Periosteal progenitors contribute to load-induced bone formation in adult mice and require primary cilia to sense mechanical stimulation

Abstract

Background: The fully developed adult skeleton adapts to mechanical forces by generating more bone, usually at the periosteal surface. Progenitor cells in the periosteum are believed to differentiate into bone-forming osteoblasts that contribute to load-induced adult bone formation, but in vivo evidence does not yet exist. Furthermore, the mechanism by which periosteal progenitors might sense physical loading and trigger differentiation is unknown. We propose that periosteal osteochondroprogenitors (OCPs) directly sense mechanical load and differentiate into bone-forming osteoblasts via their primary cilia, mechanosensory organelles known to be involved in osteogenic differentiation.

Methods: We generated a diphtheria toxin ablation mouse model and performed ulnar loading and dynamic histomorphometry to quantify the contribution of periosteal OCPs in adult bone formation in vivo. We also generated a primary cilium knockout model and isolated periosteal cells to study the role of the cilium in periosteal OCP mechanosensing in vitro. Experimental groups were compared using one-way analysis of variance or student's t test, and sample size was determined to achieve a minimum power of 80%.

Results: Mice without periosteal OCPs had severely attenuated mechanically induced bone formation and lacked the mineralization necessary for daily skeletal maintenance. Our in vitro results demonstrate that OCPs in the periosteum uniquely sense fluid shear and exhibit changes in osteogenic markers consistent with osteoblast differentiation; however, this response is essentially lost when the primary cilium is absent.

Conclusions: Combined, our data show that periosteal progenitors are a mechanosensitive cell source that significantly contribute to adult skeletal maintenance. More importantly, an OCP population persists in the adult skeleton and these cells, as well as their cilia, are promising targets for bone regeneration strategies.

Keywords: Bone regeneration; Mechanotransduction; Osteoblasts; Osteochondroprogenitors; Periosteal progenitors; Primary cilium; Prx1.

Fig. 1

Prx1 expression is confined to the adult periosteum in the ulnar midshaft. Skeletally mature 16-week-old Prx1CreER-GFP;Rosa26^tdTomato mice received a single dose of tamoxifen to induce tdTomato production and were sacrificed a week later. Recombined cells (red) were found in the periosteum (white arrows) of the ulna, but were absent from cortical bone, trabecular bone, muscle, and bone marrow. Micrographs were captured with a confocal microscope at 20X. Scale bar = 50 μm

Fig. 2

Tamoxifen treatment successfully ablates periosteal progenitors and their cilia in our transgenic animal models. Skeletally mature 16-week-old Prx1CreER-GFP;Rosa26^DTA ablation mice and Prx1CreER-GFP controls received tamoxifen injections and were sacrificed a week later. Prx1-expressing cells (green) were observed in the periosteum of Prx1CreER-GFP control ulnae (a) but were absent in Prx1CreER-GFP;Rosa26^DTA animals (b). Primary OCPs obtained from 3-week-old Prx1CreER-GFP;Ift88^fl/fl animals (c) normally contain primary cilia (red); however, the presence and length of these cilia was diminished after exposure to 5 μM 4-hydroxytamoxifen (d). Indeed, cells treated with tamoxifen have decreased Ift88 mRNA expression (e). A magnified view of box region in panels c and d (f). Micrographs of the periosteum and isolated cells were collected at 20X and 40X, respectively. Nuclei are displayed in blue. Scale bars = 50 μm. Data are reported as mean and standard error. n = 4 for each group, ***p < 0.0001

Fig. 3

Mineralization and load-induced bone formation are severely attenuated in mice lacking OCPs. Skeletally mature Rosa26^DTA control and Prx1CreER-GFP;Rosa26^DTA ablation animals injected with tamoxifen were exposed to ulnar loading and the resulting mineralizing surfaces were labeled with calcein (green) and alizarin (red) fluorochrome dyes. Mice lacking periosteal OCPs demonstrated poor mineralization, indicated by a lack of labeling at the periosteal surface in both loaded and nonloaded ulnae (a). We performed dynamic histomorphometry and confirmed this visual observation (b). Ablated animals also exhibited an inferior mineral apposition rate (c), resulting in attenuated bone formation compared with controls (d). Loaded ulnae were normalized to nonloaded contralateral limbs. Micrographs were collected at 10X. Data are reported as mean and standard error. n = 16 for each group, ***p < 0.0001. rBFR/BS relative bone formation rate/bone surface, rMAR relative mineral apposition rate, rMS/BS relative mineralizing surface/bone surface

Fig. 4

Periosteal OCPs uniquely respond to mechanical stimulation in an osteogenic manner. Primary periosteal cells isolated from 3-week-old Prx1CreER-GFP mice were exposed to 1 h of oscillatory fluid flow (OFF) and changes in mRNA expression were quantified via RT-qPCR. Prx1-expressing OCPs (green) exposed to OFF (line pattern) demonstrated increases in Cyclooxygenase-2 (COX-2) (a) and Osteopontin (OPN) expression (b) compared with controls, whereas other cells of the periosteum (white) were nonresponsive. OCPs have elevated OPN expression compared with other cells of the periosteum under static conditions (b). Additionally, the fold changes in COX-2 (c) and OPN (d) expression were significantly higher for OCPs compared with other cells of the periosteum. mRNA expression was normalized to GAPDH expression. Data are reported as mean and standard error. Other no-flow n = 5, other flow n = 7, OCP no-flow and flow n = 6, *p < 0.01, ***p < 0.0001

Fig. 5

OCP primary cilia are necessary for the flow-induced osteogenic response. Primary periosteal OCPs isolated from 3-week-old Prx1CreER-GFP;Ift88^fl/fl mice were treated with the active compound of tamoxifen or vehicle control and exposed to 1 h of oscillatory fluid flow (OFF). Vehicle control OCPs (white) demonstrated an increase in Cyclooxygenase-2 (COX-2) (a), Osteopontin (OPN) (b), and Runt-related transcription factor 2 (RUNX2) (c) expression, but a decrease in Bone gamma-carboxyglutamic acid-containing protein (BGLAP) (d) expression. These effects were lost in OCPs treated with tamoxifen (gray) to disrupt ciliogenesis. mRNA expression was normalized to GAPDH expression and OFF samples were normalized to static controls. Data are reported as mean and standard error. Vehicle n = 6, tamoxifen n = 5, ***p < 0.0001

Fig. 6

OCPs require primary cilia to respond to paracrine signals from mechanically stimulated osteocytes. Primary periosteal OCPs isolated from 3-week-old Prx1CreER-GFP;Ift88^fl/fl mice were treated with the active compound of tamoxifen to disrupt ciliogenesis or vehicle control and then treated with conditioned media from MLO-Y4s exposed to oscillatory fluid flow (OFF) or static controls. Vehicle control OCPs (white) demonstrated a nearly twofold increase in Osteopontin (OPN) (a), Runt-related transcription factor 2 (RUNX2) (b), and Bone gamma-carboxyglutamic acid-containing protein (BGLAP) expression (c). This effect was lost in OCPs treated with tamoxifen (gray). mRNA expression was normalized to GAPDH expression and OFF samples were normalized to static controls. Data are reported as mean and standard error. n = 5 for both groups, ***p < 0.0001