Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells.

PURPOSE This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. METHODS hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. RESULTS IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. CONCLUSION Cxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM

Uncovering the secretome of mesenchymal stromal cells exposed to healthy, traumatic, and degenerative intervertebral discs: a proteomic analysis

Background: Mesenchymal stromal cells (MSCs) have been introduced as promising cell source for regenerative medicine. Besides their multilineage differentiation capacity, MSCs release a wide spectrum of bioactive factors. This secretome holds immunomodulatory and regenerative capacities. In intervertebral disc (IVD) cells, application of MSC secretome has been shown to decrease the apoptosis rate, induce proliferation, and promote production of extracellular matrix (ECM). For clinical translation of secretome-based treatment, characterization of the secretome composition is needed to better understand the induced biological processes and identify potentially effective secretomes. Methods: This study aimed to investigate the proteome released by bone marrow-derived MSCs following exposure to a healthy, traumatic, or degenerative human IVD environment by mass spectroscopy and quantitative immunoassay analyses. Exposure of MSCs to the proinflammatory stimulus interleukin 1β (IL-1β) was used as control. Results: Compared to MSC baseline secretome, there were 224 significantly up- or downregulated proteins following healthy, 179 following traumatic, 223 following degenerative IVD, and 160 proteins following IL-1β stimulus. Stimulation of MSCs with IVD conditioned media induced a more complex MSC secretome, involving more biological processes, compared to stimulation with IL-1β. The MSC response to stimulation with IVD conditioned medium was dependent on their pathological status. Conclusions: The MSC secretome seemed to match the primary need of the IVD: homeostasis maintenance in the case of healthy IVDs, versus immunomodulation, adjustment of ECM synthesis and degradation disbalance, and ECM (re) organization in the case of traumatic and degenerative IVDs. These findings highlight the importance of cell preconditioning in the development of tailored secretome therapies. The secretome of human bone marrow-derived mesenchymal stromal cells (MSCs) stimulated with intervertebral disc (IVD) conditioned medium was analyzed by proteomic profiling. Depending on the pathological state of the IVD, the MSC secretome protein composition indicated immunomodulatory or anabolic activity of the secretome. These findings may have implications for tailored secretome therapy for the IVD and other tissues

3D analysis of fatty infiltration of the paravertebral lumbar muscles using T2 images - a new approach.

PURPOSE Factors influencing paraspinal muscle degeneration are still not well understood. Fatty infiltration is known to be one main feature of the degeneration cascade. The aim of this cross-sectional study was to illustrate the 3D cluster of paraspinal lumbar muscle degeneration on T2-weighted MRI images using our newly developed software application 'iSix'. METHODS Mono- (Mm. rotatores), multi- (Mm. multifidus) and pluri-segmental (M. erector spinae) lumbar muscles groups were segmented on T2-weighted MR sequences using a novel computer-assisted technique for quantitative muscle/fat discrimination. The degree of fatty infiltration of the three predefined muscle groups was compared on a 3-dimensional basis, with regard to segment involvement and age. General linear models were utilized for statistical comparison. RESULTS N = 120 segments (age: 52.7; range 16-87 years) could be included. The overall relative fatty infiltration of the mono-segmental muscles was higher (21.1 14.5%) compared to the multi-segmental (16.0 8.8% p = 0.049) and pluri-segmental muscles (8.5 8.0%; p = 0.03). Mono-segmental muscles on the levels L4/5 (22.9 ± 10.2 [CI 17.6-28.2] %) and L5/S1 (27.01 ± 15.1 [CI 21.4-32.7] %) showed a significant higher amount of fat compared to the levels L2/3 (8.2 ± 6.8 [CI 2.2-14.2] %; L4/5 vs. L2/3, p = 0.03; L5/S1 vs. L2/3, p = 0.02) and L3/4 (13.2 ± 5.4 [CI 8.6-17.7]%; L4/5 vs. L3/4, p = 0.02; L5/S1 vs. L3/4, p < 0.01). Multivariate linear regression analyses revealed age and Pfirrmann grade as independent factors for fatty muscle degeneration. CONCLUSIONS 3D analysis of fatty infiltration is an innovative tool to study lumbar muscle degeneration. Mono-segmental muscles are more severely affected by degeneration compared to multi-/pluri-segmental muscles, especially at the L4/5 and L5/S1 level. Age and disc degeneration independently correlate with muscle degeneration