Models for determining estrogen effects on meniscal fibrochondrocytes

Abstract

Abstract Introduction: An estimated 242 million people in the world are living with symptomatic and activity-limiting osteoarthritis (OA). Women are 2-6X more likely to experience musculoskeletal injury and degeneration, highlighting a sexual dimorphism in diseases such as OA. One major cause of knee osteoarthritis is damage or injury to the fibrocartilaginous meniscal discs. The most common treatment for meniscal injury is partial or complete resection of the meniscus by meniscectomy. With resection of the meniscal tissue, the meniscus structural benefits to the joint are severely limited. Estrogens are steroid hormones that function to promote gene transcription and downstream activity and to play a role in musculoskeletal cell proliferation and new extracellular matrix production. The overall purpose of this thesis is to develop models for understanding the in-vitro response of meniscal cells to estrogen treatment to both determine a mechanism for the sex differences in OA and identify therapeutic strategies to promote new meniscal tissue growth by capitalizing on estrogen signaling. Methods: Meniscus cells were harvested from the lateral, and medial meniscal discs of bovine (17 months old) and human samples (male and female, 18 years and 47 years old) and separated based on region (inner vs outer). The effect of hormone-free media composition, estrogen dosing concentration and kinetics, and 2D vs 3D on cell proliferation and extracellular matrix production was determined using assays to assess cell number and quantify RNA and protein level changes to extracellular matrix components. Specifically, the effect of hormone-free media components, namely charcoal/dextran treated FBS (Char FBS) and phenol red-free basal media (prfDMEM), on cell proliferation and ECM production was assessed to establish baseline readings prior to estrogen studies. Estrogen dosing at low and high doses and pulsed or continuous dosing kinetics was completed. Lastly, scaffold-free spheroid culture models were established using a hanging drop method to assess the role of estrogen in 3D. Results: Both 2D and 3D in-vitro models were established in hormone-free culture media for estrogen dosing studies. Proliferation at day one in both 10 % and 15 % Char FBS was not statistically different from the control group. No statistically significant changes to total GAG and collagen content were observed due to media composition or estrogen treatment. Phenol red-free DMEM (prfDMEM) significantly reduced cell proliferation compared to cells in charcoal FBS and normal FBS medium. Estrogen treatment showed no observable differences between conditions at 24 and 72 hours from inner and outer meniscus in cell proliferation. Gene expression of extracellular matrix components showed preliminary evidence that estradiol exhibited dose-dependent effects in both male and female cells. There are significant differences between sexes in cell proliferation but not significant differences in gene expression of matrix macromolecules. Conclusions: 2D and 3D culture models were established to assess the role of estrogen on bovine and human MFCs. Using these models, it was determined that E2 promotes the upregulation of ACAN and Col2a1 gene expression while decreasing Col1a1 expression, at concentrations of 10-8M. Further, in the spheroid culture model, 10-8M E2 decreased significantly less in diameter compared to the 0M E2 group. Overall, these results highlight that MFCs, although partly responsive, are likely not the cell type responsible for responding to estrogen treatment at these dosages and culture conditions. The models developed in this thesis will continue to be explored to expand our working knowledge and develop better strategies to probe the effects of E2 on other cells in the knee joint to determine their role in knee health and joint tissue homeostasis.