Kang, Yunqing

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here a 3D esophageal tumor tissue model was engineered using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, a detergent-free, rapid decellularization method was developed to decellularize porcine esophagus. The decellularized esophageal matrix (DEM) was characterized and the DEM was utilized for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. Then the expression of cancerrelated markers of KYSE30 cells was analyzed and compared with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens.

Rapid and efficient vascularization is still a considerable challenge of a tissue engineered β-tricalcium phosphate (β-TCP) scaffold. To overcome this challenge, branched channels were created in the porous scaffold to stimulate the instant flow of blood supply. The branched channeled porous β-TCP scaffold was fabricated using 3D printing and template-casting method. Human bone mesenchymal stem cells (hBMSC) and human umbilical vein endothelial cells (HUVEC) were seeded in the scaffolds and characterized through double-stranded DNA (dsDNA) assay, alkaline phosphatase (ALP) assay and cell migration. Scaffolds were then implanted in the subcutaneous pockets in mice. Hematoxylin and eosin staining and Immunohistochemical staining on vascularization, bone-related markers were carried out. Results showed that branched channels significantly promoted HUVECs’ infiltration, migration, proliferation, and angiogenesis and also promote the proliferation and osteogenesis differentiation of hBMSCs. Scaffolds did not show significant pro-inflammatory effects. In vivo results showed that in the early stage after implantation, cells significantly migrated into branched channeled scaffolds compared to non-channeled and straight channeled scaffolds. More and matured blood vessels formed in the branched channeled scaffolds compared to in non-channeled and straight channeled scaffolds. Besides promoting vascularization, the branched channels also stimulated the infiltration of bone-related cells into the scaffolds. These results suggested that the geometric design of branched channels in the porous β-TCP scaffold promoted rapid vascularization and potentially stimulated bone cell recruitment. To further enhance the function of the scaffold to promote the MSCs differentiation, MnO2 hollow and solid nanoparticles were doped into the scaffold with different concentrations.

Human esophageal squamous cell carcinoma (hESCC) is a very aggressive form of cancer due to its ability to easily metastasize into proximal lymph nodes and adjacent organs. The role of the extracellular matrix (ECM) and its stromal cells in metastasis remains unclear. To better understand the effect of the ECM and fibroblast cells on esophagus cancer cell migration and invasion, we propose a biomimetic human esophagus model cultured with hESCC and human primary fibroblast cells (fibroblast). To mimic the extracellular matrix of human esophagus we use decellularized porcine esophagus matrix (DEM) to culture with hESCC and fibroblasts in static conditions. This DEM can recapitulate the human esophagus tumor microenvironment with relevant cues. This model will provide valuable information regarding esophagus cancer cell migration with respect to the heterogeneous extracellular matrix and stromal fibroblast cells. We expect to discover the mechanisms by which extracellular matrix and stromal cells affect cancer migration and invasion in vitro. Characterizing this process will provide vital insight towards the effects of fibroblasts cells on facilitating migration and invasion of esophageal cancer cells. This esophagus cancer model also provides promising potential to study drug screening and develop new strategies against esophagus metastasis.

This thesis reports the development of a novel drug delivery system consisting of hollow nanoparticles, formed from manganese dioxide (δ-MnO2) sheets, that are coated with polydopamine and folic acid to selectively target cancer cells. The biodegradability and colloidal stability of the uncoated hollow nanoparticles were investigated in comparison to solid MnO2 nanoparticles and graphene oxide sheets. The MnO2 hollow nanoparticles degraded at a faster rate and seem to have a higher surface area and better colloidal dispersion than solid MnO2 nanoparticles. Xanthan gum was proven to improve colloidal dispersion of these hollow nanoparticles and were used for further cell studies. In this study, cancer and healthy cells were treated with coated hollow nanoparticles, and results indicate that this novel hollow nanoparticle may preferentially target and kill cancer cells. Particle aggregation has shown to be toxic to cells. Further studies with this novel drug delivery system may lead to a groundbreaking solution to targeted cancer therapy.

Inadequate nutrition exchange and slow transportation in a porous scaffold often resulted in insufficient vasculature formation, which hindered rapid bone regeneration. In this study, interconnected porous beta-tricalcium phosphate (b-TCP) scaffolds with channeled geometry were fabricated. In vitro fluid transportation and degradation of the scaffolds were performed. Cell attachment, migration, proliferation, and differentiation were carried out under both static and dynamic culturing conditions. A computational simulation model and a series of immunofluorescent staining were implemented to understand the mechanism of cell behavior in respond to different scaffolds geometry. We then implanted scaffolds into rat critical-sized calvarial defects to further evaluate channels’ function on bone regeneration in vivo. Results showed that multiple channeled geometry significantly accelerated the release of Ca2+ and increased the fluid diffusion efficiency. Moreover, multiple channels promoted human umbilical vein endothelial cells (HUVECs) infiltration, migration, besides prominently promoted alkaline phosphatase (ALP) activity, and up-regulated osteogenic gene expression in human bone marrow mesenchymal stem cells (hBMSCs) at both static and dynamic culturing conditions in vitro. The expression of both cell migration related protein a5 and angiogenesis related protein CD31 were upregulated by multiple channels in HUVECs. And the expression of mechanosensing markers, focal adhesion kinase (FAK), polymeric filamentous actin (Factin), and Yes-associated protein-1 (YAP-1) were highly stimulated by multiple channels in hBMSCs. The in vivo implantation and characterization results demonstrated more bone formation inside multiple-channeled scaffolds compared to non-channeled scaffolds. Multiple channels accelerated collagen type I, Bone Sialoprotein (Bsp), Osteocalcin (OC) protein expression prominently. The angiogenesis related protein CD31 staining displayed longer and more vasculature structures on multiple-channeled scaffolds compared to nonchanneled scaffolds. Fluorescent images of the fluorochrome labeled samples exhibited considerably more mineral deposition on multiple-channeled scaffolds than non-channeled scaffolds. All the findings suggested that the addition of multiple channels in the porous b-TCP scaffold is very promising approach to promote vascularization and bone tissue regeneration.

In spite of the vast research on polymer-based tissue regeneration, extensive studies to develop an elastic and cell-promoting polymer biomaterial are still ongoing. However, using a renewable resource and a simple, environment-friendly synthesis route to synthesize an elastic polymer has not been successfully achieved yet.
The objective of this work was to develop an elastic polymer for tissue engineering and drug delivery applications by using non-toxic, inexpensive and renewable monomers. A new nature-derived renewable material, xylitol, was used to synthesize an elastic polymer with the presence of a crosslinking agent, dodecanedioic acid. Here a simple melt condensation polymerization method was used to synthesize the poly(xylitoldodecanedioic acid)(PXDDA). The physicochemical and biological properties of the new PXDDA polymer were characterized. Fourier transform infrared (FTIR) confirmed the formation of ester bonding in the polymer structure, and thermal analysis demonstrated that the polymer was completely amorphous. The polymer shows high elasticity. Increasing the molar ratio of dodecanedioic acid resulted in higher hydrophobicity and lower glass transition temperature. Further, the polymer degradation and in vitro dye release studies revealed that the degradation and dye release from the polymer became slower when the amount of dodecanedioic acid in the composite increased.

Palliation therapy for dysphagia using esophageal stents is the current treatment of choice for those patients with inoperable esophageal malignancies. However, the stents currently used in the clinical setting, regardless of the type of metal mesh or plastic mesh stents (covered/uncovered), may cause complications, such as tumor ingrowth and stent migration into the stomach. Furthermore, metal mesh stents have limited capacities for loading anti-cancer drugs. To effectively reduce/overcome those complications and enhance the efficacy of drug release, we designed and 3D-printed a tubular, flexible polymer stent with spirals, and then load anti-cancer drug, paclitaxel, on the stent for drug release. Non- spiral 3D-printed tubular and mesh polymer stents served as controls. The self-expansion and anti migration properties, cytotoxicity, drug release profile, and cancer cell inhibition of the 3D-printed stent were fully characterized. Results showed the self-expansion force of the 3D-printed polymer stent with spirals was slightly higher than the stent without spirals. The anti-migration force of the 3D-printed stent with spirals was significantly higher than the anti-migration force of a non-spiral stent. Furthermore, the stent with spirals significantly decreased the migration distance compared to the migration distance of the non-spiral 3D-printed polymer stent. The in vitro cytotoxicity of the new stent was examined through the viability test of human esophagus epithelial cells, and results indicated that the polymer stent does not have any cytotoxicity. The results of in vitro cell viability of esophageal cancer cells further indicated that the paclitaxel in the spiral stent treated esophageal cancer cells much more efficiently than that in the mesh stent. Furthermore, the results of the in vitro drug release profile and drug permeation showed that the dense tubular drug-loaded stent could efficiently be delivered more paclitaxel through the esophageal mucosa/submucosa layers in a unidirectional way than mesh stent that delivered less paclitaxel to the esophageal mucosa/submucosa but more to the lumen. In summary, these results showed that the 3D-printed dense polymer stent with spirals has promising potential to treat esophageal malignancies.

Chitosan was widely studied for applications in tissue regeneration, because of its biodegradability and biocompatibility. However, its insolubility in a neutral solution and long gelation time limit its wide application in tissue engineering. In this thesis, a new chitosan-based biomaterial was synthesized, and its chemical structure and solubility were characterized. Afterwards, the gelation properties (crosslinker, crosslink time, swelling ratio, drug release and biocompatibility) of TMC material was investigated. Results show that TMC has higher water solubility than chitosan. The TMC liquid solution can transform to a hydrogel quickly at body temperature. The formed hydrogel controlled the release of the model protein. Cytotoxicity result shows the cationic TMC hydrogel brings a toxic effect on stromal cells but it may have the potential to inhibit bacteria or cancer cells, although more studied are required to confirm its potential functions. In summary, this new TMC hydrogel has a promising potential in biomedical fields.