PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling

Abstract

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis.

Keywords: allograft; angiogenesis; calvarial bone repair; fracture healing; osteogenesis; parathyroid hormone.

Figure 1

Experimental overview. The aim of this study was to further elucidate the mechanism of the PTH effect on osteogenesis and angiogenesis, specifically in the context of allograft-mediated cranial defect healing. We hypothesize that the PTHR1 in endothelial cells plays a critical role in PTH-induced bone formation, as well as in the hormone’s ability to modulate the vascular tree feeding the graft. To evaluate this hypothesis, we used several lines of transgenic mice: (1) mice in which a CRE-flox system was implemented to delete the PTHR1 in endothelial VE-cadherin+ cells, (2) mice that exclusively over-express constitutively active PTHR1 in collagen-1α+ osteoprogenitors, and (3) mice expressing the Luciferase reporter gene in osteocalcin+ osteoprogenitor cells.

Figure 2

The deletion of PTHR in endothelial cells diminished the hormone’s osteogenic effect. Wild-type C57BL/6 mice (C57) and C57BL/6 mice engineered with CRE-recombinase under the endothelial VE-cadherin promoter and floxed PTH-Receptor 1 gene (VEcad/PTHRfx) underwent a circular 5 mm defect in the skull, followed by bone allograft implantation. Four weeks later, and one week after a 3-week treatment with either PTH or PBS (control group), significantly greater bone volume was observed in the wild-type C57 mice that received PTH, but no difference in bone volume was observed between the transgenic mice treatment groups (A,B), n = 6, * p-value < 0.05, t-test. Bone connectivity and density was higher in wild-type C57 mice given PTH, compared to the counterpart control group (C), n = 6, p-value = 0.057, t-test. Bone mineral density (BMD) was similar across all groups (D).

Figure 3

PTHR signaling activated in osteoprogenitors induced osteogenesis to a lesser degree compared to systemic PTH administration. Bone allografts were implanted in wild-type FVB/N mice given PTH (FVBn + PTH) and in FVB/N mice engineered with constitutively active intramembranous portions of the PTHR under collagen-1α (Col1ca-PTHR). Mice were euthanized four weeks later, and bone volume was significantly higher in the wild-type mice treated with systemic PTH, compared to Col1ca-PTHR mice (A,B), n = 6, * p-value < 0.05, t-test. Furthermore, bone connectivity and density were significantly higher in the FVBn + PTH group (C), n = 6, * p-value < 0.05, t-test. Bone mineral density (BMD) was similar in both groups (D).

Figure 4

The induction of host cell differentiation is greater when PTH is administered systemically, compared to PTHR activation in osteoprogenitors alone. The Col1ca-PTHR mice were crossed with Osteocalcin-Luciferase (Oc-Luc) mice. Bioluminescence imaging was performed at designated time points following allograft implantation, using the beetle luciferin substrate for luminescence (A). Quantitative analysis was performed first in the cranial region (B), n = 7, ** p-value < 0.01, **** p-value < 0.001, two-way ANOVA, and then normalized to each animal’s tail vertebrae, which acted as a non-fractured anatomical site to normalize luminescence (C), n = 7, ** p-value < 0.01, **** p-value < 0.001, two-way ANOVA.

Figure 5

Systemic PTH administration increases the formation of small-diameter blood vessels, while PTHR deletion in endothelial cells attenuates this PTH-induced effect, but PTHR activation in osteoprogenitors alone is sufficient to re-establish this effect. One week following the calvarial graft implantation, mice were anesthetized, and a radio-opaque contrast agent was systemically administrated. After the rubbery contrast agent was set to cure, the calvarial region was harvested and decalcified, so the vasculature could be analyzed by µCT. First, a 3-dimensional thickness map was generated (A), and the frequency of each vessel-diameter was calculated (B), n = 8, * p-value < 0.05, two-way ANOVA.