Spinal pain is a major clinical problem, however, its origins and underlying mechanisms remain unclear. Here we report that in mice, osteoclasts induce sensory innervation in the porous endplates which contributes to spinal hypersensitivity in mice. Sensory innervation of the porous areas of sclerotic endplates in mice was confirmed. Lumbar spine instability (LSI), or aging, induces spinal hypersensitivity in mice. In these conditions, we show that there are elevated levels of PGE2 which activate sensory nerves, leading to sodium influx through Na v 1.8 channels. We show that knockout of PGE2 receptor 4 in sensory nerves significantly reduces spinal hypersensitivity. Inhibition of osteoclast formation by knockout Rankl in the osteocytes significantly inhibits LSI-induced porosity of endplates, sensory innervation, and spinal hypersensitivity. Knockout of Netrin-1 in osteoclasts abrogates sensory innervation into porous endplates and spinal hypersensitivity. These findings suggest that osteoclast-initiated porosity of endplates and sensory innervation are potential therapeutic targets for spinal pain.
Functional fibrocartilage regeneration is a bottleneck during bone–tendon healing, and the currently available tissue-engineering strategies for fibrocartilage regeneration are insufficient because of a lack of appropriate scaffold that can load large seeding-cells and induce chondrogenesis of stem cells. The acellular fibrocartilage scaffold (AFS) contains active growth factors as well as tissue-specific epitopes for cell-matrix interactions, which make it a potential scaffold for tissue-engineered fibrocartilage. A limitation to this scaffold is that its low porosity inhibits cells loading and infiltration. Here, inspired by book appearance, we sectioned native fibrocartilage tissue (NFT) into book-shape to improve cells loading and infiltration, and then decellularized with four protocols: (1) 2% SDS for 6-h, (2) 2% SDS for 24-h, (3) 4 SDS for 6-h, (4) 4% SDS for 24-h, followed by nuclease digestion. The optimal protocol was screened with respect to microstructures, DNA residence, native ingredients reservation, and chondrogenic inducibility of the AFS. In vitro studies demonstrated that this screened scaffold is noncytotoxicity and low-immunogenicity, allows adipose-derived stromal cells (ASCs) attachment and proliferation, shows superior chondrogenic inducibility, and stimulates collagen or glycosaminoglycans secretion. The underlying mechanism for this chondrogenic inducibility may be related to hedgehog pathway activating. Additionally, a novel pattern for fabricating tissue-engineered fibrocartilage was developed to enlarge seeding-cells loading, namely, cell-sheets sandwiched by book-shaped scaffold. In-vivo studies indicate that this screened scaffold alone could induce endogenous cells to satisfactorily regenerate fibrocartilage at 16-week, as characterized by fibrocartilaginous extracellular matrix (ECM) deposition and good interface integration. Interleaving this book-shaped AFS with autologous ASCs-sheets significantly enhanced its ability to regenerate fibrocartilage. Cell tracking demonstrated that fibrochondrocytes, osteoblasts, and osteocytes in the healing interface at postoperative 8-week partly originated from the sandwiched ASCs-sheets. On that basis, we propose the use of this book-shaped AFS and cell sheet technique for fabricating tissue-engineered fibrocartilage to improve bone–tendon healing.
Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin αvβ6 to activate the transforming growth factor (TGF)-β-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-β activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.
The regeneration of the blood vessel system post spinal cord injury (SCI) is essential for the repair of neurological function. As a significant means to regulate gene expression, epigenetic regulation of angiogenesis in SCI is still largely unknown. Here, we found that Ubiquitously Transcribed tetratricopeptide repeat on chromosome X (UTX), the histone H3K27 demethylase, increased significantly in endothelial cells post SCI. Knockdown of UTX can promote the migration and tube formation of endothelial cells. The specific knockout of UTX in endothelial cells enhanced angiogenesis post SCI accompanied with improved neurological function. In addition, we found regulation of UTX expression can change the level of microRNA 24 (miR-24) in vitro . The physical binding of UTX to the promotor of miR-24 was indicated by chromatin immunoprecipitation (ChIP) assay. Meanwhile, methylation sequencing of endothelial cells demonstrated that UTX could significantly decrease the level of methylation in the miR-24 promotor. Furthermore, miR-24 significantly abolished the promoting effect of UTX deletion on angiogenesis in vitro and in vivo . Finally, we predicted the potential target mRNAs of miR-24 related to angiogenesis. We indicate that UTX deletion can epigenetically promote the vascular regeneration and functional recovery post SCI by forming a regulatory network with miR-24.
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