The aim of this study was to systematically investigate the role of necroptosis in compression-induced rat nucleus pulposus (NP) cells death, as well as explore the underlying mechanisms involved. Rat NP cells underwent various periods of exposure to 1.0 MPa pressure. Cell viability and cell death were quantified by using cell counting kit-8 (CCK-8), and Calcein-AM/propidium iodine (PI) staining respectively. Necroptosis-associated target molecules receptor-interacing protein kinase 1 (RIPK1), phosphorylated RIPK1 (pRIPK1), receptor-interacing protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3) and mixed lineage kinase domain-like (MLKL) were analyzed by Western-blot and RT-PCR. NP cells were also examined for morphological and ultrastructural changes, which can indicate necroptosis. To indirectly establish the presence of necroptosis, the RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK'872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) were utilized. The results established necroptosis was taking place in NP cells. The level of necroptosis increased in a time-dependent manner, and this effect was reduced by Nec-1 in vitro. Additionally, NP cells death were significantly attenuated following treatment with Nec-1, GSK'872 or NSA. SiRNA-induced knockdown of RIPK3 or MLKL increased cell survival rate, while knockdown of RIPK1 resulted in a decreased cell survival rate. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis may play an important role in NP cells death induced by continuous mechanical stress. Treatment strategies which aim to regulate necroptosis may prove beneficial, by both reducing NP cells death and slowing IVD degeneration.

Objective: This paper aims to provide a comprehensive review of the changing role of transforming growth factor-b (TGF-b) signaling in intervertebral disc (IVD) health and disease. Methods: A comprehensive literature search was performed using PubMed terms 'TGF-b' and 'IVD'. Results: TGF-b signaling is necessary for the development and growth of IVD, and can play a protective role in the restoration of IVD tissues by stimulating matrix synthesis, inhibiting matrix catabolism, inflammatory response and cell loss. However, excessive activation of TGF-b signaling is detrimental to the IVD, and inhibition of the aberrant TGF-b signaling can delay IVD degeneration. Conclusions: Activation of TGF-b signaling has a promising treatment prospect for IVD degeneration, while excessive activation of TGF-b signaling may contribute to the progression of IVD degeneration. Studies aimed at elucidating the changing role of TGF-b signaling in IVD at different pathophysiological stages and its specific molecular mechanisms are needed, and these studies will contribute to safe and effective TGF-b signaling-based treatments for IVD degeneration.

Excessive compression, the main cause of intervertebral disc (IVD) degeneration, affected endogenous repair of the intervertebral disc. Pioglitazone (PGZ) is the agonist of peroxisome proliferator-activated receptor γ, which has been widely used in the treatment of diabetes mellitus. The present study aim at investigating whether pioglitazone has protective effects on compression-mediated cell apoptosis in nucleus pulposus mesenchymal stem cells (NP-MSCs) and further exploring the possible underlying mechanism. Our results indicated that the isolated cells satisfied the criteria of MSC stated by the International Society for Cellular Therapy. Besides, our research revealed that pioglitazone could protect cell viability, cell proliferation of NP-MSCs and alleviated the toxic effects caused by compression. The actin stress fibers was suppressed obviously under compression, and pioglitazone alleviated the adverse outcomes. Pioglitazone exerted protective effects on compression-induced NP-MSCs apoptosis according to annexin V/PI double-staining and TUNEL assays. Pioglitazone suppressed compression-induced NP-MSCs oxidative stress, including decreasing compression-induced overproduction of reactive oxygen species (ROS) and malondialdehyde (MDA), and alleviated compression-induced mitochondrial membrane potential (MMP) decrease. Ultrastructure collapse of the mitochondria exhibited a notable improvement by pioglitazone in compression-induced NP-MSCs according to transmission electron microscopy (TEM). Furthermore, the molecular results showed that pioglitazone significantly decreased the expression of apoptosis-associated proteins, including cyto.cytochrome c, Bax, cleaved caspase-9, and cleaved caspase-3, and promoted Bcl-2 expression. These results indicated that pioglitazone alleviated compression-induced NP-MSCs apoptosis by suppressing oxidative stress and the mitochondrial apoptosis pathway, which may be a valuable candidate for the treatment of IVD degeneration.

Intervertebral disc (IVD) degeneration is considered to be the primary reason for low back pain. Despite remarkable improvements in both pharmacological and surgical management of IVD degeneration (IVDD), therapeutic effects are still unsatisfactory. It is because of the fact that these therapies are mainly focused on alleviating the symptoms rather than treating the underlying cause or restoring the structure and biomechanical function of the IVD. Accumulating evidence has revealed that the endogenous stem/progenitor cells exist in the IVD, and these cells might be a promising cell source in the regeneration of degenerated IVD. However, the biological characteristics and potential application of IVD-derived stem/progenitor cells (IVDSCs) have yet to be investigated in detail. In this review, the authors aim to perform a review to systematically discuss (1) the isolation, surface markers, classification, and biological characteristics of IVDSCs; (2) the aging- and degeneration-related changes of IVDSCs and the influences of IVD microenvironment on IVDSCs; and (3) the potential for IVDSCs to promote regeneration of degenerated IVD. The authors believe that this review exclusively address the current understanding of IVDSCs and provide a novel approach for the IVD regeneration.

Tissue specificity, a key factor in the decellularized tissue matrix (DTM), has shown bioactive functionalities in tuning cell fate—e.g., the differentiation of mesenchymal stem cells. Notably, cell fate is also determined by the living microenvironment, including material composition and spatial characteristics. Herein, two neighboring tissues within intervertebral discs, the nucleus pulposus (NP) and annulus fibrosus (AF), were carefully processed into DTM hydrogels (abbreviated DNP-G and DAF-G, respectively) to determine the tissue-specific effects on stem cell fate, such as specific components and different culturing methods, as well as in vivo regeneration. Distinct differences in their protein compositions were identified by proteomic analysis. Interestingly, the fate of human bone marrow mesenchymal stem cells (hBMSCs) also responds to both culturing methods and composition. Generally, hBMSCs cultured with DNP-G (3D) differentiated into NP-like cells, while hBMSCs cultured with DAF-G (2D) underwent AF-like differentiation, indicating a close correlation with the native microenvironments of NP and AF cells, respectively. Furthermore, we found that the integrin-mediated RhoA/LATS/YAP1 signaling pathway was activated in DAF-G (2D)-induced AF-specific differentiation. Additionally, the activation of YAP1 determined the tendency of NP- or AF-specific differentiation and played opposite regulatory effects. Finally, DNP-G and DAF-G specifically promoted tissue regeneration in NP degeneration and AF defect rat models, respectively. In conclusion, DNP-G and DAF-G can specifically determine the fate of stem cells through the integrin-mediated RhoA/LATS/YAP1 signaling pathway, and this tissue specificity is both compositional and spatial, supporting the utilization of tissue-specific DTM in advanced treatments of intervertebral disc degeneration.

Purpose Mechanical loading plays a vital role in the progression of intervertebral disc (IVD) degeneration, but little is known about the effect of compression loading on human nucleus pulposus-derived mesenchymal stem cells (NP-MSCs). Thus, this study is aimed at investigating the effect of compression on the biological behavior of NP-MSCs in vitro. Methods Human NP-MSCs were isolated from patients undergoing lumbar discectomy for IVD degeneration and were identified by immunophenotypes and multilineage differentiation. Then, cells were cultured in the compression apparatus at 1.0 MPa for different times (0 h, 24 h, 36 h, and 48 h). The viability-, differentiation-, and differentiation-related genes (Runx2, APP, and Col2) and colony formation-, migration-, and stem cell-related proteins (Sox2 and Oct4) were evaluated. Results The results showed that the isolated cells fulfilled the criteria of MSC stated by the International Society for Cellular Therapy (ISCT). And our results also indicated that compression loading significantly inhibited cell viability, differentiation, colony formation, and migration. Furthermore, gene expression suggested that compression loading could downregulate the expression of stem cell-related proteins and lead to NP-MSC stemness losses. Conclusions Our results suggested that the biological behavior of NP-MSCs could be inhibited by compression loading and therefore enhanced our understanding on the compression-induced endogenous repair failure of NP-MSCs during IVDD.

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Pain is the major reason that patients suffering from osteoarthritis (OA) seek medical care. We found that vascular endothelial growth factors (VEGFs) mediate signaling in OA pain pathways. To determine the specific contributions of VEGFs and their receptors (VEGFRs) to joint pathology and pain transmission during OA progression, we studied intra-articular (IA) injections of VEGF ligands into murine knee joints. Only VEGF ligands specific for the activation of VEGFR1, but not VEGFR2, induced allodynia within 30 min. Interventions in OA by inhibitors of VEGFRs were done in vivo using a preclinical murine OA model by IA injections of selective inhibitors of VEGFR1/VEGFR2 kinase (pazopanib) or VEGFR2 kinase (vandetanib). OA phenotypes were evaluated using pain-associated murine behavioral tests and histopathologic analyses. Alterations in VEGF/VEGFR signaling by drugs were determined in knee joints, dorsal root ganglia, and spinal cord by immunofluorescence microscopy. Pazopanib immediately relieved OA pain by interfering with pain transmission pathways. Pain reduction by vandetanib was mainly due to the inhibition of cartilage degeneration by suppressing VEGFR2 expression. In conclusion, IA administration of pazopanib, which simultaneously inhibits VEGFR1 and VEGFR2, can be developed as an ideal OA disease-modifying drug that rapidly reduces joint pain and simultaneously inhibits cartilage degeneration.