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Objective: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD)., Methods: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD., Results: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD., Conclusions: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Macrophage infiltration and polarization during lumbar intervertebral disc herniation (LDH) have attracted increased attention but their role remains unclear. To explore macrophage polarization in herniated nucleus pulposus (NP) tissue of patients with LDH and investigate the association between cell frequency and different clinical characteristics or symptoms, we conducted a retrospective study by analyzing NP tissue samples from 79 patients. Clinical features and symptoms, using the visual analog scale (VAS) and Oswestry disability index (ODI), were collected. The macrophage markers CD68, CCR7, CD163, and CD206; pro-inflammatory cytokine TNF-α; and anti-inflammatory factor IL-4 were analyzed by immunohistochemistry. The frequency of polarized macrophages and positivity rate of pro- and anti-inflammatory cytokines showed significant differences in some of clinical characteristics. Specifically, higher CCR7 + and TNF-α + proportions were identified in the high-intensity zone (HIZ) and the type of extrusion and sequestration NP tissue than in non-HIZ and protrude NP tissue. Higher CD206 + and IL-4+ proportion were detected in Modic changes. However, no differences in gender, age, smoking status, Pfirrmann grade, analgesic use, leg pain duration, and segments were found between groups. CD68 + , CCR7 + , and CD206 + cell proportions, and TNF-α and IL-4 showed positive associations with VAS scores preoperation. Associations between ODI and the macrophages markers were weak/insignificant. Our results indicated that macrophage polarization or macrophage-like cells contribute to LDH pathological features. Macrophage populations displaying significant associations with VAS score reflected continuous M1/M2 transition contributing to pain during LDH. These findings may contribute to enhanced/personalized pharmacological interventions for patients with LDH considering pain heterogeneity., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Inflammation is the primary pathological phenomenon associated with disc degeneration; the inflammatory cytokine tumor necrosis factor (TNF-α) plays a crucial role in this pathology. The anti-inflammatory and regenerative effects of M2 macrophages on nucleus pulposus cells (NPCs) in intervertebral disc degeneration (IDD) progression remain unknown. Here, M2 conditioned medium (M2CM) was harvested and purified from human acute monocytic leukaemia cell line (THP-1) cells and mouse peritoneal macrophages, respectively; it was used for culturing human NPCs and a mouse intervertebral disc (IVD) organ culture model. NPCs and IVD organ models were divided into three groups: group 1 treated with 10% fetal bovine serum (control); group 2 treated with 10 ng/ml TNF-α; and group 3 treated with 10 ng/ml TNF-α and M2CM (coculture group). After 2-14 days, cell proliferation, extracellular matrix synthesis, apoptosis, and NPC senescence were assessed. Cell proliferation was reduced in TNF-α-treated NPCs and inhibited in the M2CM co-culture treatment. Moreover, TNF-α treatment enhanced apoptosis, senescence, and expression of inflammatory factor-related genes, including interleukin-6, MMP-13, ADAMTS-4, and ADAMTS-5, whereas M2CM coculture significantly reversed these effects. In addition, co-culture with M2CM promoted aggrecan and collagen II synthesis, but reduced collagen Iα1 levels in TNF-α treatment groups. Using our established three-dimensional murine IVD organ culture model, we show that M2CM suppressed the inhibitory effect of TNF-α-rich environment. Therefore, co-culture with M2CM promotes cell proliferation and extracellular matrix synthesis and inhibits inflammation, apoptosis, and NPC senescence. This study highlights the therapeutic potential of M2CM for IDD., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: Cell therapy for the treatment of intervertebral disc degeneration (IDD) faces serious barriers since tissue-specific adult cells such as nucleus pulposus cells (NPCs) have limited proliferative ability and poor regenerative potential; in addition, it is difficult for exogenous adult stem cells to survive the harsh environment of the degenerated intervertebral disc. Endogenous repair by nucleus pulposus mesenchymal stem cells (NPMSCs) has recently shown promising regenerative potential for the treatment of IDD. Notochordal cells (NCs) and NC-conditioned medium (NCCM) have been proven to possess regenerative ability for the treatment of IDD, but this approach is limited by the isolation and passaging of NCs. Our previous study demonstrated that modified notochordal cell-rich nucleus pulposus (NC-rich NP) has potential for the repair of IDD. However, whether this can protect NPMSCs during IDD has not been evaluated., Methods: In the current study, tumor necrosis factor (TNF)-α was used to mimic the inflammatory environment of IDD. Human NPMSCs were cocultured with NC-rich NP explants from healthy rabbit lumbar spine with or without TNF-α. Cell proliferation and senescence were analyzed to investigate the effect of NC-rich NP explants on TNF-α-treated NPMSCs. The expression of mRNA encoding proteins related to matrix macromolecules (such as aggrecan, Sox-9, collagen Iα, and collagen IIα), markers related to the nucleus pulposus cell phenotype (including CA12, FOXF1, PAX1, and HIF-1α), and senescence markers (such as p16, p21, and p53), senescence-associated proinflammatory cytokines (IL-6), and extracellular proteases (MMP-13, ADAMTS-5) was assessed. The protein expression of CA12 and collagen II was also evaluated., Results: After a 7-day treatment, the NC-rich NP explant was found to enhance cell proliferation, decrease cellular senescence, promote glycosaminoglycan (GAG), collagen II, and CA12 production, upregulate the expression of extracellular matrix (ECM)-related genes (collagen I, collagen II, SOX9, and ACAN), and enhance the expression of nucleus pulposus cell (NPC) markers (HIF-1α, FOXF1, PAX1, and CA12)., Conclusion: Modified NC-rich NP explants can attenuate TNF-α-induced degeneration and senescence of NPMSCs in vitro. Our findings provide new insights into the therapeutic potential of NC-rich NP for the treatment of IDD.

Recent studies suggested that notochordal cells (NCs) and NC-conditioned medium (NCCM) can stimulate cell viability and matrix production of nucleus pulposus cells (NPCs). However, the potential of notochordal cell-rich nucleus pulposus (NRNP) incorporating the native environment of the intervertebral disc (IVD) has not been evaluated. The objective of this study was to develop an optimal NRNP model and test whether it can allow a significant level of NPC activation in vitro. Rabbit NRNP explants were divided into three groups according to different digestion time: digestion NRNP of 8 h, partial digestion NRNP of 2 h, and natural NRNP. Cell viability and NC phenotype were compared between these groups after 14 days of incubation. The products of the selected partial digestion NRNP group were then cocultured with human degenerated NPCs for 14 days. NPC viability, cell proliferation and senescence, the production of glycosaminoglycan (GAG) found in extracellular matrix, and NP matrix production by NPCs were assessed. The results showed that coculturing with partial digestion NRNP significantly improved the cell proliferation, cell senescence, and disc matrix gene expression of NPCs compared with those in the monoculture group. In addition, GAG/DNA ratio in the coculture group increased significantly, while the level of collagen II protein remained unchanged. In this study, we demonstrated that partial digestion NRNP may show a promising potential for NPC regeneration in IVD tissue engineering.

Background: Eliminating the symptoms during treatment of intervertebral disc degeneration (IVDD) is only a temporary solution that does not cure the underlying cause. A biological method to treat this disorder may be possible by the newly discovered nucleus pulposus derived stem cells (NPDCs). However, the uncertain characteristics and potential of NPDCs calls for a comprehensive study., Methods: In the present study, nucleus pulposus samples were obtained from 5 patients with IVDD undergoing discectomy procedure and NPDCs were harvested using fluorescence activated cell sorting (FACS) by the co-expression of GD2 + and Tie2 + . After in vitro expansion, the properties of NPDCs were compared with those of bone marrow mesenchyme stem cells (BMSCs) from the same subjects., Results: NPDCs performed similar properties in cell colony-forming ability, cell proliferation rate, cell cycle and stem cell gene expression similar to those of BMSCs. In addition, NPDCs could be differentiated into osteoblasts, adipocytes, and chondrocytes, and are found to be superior in chondrogenesis but inferior in adipocyte differentiation., Conclusions: NPDCs derived from the degenerated intervertebral disc still keep the regeneration ability similar to BMSCs. Besides, the superior capacity in chondrogenesis may provide a promising cell candidate for cell-based regenerative medicine and tissue engineering in IVDD.

Background: Low back pain or sciatic pain because of lumbar intervertebral disc herniation (LDH) is caused by mechanical compression and/or an inflammatory component on the nerve root. However, it is difficult to define to what extent each component contributes to the pain. This study attempted to explore the effects of macrophage polarization on clinical symptoms in patients experiencing LDH after surgery, and investigated the association between macrophage cell percentages and clinical efficacy. Methods: This study retrospectively harvested nucleus pulposus (NP) tissue samples from 117 patients. Clinical symptoms and efficacy using the visual analog scale (VAS) and Oswestry Disability Index (ODI) were evaluated at different time points preoperatively and postoperatively. CD68, CCR7, CD163, and CD206 were selected as macrophage phenotypic markers. Results: Seventy‐six samples showed positive expression of macrophage markers in NP samples of patients with LDH, whereas 41 patients displayed negative results. No significant differences were detected between the two groups, involvement of several demographic data, and preoperative clinical findings. With respect to the macrophage‐positive group, no significant correlation was detected between the positive rate of the four markers and the VAS score or ODI after surgery. However, patients with NP samples positive for CD68 and CCR7 expression showed significantly lower VAS scores 1 week after surgery compared with those in the negative group. Moreover, the improvement in VAS score showed a strong positive correlation with CD68‐ and CCR7‐positive cell percentages. Conclusions: Our results indicated that pro‐inflammatory M1 macrophages may be associated with the reduction of chronic pain after surgery. Therefore, these findings contribute to better personalized pharmacological interventions for patients with LDH, considering the heterogeneity of pain. [ABSTRACT FROM AUTHOR]

Background: Eliminating the symptoms during treatment of intervertebral disc degeneration (IVDD) is only a temporary solution that does not cure the underlying cause. A biological method to treat this disorder may be possible by the newly discovered nucleus pulposus derived stem cells (NPDCs). However, the uncertain characteristics and potential of NPDCs calls for a comprehensive study.Methods: In the present study, nucleus pulposus samples were obtained from 5 patients with IVDD undergoing discectomy procedure and NPDCs were harvested using fluorescence activated cell sorting (FACS) by the co-expression of GD2+ and Tie2+. After in vitro expansion, the properties of NPDCs were compared with those of bone marrow mesenchyme stem cells (BMSCs) from the same subjects.Results: NPDCs performed similar properties in cell colony-forming ability, cell proliferation rate, cell cycle and stem cell gene expression similar to those of BMSCs. In addition, NPDCs could be differentiated into osteoblasts, adipocytes, and chondrocytes, and are found to be superior in chondrogenesis but inferior in adipocyte differentiation.Conclusions: NPDCs derived from the degenerated intervertebral disc still keep the regeneration ability similar to BMSCs. Besides, the superior capacity in chondrogenesis may provide a promising cell candidate for cell-based regenerative medicine and tissue engineering in IVDD. [ABSTRACT FROM AUTHOR]