Low‐intensity pulsed ultrasound stimulation enhances TIMP‐1 in nucleus pulposus cells and MCP‐1 in macrophages in the rat

Recent studies have reported that low‐intensity pulsed ultrasound (LIPUS) stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells, and moreover promotes the secretion of MCP‐1 (monocyte chemotaxis protein‐1) from macrophages in a disc organ culture model. These findings suggest the possible application of LIPUS for biological repair of disc degeneration and herniation. Although the mechanisms involved are not well understood, several cytokine pathways may play a role. Therefore, in order to evaluate the effect of LIPUS stimulation on cytokine production by nucleus pulposus cells and macrophages, in vitro culture studies were designed. Nucleus pulposus cells and macrophages were collected from Sprague‐Dawley rats, cultured separately in a monolayer, and stimulated with LIPUS for 7 days. After culture, the culture medium and the cells were analyzed by cytokine array, RT‐PCR, and ELISA. Cytokine array showed that LIPUS stimulation significantly upregulated TIMP‐1 (tissue inhibitor of metalloproteinase‐1) in the nucleus pulposus and MCP‐1 in macrophages in comparison with the control. This was confirmed at the gene level by RT‐PCR in nucleus pulposus cells and macrophages after stimulation with LIPUS. Quantitative evaluation of these proteins by ELISA showed higher levels in nucleus pulposus cells and macrophages stimulated by LIPUS than in controls. These results showed that LIPUS stimulation significantly activated TIMP‐1 and MCP‐1 in nucleus pulposus cells and macrophages at both the protein and gene levels, suggesting that LIPUS may be a promising supplemental treatment for intervertebral disc herniation. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:865–871, 2008     

Hepatocyte growth factor/c‐met promotes proliferation,suppresses apoptosis,and improves matrix metabolism in rabbit nucleus pulposus cells in vitro

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti‐apoptotic and anti‐inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H₂O₂ or TNF‐α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase‐2 and matrix metalloproteinase‐3 and ‐9 by TNF‐α was significantly suppressed by HGF treatment. Expression of c‐Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF‐α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c‐Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:709–716, 2016.     

Human nucleus pulposus cells significantly enhanced biological properties in a coculture system with direct cell‐to‐cell contact with autologous mesenchymal stem cells

Activated nucleus pulposus (NP) cells can be reinserted into the disc to inhibit intervertebral disc degeneration. Experimental studies in animals showed that using a coculture system with direct cell‐to‐cell contact with mesenchymal stem cells (MSCs) significantly upregulated the biological activity of NP cells. The purpose of this study is to determine whether this activation of NP cells by autologous MSCs is applicable to human cells in vitro. Human NP tissue was obtained from surgical specimens and MSCs from bone marrow of 10 subjects. Six‐well culture plates and inserts were used for culture; 1.0 × 10⁴ NP cells were seeded onto each insert and incubated alone, in standard coculture with 1.0 × 10⁴ MSCs, or cocultured with direct cell‐to‐cell contact. NP cell proliferation, DNA synthesis, and proteoglycan (PG) synthesis were evaluated. Chromosome abnormalities in the activated NP cells and tumorigenesis of the cells were evaluated in an additional 10 patients by microscopic examination for segmented cells and histological assessment of activated cells transplanted into nude mice. Cell proliferation, DNA synthesis, and PG synthesis were significantly upregulated. The positive effects of the coculture system with direct cell‐to‐cell contact seen in animal studies were also confirmed in human cells. Chromosome abnormalities and tumorigenesis were not observed in the activated NP cells. In conclusion, a coculture system with direct cell‐to‐cell contact demonstrated a significant positive effect, enhancing the biological properties of human NP cells, as it did in animal models. These results should prove useful for conducting trials leading to the clinical use of activated NP cell transplantation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:623–630, 2010     

Impact of direct cell co‐cultures on human adipose‐derived stromal cells and nucleus pulposus cells

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose‐derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co‐cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co‐cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co‐culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up‐regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP‐1, TGF‐β1, IGF‐1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up‐regulated. Collectively, these results suggest that direct co‐cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1804–1813, 2013     

Response to tumor necrosis factor‐α mediated inflammation involving activation of prostaglandin E2 and Wnt signaling in nucleus pulposus cells

The cyclooxygenase 2 (COX‐2) product, prostaglandin E₂ (PGE₂), acts through a family of G protein‐coupled receptors designated E‐prostanoid (EP) receptors that mediate intracellular signaling by multiple pathways. However, it is not known whether crosstalk between tumor necrosis factor‐α(TNF‐α)–PGE₂‐mediated signaling and Wnt signaling plays a role in the regulation of intervertebral disc (IVD) cells. In this study, we investigated the relationship between TNF‐α–PGE₂ signaling and Wnt signaling in IVD cells. TNF‐α increased the expression of COX‐2 in IVD cells. The EP receptors EP1, EP3, and EP4 were expressed in IVD cells, and TNF‐α significantly increased PGE₂ production. Stimulation with TNF‐α also upregulated EP3 and EP4 mRNA and protein expression in IVD cells. The inductive effect of the EP3 and EP4 receptors on Topflash promoter activity was confirmed through gain‐ and loss‐of‐function studies using selective EP agonists and antagonists. PGE₂ treatment activated Wnt–β‐catenin signaling through activation of EP3. We conclude that TNF‐α‐induced COX‐2 and PGE₂ stimulate Wnt signaling and activate Wnt target genes. Suppression of the EP3 receptor via TNF‐α–PGE₂ signaling seems to suppress IVD degeneration by controlling the activation of Wnt signaling. These findings may help identify the underlying mechanism and role of Wnt signaling in IVD degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1756–1768, 2015.     

The role of leptin on the organization and expression of cytoskeleton elements in nucleus pulposus cells

Obesity is an important risk factor for intervertebral disc degeneration and leptin is a biomarker of obesity. However, the expression of leptin receptors has not been determined in disc tissue. It is not known whether leptin has a direct effect on the nucleus pulposus (NP) cells. To determine whether the NP tissues and cells express leptin receptors (OBRa and OBRb) and whether leptin affects the organization and the expression of major cytoskeletal elements in NP cells. Messenger RNA (mRNA) and protein levels of OBRa and OBRb were measured by real‐time PCR and Western blot, respectively, in NP tissues and cells. Immunofluorescence and real‐time PCR and Western blot were performed to investigate the effect of leptin on cytoskeleton reorganization and expression. Results show that mRNA and proteins of OBRa and OBRb were expressed in all NP tissues and cells, and that OBRb expression was correlated with patients' body weight. Increased expression of β‐actin and reorganization of F‐actin were evident in leptin‐stimulated NP cells. Leptin also induced vimentin expression but had no effect on β‐tubulin in NP cells. These findings provide novel evidence supporting the possible involvement of leptin in the pathogenesis of intervertebral disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 847–857, 2013     

Activation of rat nucleus pulposus cells by coculture with whole bone marrow cells collected by the perfusion method

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor‐β in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [³⁵S]‐sulfate and [³H]‐thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF‐β secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:222–228, 2009     

Integrin‐mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin‐mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM‐111 and LM‐511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM‐binding integrins was quantified for human NP cells using flow cytometry. The cell‐ECM attachment mechanism was determined by quantifying cell attachment to LM‐111, LM‐511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM‐111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell‐based therapeutics. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1661–1667, 2013     

水凝胶再生修复退变椎间盘的研究进展

目的总结生物材料水凝胶治疗椎间盘退变的研究进展,探讨水凝胶再生修复退变椎间盘的临床应用潜能。方法广泛查阅国内外有关水凝胶修复退变椎间盘尤其是髓核组织的研究文献,并进行总结和分析。结果水凝胶与髓核组织具有相似特性,在退变椎间盘再生修复中扮演重要角色,主要可体现在人工髓核假体、以水凝胶为载体的细胞治疗、非细胞治疗和组织工程修复。结论水凝胶作为生物材料广泛应用于椎间盘的再生修复治疗,为治疗椎间盘退变提供了新方向。     

Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR‐dependent pathway

Although glucosamine has been suggested to be effective in the treatment of osteoarthritis, its effect on disc degeneration remains unclear. We sought to explore whether glucosamine can activate autophagy in rat nucleus pulposus (NP) cells and protect cells treated with IL‐1β or hydrogen peroxide (H₂O₂). Autophagy in cells was examined by detecting for LC3, Beclin‐1, m‐TOR, and p70S6K, as well as by analyzing autophagosomes. To inhibit autophagy, 3‐methyladenine (3‐MA) was used. In the cells treated with IL‐1β, the levels of Adamts‐4, Mmp‐13, aggrecan, and Col2a1 were analyzed by real‐time PCR and immunofluorescence. Apoptosis was analyzed by TUNEL. Cell senescence under H₂O₂ was revealed by SA‐β‐Gal staining. Glucosamine could activate autophagy in a dose‐dependent manner within 24 h and inhibit the phosphorylation of m‐TOR and p70S6K. Autophagy in IL‐1β or H₂O₂‐treated cells was increased by glucosamine. Glucosamine attenuated the decrease of aggrecan and prevented the apoptosis of the NP cells induced by IL‐1β, whereas 3‐MA partly reversed these effects. The percentage of SA‐β‐Gal‐positive cells induced by H₂O₂ treatment was decreased by glucosamine, accompanied by the decline of p70S6K phosphorylation. Glucosamine protects NP cells and up‐regulates autophagy by inhibiting the m‐TOR pathway, which might point a potential therapeutic agent for disc degeneration. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1532–1542, 2014.     

Alterations of ADAMTSs and TIMP‐3 in human nucleus pulposus cells subjected to compressive load: Implications in the pathogenesis of human intervertebral disc degeneration

Intervertebral disc degeneration (IDD) pertains to the loss of extracellular matrix (ECM), particularly the early loss of aggrecan, the turnover of which is regulated by ADAMTSs. Amongst the etiological factors of IDD, mechanical stress plays an important role in the physiological and pathological processes of nucleus pulposus (NP) cells. However, the role of ADAMTSs and their inhibitor in human NP cells under mechanical stress has not been elucidated to date. The purpose of this study was to investigate the role of ADAMTSs and TIMP‐3 in NP cells under mechanical stress. Human NP cells isolated from non‐degenerative and degenerative discs were subjected to dynamic compressive load. The expression of ADAMTSs, aggrecan, and TIMP‐3 was detected by quantitative real‐time PCR and/or Western blot. Consequently, the gene expression of ADAMTS‐1, 4, and 5 increased significantly in loaded NP cells compared with not‐loaded cells from either non‐degenerative or degenerative discs, whereas the gene expression of aggrecan decreased significantly. Moreover, Western blot indicated increased protein levels of ADAMTSs‐1, 4, and 5. However, the expression of TIMP‐3 altered insignificantly. Together, this study is the first addressing the underlying mechanisms of compressive load as a contributing factor to IDD in terms of ADAMTSs. Our results suggest that compressive load leads to the increase in ADAMTS‐1, 4, and 5 that contributes to the decrease of aggrecan and IDD via TIMP‐3 independent machinery. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:267–273, 2012     

Prostaglandin E2 and prostaglandin F2α differentially modulate matrix metabolism of human nucleus pulposus cells

Prostaglandin (PG) actions on disc metabolism are unclear even though certain PGs are highly expressed by disc cells under inflammatory conditions and nonsteroidal anti‐inflammatory drugs (NSAIDs) are frequently used to block PG production to treat back pain. Hence this study aimed to (1) quantify gene expression of arachidonic acid cascade components responsible for PG synthesis and (2) examine the effects of key PGs on disc matrix homeostasis. Microarray analysis revealed that inflammatory stress increases expression of synthases and receptors for prostaglandin E2 (PGE₂) and prostaglandin F2α (PGF_2α), resulting in elevated PGE₂ and PGF_2α production in conditioned media of disc cells. PGE₂ diminished disc cell proteoglycan synthesis, in a dose‐dependent manner. Semiquantitative RT‐PCR revealed differential effects of PGE₂ and PGF_2α on disc cell expression of key matrix structural genes, aggrecan, versican, collagens type I and II. PGE₂ and PGF_2α also decreased message for the anabolic factor, IGF‐1. PGE₂ decreased mRNA expression for the anti‐catabolic factor TIMP‐1 while PGF_2α increased mRNAs for catabolic factors MMP‐1 and MMP‐3. Thus, PGE₂ and PGF_2α may have an overall negative impact on disc matrix homeostasis, and the use of NSAIDs may impact disc metabolism as well as treat back pain. Published by Wiley Periodicals, Inc. J Orthop Res 28:1259–1266, 2010     

Development of a bovine decellularized extracellular matrix‐biomaterial for nucleus pulposus regeneration

Painful intervertebral disc (IVD) degeneration is a common cause for spinal surgery. There is a clinical need to develop injectable biomaterials capable of promoting IVD regeneration, yet many available biomaterials do not mimic the native extracellular matrix (ECM) or promote matrix production. This study aimed to develop a decellularized injectable bovine ECM material that maintains structural and compositional features of native tissue and promotes nucleus pulposus (NP) cell (NPC) and mesenchymal stem cell (MSC) adaption. Injectable decellularized ECM constructs were created using 3 NP tissue decellularization methods (con.A: sodium deoxycholate, con.B: sodium deoxycholate & sodium dodecyl sulfate, con.C: sodium deoxycholate, sodium dodecyl sulfate & TritonX‐100) and evaluated for protein, microstructure, and for cell adaptation in 21 day human NPC and MSC culture experiments. Con.A was most efficient at DNA depletion, preserved best collagen microstructure and content, and maintained the highest glycosaminoglycan (GAG) content. NPCs in decellularized constructs of con.A&B demonstrated newly synthesized GAG production, which was apparent from “halos” of GAG staining surrounding seeded NPCs. Con.A also promoted MSC adaption with high cell viability and ECM production. The injectable decellularized NP biomaterial that used sodium deoxycholate without additional decellularization steps maintained native NP tissue structure and composition closest to natural ECM and promoted cellular adaptation of NP cells and MSCs. This natural decellularized biomaterial warrants further investigation for its potential as an injectable cell seeded supplement to augment NP replacement biomaterials and deliver NPCs or MSCs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:876–888, 2016.     

Rock信号通路抑制剂对大鼠髓核细胞生物学特性的影响

【摘要】 目的 探讨Rock信号通路抑制剂对大鼠髓核细胞的基质代谢及凋亡等生物学特性的影响。方法〓qPCR和western blot检测TNF-α与Rock通路抑制剂Y-27632对大鼠原代髓核细胞Cox2、MMP3、MMP13的表达调控;MTS细胞增殖试验检测不同浓度Y-27632刺激下对髓核细胞增殖的影响;Annexin/PI法检测Y-27632对髓核细胞凋亡的影响。结果〓予Rock抑制剂Y-27632刺激后,髓核细胞MMP13的表达水平上调(P<0.05),髓核细胞的凋亡率增加(P<0.05),但予Y-27632刺激后髓核细胞的增殖速度无明显差异(P>0.05)。结论〓Rock信号通路可能通过调控髓核细胞MMP13表达水平及其凋亡等生物学特性从而参与了椎间盘的退变。     

Autologous nucleus pulposus primes T cells to develop into interleukin‐4‐producing effector cells: An experimental study on the autoimmune properties of nucleus pulposus

An autoimmune response to herniated nucleus pulposus has been proposed to constitute a pathophysiologic mechanism for inducing sciatica based on the fact that nucleus pulposus under normal conditions is excluded from the development of immunological tolerance. The manifestation of an autoimmune response comprises different steps starting with antigen capture, continuing with activation of T helper (T_H) cells and ending with production of autoantibodies. Activated T_H cells differentiate into either T_H1 cells, predominately producing proinflammatory cytokines such as interferon γ (IFNγ) or a T_H2 subset mainly producing anti‐inflammatory cytokines such as interleukin‐4 (IL‐4). The aim of the present study was to examine if exposure of autologous nucleus pulposus (NP) to the immune system for 3 weeks is potent enough to prime T_H cells to differentiate into T_H2 cells. The study was performed in a pig model allowing the exposure of NP to the immune system. To assess the polarization of T_H cells the intracellular production of IFNγ and IL‐4 was measured in T cells by using flow cytometry. The revealed predominant production of IL‐4 together with low production of IFNγ in T cells after NP exposure to the immune system indicates that nucleus pulposus may prime T_H cells to develop into IL‐4‐producing T_H2 cells after being exposed to the immune system, for example, in association with disc herniation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:97–103, 2009