysed through in vitro muscle and neural cultures. Work detailed through this thesis aims to vigorously characterise Fmoc-SAP hydrogels and bioinks, providing the foundations for further biological studies and material optimisation

Biomaterials‐Based Approaches to Tumor Spheroid and Organoid Modeling

Evolving understanding of structural and biological complexity of tumors has stimulated development of physiologically relevant tumor models for cancer research and drug discovery. A major motivation for developing new tumor models is to recreate the 3D environment of tumors and context‐mediated functional regulation of cancer cells. Such models overcome many limitations of standard monolayer cancer cell cultures. Under defined culture conditions, cancer cells self‐assemble into 3D constructs known as spheroids. Additionally, cancer cells may recapitulate steps in embryonic development to self‐organize into 3D cultures known as organoids. Importantly, spheroids and organoids reproduce morphology and biologic properties of tumors, providing valuable new tools for research, drug discovery, and precision medicine in cancer. This Progress Report discusses uses of both natural and synthetic biomaterials to culture cancer cells as spheroids or organoids, specifically highlighting studies that demonstrate how these models recapitulate key properties of native tumors. The report concludes with the perspectives on the utility of these models and areas of need for future developments to more closely mimic pathologic events in tumors.State‐of‐the‐art approaches using natural, synthetic, and composite biomaterials for 3D tumor modeling are presented in this Progress Report. Furthermore, it is discussed how these models uniquely reproduce key properties of native tumors to facilitate basic and applied cancer research and cancer drug discovery efforts.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/1/adhm201700980.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/2/adhm201700980-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/3/adhm201700980_am.pd

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Engineering a Tissue Mimic for Predictive Nanoparticle Assessment

Bio-scientific research has relied heavily on models of cell monolayers cultured on plastic. For most cell types, this does not represent their in vivo tissue organisation well. As a result they behave differently in vitro from in vivo, leading to poorly predictive data. Plastic compression (PC) of collagen is used to engineer constructs with more tissue-like conditions. The aim of this study was to test the feasibility of using these constructs as a three-dimensional tissue model for assessing the fate of hyaluronan nanoparticles (HA-NP). Collagen hydrogels were seeded with cells and HA-NP and subjected to PC. Due to their small size, HA-NP retention following PC was investigated. HA-NP uptake by cells was then compared to conventional monolayer cell cultures. 19.1±1.2% of the initial HA-NP load was retained following PC, which could be increased to 31.1±3.1% by multi-layering. This entrapment was found to be largely physical as HA-NPs were released from the construct following cellular remodeling, but not without it. Cells in monolayer reached their maximum HA-NP uptake in 3 days whilst cells in collagen peaked at 7 days. This maximum uptake was 60.1 a.u., twice as large as that of 3D-cultured cells (32.8 a.u). A novel method was developed to analyse local collagen densities which revealed particular collagen distributions in micro-patterned constructs depending on the shape of template used; round grooves had a 21.4±4% increase in collagen density at their bases, whilst rectangular grooves displayed two peaks corresponding to their internal corners, which were 15.2±4% and 16.9±3% denser than the unpatterned regions. This work has enabled greater understanding of the PC and micro-moudling which will aid in creating more complex tissue constructs in a predictable and controlled way. The importance of 3D tissue organisation in in vitro models, particularly for nanoparticle testing, has also been demonstrated in this work

From Protein Building Blocks to Functional Materials

Proteins are the fundamental building blocks for high performance materials in nature. Such materials fulfil structural roles, as in the case of silk and collagen, and can generate active structures including the cytoskeleton. Attention is increasingly turning to this versatile class of molecules for the synthesis of next generation green functional materials for a range of applications. Protein nanofibrils are a fundamental supramolecular unit from which many macroscopic protein materials are formed. In this review, we focus on the multiscale assembly of such protein nanofibrils formed from naturally occurring proteins into new supramolecular architectures and discuss how they can form the basis of material systems ranging from bulk gels, films, fibers, micro/nanogels, condensates and active materials. We review current and emerging approaches to process and assemble these building blocks in a manner which is different to their natural evolutionarily selected role, but allows the generation of tailored functionality, with a focus on microfluidic approaches. We finally discuss opportunities and challenges for this class of materials, including applications that can be involved in this material system which consists of fully natural, biocompatible and biodegradable feedstocks yet has the potential to generate materials with performance and versatility rivalling that of the best synthetic polymers

Bioprinting: Uncovering the utility layer-by-layer

Bioprinting is becoming a must have capability in tissue engineering research. Key to the growth of the field is the inherent flexibility, which can be used to answer basic scientific questions that can only be addressed under 3D culture conditions, or organ-on-chip systems that could quickly replace underperforming animal models. Almost certainly the most challenging application of bioprinting will be for bottom-up tissue construction, which faces many of the same challenges as scaffold-based tissue engineering. In this review, the current state-of-the-art approaches to 3D bioprinting are discussed in terms of performance and suitability. This is complemented by an overview of hydrogel-based bioinks, with a special emphasis on composite biomaterial systems. </jats:p

Engineering Functional Capillary Networks.

A major translational challenge in the fields of therapeutic angiogenesis and tissue engineering is the ability to form functional networks of blood vessels. Cell-based strategies to promote neovascularization have led to the consensus that co-delivery of endothelial cells (ECs) with a supporting stromal cell type is the most effective approach. However, the choice of stromal cells has varied across studies, and their impact on the functional qualities of the capillaries produced has not been examined. Our lab has developed methods to form interconnected networks of pericyte-invested capillaries both in vitro in a 3D cell culture model and in vivo. However, if the engineered vessels contain ECs that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this thesis was to determine if stromal cells of different origins yield capillaries with different functional properties, in complementary in vitro and in vivo models. In vitro, a fluorescent dextran tracer was used to visualize and quantify transport across the endothelium. In EC-fibroblast co-cultures, the dextran tracer penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, tracer accumulated at the vessel periphery, with very little transported across the endothelium. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), tighter control of permeability was achieved. In vivo, all conditions yielded new vessels that inosculated with mouse dorsal vasculature and perfused the implant, but there were significant functional differences, depending on the identity of the co-delivered stromal cells. EC alone and EC-fibroblast implants yielded immature capillary beds characterized by high levels of erythrocyte pooling in the surrounding matrix, while EC-MSC and EC-AdSC implants produced more mature capillaries characterized by less extravascular leakage and expression of mature pericyte markers. Injection of dextran tracer into the circulation also showed that EC-MSC and EC-AdSC implants formed vasculature with more tightly regulated permeability. These results suggest that the identity of the stromal cells is key to controlling the functional properties of engineered capillary networks.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91412/1/graingst_1.pd

Contribution of the ELRs to the development of advanced in vitro models

Developing in vitro models that accurately mimic the microenvironment of biological structures or processes holds substantial promise for gaining insights into specific biological functions. In the field of tissue engineering and regenerative medicine, in vitro models able to capture the precise structural, topographical, and functional complexity of living tissues, prove to be valuable tools for comprehending disease mechanisms, assessing drug responses, and serving as alternatives or complements to animal testing. The choice of the right biomaterial and fabrication technique for the development of these in vitro models plays an important role in their functionality. In this sense, elastin-like recombinamers (ELRs) have emerged as an important tool for the fabrication of in vitro models overcoming the challenges encountered in natural and synthetic materials due to their intrinsic properties, such as phase transition behavior, tunable biological properties, viscoelasticity, and easy processability. In this review article, we will delve into the use of ELRs for molecular models of intrinsically disordered proteins (IDPs), as well as for the development of in vitro 3D models for regenerative medicine. The easy processability of the ELRs and their rational design has allowed their use for the development of spheroids and organoids, or bioinks for 3D bioprinting. Thus, incorporating ELRs into the toolkit of biomaterials used for the fabrication of in vitro models, represents a transformative step forward in improving the accuracy, efficiency, and functionality of these models, and opening up a wide range of possibilities in combination with advanced biofabrication techniques that remains to be explored

Tissue engineered micro and macrovasculature utilizing stromal vascular fraction.

This dissertation describes the use of stromal vascular fraction to tissue engineer 3D microvasculature and macrovasculature. Stromal vascular fraction is an easily isolatable cell source from adipose tissue depots. It has demonstrated remarkable potential both in vitro and in vivo for forming microcirculation capable of perfusion upon implantation. SVF is clinically utilized as a therapeutic cell source for anti-inflammation for osteoarthritis and is being studied for ischemic tissue application to stimulate revascularization. The work described herein is divided within four chapters. Chapter I provides an introductory overview and lists the aims and hypothesis for the dissertation. Chapter II describes experiments towards elucidating specific aim 1: determine the mechanism by which SVF forms neovascular networks in 3D fibrin gels in vitro. This was accomplished through a multitude of experiments describing SVF undergoing vasculogenesis and angiogenesis in a 2D automated in vitro assay, and the ability to inhibit these processes via NOTCH and PDGF-B/PDGFR-b interruption. These mechanisms, as well as integrin dependent mechanisms, were analyzed within 3D fibrin and 3D collagen I culture systems as well. It is believed that the activation of the fibrin specific integrin aVb3 plays a role in hyper-stimulating fibrin-embedded endothelial cells in a VEGF dependent manner. Chapter II describes experiments towards understanding specific aim 2: create deliverable tissue units of SVF-derived microvasculature or macrovasculature utilizing bioprinting, and electrospinning technologies. This was accomplished through bioprinting spheroids containing cells embedded in collagen I or fibrin using superhydrophobic surface technology or electrospinning varying porosities of PCL and pressure sodding SVF cells into the material. It is possible to automate and create dosable units of microvascular tissue in spheroid format using SVF cells, ECM such as fibrin or collagen I, and bioprinting technologies. Additionally, it is possible to create blood vessel mimics of multiple porosities in order to retain and allow cellular infiltration within the biomaterial. Chapter IV is an overall summary and conclusion of the dissertation. These studies could hopefully generate more knowledge on the creation of tissue engineered microvasculature and microvasculature for use in treating ischemic cardiomyopathies

Recapitulating the tumor ecosystem along the metastatic cascade using 3D culture models

Advances in cancer research have shown that a tumor can be likened to a foreign species that disrupts delicately balanced ecological interactions, compromising the survival of normal tissue ecosystems. In efforts to mitigate tumor expansion and metastasis, experimental approaches from ecology are becoming more frequently and successfully applied by researchers from diverse disciplines to reverse engineer and re-engineer biological systems in order to normalize the tumor ecosystem. We present a review on the use of 3D biomimetic platforms to recapitulate biotic and abiotic components of the tumor ecosystem, in efforts to delineate the underlying mechanisms that drive evolution of tumor heterogeneity, tumor dissemination, and acquisition of drug resistance.ope