Fischell Department of Bioengineering

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Additive Manufacturing for Recapitulating Biology in vitro and Establishing Cellular & Molecular Communication
(2023) Chen, Chen-Yu; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Recapitulating biological systems within laboratory devices, particularly those with analytical instrumentation, has enhanced our ability to understand biology. Especially useful are systems that provide data at the length and time scales characteristic of the assembled biological systems. In this dissertation, we have employed two advanced technologies — additive manufacturing and electrobiofabrication to create systems that both recapitulate biology and provide ready access to molecular data. First, we utilized two-photon direct laser writing (DLW) and digital light processing (DLP) 3D printing to reconstruct morphologies of human gut villi. Our constructs enable small molecule diffusion through pores and enable epithelial cell growth and differentiation, as in the gastrointestinal (GI) tract. We also developed a cell/particle alignment methodology that applies a vacuum on the underside of a device to rapidly facilitate attachment to 3D printed scaffolds. These simple demonstrations of additive manufacturing show how one can better tailor geometric features of organ-on-a-chip and other in vitro models. We then added electrobiofabrication as a means create functionalized surfaces that rapidly assemble biological components, noted for their labile nature, onto devices with just an applied voltage. In one example, we show how a thiolated polyethylene glycol (PEG) can be electroassembled as a sensor interface that includes antibody binding proteins for both titer and glycan analysis. Rapid assessment of titer and glycan structure is important for biopharmaceuticals development and manufacture. While the interface and sensing methodology was performed using standard laboratory instrumentation, we show that the methodology can be streamlined and operated in parallel by incorporating into a microfluidic sensor platform. Additionally, we show how the combination of optical and electrochemical (redox) based measurements can be combined in a simplified insert that “fits” nearly any microplate reader or other fairly standardized laboratory spectrophotometric unit. We believe that by adapting transformative electrochemical analytical methods so they can augment more traditional optical techniques, we might ultimately generate devices that provide a far more comprehensive picture of the target, promoting better investigation. Specifically, we show how three important biological and chemical systems can be interrogated using both optical measurements and electrochemistry: the oxidation state of proteins including monoclonal antibodies, redox status of hydrogel materials, and electrobiofabrication and electrogenetic induction. Lastly, we demonstrate how electrobiofabrication can be used to create designer communities of bacteria — artificial biofilms — the study of which is important for understanding phenomena from infectious disease to food contamination. That is, we discovered that by varying the applied voltage, surface area, and composition of the to-be-assembled hydrogel solution, we can precisely control the intercellular environment among bacterial populations. In sum, this dissertation integrates advances in assembly, through additive manufacturing, electrobiofabrication, with advances in electrochemical analysis to bring to the fore an electronic understanding of complex biological phenomena. We believe that the capability of translating biological information into a processible digital language opens tremendous opportunities for advancing our understanding of nature’s amazing systems, potentially enabling electronic means to control her subsystems.
DEVELOPMENT OF A HYBRID 3D PRINTING STRATEGY FOR NIPPLE RECONSTRUCTION
(2020) Van Belleghem, Sarah Miho; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Breast cancer and its most radical treatment, the mastectomy, significantly impose both physical transformations and emotional pain in thousands of women across the globe. Although reconstructive surgery is viewed as a possible recovery route for a lost symmetry and gender identity, it provides these patients with a breast mound whose most notable feature is scarring from the initial invasive procedure. Restoring the appearance of a nipple-areola complex directly on the breast represents an important psychological healing experience for these women and remains an unresolved clinical challenge, as current restorative techniques using Skin Flap Suturing (SFS) renders a flattened disfigured skin tab within a single year and requires subsequent surgeries. A tissue-engineered scaffold designed to integrate with the breast skin can not only aid in the development of a more robust and aesthetically pleasing nipple but can also aid in minimizing the patients’ prominent mastectomy scars. As 3D printing has become a popular and advantageous way to produce scaffolds with complex, patient-specific structures, this technology holds great promise for the fabrication of custom shaped nipple-areola grafts per any breast size. The work presented here is aimed at the development of a hybrid scaffold, composed of complementary biodegradable and synthetic hydrogels, that fosters the regeneration of a viable dermal layer in the form of a nipple-areola complex. The first aim of this research defined a dynamic dual bioink 3D printing strategy to produce soft tissue grafts that allow for enhanced host integration and volume retention. A new shape analysis technique utilizing CloudCompare software was also demonstrated to expand our available toolbox for assessing scaffold aesthetic properties. We then extended both modular printing and shape assessment techniques to the fabrication of a nipple-areola scaffold in the second aim, where both structural and bioactive components of the design were further adjusted. Lastly, the third aim explored the immune and vascular responses to these hybrid materials in a rigorous evaluation of an in vivo rat subcutaneous implantation study. Envisioned as subdermal implants, these nipple-areola bioprinted scaffolds have the potential to reduce subsequent surgical intervention by creating a lasting nipple-areola structure that harmoniously coexists with the patient’s breast skin. The proposed system can be applied to current breast reconstruction practices post patient healing of silicone implantation.
DEVELOPMENT OF AN ENDOTHELIAL CELL/MESENCHYMAL STEM CELL COCULTURE STRATEGY FOR THE VASCULARIZATION OF ENGINEERED BONE TISSUE.
(2019) Piard, Charlotte Marianne; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In the past two decades, remarkable progress has been made in the development of surgical techniques for bone reconstruction, significantly improving clinical outcomes. However, major reconstruction after trauma or cancer is still limited by the paucity of autologous material and donor site morbidity. Recent advances in the field of tissue engineering have generated new approaches for restoring bone defects. In spite of this progress, the necessity of suitable blood supply to ensure cell function is a major challenge in the development of more complex and functional grafts. Many investigators have successfully demonstrated the use of different strategies including growth factor delivery and in vitro coculture of ECs and MSCs to develop vascular structures. MSCs have the ability to secrete a wide range of bioactive cytokines and growth factors that can influence nearby cells via paracrine signaling. This crosstalk between ECs and MSCs is mutually beneficial, as ECs enhance osteogenic differentiation of hMSCs through direct cell-cell contact and paracrine signaling. In the native environment of cortical bone, both cell populations, osteogenic and vasculogenic, follow a unique well-defined pattern, called osteons. The goal of this proposed study was to develop a novel bio-inspired and vascularized bone construct, harvesting the synergistic effects of pro-angiogenic growth factor delivery and coculture of ECs and MSCs. To address this goal, we first developed mesoporous calcium deficient hydroxyapatite apatite microparticles, with biological properties closer to bone than commercially available hydroxyapatite, and capable of efficiently loading and sustainably releasing pro-angiogenic growth factors. We then demonstrated the successful fabrication of a novel bio-inspired 3DP fibrin-PCL composite scaffold, with mechanical strength comparable to bone. The utilization of these scaffolds in constructing osteons for bone regeneration demonstrated the promising capacity of the construct to improve neovascularization. In light of these results, we hypothesized that cell placement or patterning could play a critical role in neovascularization. Which lead us to investigate the role of distance between cell populations, introduced via 3D printing, in ECs/MSCs crosstalk. Our results suggested that controlling the distance between ECs and MSCs in coculture, using 3D printing, could influence angiogenesis.
Dual-Chambered Membrane Bioreactor for the Dynamic Co-Culture of Dermal Stratified Tissues
(2019) Navarro Rueda, Javier; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Every year over 11 million patients suffer severe burns worldwide. Facial burn statistics include victims of violence (warfare, acid attacks, scalding) and trauma (flame, electrical, chemical). Skin is the first barrier against external mechanical and biochemical factors, such as burning agents, and is composed of the epidermis, dermis, and hypodermis layers. When burned, skin cannot regulate temperature or fluid transport, or stop bacterial infection. Due to the importance of the skin barrier, natural healing and grafting treatments aim to quickly close the wounds with fast proliferation of fibroblasts and collagen deposition, a process that results in scarring, loss of function, and disfigurement. Tissue engineering has produced epidermis-dermis skin scaffolds for clinical use and in vitro dermal models. Throughout this work we studied 3D printing and bioreactor strategies for the simultaneous physiologic and topographic reconstruction of burnt facial skin tissues. First, we formulated a keratin-based bioink that can be used for 3D printing on a lithography-based 3D printer. Second, we implemented the keratin bioink in the production of Halofuginone-laden face masks for the improvement of contracture, scarring, and aesthetics in severe skin wound healing in an animal model. Due to lack of collagen organization and microstructural development, we introduced a novel dual-chambered (DCB) bioreactor system to study stratified tissues. For this, crosslinking density of the keratin-based hydrogels was used to fine tune the transport properties of membranes for potential use in guided tissue regeneration applications. Then, we assessed the viability of our novel DCB for co-culturing adjacent cell populations with the inclusion of a regulatory keratin membrane. Last, having studied the DCB with flat interfaces, we assessed its viability for perfusing curved interfaces. The integration of both curvature and cell populations allowed to assess the synergistic development of adjacent dermis fibroblasts and hypodermis stem-cell-derived adipocytes and evaluate whether including topography parameters would alter cell viability in the DCB. The strategies developed here elucidate on tissue stratification and aesthetic reconstruction. Furthermore, the keratin-based bioink, the engineered membranes, and the DCBs can be extended to study other stratified or gradient tissues and to fine-tune communication between cell populations in complex 3D constructs.
The Effect of Architecture and Shear Stress on Endothelialization of 3D Printed Vascular Networks
(2016) Talaie, Tara; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Despite significant progress in the field of tissue engineering within the last decade, a number of unsolved problems still remain. One of the most relevant issues is the lack of proper vascularization that limits the size of engineered tissues to smaller than clinically relevant dimensions. In particular, the growth of engineered tissue in vitro within bioreactors is plagued with this challenge. Specifically, the tubular perfusion system bioreactor has been used for large scale bone constructs; however these engineered constructs lack inherent vasculature and quickly develop a hypoxic core, where no nutrient exchange can occur, thus leading to cell death. Through the use of 3D printed vascular templates in conjunction with a tubular perfusion system bioreactor, we attempt to create an endothelial cell monolayer on 3D scaffolds that could potentially serve as the foundation of inherent vasculature within these engineered bone grafts.
EFFECTS OF 3D PRINTED VASCULAR NETWORKS ON HUMAN MESENCHYMAL STEM CELL VIABILITY IN LARGE BONE TISSUE CONSTRUCTS
(2015) Ball, Owen Matthew; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
There is a significant clinical need for engineered bone graft substitutes that can quickly, effectively, and safely repair segmental bone defects. One emerging field of interest involves the growth of engineered bone tissue in vitro within bioreactors, the most promising of which, are perfusion bioreactors. Utilizing a tubular perfusion system bioreactor, which allows media to perfuse freely around alginate scaffolds laden with human mesenchymal stem cells, large-scale bone constructs can be created by simply aggregating these beads together in the desired shape. However, these engineered constructs lack inherent vasculature and quickly develop a necrotic core, where no nutrient exchange occurs. Through the use of 3D printed vascular structures, used in conjunction with a TPS bioreactor, cell viability after just one day of aggregation was found to increase by as much as 50 percent in the core of these constructs, with in silico modeling predicting construct viability at steady state.
Engineering Biodegradable Vascular Scaffolds for Congenital Heart Disease
(2015) Melchiorri, Anthony John; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The most common birth defects worldwide are congenital heart defects. To treat these malformations in a child’s cardiovascular system, synthetic grafts have been used as a primary intervention. However, current grafts suffer from deficiencies such as minimal biological compatibility, inability to grow and adapt, and high failure rates. Additionally, the grafts are not customized to the patient, which may lead to graft failure given that defects may vary significantly from patient to patient. The work presented here aims to adapt tissue engineering paradigms to develop customizable vascular grafts for congenital heart defects using to reduce the long-term risk and the number of surgeries experienced by patients. The first component of this research focuses on solvent-cast vascular grafts. This system of fabrication was used to explore how various strategies and graft modifications affect the graft’s performance in vivo. Grafts were fabricated with the mechanical properties necessary to withstand the stresses of a physiological environment and support neotissue formation. To improve tissue formation, the grafts were modified with bioactive molecules to improve vascular tissue growth. In addition, the grafts were combined with a tissue perfusion bioreactor. The bioreactor applied fluid flow to support cell seeding, differentiation, and growth of endothelial progenitor cells on the grafts, demonstrating a robust strategy for tissue formation prior to implantation. The second component of this research centers on the development of a biomaterial for 3D printing. 3D printing offers unparalleled customizability, as a graft can be designed based on medical images of a patient, tested via computer modeling, and then printed for implantation. A resin was developed consisting to produce grafts that were mechanically compatible with native blood vessels and maintained patency and tissue formation six months after implantation. The library of 3D printed vascular graft materials was also expanded by creating a novel copolymer resins, which varied in mechanical properties and degradation profiles. In addition, the concepts and strategies of biofunctionalization developed in the solvent-cast vascular grafts can be combined with the 3D printed graft strategies. Grafts designed, printed, and modified using these combinatorial approaches can greatly improve the long-term outcomes of treating congenital heart disease.
Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity
(Wiley, 2023-03-17) Kronstadt, Stephanie M.; Patel, Divya B.; Born, Louis J.; Levy, Daniel; Lerman, Max J.; Mahadik, Bhushan; McLoughlin, Shannon T.; Fasuyi, Arafat; Fowlkes, Lauren; Van Heyningen, Lauren Hoorens; Aranda, Amaya; Abadchi, Sanaz Nourmohammadi; Chang, Kai-Hua; Hsu, Angela Ting Wei; Bengali, Sameer; Harmon, John W.; Fisher, John P.; Jay, Steven M.
Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.
TOWARD PHANTOM DEVELOPMENT FOR MEDICAL IMAGING USING DIRECT LASER WRITING
(2020) Lamont, Andrew Carl; Sochol, Ryan D.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
An important tool for the performance analysis and standardization of medical imaging technologies is the phantom, which offers specifically defined properties that mimic the structural and optical characteristics of a tissue of interest. The development of phantoms for high-resolution (i.e., micro-scale) three-dimensional (3D) imaging modalities can be challenging, however, as few manufacturing techniques can capture the architectural complexity of biological tissues at such scales. Direct Laser Writing (DLW) is an evolving additive manufacturing technique with nano-scale precision that can fabricate micro and nanostructures with unparalleled geometric complexity. This dissertation outlines the unique microfluidic-based DLW strategies that have been developed for novel micro-scale phantom production. First, I will outline the development and characterization of an in situ DLW strategy used to adhere printed components to the surfaces of a microchannel. I will then explain how we have leveraged this strategy for a proof-of-concept retinal cone outer segment phantom that is laden with light-scattering Titanium (IV) Dioxide nanoparticles. This phantom has valuable implications for the performance analysis of the emerging ophthalmological modality, adaptive optics-optical coherence tomography (AO-OCT). Next, I will describe the development and characterization of a microfluidic-based multi-material DLW strategy to fabricate single components from multiple materials with minimal registration error between the materials. Ultimately, we intend to use this method to develop multi-material platforms and phantoms, including high-aspect-ratio multi-material retinal cone phantoms for AO-OCT. Finally, to demonstrate the applicability of this method for applications beyond AO-OCT, I present a preliminary phantom production strategy for the light microscopy-based modality, whole slide imaging (WSI). Specifically, we assess the DLW and light microscopy performance of dyed photoresists and offer a preliminary multi-material demonstration, which are pivotal first steps toward the creation of a first-generation multi-material WSI phantom. This work provides valuable insights and strategies that leverage microfluidic-based DLW techniques to fabricate novel micro-scale phantoms. It is anticipated that these strategies will have a lasting impact, not only on the production of phantoms for medical imaging modalities, but also for the fabrication of advanced microfluidic and multi-material microstructures for fields such as meta-materials, micro-optics, lab-on-a-chip, and organ-on-a-chip.
USE OF 3D PRINTED POLY(PROPYLENE FUMARATE) SCAFFOLDS FOR THE DELIVERY OF DYNAMICALLY CULTURED HUMAN MESENCHYMAL STEM CELLS AS A MODEL METHOD TO TREAT BONE DEFECTS
(2014) Wang, Martha Elizabeth Ottenberg; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
This project investigates the use of a tissue engineering approach of an absorbable polymer, poly(propylene fumarate) (PPF) to provide long term mechanical stability while delivering a bioactive material, precultured human mesenchymal stem cells (hMSC) encapsulated in hydrogel, to repair bone defects. Annually over 2.2 million bone grafting procedures are performed worldwide; however, current treatment options are limited for critically sized and load bearing bone defects. Much progress has been made in development of bone tissue replacements within the field of bone tissue engineering. The combination of a polymer scaffold seeded with cells for the eventual replacement by host tissue has shown significant promise. One such polymer is PPF, a synthetic linear polyester. PPF has been shown to be biocompatible, biodegradable and provide sufficient mechanical strength for bone tissue engineering applications. Additionally PPF is able to be photocrosslinked and therefore can be fabricated into specific geometries using advanced three-dimensional (3-D) rapid prototyping. Current technology to culture and differentiate hMSCs into osteoblasts has been enhanced with the development of the tubular perfusion system (TPS). The TPS bioreactor has been shown to enhance osteoblastic differentiation in hMSCs when encapsulated in alginate beads. Although this system is effective in differentiating hMSCs it lacks the sufficient mechanical strength for the treatment of bone defects. Therefore this work suggests a combination strategy of harnessing the ability of the TPS bioreactor to enhance osteoblastic differentiation with the mechanical properties of poly(propylene fumarate) to develop a porous PPF sleeve scaffold for the treatment of bone defects. This is accomplished through four steps. The first step investigates the cytotoxicity of the polymer PPF. Concurrently the second step focuses on designing, fabricating and characterizing PPF scaffolds. The third step investigates the degradation properties of 3D printed porous PPF scaffolds. The fourth step characterizes alginate bead size and composition for use within the PPF sleeve scaffolds. The successful completion of these aims will develop a functional biodegradable bone tissue engineering strategy that utilizes PPF fabricated scaffolds for use with the TPS bioreactor.

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