Browsing by Author "Grande-Allen, Kathryn J"

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    Development of Cell-laden Hydrogel Composites for Osteochondral Tissue Engineering
    (2015-12-14) Lam, Johnny; Mikos, Antonios G.; Grande-Allen, Kathryn J; Zygourakis, Kyriacos; Kasper, Fred K
    Articular cartilage is a flexible connective tissue that enables the frictionless and painless articulation of bones in synovial joints throughout the body. Given its avascular nature, articular cartilage tissue inherently exhibits a compromised endogenous capacity for regeneration upon damage. Defects caused by disease or trauma often lead to chronic pain and osteoarthritis as current clinical treatments are still unable to achieve long-term repair. Hence, tissue engineers are developing innovative technologies to provide strategies for successful cartilage repair and regeneration. This thesis focuses on the development and evaluation of cell-based osteochondral tissue engineering solutions based on injectable and biodegradable polymer biomaterials for bone and cartilage repair. First, we developed and characterized oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels for osteochondral tissue engineering applications. We investigated the main effects of five main hydrogel fabrication parameters (the poly(ethylene glycol) molecular weight (PEG MW), the crosslinker-to-OPF carbon-carbon double bond ratio (DBR), the crosslinker type, the crosslinking density of encapsulated gelatin microparticles, and the incubation medium composition) as well as their interaction effects on the swelling behavior and degradation of OPF hydrogel composites. We found that increasing the PEG MW increased the mean swelling ratio and decreased the mean mass remaining %, while changing the crosslinker type from methylene bisacrylamide (MB) to PEG diacrylate yielded the opposite effect. Additionally, we found that the swelling of hydrogels fabricated with higher PEG MW or with MB were more sensitive to increases in DBR. From these results, we showed that the swelling and degradation properties of OPF-based hydrogels can be precisely tuned through the modulation of these five fabrication parameters. The second part of this thesis investigated the potential of bilayered OPF hydrogel composites encapsulating chondrogenically and osteogenically pre- differentiated MSCs in a spatially controlled fashion for osteochondral tissue repair. We demonstrated that MSCs that underwent 7 days (CG7), but not 14 days (CG14), of chondrogenic pre-differentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenically pre-differentiated (OS) cells in a bilayered OPF hydrogel. We found that the respective chondrogenic and osteogenic phenotypes of encapsulated MSCs were maintained for up to 28 days in vitro without the need for external growth factors. When taken in vivo, the delivery of CG7 cells, as opposed to CG14 cells, in combination with OS cells via a bilayered OPF hydrogel composite stimulated morphologically superior cartilage repair. Indeed, the present work showed that cartilage regeneration in osteochondral defects can be enhanced by MSCs that are chondrogenically and osteogenically pre-differentiated prior to implantation. Longer chondrogenic pre-differentiation periods, however, resulted in diminished cartilage repair. The final section of this thesis investigated the use of poly(L-lysine) (PLL), previously shown to up-regulate condensation during cartilage development in vitro, as an early chondrogenic stimulant of MSCs encapsulated in OPF hydrogels. We showed that PLL incorporation resulted in early enhancements of type II collagen and aggrecan gene expression as well as increased type II/type I collagen expression ratios when compared to blank controls. We also demonstrated that PLL enhanced N-cadherin gene expression of encapsulated MSCs under certain conditions, suggesting that PLL also likely induced pre-cartilaginous condensation.
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    Directing Collective Epithelial Morphology Using a Light-Based Carving Tool
    (2022-08-11) Trubelja, Alen; Grande-Allen, Kathryn J; Harrington, Daniel A
    Head and Neck Cancer (HNC) refers to tumors that originate predominantly in the mouth, nose, and throat, accounting for more than 10,000 deaths yearly in the US. Radiotherapy is a common treatment for HNC. Patients that undergo radiotherapy (RT) oftentimes develop Xerostomia (dry mouth). This is an iatrogenic disorder with no cure, which significantly impacts quality of life. Xerostomia results from irreversible damage to the salivary glands (SG), which are complex branched organs with an unmet need for regenerative therapy. RT causes apoptosis of secretory salivary acinar cells and their progenitor source in the ducts. Tissue engineering could offer a therapeutic solution by harvesting healthy SG tissue prior to RT, expanding these cells in vitro to form 3D spheroids, then creating functional tissue for implantation post-RT. During development, salivary glands form by repeated cleft and bud formation, forming a divergent, duct-to-acini architecture difficult to recapitulate with standard gel scaffolds. Recent advances in biofabrication have enabled high-resolution control over user-defined architectures within 3D tissue constructs. In this work, we leverage a subtractive manufacturing technique known as laser-based hydrogel degradation (LBHD) to guide collective epithelial morphology and exert spatiotemporal control over cell differentiation. We demonstrate the ability to carve features at high resolution within 3D tissue constructs. This thesis demonstrates the potential to direct clusters of salivary cells to migrate through the tunnels carved into hydrogels, form open lumens, and obey branching cues to form a rudimentary gland. This work has the potential to contribute to our understanding of how to create microscale glandular tissues.
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    ELUCIDATION OF THE UNIQUE TRANSLATION OF ANGIOGENIC SIGNALING BY AORTIC VALVE CELLS
    (2015-12-04) Arevalos, Alex; Grande-Allen, Kathryn J
    Angiogenesis is a fundamental biological process but is a critical step in the progression of calcific aortic valve disease (CAVD). However, the process through which native valve cells, valve endothelial cells (VECs) and valve interstitial cells (VICs), form the neovascularization exhibited during CAVD is unclear due to their atypical translation of several angiogenesis related signals. Therefore, the in vitro angiogenic capacity of valve endothelial cells was characterized and compared to vascular derived endothelial cells. Their vasculogenic networks were demonstrated to be quantitatively and morphologically different from the networks generated by a vascular endothelial cell line, but the geometry of the VECs’ networks could be manipulated with small molecule Rho GTPase inhibitors, similar to previous studies of vascular endothelial cells, thus demonstrating typical and atypical ways in which VECs translate angiogenic signals. Next, the pericyte-like capacity of VICs was demonstrated by tracking fluorescently marked VECs and VICs in a long term in vitro angiogenesis co-culture assay. VICs regulated early VEC network organization in a ROCK-dependent manner, wrapping themselves around VEC network edges in a manner similar to a pericyte cell line. Using a novel method to quantify the Lagrangian-corrected chemoattraction of one cell type towards another in a mixed population, we identified and quantified a subpopulation of VICs that demonstrated a pericyte-like chemoattraction towards VECs. Directly comparing valve cell co-cultures to vascular cell co-cultures revealed that unlike vascular control cells, the valve cell cultures ultimately formed invasive spheroids with 3D sprouts. These 3D sprouts were found to have several markers typical of in vivo angiogenic root sprouts such as delta-like ligand 4 and β-catenin polarity. VECs co-cultured with VICs displayed significantly more invasion than VECs alone; interestingly, VICs were found leading and wrapping around VEC invasive sprouts demonstrating both tip cell and pericyte behaviors. Angiopoietin1-Tie2 signaling was found to regulate valve cell organization during VEC/VIC spheroid formation and invasion. Long term co-cultures demonstrated pronounced deviation of several angiogenesis and pericyte markers when measured with qRT-PCR. In the next study, mechanical stimulation is known to be a strong regulator of vascular endothelial cell angiogenic capacity, but its role in regulating VEC angiogenic capacity physiologically or pathologically was unknown. Therefore, experiments were performed to examine the effect of cyclic uni-axial strain on regulating the response of valve endothelial cells to an in vitro angiogenesis model. Network analysis revealed a strong pattern that strain decreases the propensity of VECs to form networks. Finally, the factors that govern VEC angiogenesis were investigated from the network scale down using tissue engineering strategies. Vascular networks of varying complexity can be designed within engineered tissues, but the amount of biological complexity necessary for proper biological functionality is unclear, as more complex is not necessarily better or necessary. Since VEC network complexity had been demonstrated to be sensitive to changes in actin regulators, this study used VECs as a framework to examine the fundamental relationship between network structure and endothelial cell biology more specifically it was tested whether the internal signaling biology of endothelial cells could be tuned based upon spatially-defined synthetic networks. Notable differences in several angiogenesis related markers were found as a function of the pattern the cells were seeded on, including several markers for actin activity regulators as well as changes in actin alignment, mimicking changes in signaling previously observed in in vitro angiogenesis models with VECs. Overall, this work has contributed to the understanding of the translation of angiogenic signals by valve cells and its potential role in the pathogenesis of valvular disease. Understanding from these studies can be applied to future studies of valve diseases with a similar framework to clarify the role of angiogenic signaling in the pathology of CAVD. This insight will allow development of targeted therapeutic strategies for the treatment of valvular diseases, as well as strategies to assess what level of complexity is sufficient to induce functional angiogenesis in tissue engineered constructs, such as those needed in pediatric aortic valve replacements.
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    Evaluation of Valvular Endothelial Cell Hemostatic Behavior in Native Valves and Novel Co-culture Models
    (2014-12-03) Balaoing, Liezl Rae; Grande-Allen, Kathryn J; Moake, Joel L; Kiang, Ching-Hwa; Qutub, Amina; Harrington, Daniel
    The endothelial cell-mediated process of hemostasis is critical in all living heart valve tissues. As these tissues undergo changes with age and disease, the ability for valvular endothelial cells (VECs) to manage anti- and pro-thrombotic mechanisms may also change. Furthermore, degeneration- and thrombosis-related failures in artificial valves emphasizes the need to understand the anti-thrombotic mechanisms of VECs in order to develop effective strategies to endothelialize implants and tissue-engineered heart valves. Therefore, a study was performed to evaluate the regulation and function of von Willebrand Factor (VWF), ADAMTS-13 (VWF cleaving enzyme), and other thrombotic and anti-thrombotic mediators secreted from VECs from different aged valves. This work identified age-related differences in VEC hemostatic protein regulation, and an increased capacity of specific proteins to aggregate within regions of elderly valves, which are known to have age-associated loss of extracellular matrix (ECM) organization that are linked to calcific aortic valve disease. With the knowledge that ECM can influence hemostasis, we then studied changes in VEC hemostatic regulation using synthetic culture conditions that modulated substrate stiffness and adhesive ligands. RKRLQVQLSIRT (RKR), a syndecan binding cell adhesive peptide derived from laminin-α1 G-domain, was optimal for promoting strong VEC adhesion and balanced hemostatic function on hydrogel constructs of various stiffness in comparison to the commonly used integrin binding peptide RGDS. Next, to evaluate interactions between valve cells, magnetic levitation technology was used to co-culture VECs with valvular interstitial cells (VICs) in a 3D scaffoldless aortic valve co-culture (AVCC). The cell-based AVCC design allowed for synthesis of multiple constructs within a few hours. AVCCs had regional localization of CD31 positive VECs at construct surface. Cells in the AVCC interior (including VECs) expressed low levels of α-smooth muscle actin (αSMA), suggesting maintenance of quiescent VIC phenotype, but potential endothelial to mesenchymal differentiation in interior-localized VECs. In addition, AVCCs produced ECM and expressed hemostatic proteins such as endothelial nitric oxide synthase (eNOS) and VWF. In light of the VEC localization within the AVCC potentially affecting healthy phenotype, a more physiologically organized and customizable scaffold model was needed for further evaluation of direct interactions between VECs and VICs. Therefore, previous RKR-functionalization work was combined with strategies for VIC encapsulation in biofunctionalized-MMP degradable hydrogels to develop a 3D adhesive ligand localized hydrogel scaffold for an endothelialized aortic valve co-culture model. The resulting hydrogel-based endothelialized aortic valve model (HEAVM) promoted the formation of a stable VEC monolayer at the scaffold surface, and supported the maintenance of VIC quiescent phenotypes within the scaffold, thereby mimicking physiological valve cell organization in aortic valves. Platelet adhesion and nitric oxide functional assays confirmed healthy VEC cell behavior, while immunohistochemistry and qRT-PCR were used to asses VIC and VEC phenotype and extracellular matrix (ECM) production. Overall, by utilizing principles from cell and extracellular matrix biology, biomechanics, and biomaterials, this work was able to improve the understanding of the VEC roles in valve homeostasis and the pathogenesis of valvular disease. Furthermore, new biomaterial-based models were designed to enhance the field’s understanding of VEC functions and communication with VICs. The knowledge learned from these models may be applied to future evaluation of various valve diseases, as well as endothelialization strategies for valve implants.
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    Interfacial Hydrogel Coatings to Improve the Biocompatibility of Bioprosthetic Valves
    (2020-05-21) Roseen, Madeleine Ava; Grande-Allen, Kathryn J
    Bioprosthetic valves (BPVs) recapitulate physiological blood flow for patients with late-stage heart valve failure, but the fixed xenograft tissues deteriorate over time. BPVs fail in roughly 15 years, requiring over 300,000 replacement surgeries in the US per year. Technologies that increase the lifespan of these valves would decrease the frequency of replacement surgeries and increase patient quality of life. Failure of BPVs is not well understood but is thought to be due to the altered properties of BPVs compared to native valves and their interactions with the body. BPVs are fixed with glutaraldehyde before implantation, which is necessary to minimize host immune response, but alters the material and mechanical properties of BPVs. To improve their longevity, BPVs need to be shielded from the degrading bodily interactions and their physiological properties restored. Towards this goal, a hydrogel coating method was optimized and applied to BPV tissue. Hydrogels are well-characterized for their biocompatibility and tunable characteristics. Herein we evaluated the use of two hydrogels – poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) diacrylamide – for their ability to bind to the surface and improve the characteristics of BPVs. Initially, the coating method was verified for efficacy on a BPV model system using only PEGDA. The model demonstrated that the coating method was successful in depositing a hydrogel layer which significantly reduced protein adhesion compared to uncoated controls, an important improvement. The coating was then transitioned to glutaraldehyde-fixed tissue to assess its efficacy on BPVs. Both hydrogels showed significant, near-continuous coating along the surface, as well as the restoration of physiologically lower protein adhesion levels and mechanical stiffness. These results confirm that the coating method optimized during this research can alter BPVs and has the promise to transform this important medical device for the better.
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    Magnetic Nanoparticle-based Immunoassays for Rapid, High-Sensitivity Detection of Protein Biomarkers
    (2024-07-29) Singampalli, Kavya Lahari; Lillehoj, Peter B.; Grande-Allen, Kathryn J; Yee, Cassian
    The detection of protein biomarkers in human biofluids is a standard clinical practice for diagnosis, and monitoring of disease progression and therapeutic response. Current gold standard techniques for protein measurement are based on immunoassays, in which antibodies specific to the analyte of interest are used to bind and isolate the target protein. The most commonly used immunoassay is the enzyme-linked immunosorbent assay (ELISA), which produces a color change proportional to the concentration of the target analyte. A point-of-care friendly alternative to ELISA is an electrochemical amperometric immunosensor, which measures a current change. These techniques are highly sensitive, often detecting target analyte levels in the pg’s/mL, and specific due to their use of antibodies. However, they require long (3-4 hr) incubation times, skilled laboratory personnel, single-use substrates, and may not achieve the sensitivity necessary to detect ultralow levels of protein biomarkers. To address these challenges, we have employed magnetic nanoparticles to amplify the detection signal produced by immunoassays and enhance binding kinetics in both ELISA and electrochemical sensing. We have developed a magneto-ELISA employing dually-labelled magnetic nanoparticles (DMPs), which are bound to an excess of detection antibody (dAb) and enzymatic reporter, in order to increase the binding of target analytes, enhance signal amplification, and reduce incubation times within the assay. By using an external magnetic field, DMP-immunocomplexes are concentrated at the bottom of each well, facilitating binding with the capture antibody (cAb). Using Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a marker for malaria, as a proof-of-concept biomarker, we found that this assay can detect proteins in the 10’s pg/mL range within 30 minutes, maintaining the sensitivity of a standard ELISA and producing results up to 4-fold faster. The magneto-ELISA was further adapted for serological detection of antibodies against Trypanosoma cruzi (T. cruzi), as would be found in chronic Chagas disease. Using DMPs conjugated to both Tc24, a protein specific to T. cruzi, and an enzymatic reporter, we show that anti-Tc24 antibodies can be identified in 6400x diluted clinical serum samples with an equivalent accuracy to a standard ELISA. We have also leveraged the capability of magnetic nanoparticles to be positioned using an external magnetic field to develop a reusable electrochemical sensor. Standard electrochemical immunoassay techniques render the sensors to be single-use consumables, increasing waste and costs associated with point-of-care quantitative protein biomarker detection. By using cAb-labelled magnetic nanoparticles (MNPs), we are able to temporarily immobilize immunocomplexes onto the working electrode, which can subsequently be washed away using a mild detergent. Furthermore, the addition of dually-labelled gold nanoparticles, bound to both dAb and a reporter molecule, allows for signal amplification. Together, these modifications to standard electrochemical sensing allow the sensor to be reused up to 100 times with a minimal reduction in analytical performance, while allowing for the detection of pg’s/mL of protein biomarkers in approximately one hour using point-of-care friendly instrumentation. Overall, we demonstrate the benefits of dually-labelled nanoparticles and MNPs in enhancing the sensitivity and reducing incubation times required for protein biomarker detection. The techniques described here are done with proof-of-concept biomarkers and biofluids, but can be easily adapted for the detection of other biomarkers. Furthermore, these techniques do not require additional laboratory equipment for protein detection, thus facilitating their adoption into clinical practice.