COBRE in Mesenchymal and Neural Regulation of Metabolic Networks


Project 2:  Novel mechanisms of osteocyte control of marrow adiposity.  Dr. Michaela Reagan

Project significance – Within the bone marrow, there is a population of adipocytes that changes with disease and under stress conditions. For example, high levels of bone marrow adipose cells are associated with increased fracture risk in individuals with type 2 diabetes and anorexia nervosa. Obesity also increases bone marrow adipose tissue, and elevated fat in the bone marrow is associated with bone loss. Thus, in the bone marrow microenvironment, the level of fat and bone are intricately linked, and this project studies the factors that regulate this balance during disease. Part of our strategy to understand these factors includes establishing three-dimensional scaffolds made of silk to allow bone cells and fat cells to interact in an environment more similar to the bone marrow in the body.

Silk scaffolds to model cell interactions. (a) Silk worms (Bombyx mori) are grown on mulberry leaves and produce cocoons to begin metamorphosis. (b) Cleaned cocoons are boiled and dried to produce silk fibroin with a cottony texture, which is dissolved to produce a silk solution (c). (d) This solution is then dialyzed to purify, and can then be poured into molds and crossslinked to create a porous scaffold (e). (f) A silk scaffold viewed with a scanning electron microscope. (g) Schematic of the process to make silk scaffolds. (h) Scaffolds are then soaked in media, and cells can be seeded into the surface to grow. (i) Whole flushed bone marrow cells from C3H mice were seeded onto silk scaffolds and  imaged with live/dead confocal imaging 3 days after seeding. (1) Silk autofluorescence is seen in the blue channel, dead cells (ethidium homodimer-1) and autofluorescence of silk is seen in the red channel, and live cells (calcein) are see in the green channel. (2) Magnification of the overlay from shows scaffold in purple/pink, live cells in green, and a few dead cells, indicated in red. (3) Live-dead (calcein-green/ethidium homodimer-1/red) staining confocal imaging of mouse mesenchymal stem cells (green) first expanded in vitro on tissue culture plastic, and then seeded on silk scaffolds (red) and cultured for 9 days. Cells can be seen growing off the scaffold and in the pores throughout the scaffold.

From Dadwal et al. 2016

Figure 1. Signals between the brain, the body, and bone. The skeleton is emerging as a key regulator of complex biological processes, including the sending and receiving of endocrine signals. In response to biochemical stimuli, mesenchymal stem cells (MSCs) differentiate into mature, functioning cells. In healthy bone marrow (BM), MSCs differentiate into BM-adipocytes (BMAs) or osteoblasts (OBs) in response to the addition or removal of WNT signaling, respectively. WNT signaling induces RUNX2 expression driving OB differentiation, but inhibition of WNT signaling is required for the differentiation into adipocytes. Sclerostin (SOST), a WNT inhibitor, is emerging as a potential player in the differentiation of BM-MSCs, adding complexity to the regulation of bone marrow adipose tissue (MAT) in response to both adipose (energetic), and bone-derived signals.

Fairfield et al 2017, Current Molecular Biology Reports