Local injections of \u03b2-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion

Kevin O Rivera,Ryan E Tomlinson,Chelsea S Bahney,Ryan M Boileau,Theodore Miclau,Tejal A Desai,Ralph S Marcucio,Fabrizio Russo

doi:10.1038/s41598-020-78983-y

Abstract

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local β-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from β-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local β-NGF injections. Finally, we demonstrate functional improvements to bone healing following local β-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate β-NGF’s ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation.

Highlights

IntroductionOsteoprogenitor cells along the bone surfaces undergo direct osteogenic differentiation (intramembranous ossification) to form new bone along the existing bone adjacent to the fracture site
Osteoprogenitor cells along the bone surfaces undergo direct osteogenic differentiation to form new bone along the existing bone adjacent to the fracture site
The process of chondrocyte hypertrophy is pivotal in the endochondral conversion of cartilage to bone and is a process tightly regulated through a complex negative feedback loop between Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP)[20]

Summary

Introduction

Osteoprogenitor cells along the bone surfaces undergo direct osteogenic differentiation (intramembranous ossification) to form new bone along the existing bone adjacent to the fracture site. Current evidence suggests that subsequent transformation of these hypertrophic chondrocytes into osteoblasts is regulated by the loss of Sox[9] expression and activation of canonical Wnt signaling[25,26,27,28,29]. Despite the importance of endochondral ossification to successful fracture repair, therapeutic approaches to bone regeneration have traditionally focused on promoting intramembranous ossification through the use of bone morphogenetic proteins (BMPs), which forms bone through direct osteoblast differentiation of osteochondroprogenitors[30]. The NGF-TrkA pathway is essential for load-induced bone formation in mice and exogenous NGF delivery was shown to stimulate angiogenesis and activate Wnt/β-catenin signaling to promote osteogenic lineage p rogression[37,38]. We aimed to understand endogenous signaling patterns of NGF-TrkA during endochondral ossification and test whether NGF could be used therapeutically to promote healing. Our data suggest NGF acts mechanistically to promote cartilage to bone conversion improving fracture repair

Objectives

Methods

Results

Conclusion