Alginate Hydrogels as Three-Dimensional Scaffolds for in Vitro Culture Models of Growth Plate Cartilage Development and Porcine Embryo Elongation

Authors

Taylor D. Laughlin, University of Nebraska - LincolnFollow

First Advisor

Angela K. Pannier

Date of this Version

Summer 7-27-2016

Citation

Laughlin T D: Alginate Hydrogels as Three-Dimensional Scaffold for In Vitro Culture Models of Growth Plate Cartilage Development and Porcine Embryo Elongation. Lincoln, NE: MS Thesis, University of Nebraska; 2013.

Comments

A THESIS Presented to the Faculty of The Graduate College at University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Agricultural and Biological Systems Engineering, Under the supervision of Professor Angela K. Pannier. Lincoln, Nebraska: July 2016

Copyright (c) 2016 Taylor D. Laughlin

Abstract

The establishment of in vitro culture models utilizes tissue engineering principles to design functional mimics of in vivo environments in vitro. Advantages for the use of in vitro culture models include ethical alleviation of animal models for therapeutic testing, cost efficiency, and a greater ability to study specific mechanisms via a systematic, ground-up approach to development. In this thesis, alginate hydrogels are utilized in the development of in vitro culture models of porcine embryo elongation and growth plate cartilage development. First, the effect of scaffold and modifications to the scaffold were explored in both projects. In order to modulate cell-scaffold interactions, the alginate molecule was covalently modified with the arginine-glycine-aspartic acid (RGD) peptide, which has been extensively used to promote cell adhesion in vitro. In the porcine embryo elongation studies, a previously established culture system was used as a tool to encapsulate embryos and explore the effect of soluble factors on embryo survivability, rate of morphological changes, gene expression and hormone production by using RGD-modified alginate and alginate mixed with osteopontin (SPP1), a glycoprotein known to be secreted in the uterus. RGD-alginate encapsulation led to significant increases in embryo survivability and morphological changes along with corresponding cellular outputs. In the growth plate cartilage development model, chondrocytes were encapsulated in alginate and RGD-alginate to elucidate effects of cell adhesion, mimicking cell-matrix interactions within the growth plate, on chondrocyte phenotype. Additionally, alginate encapsulation was utilized to culture chondrocytes in media supplemented with parathyroid hormone (PTH) or Indian hedgehog (IHH) to begin to study the mechanisms behind these two molecules on the establishment of the zonal architecture and column formation that is unique to the growth plate. Cell cycle distribution, gene expression immunofluorescence and fluorescent in situ hybridization techniques together indicated our ability to suppress hypertrophy using alginate hydrogel scaffolds and/or signaling molecules. Overall, utilization of these in vitro culture models as tools to study developmental mechanisms can be used to advance both the fields of porcine embryology and cartilage developmental biology to improve pregnancy outcomes in pigs and to engineer therapies for the treatment of growth plate injuries and diseases.

Advisor: Angela K. Pannier