A 3D Cancer Cell Migration Assay on a 384-Pillar Plate with Sidewalls

Hepatocellular carcinoma (HCC) is an aggressive liver cancer where prognosis is heavily tied to metastasis progression. Researchers look to determine the triggers for metastasis to control its spread. The goal of this project is to determine these triggers by quantifying Hep3B cell migration on a high-throughput platform. We infected Hep3B cells with lentiviruses containing mCherry to produce stable fluorescent cells. Next, we determined the stability of growth factors in oxidized, methacrylated alginate (OMA) hydrogel by binding growth factors with methacrylated heparin sulfate (MHS) before encapsulating in OMA, printing onto the 384-pillar plate with sidewalls, and quantifying growth factor release via ELISA. Finally, we printed layer-by-layer migration assays, in which bottom layers of fluorescent cells would migrate in response to top layers of growth factors and quantified migration and proliferation using previously developed macros. Initially, there was a strong release of growth factor, but the release rate was retarded by binding to MHS, meaning growth factors were stable. Cells proliferated in response to growth factors that encourage proliferation, while migration occurred towards growth factors that upregulate angiogenesis. These results show that we have successfully developed a 3D-cancer cell migration assay which has implications in the characterization of other cancers.https://engagedscholarship.csuohio.edu/u_poster_2018/1067/thumbnail.jp

A 3D Cancer Cell Migration Assay on a 384-Pillar Plate with Sidewalls

Hepatocellular carcinoma (HCC) is an aggressive liver cancer where prognosis is heavily tied to metastasis progression. Researchers look to determine the triggers for metastasis to control its spread. The goal of this project is to determine these triggers by quantifying Hep3B cell migration on a high-throughput platform. We infected Hep3B cells with lentiviruses containing mCherry to produce stable fluorescent cells. Next, we determined the stability of growth factors in oxidized, methacrylated alginate (OMA) hydrogel by binding growth factors with methacrylated heparin sulfate (MHS) before encapsulating in OMA, printing onto the 384-pillar plate with sidewalls, and quantifying growth factor release via ELISA. Finally, we printed layer-by-layer migration assays, in which bottom layers of fluorescent cells would migrate in response to top layers of growth factors and quantified migration and proliferation using previously developed macros. Initially, there was a strong release of growth factor, but the release rate was retarded by binding to MHS, meaning growth factors were stable. Cells proliferated in response to growth factors that encourage proliferation, while migration occurred towards growth factors that upregulate angiogenesis. These results show that we have successfully developed a 3D-cancer cell migration assay which has implications in the characterization of other cancers.https://engagedscholarship.csuohio.edu/u_poster_2018/1067/thumbnail.jp

Modeling and experimental methods to predict oxygen distribution in bone defects following cell transplantation

We have developed a mathematical model that allows simulation of oxygen distribution in a bone defect as a tool to explore the likely effects of local changes in cell concentration, defect size or geometry, local oxygen delivery with oxygen-generating biomaterials (OGBs), and changes in the rate of oxygen consumption by cells within a defect. Experimental data for the oxygen release rate from an OGB and the oxygen consumption rate of a transplanted cell population are incorporated into the model. With these data, model simulations allow prediction of spatiotemporal oxygen concentration within a given defect and the sensitivity of oxygen tension to changes in critical variables. This information may help to minimize the number of experiments in animal models that determine the optimal combinations of cells, scaffolds, and OGBs in the design of current and future bone regeneration strategies. Bone marrow-derived nucleated cell data suggest that oxygen consumption is dependent on oxygen concentration. OGB oxygen release is shown to be a time-dependent function that must be measured for accurate simulation. Simulations quantify the dependency of oxygen gradients in an avascular defect on cell concentration, cell oxygen consumption rate, OGB oxygen generation rate, and OGB geometry

Targeted Delivery of Bioactive Molecules for Vascular Intervention and Tissue Engineering

Cardiovascular diseases are the leading cause of death in the United States. Treatment often requires surgical interventions to re-open occluded vessels, bypass severe occlusions, or stabilize aneurysms. Despite the short-term success of such interventions, many ultimately fail due to thrombosis or restenosis (following stent placement), or incomplete healing (such as after aneurysm coil placement). Bioactive molecules capable of modulating host tissue responses and preventing these complications have been identified, but systemic delivery is often harmful or ineffective. This review discusses the use of localized bioactive molecule delivery methods to enhance the long-term success of vascular interventions, such as drug-eluting stents and aneurysm coils, as well as nanoparticles for targeted molecule delivery. Vascular grafts in particular have poor patency in small diameter, high flow applications, such as coronary artery bypass grafting (CABG). Grafts fabricated from a variety of approaches may benefit from bioactive molecule incorporation to improve patency. Tissue engineering is an especially promising approach for vascular graft fabrication that may be conducive to incorporation of drugs or growth factors. Overall, localized and targeted delivery of bioactive molecules has shown promise for improving the outcomes of vascular interventions, with technologies such as drug-eluting stents showing excellent clinical success. However, many targeted vascular drug delivery systems have yet to reach the clinic. There is still a need to better optimize bioactive molecule release kinetics and identify synergistic biomolecule combinations before the clinical impact of these technologies can be realized

Intradiscal treatment of the cartilage endplate for improving solute transport and disc nutrition

Poor nutrient transport through the cartilage endplate (CEP) is a key factor in the etiology of intervertebral disc degeneration and may hinder the efficacy of biologic strategies for disc regeneration. Yet, there are currently no treatments for improving nutrient transport through the CEP. In this study we tested whether intradiscal delivery of a matrix-modifying enzyme to the CEP improves solute transport into whole human and bovine discs. Ten human lumbar motion segments harvested from five fresh cadaveric spines (38–66 years old) and nine bovine coccygeal motion segments harvested from three adult steers were treated intradiscally either with collagenase enzyme or control buffer that was loaded in alginate carrier. Motion segments were then incubated for 18 h at 37 °C, the bony endplates removed, and the isolated discs were compressed under static (0.2 MPa) and cyclic (0.4–0.8 MPa, 0.2 Hz) loads while submerged in fluorescein tracer solution (376 Da; 0.1 mg/ml). Fluorescein concentrations from site-matched nucleus pulposus (NP) samples were compared between discs. CEP samples from each disc were digested and assayed for sulfated glycosaminoglycan (sGAG) and collagen contents. Results showed that enzymatic treatment of the CEP dramatically enhanced small solute transport into the disc. Discs with enzyme-treated CEPs had up to 10.8-fold (human) and 14.0-fold (bovine) higher fluorescein concentration in the NP compared to site-matched locations in discs with buffer-treated CEPs (p < 0.0001). Increases in solute transport were consistent with the effects of enzymatic treatment on CEP composition, which included reductions in sGAG content of 33.5% (human) and 40% (bovine). Whole disc biomechanical behavior—namely, creep strain and disc modulus—was similar between discs with enzyme- and buffer-treated CEPs. Taken together, these findings demonstrate the potential for matrix modification of the CEP to improve the transport of small solutes into whole intact discs

Three-Dimensional Cell and Tissue Patterning in a Strained Fibrin Gel System

Techniques developed for the in vitro reproduction of three-dimensional (3D) biomimetic tissue will be valuable for investigating changes in cell function in tissues and for fabricating cell/matrix composites for applications in tissue engineering techniques. In this study, we show that the simple application of a continuous strain to a fibrin gel facilitates the development of fibril alignment and bundle-like structures in the fibrin gel in the direction of the applied strain. Myoblasts cultured in this gel also exhibited well-aligned cell patterning in a direction parallel to the direction of the strain. Interestingly, the direction of cell proliferation was identical to that of cell alignment. Finally, the oriented cells formed linear groups that were aligned parallel to the direction of the strain and replicated the native skeletal muscle cell patterning. In addition, vein endothelial cells formed a linear, aligned vessel-like structure in this system. Thus, the system enables the in vitro reproduction of 3D aligned cell sets replicating biological tissue patterns

Development of novel hydrogels with controlled adhesion and degradation properties for bone tissue engineering.

Tissue engineering, which is the regeneration of tissues to replace those damaged or lost as a result of disease, trauma, or congenital abnormalities, has the potential to restore function and health to millions of people. Specific control over cell behavior may be necessary to guide the process of tissue formation. Thus, the hypothesis of this thesis is that osteoblast cellular behavior may be positively modulated when engineering bone tissue by regulating adhesion ligand presentation and controlling polymer degradation. The hypothesis was investigated using alginate, which is naturally non-adhesive to cells. Alginate was covalently modified with specific adhesive properties and ionically crosslinked to form hydrogels. Varying the adhesion peptide sequence type and density controlled osteoblast proliferation and differentiation in vitro. Compared to unmodified alginate controls, peptide-modified cell delivery vehicles significantly increased the amount of bone tissue formed in vivo following implantation with cells. This finding marks the first time regulation of biomaterial adhesive properties has been shown to control bone tissue regeneration in vivo. Chondrocytes were then transplanted in vivo using this peptide-modified alginate system to test whether controlling biomaterial adhesion characteristics could improve the formation of another tissue type. Cartilaginous tissue was formed that grew in volume over time. Once a growing cartilaginous anlage was engineered, osteoblasts and chondrocytes were co-transplanted within peptide-modified alginate to partially recreate the cellular milieu present in endochondral ossification. Growing bony tissues resulted with regions resembling growth plate-like structures. This result suggests that co-transplantation of several cell types may be required in order to fully replicate the structure and function of many complicated tissues. Once some control over cell behavior and new tissue formation was achieved using this system, the effect of improved biomaterial degradation rate on tissue formation was examined. Increasing the rate of biomaterial biodegradation resulted in improved rate, quality, and quantity of engineered bone tissue formation. The results of this thesis provide convincing evidence supporting the design of biomaterials that are bioactive and provide stimulatory signals to transplanted cells and surrounding host tissue. Enhanced tissue regeneration may also be achieved with biomaterials that degrade in concert with the formation of new tissue.Ph.D.Applied SciencesBiomedical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123119/2/3068817.pd

Functionalized, biodegradable hydrogels for control over sustained and localized siRNA delivery to incorporated and surrounding cells

a b s t r a c t Currently, the most severe limitation to applying RNA interference technology is delivery, including localizing the molecules to a specific site of interest to target a specific cell population and sustaining the presentation of these molecules for a controlled period of time. In this study, we engineered a functionalized, biodegradable system created by covalent incorporation of cationic linear polyethyleneimine (LPEI) into photocrosslinked dextran (DEX) hydrogels through a biodegradable ester linkage. The key innovation of this system is that control over the sustained release of short interference RNA (siRNA) was achieved, as LPEI could electrostatically interact with siRNA to maintain siRNA within the hydrogels and degradation of the covalent ester linkages between the LPEI and the hydrogels led to tunable release of LPEI/siRNA complexes over time. The covalent conjugation of LPEI did not affect the swelling or degradation properties of the hydrogels, and the addition of siRNA and LPEI had minimal effect on their mechanical properties. These hydrogels exhibited low cytotoxicity against human embryonic kidney 293 cells (HEK293). The release profiles could be tailored by varying DEX (8 and 12% w/w) and LPEI (0, 5, 10 lg/100 ll gel) concentrations with nearly 100% cumulative release achieved at day 9 (8% w/w gel) and day 17 (12% w/w gel). The released siRNA exhibited high bioactivity with cells surrounding and inside the hydrogels over an extended time period. This controllable and sustained siRNA delivery hydrogel system that permits tailored siRNA release profiles may be valuable to guide cell fate for regenerative medicine and other therapeutic applications such as cancer treatment