Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte-like cells (CLCs). Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non-chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non-chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/β-catenin signaling in the healthy, NC-rich NP and early degenerated, CLC-rich NP of both breed types by immunohistochemistry of β-catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c-myc. Both NCs and CLCs showed nuclear and cytoplasmic β-catenin protein expression and axin2 gene expression, but β-catenin signal intensity and Wnt target gene expression were higher in the CLC-rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/β-catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c-myc gene expression. In conclusion, Wnt/β-catenin signaling activity in the NC within the NC-rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/β-catenin signaling activity in early IVD degeneration may be a reflection of its dual role.     

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types—notochordal cells (NCs) and non‐notochordal chondrocyte‐like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI‐positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin‐8‐ and galectin‐3‐co‐positive cells, identified as NCs. The proportion of TUNEL‐positive cells, which predominantly comprised non‐NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up‐regulation of ADAMTS‐5 in the 1‐day loaded group and MMP‐3 in the 7‐day loaded group. Aggrecan‐1 and collagen type 2α‐1 mRNA levels were down‐regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:455–463, 2014.     

Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL‐1β and p38 MAPK signal

Nucleus pulposus cells (NPCs) from degenerating disks produce catabolic and inflammatory factors, including interleukin (IL)‐1, nitric oxide (NO), prostaglandin E2 (PGE‐2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disk. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O₂ at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time quantitative polymerase chain reaction (Q‐PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL‐1β, NO, PGE‐2, MMP‐3, and TIMP‐1 in the conditioned media were quantified by enzyme‐linked immunosorbent assay (ELISA). Our data showed that HBO treatment decreased expression of IL‐1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE‐2, and MMP‐3, and increased TIMP‐1 expression in NPCs as compared with the atmospheric treatment. These results support the hypothesis that IL‐1β and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disk degeneration, and support our proposal that HBO treatment‐induced increase of the anabolic factor (TIMP‐1)/catabolic factor (MMP‐3) ratio may provide a therapeutic approach to slow the course of intervertebral disk degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:14–19, 2011     

Influence of age‐related degeneration on regenerative potential of human nucleus pulposus cells

Nucleus pulposus (NP) cells, sourced from herniation surgeries, may be used as a cell‐based therapy for intervertebral disc (IVD) degeneration. But, both the regenerative potential of these degenerative adult NP cells and how to stimulate optimum matrix synthesis is not yet clear. The purpose of the current study was to understand the different phenotypic behaviors between degenerative adult NP cells and normal adolescent NP cells. Degenerative adult NP cells produced a significantly higher amount of proteoglycans and collagens than adolescent cells. Insulin‐like growth factor‐1 was the only anabolic cytokine with increased endogenous expression in degenerative adult NP cells. TGF‐β1 treatment of degenerative NP cells promoted matrix synthesis but stimulated too much type I collagen and suppressed type II collagen and aggrecan. Adult degenerative NP cells possess upregulated regenerative potential, but stimulation in addition to TGF‐β1 is needed to enhance matrix productivity and optimize the collagen expression profile. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:379–383, 2010     

Fibronectin fragment activation of ERK increasing integrin α5 and β1 subunit expression to degenerate nucleus pulposus cells

Fibronectin fragments (Fn‐f), which are the breakdown products of fibronectin, accumulate in the disc during degeneration and are proved to induce the degeneration of intervertebral disc. The goal of this investigation was to determine the functional role of integrin α₅β₁, extracellular signal‐regulated kinase (ERK), and protein kinase C (PKC) in the process of Fn‐f degeneration nucleus pulposus (NP) cells. We found that Fn‐f (100 nM, 30 kDa) exposure led to degeneration of NP cells, up‐regulation of integrin α₅β₁ expression and phosphorylation of the ERK_1/2. After the expression of integrin α₅β₁ was silenced in NP cells, the phosphorylation of ERK_1/2 and the expression of MMP9, MMP13, and collagen II had no difference with control under the treatment of Fn‐f. Finally, when the inhibitor of ERK_1/2 and the inhibitor of PKC were added into the medium of NP cells; we found these two inhibitors could eliminate the effect of Fn‐f on NP cells. It is concluded that Fn‐f had the potential to enhance the NP cell degeneration in a vicious circle. And the integrin α₅β₁ subunit, ERK, and PKC were all included in this loop. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:556–561, 2011     

Synoviocytes are more sensitive than cartilage to the effects of minocycline and doxycycline on IL‐1α and MMP‐13‐induced catabolic gene responses

The objective of this study was to determine the primary articular tissue target of doxycycline and minocycline. Synoviocytes—cartilage cocultures (n = 4) were treated with MMP‐13 (25 ng/mL medium) or IL‐1 (1.0 ng/mL medium) for 24 h. Doxycycline (4.3, 0.43, 0.043 µM) or minocycline (10, 1.0 or 0.1 µM) were then added and cultures were continued for 96 h. Cartilage and media were analyzed for GAG content. Quantitative PCR was used to measure cartilage MMP‐3, MMP‐13, aggrecan, COL2A1, ADAMTS‐4, and ADAMTS‐5 expression, and synoviocyte MMP‐3, MMP‐13, ADAMTS‐4, and ADMATS‐5 expression. Total and active MMP‐3, MMP‐13, and ADAMTS 4/5 enzymes were measured in culture medium. All concentrations of doxycycline and minocycline diminished GAG accumulation in the media. All concentrations of minocycline, but only the highest concentration of doxycycline decreased MMP‐3 and MMP‐13 expression in synoviocytes but not cartilage, and basal ADAMTS‐5 mRNA levels in both synoviocytes and cartilage. Only minocycline decreased active MMP‐13 protein in synoviocytes. In summary, the protective effects of tetracycline compounds are more pronounced in synoviocytes than cartilage, and following minocycline compared to doxycycline. Studies to determine the molecular mechanism of action of the tetracyclines in synoviocytes might lead to the design of targeted therapeutics for the treatment of OA or RA. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:522–528, 2010     

Transdifferentiated circulating monocytes release exosomes containing 14‐3‐3 proteins with matrix metalloproteinase‐1 stimulating effect for dermal fibroblasts

Fibroblasts are major cellular components of healing wounds. In this regard, it remains to be fully understood how different paracrine signals may influence the final collagen/matrix metalloproteinase (MMP) balance in resident fibroblasts. Our previous reports have demonstrated that circulating stem cells and monocytes can be transdifferentiated into “keratinocyte‐like cells” under certain culture conditions. These transformed cells are able to stimulate MMP‐1 expression in dermal fibroblasts. However, the underlying mechanism of this cell‐to‐cell interaction is unknown. This study describes exosomes as a major delivery system that keratinocyte‐like cells use to release proteins into the conditioned media. The exosomes exhibited distinctive size, density, and saucer‐like morphology. Using PKH‐26 and GFP‐adenovirus infection, we demonstrated that exosomes are able to fuse and then release their protein content into dermal fibroblasts. Mass spectrometry and Western blotting identified five 14‐3‐3 isoforms (β, γ, ?, τ, and ζ) as MMP‐1 stimulating factors for dermal fibroblasts. Immunoprecipation assays confirmed that these 14‐3‐3 isoforms account for almost the entire MMP‐1 up‐regulation induced by exosomes. In summary, our results demonstrated that circulating monocytes stimulated to be transformed into “keratinocyte‐like cells” could promote an anti‐fibrogenic commitment of dermal fibroblasts via exosomal 14‐3‐3 proteins.     

Transforming Growth Factor β Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro

The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor β (TGFβ) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFβ1 or TGFβ3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFβ1 nor TGFβ3 were able to restore the notochordal phenotype. Although TGFβ1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020     

人转化生长因子β1重组腺病毒转染传代髓核细胞对细胞外基质合成的影响

目的探讨人转化生长因子融（transforming growthfactorβ，TGF-β1）对传代羊髓核细胞的细胞外基质（extracellular matrix，ECM）和DNA合成调节因子的作用。方法取1岁龄成年山羊腰椎间盘，体外分离培养羊髓核细胞，传至第3代后以携人TGF-β1（humanTGF-β1，hTGF-β1）或lacZ基因的复制缺陷型腺病毒（Ad／hTGF—β1及Ad／lacZ）感染，分别为实验组和阴性对照组；未加病毒液的细胞为空白对照组；原代髓核细胞为原代组。然后继续单层或藻酸钙凝胶三维（3-D）培养10d。对两种系统培养的细胞分别行DNA荧光定量、Westernblot分析和蛋白多糖（glycosaminoglycan，GAG）定量检测。结果DNA荧光定量显示，单层培养时实验组细胞的DNA合成显著高于两对照组（P〈0．05），藻酸钙凝胶3-D培养各组间比较差异无统计学意义（P〉0．05）。Western blot检测hTGF—β1、Ⅱ型胶原、Ⅰ型胶原和Aggrecan的表达显示，两种培养系统中，实验组hTGF—β1、Ⅱ型胶原和Aggrecan的表达均显著高于两对照组（P〈0．05），Ⅰ型胶原的表达显著低于两对照组（P〈0．05），实验组Ⅱ型胶原／Ⅰ型胶原比值较两对照组显著增高（P〈0．05）。GAG定量结果显示，两种培养系统中实验组细胞的GAG合成均显著高于两对照组（P〈0．05）。结论hTGF-β1在很大程度上可起到维持髓核细胞表型，并在细胞传代后仍发挥表型的调节作用。通过基因工程方法使髓核细胞表达hTGF—β1，有望遏制、甚至逆转椎间盘退变；而以Ad／hTGF—β1感染过的髓核细胞，在藻酸钙凝胶3-D培养系统中培养则表现出原始表型。     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

Effect of dose and release rate of CTGF and TGFβ3 on avascular meniscus healing

Meniscus tears in the avascular region rarely functionally heal due to poor intrinsic healing capacity, frequently resulting in tear propagation, followed by meniscus deterioration. Recently, we have reported that time‐controlled application of connective tissue growth factor (CTGF) and transforming tissue growth factor β3 (TGFβ3) significantly improved healing of avascular meniscus tears by inducing recruitment and step‐wise fibrocartilaginous differentiation of mesenchymal stem/progenitor cells (MSCs). In this study, we investigated effects of the dose of CTGF and the release rate of TGFβ3 on avascular meniscus healing in our existing explant model. Our hypothesis was that dose and release rate of CTGF and TGFβ3 are contributing factors for functional outcome in avascular meniscus healing by stem cell recruitment. Low (100 ng/ml) and high (1,000 ng/ml) doses of CTGF as well as fast (0.46 ± 0.2 ng/day) and slow (0.29 ± 0.1 ng/day) release rates of TGFβ3 were applied to our established meniscus explant model for meniscus tears in the inner‐third avascular region. The release rate of TGFβ3 was controlled by varying compositions of poly(lactic‐co‐glycolic acids) (PLGA) microspheres. The meniscus explants were then cultured for 8 weeks on top of mesenchymal stem/progenitor cells (MSCs). Among the tested combinations, we found that a high CTGF dose and slow TGFβ3 release are most effective for integrated healing of avascular meniscus, demonstrating improvements in alignment of collagen fibers, fibrocartilaginous matrix elaboration and mechanical properties. This study may represent an important step toward the development of a regenerative therapy to improve healing of avascular meniscus tears by stem cell recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1555–1562, 2019.     

非接触共培养脊索细胞诱导骨髓间充质干细胞向类软骨细胞分化的实验研究

目的:分离兔髓核脊索细胞(notochordal cells,NC)及骨髓间充质干细胞(mesenchymal stem cell,MSC),通过非接触共培养探讨NC对MSC细胞表型的影响。方法:4～6周龄新西兰兔8只,取胸腰段脊柱的髓核,用密度梯度离心提取NC,同时取其股骨骨髓用FICOLL液分离MSC,将NC和MSC等比例(1∶1)通过transwell培养板进行非接触共培养作为实验组,单纯MSC细胞培养作为对照组,光镜下观察细胞的生长情况。对两组的MSC行免疫组化及RT-PCR、Western-blot检测MSC细胞表型的改变情况。结果:原代NC呈圆形或椭圆形,细胞体积大,细胞增殖不明显;MSC贴壁生长,呈三角形或梭形,漩涡状排列。甲苯胺蓝染色:对照组MSC细胞核淡染,胞体染色不明显,染色阴性;实验组MSC可见从第3天开始胞体及胞外基质出现紫红色,第5天染色更加明显。Ⅱ型胶原免疫组化对照组MSC淡染,细胞形态不清楚;实验组第3天出现MSC内出现棕黄色深染,随着时间推移细胞染色加深呈阳性表现。RT-PCR检测,经过5d非接触共培养后实验组蛋白聚糖的基因表达为对照组的2.35倍(P<0.05),Ⅱ型胶原的基因表达为对照组的1.61倍(P<0.05),对照组Ⅰ型胶原的基因表达为实验组的2.56倍(P<0.05)。Western-blot检测后发现:经过5d非接触共培养,实验组蛋白聚糖的含量为对照组的1.61倍(P<0.05),Ⅱ型胶原的表达为对照组的10.04倍(P<0.05)(P<0.05)。结论:在非接触共培养条件下脊索细胞可以诱导骨髓间充质干细胞表型发生变化,向类软骨细胞方向分化,这将为组织工程化髓核的种子细胞筛选提供新选择。     

Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-α

STUDY DESIGN.: Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1β or TNF-α. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. OBJECTIVE.: To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-α actions in these cells. SUMMARY OF BACKGROUND DATA.: Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. METHODS.: NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1β or TNF-α. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. RESULTS.: IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-α stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-α. MMP-13 induction by TNF-α was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-α fourfold. CONCLUSION.: The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-α, thus, helping preserve disc composition, structure, and function.     

TGF‐β signaling regulates fibrotic expression and activity in carpal tunnel syndrome

Fibrosis of the subsynovial connective tissue (SSCT) is a predominant feature of carpal tunnel syndrome (CTS). While the nature of CTS has been extensively studied, little is known about the etiology of this disease. We investigated SSCT tissue from patients with CTS and control subjects using fibrosis arrays and cell culture analysis. Twofold changes in fibrotic gene expression were found in multiple genes from patient SSCT using fibrosis arrays. This data was confirmed via qRT‐PCR on a subset of genes; collagen I (Col1), collagen III (Col3), connective tissue growth factor (CTGF), transforming growth factor β (TGF‐β), and SMAD3 (P < 0.05) which significantly corroborate the fold changes found in the fibrosis arrays. To further explore the nature of SSCT fibrosis, cells were isolated from patient and control tissue. Col1, Col3, TGF‐β, and SMAD3 were highly expressed in patient SSCT fibroblasts as compared to control (P < 0.05). Further, fibrotic genes expression was decreased by inhibiting TGF‐β receptor I (TβRI) activity (P < 0.05). TGF‐β second messenger SMAD activity was significantly activated in SSCT fibroblasts from patients and this activation was abrogated by inhibiting TβRI signaling (P < 0.05). These findings suggest that blocking TGF‐β signaling may be an important therapeutic approach to treating the underlying fibrosis of SSCT in CTS patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1444–1450, 2014.     

Simulation of biological therapies for degenerated intervertebral discs

The efficacy of biological therapies on intervertebral disc repair was quantitatively studied using a three‐dimensional finite element model based on a cell‐activity coupled multiphasic mixture theory. In this model, cell metabolism and matrix synthesis and degradation were considered. Three types of biological therapies‐increasing the cell density (Case I), increasing the glycosaminoglycan (GAG) synthesis rate (Case II), and decreasing the GAG degradation rate (Case III)‐to the nucleus pulposus (NP) of each of two degenerated discs [one mildly degenerated (e.g., 80% viable cells in the NP) and one severely degenerated (e.g., 30% viable cells in the NP)] were simulated. Degenerated discs without treatment were also simulated as a control. The cell number needed, nutrition level demanded, time required for the repair, and the long‐term outcomes of these therapies were analyzed. For Case I, the repair process was predicted to be dependent on the cell density implanted and the nutrition level at disc boundaries. With sufficient nutrition supply, this method was predicted to be effective for treating both mildly and severely degenerated discs. For Case II, the therapy was predicted to be effective for repairing the mildly degenerated disc, but not for the severely degenerated disc. Similar results were predicted for Case III. No change in cell density for Cases II and III were predicted under normal nutrition level. This study provides a quantitative guide for choosing proper strategies of biological therapies for different degenerated discs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:699–708, 2016.