Osteoblasts are inherently programmed to repel sensory innervation

Bone Research ARTICLE www.nature.com/boneres OPEN Osteoblasts are inherently programmed to repel sensory innervation Luís Leitão 1,2,3, Estrela Neto 1,2, Francisco Conceição1,2,3, Ana Monteiro1,2, Marina Couto1,2, Cecília J. Alves1,2, Daniela M. Sousa1,2 and Meriem Lamghari1,2,3 1234567890();,: Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation. Bone Research (2020)8:20 ; https://doi.org/10.1038/s41413-020-0096-1 INTRODUCTION Peripheral innervation is a critical component of tissues’ structure and function. Neuronal signaling has been implicated as a regulatory mechanism of tissue homeostasis and regeneration.1–6 During the development of the peripheral nervous system, neurons project axons to reach their target tissues and form functional circuits. The amount, type, and patterning of innervation is achieved through the tissue-specific expression in space and time of attractive or repulsive axonal guidance molecules.7,8 Axonal terminals have the molecular mechanisms to accurately react to these guidance cues, ultimately ensuring the establishment of intricate patterns of neuronal networks.7,8 An increasing body of evidence has indicated the neuro-skeletal liaison as an important regulatory mechanism for bone development, turnover, and regeneration.5,9 In pathological scenarios such as fracture, bone cancer, or osteoporosis, where a deregulation of the bone homeostasis and/or regeneration processes occurs, changes in the pattern of bone innervation are also often observed, suggesting a disturbance in the neuronal signaling to the bone.10–14 In fact, the healthy bone is highly innervated by primary afferent sensory and sympathetic fibers, branching densely in the periosteum and, to a lesser extent, mineralized bone, and bone marrow.9,15 Importantly, anatomical mapping of innervation during skeletal development shows that sensory nerve fibers are the first to be detected in the bone microenvironment, particularly in areas with high osteogenic activity,16–18 which has attracted particular interest concerning bone formation.19–23 Indeed, the osteoprogenitor mesenchymal stem cells (MSC) have been reported to support neuronal survival and promote axonal outgrowth and regeneration through the expression of neurotrophic factors.5,24–26 In the development of mouse femur, nerve growth factor (NGF) expressed by MSC has been described as a skeletal neurotrophin, promoting and directing the outgrowth of sensory axons to primary and secondary centers of incipient ossification.27 However, the role of MSC-derived mature osteoblasts (OB) in controling sensory innervation in the bone is still unclear. OB have been described to promote axonal growth through the expression of NGF when submitted to mechanical loading, but not under static conditions,28 which might be an indication that OB no longer have the ability to control bone innervation. Therefore, it remains unknown whether the signaling profile associated with the OB phenotype differentiating program impacts the patterning of sensory innervation in bone. In this study, we analyzed the paracrine signaling of OB-lineage cells throughout osteoblastogenesis and correlated it with sensory axonal behavior. We show that differentiation of human MSC to OB phenotype leads to marked impairment of their ability to promote axonal growth and creates a nonpermissive environment for the sensory nerve fibers through the expression of axonal repulsive cues. Overall, we provide valuable data on the contribution of OB-lineage cells in the regulation and maintenance of innervation in the bone. 1 Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal; 2Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200135 Porto, Portugal and 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4050-313 Porto, Portugal Correspondence: Meriem Lamghari () Received: 21 October 2019 Revised: 19 February 2020 Accepted: 24 March 2020 © The Author(s) 2020 Osteoblasts are programmed to repel sensory axons L Leitão et al. 2 RESULTS The secretome of OB-lineage cells impairs the development of sensory axonal networks To explore if the MSC commitment to OB alters its neurotrophic ability, we exposed dorsal root ganglia (DRG) to the secretome of OB at different times of differentiation. OB were differentiated from MSC as previously described,29 and the conditioned medium was collected at day 0 (MSC CM), day 7 (D7 OB CM), day 14 (D14 OB CM), and day 21 (D21 OB CM). Under osteoinductive conditions, MSC showed transient expression of the early OB marker alkaline phosphatase and acquired the full OB phenotype after 21 days in culture, characterized by high osteocalcin gene expression levels and intense calcium deposition (Supplementary Fig. 1). The neurotrophic potential of the distinct conditioned media was tested on organotypic explants of DRG cultures, and the axonal outgrowth calculated using a Matlab-based algorithm30 (Fig. 1a). Our results showed that DRG treated with the conditioned medium of differentiating OB have significantly smaller axonal networks when compared with undifferentiated MSC. DRG treated with D7 OB and D21 OB CM exhibited a 17% and 34% reduction on the axonal mesh area, respectively, when compared with MSC CM, suggesting a gradual loss of the axonal growth potential with the maturation stage of OB (Fig. 1b, c). A significant decrease was also (...truncated)