A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function

Abstract

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors.

Keywords: BMP signaling; TGFβ signaling; cell line; mechanotransduction; periosteum.

FIGURE 1

Generating a periosteum-derived cell line. (A) Schematic summarizing the steps to generate a line of periosteum-derived cells (PDCs). Created with BioRender.com. Abbreviations: periosteum (P), muscle and connective tissue (MCT), and cortical bone (CB). (B) Femur isolated from a P14 Bmp2 ^LacZ mouse and corresponding histology stained with Hematoxylin Van Gieson’s to indicate P, MCT, CB, and bone marrow (BM). (C) Femur from (B) after the epiphyses and most of the MCT were removed under a dissecting microscope. (D) Femur from (C) after the periosteum was peeled under a dissecting microscope for the cell digest. Images were taken at ×20 magnification and scale bars indicate 100 μm. (E) mRNA expression of markers used to identify periosteum-derived clones. Expression was examined in freshly isolated periosteum (Peri) from P14 Bmp2 ^LacZ mice and candidate clones at passages 24 (P-24) and 34 (P-34). Denotes markers that are expressed (+) or not detected (−). n = 3–4 biological replicates for each group. (F) Cell morphology of the clone selected for the PDC line. Image was taken at ×10 magnification and scale bar indicates 100 μm.

FIGURE 2

The PDC line is multipotent. (A) Alizarin Red staining of PDCs incubated in culture media and osteogenic differentiation media (ODM). (B) Alcian Blue staining of PDCs incubated in culture media and chondrogenic differentiation media (CDM). (C) Oil Red O staining of PDCs incubated in culture media and adipogenic differentiation media (ADM). Images were collected at ×4 magnification and n = 3–4 technical replicates in 3 biological replicates for each group in (A–C). (D) mRNA expression of genes associated with differentiation. Values are represented as fold changes with DM treatment compared to regular culture media controls. Osteogenic and Chondrogenic values are normalized to Gapdh expression, Adipogenic values are normalized to β-actin expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 6-8 technical replicates in 3 biological replicates for each group. Replicates are represented as individual dots on bar graphs.

FIGURE 3

The PDC line engages in BMP/TGFβ signaling. (A) Expression of genes associated with the BMP/TGFβ superfamily in freshly isolated periosteum (Peri) from P14 Bmp2 ^LacZ mice and PDCs at passage 24 (P-24) and 34 (P-34). Denotes markers that are expressed (+) or not detected (−). n = 4–6 technical replicates in 3 biological replicates for each group. (B) Representative Western blot image and quantification of changes in total and phosphorylated (p) Smad1 and Smad2/3 with 100 ng/mL BMP2 or 1 ng/mL TGFβ-1 treatment. All values were normalized to Actin expression. *p < 0.05, ****p < 0.0001, n = 4-5 technical replicates in 3 biological replicates for each group. (C) Fold changes in mRNA expression of genes associated with activation of BMP and TGFβ signaling in PDCs treated with 100 ng/mL BMP2 or 1 ng/mL TGFβ-1. Changes in expression are represented as fold changes compared to static controls and all values are normalized to Gapdh expression. **p < 0.01, ****p < 0.0001, n = 3–4 technical replicates in 3 biological replicates for each group. Replicates are represented as individual dots on bar graphs.

FIGURE 4

The PDC line is mechanoresponsive. (A) Fold change in mRNA expression of genes associated with mechanically-induced osteogenesis under static or fluid flow (FF) conditions. (B) Representative Western blot image and quantification of changes in total and phosphorylated (p) Smad1 and Smad2/3 under static or FF conditions. All values are normalized to Actin expression and fold changes are normalized to static controls. *p < 0.05, **p < 0.01, n = 3–4 technical replicates in 3 biological replicates for each group. (C) Fold change in mRNA expression of genes associated with BMP and TGFβ signaling. Changes in mRNA expression (A,C) are represented as fold changes compared to static controls and all values are normalized to Gapdh expression. n = 4–6 technical replicates in 3 biological replicates for all groups. **p < 0.01, ***p < 0.001, ****p < 0.0001. Replicates are represented as individual dots on bar graphs.