Dose-Dependent Effects of T3 on Osteogenic Differentiation of Human Mesenchymal Stem Cells By Megan Scott

Abstract: Regeneration of large bone defects is considered a daunting task to many orthopedic surgeons. Currently, bone grafts or metal implants are ways to correct bone defects. The inherent limitations associated with such methods have led to investigation into bone tissue engineering. The objective of this study was to design appropriate conditions, using triiodothronine(T3) hormone and XT199, that would induce osteogenic differentiation of human mesenchymal stem cells to eventually render applicable methods for clinical use. Stem cells are a multipotent cell, meaning, they have the ability to differentiate into any of the somatic cells found in organisms. Human mesenchymal stem cells, hMSCs, are a type of stem cell derived from the bone marrow. In this project, hMSCs were induced into osteogenic differentiation. hMSCs were plated with osteogenic medium and treated with differing concentrations of T3 and the same concentrations of T3 paired with XT199. Controls were be regular medium, DMEM, with and without XT199 and osteogenic medium with and without XT199. These varying concentrations were utilized to test which most effectively induces osteogenic differentiation. The effect of T3 was analyzed by comparing various stains and analyzing the number of mineralized nodules. The stains used are Von Kossa Stain and Alizarin Red Stain which both identify calcium. The presence of

calcium indicates osteogenic differentiation of hMSCs because osteoblasts, a type of bone cell, secrete calcium. Von Kossa stain is a technique used for demonstrating deposits of calcium or calcium salt by treating cells with a silver nitrate solution and exposing the cells to strong light. The silver replaces calcium, thereby visualizing calcium nodules as metallic silver. Alizarin Red stain was also performed to identify calcium produced by cells. Calcium forms an Alizarin Red S-calcium complex in a chelation process. The end result is a birefringence, or double refraction, which is the decomposition of a ray of light into two rays. This was seen through a microscope. It was found that 1 nM and 0.1 nM demonstrated the highest levels of osteogenic differentiation of hMSCs and .001nm had the next best results. It should be noted that 0.1 to 1 nM are near physiological conditions. Also, XT199 proved to inhibit osteogenic differentiation and overall reduce cell division. This study used concentrations higher and lower than physiological levels. Therefore, this study provides the first impression in explain the mechanisms by which thyroid hormones produce different dose-dependent effects in vivo. Thus, it can be concluded that hyperthyroidism and hypothyroidism can pose complications because of the inhibition of osteogenic differentiation of hMSCs in a person s bone marrow.

Introduction: Human mesenchymal stem cells are characterized by two properties. First, is the ability to extensively replicate. The second property is the capacity for hMSCs to differentiate into multiple cell lines [1]. These cells are resident in many tissues in

adults, including bone marrow. The notion that bone marrow houses osteogenic precursor cells was first proposed by Petrakova et al, 1963. For this reason, hMSCs have been studied in bone tissue engineering. Also, since hMSCs are usually obtained from the patient s own bone marrow, their transplantation is not associated with host versus graft reaction. Furthermore, some research has indicated that hMSCs express no HLA class II receptor indicating that even nonautologous transplantation would not trigger host immune response [2]. In this experiment, human mesenchymal stem cells were plated with varying concentrations of thyroxine (T3) in order to induce osteogenic differentiation. T3 is essential for the growth and development of many organs and tissues in utero, most importantly, bone tissue. The effects of these thyroid hormones are mainly due to the binding of T3 to the receptors TR and TR [2]. These receptors are present in the cells of many tissues including osteoblasts and mesenchymal stem cells [1-3]. Another set of hMSCs were treated with the same differing concentrations of T3 paired with a constant concentration of XT199. XT199 is the name of a synthesized chemical compound that acts as an integrin protein thus inhibits cell attachment. The effect of a constant concentration of XT199 and different concentrations of T3 will also be analyzed. In osteogenic differentiation, hMSCs differentiate into osteoblasts. Osteoblasts are one of three types of cells in bone tissue. The other two types are osteoclasts and osteocytes. Osteoblasts are bone-forming cells which have the high alkaline phosphatase activity required for the mineralization of bones [3]. Osteoclasts are bone macrophages specialized in bone reabsorbtion. Lastly, osteocytes are

ostoblastic cells that are fixed within their own deposited matrix during the osteogenesis process [4]. It is well known that the proliferation and differentiation of hMSCs are under the command of many factors. These factors include external cellular signals, growth factors, cellular density and physical contact with neighboring cells [5]. Despite the diversity of the studies that have been performed to use stem cells to treat a wide variety of diseases, there are many aspects of human mesenchymal stem cell biology that has been ignored. An example of this lacking knowledge is regarding the effects of thyroid hormones on osteogenic differentiation. A similar study has been performed by Ishida et al, 1995, evaluating the effect of T3 in regulating the differentiation of rat calvaria cells. These cells are differentiated, unlike the undifferentiated stem cells used in this study. Thus, until this study, there have been few documented studies on the dose-dependent effect of T3 on osteogenic differentiation of human mesenchymal stem cells.

References:

1. Petrakova KV, Tolmachava AA, Friedenstein AJ. Bone formation occurring in bone marrow transplantation in diffusion chambers. Bull exp Biol Med. 1963; 56(12):87-91

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