葡萄糖对体外培养椎间盘髓核细胞的影响

目的:探讨葡萄糖对体外培养髓核细胞的生物学特性的影响。方法:酶消化法分离培养正常椎间盘髓核细胞。对照组:DF12+20%FBS培养液(葡萄糖浓度1000mg/L)、无糖组:无糖DMEM+20%FBS(葡萄糖浓度0mg/L)培养液培养髓核细胞。HE染色观察细胞形态变化,计数板计数细胞总数,台盼蓝染色计算髓核细胞活性比率,流式细胞仪检测细胞凋亡率,Hoechst33258染色观察凋亡细胞核的变化。结果:两组培养液培养细胞形态大体正常,并无明显变化。对照组细胞总数明显多于无糖组。细胞活性率对照组也高于无糖组。Hoechst33258染色凋亡细胞,凋亡细胞核内可见致密的颗粒状和块状荧光,细胞核形态不规则,少数细胞核碎裂,部分细胞核呈月牙形。结论:葡萄糖对椎间盘髓核细胞的增殖及凋亡有显著的影响。     

PTEN promotes intervertebral disc degeneration by regulating nucleus pulposus cell behaviors

Intervertebral disc degeneration (IDD) is induced by multiple factors including increased apoptosis, decreased survival, and reduced extracellular matrix (ECM) synthesis in the nucleus pulposus (NP) cells. The tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the only known lipid phosphatase counteracting the PI3K/AKT pathway. Loss of PTEN leads to activated PI3K/AKT signaling, which plays a key role in a variety of cancers. However, the role of PTEN/PI3K/AKT signaling nexus in IDD remains unknown. Here, we report that PTEN is overexpressed in degenerative NP, which correlates with inactivated AKT. Using the PTEN knockdown approach by lentivirus‐mediated short interfering RNA gene transfer technique, we report that PTEN decreases survival but induces apoptosis and senescence of NP cells. PTEN also inhibits expression and production of ECM components including collagen II, aggrecan, and proteoglycan. Furthermore, PTEN modulates the expression of ECM regulatory molecules SOX‐9 and matrix metalloproteinase‐3 (MMP‐3). Using small‐molecule AKT inhibitor GDC‐0068, we confirm that PTEN regulates NP cell behaviors through its direct targeting of PI3K/AKT. These findings demonstrate for the first time that PTEN/PI3K/AKT signaling axis plays an important role in the pathogenesis of IDD. Targeting PTEN using gene therapy may represent a promising therapeutic approach against disc degenerative diseases.     

人颈椎间盘髓核细胞的体外培养和鉴定

目的：建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法：采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果：原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论：成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

人颈椎间盘髓核细胞的体外培养和鉴定

目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

Upregulation of P53 promotes nucleus pulposus cell apoptosis in intervertebral disc degeneration through upregulating NDRG2

P53 is an apoptosis marker which is involved in determining nucleus pulposus (NP) cell fate. Little is known about P53 interaction with N-Myc downstream-regulated gene 2 (NDRG2) in intervertebral disc degeneration (IVDD). Here, we studied the role of the P53-NDRG2 axis in IVDD. We found that NDRG2 was expressed in NP tissue obtained from patients with IVDD. The level of NDRG2 was positively related to the severity of IVDD, as determined by Pfirrmann grading. Subsequently, we overexpressed NDRG2 in human NP cells by adenoviral transfection and studied the effects of increased levels of NDRG2 on the viability and apoptosis of these cells. NDRG2 overexpression induced NP cell apoptosis and reduced viability in NP cells obtained from patient with IVDD. We also found that the level of P53 was elevated in NP cells from patients with IVDD and treatment with exogenous P53 upregulated NDRG2 in NP cells. Last, IVDD model was established in P53 knockout mice and the pathological changes in the intervertebral discs and NDRG2 expression were examined. P53 knockout can reduce the damage of NP tissues after IVDD surgery to some extent. Restoration of NDRG2 antagonized the effect of P53 knockout on IVDD. Collectively, this study suggests that elevated P53 in NP cells stimulates apoptosis of the cells by upregulating NDRG2 expression, thereby exacerbating IVDD.     

Retracted: HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.     

Overexpressed IGFBP5 promotes cell proliferation and inhibits apoptosis of nucleus pulposus derived from rats with disc degeneration through inactivating the ERK/MAPK axis

It is previously suggested that insulin-like growth factor binding proteins (IGFBPs) potentially share an association with disc degeneration (DD) that causes back pain. This study aimed at exploring the functional relevance of IGFBP5 in DD by establishing a rat model of DD. The nucleus pulposus (NP) cells were transduced with IGFBP5-shRNA or IGFBP5 overexpression to determine the cellular processes (proliferation, apoptosis, as well as colony formation). The protein levels of apoptosis-related proteins were evaluated. Furthermore, NP cells were treated with the extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK/MAPK) pathway inhibitor (PD98059) followed by measurement of ERK protein level and ERK phosphorylation content. The NP cells showed suppressed proliferation and colony formation ability, yet promoted apoptosis after transfection with IGFBP5-shRNA. It was found that silencing of IGFBP5 could lead to the ERK/MAPK axis activation, as indicated by an elevated ERK protein level and ERK phosphorylation content. However, overexpression of IGFBP5 could reverse all the reaction induced by silenced IGFBP5. These key findings demonstrate that overexpressed IGFBP5 inactivates the ERK/MAPK axis to stimulate the proliferation and inhibit apoptosis of NP cells in a rat model of DD.     

Mesenchymal stem cells deliver exogenous miR‐21 via exosomes to inhibit nucleus pulposus cell apoptosis and reduce intervertebral disc degeneration

Although mesenchymal stem cells (MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells (NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC‐derived exosomes (MSC‐exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of miRNAs in MSC‐exosomes and associated mechanisms for NPC apoptosis. MSC‐exosomes were isolated from MSC medium, and its anti‐apoptotic effect was assessed in a cell and rat model. The down‐regulated miRNAs in apoptotic NPCs were identified, and their contents in MSC‐exosomes were detected. The target genes of eligible miRNAs and possible downstream pathway were investigated. Purified MSC‐exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR‐21 were down‐regulated in apoptotic NPCs while MSC‐exosomes were enriched in miR‐21. The exosomal miR‐21 could be transferred into NPCs and alleviated TNF‐α induced NPC apoptosis by targeting phosphatase and tensin homolog (PTEN) through phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Intradiscal injection of MSC‐exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC‐derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR‐21 contained in exosomes. Exosomal miR‐21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration.     

Numerical analysis of the influence of nucleus pulposus removal on the biomechanical behavior of a lumbar motion segment

Nucleus replacement was deemed to have therapeutic potential for patients with intervertebral disc herniation. However, whether a patient would benefit from nucleus replacement is technically unclear. This study aimed to investigate the influence of nucleus pulposus (NP) removal on the biomechanical behavior of a lumbar motion segment and to further explore a computational method of biomechanical characteristics of NP removal, which can evaluate the mechanical stability of pulposus replacement. We, respectively, reconstructed three types of models for a mildly herniated disc and three types of models for a severely herniated disc based on a L4–L5 segment finite element model with computed tomography image data from a healthy adult. First, the NP was removed from the herniated disc models, and the biomechanical behavior of NP removal was simulated. Second, the NP cavities were filled with an experimental material (Poisson's ratio = 0.3; elastic modulus = 3 MPa), and the biomechanical behavior of pulposus replacement was simulated. The simulations were carried out under the five loadings of axial compression, flexion, lateral bending, extension, and axial rotation. The changes of the four biomechanical characteristics, i.e. the rotation degree, the maximum stress in the annulus fibrosus (AF), joint facet contact forces, and the maximum disc deformation, were computed for all models. Experimental results showed that the rotation range, the maximum AF stress, and joint facet contact forces increased, and the maximum disc deformation decreased after NP removal, while they changed in the opposite way after the nucleus cavities were filled with the experimental material.     

Characterization of Krt19CreERT allele for targeting the nucleus pulposus cells in the postnatal mouse intervertebral disc

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)-inducible Krt19^CreERT allele is available. The current study describes the characterization of Krt19^CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle-aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19^CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam-treated Krt19^CreERT/+; R26^tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19^CreERT allele for genetic characterization of NP cells at different stages of the mouse life.     

Chondrocyte‐like nested cells in the aged intervertebral disc are late‐stage nucleus pulposus cells

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.     

Carbohydrate sulfotransferase 3 (CHST3) overexpression promotes cartilage endplate-derived stem cells (CESCs) to regulate molecular mechanisms related to repair of intervertebral disc degeneration by rat nucleus pulposus

To investigate the regulatory effect of carbohydrate sulfotransferase 3 (CHST3) in cartilage endplate-derived stem cells (CESCs) on the molecular mechanism of intervertebral disc degeneration after nucleus pulposus repair in rats. We performed GO and KEGG analysis of GSE15227 database to select the differential genes CHST3 and CSPG4 in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration, IHC and WB to detect the protein profile of CHST3 and CSPG4, Co-IP for the interaction between CHST3 and CSPG4. Then, immunofluorescence was applied to measure the level of CD90 and CD105, and flow cytometry indicated the level of CD73, CD90 and CD105 in CESCs. Next, Alizarin red staining, Alcian blue staining and TEM were performed to evaluate the effects of CESCs into osteoblasts and chondroblasts, respectively, CCK8 for the cell proliferation of osteoblasts and chondroblasts after induction for different times; cell cycle of osteoblasts or chondroblasts was measured by flow cytometry after induction, and WB for the measurement of specific biomarkers of OC and RUNX in osteoblasts and aggrecan, collagen II in chondroblasts. Finally, colony formation was applied to measure the cell proliferation of CESCs transfected with ov-CHST3 or sh-CHST3 when cocultured with bone marrow cells, WB for the protein expression of CHST3, CSPG4 and ELAVL1 in CSECs, transwell assay for the migration of CESCs to bone marrow cells, TEM image for the cellular characteristics of bone marrow cells, and WB for the protein profile of VCAN, VASP, NCAN and OFD1 in bone marrow cells. CHST3 and CSPG4 were differentially expressed and interacted in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration; CD73, CD90 and CD105 were lowly expressed in CESCs, osteogenic or chondroblastic induction changed the characteristics, proliferation, cell cycle and specific biomarkers of osteoblasts and chondroblasts after 14 or 21 days,; CHST3 affected the cell proliferation, protein profile, migration and cellular features of cocultured CESCs or bone marrow cells. CHST3 overexpression promoted CESCs to regulate bone marrow cells through interaction with CSPG4 to repair the grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration.     

Bu-Shen-Huo-Xue-Fang modulates nucleus pulposus cell proliferation and extracellular matrix remodeling in intervertebral disk degeneration through miR-483 regulation of Wnt pathway

Intervertebral disk degeneration (IDD) has been widely considered as one of the main causes for low back pain, which can cause a severe impact to human health and huge economic burden to worldwide society. IDD pathogenesis can be affected by extensive degradation of extracellular matrix (ECM) and the hyperproliferation of nucleus pulposus (NP) cells. During the IDD process, expression of the ECM degradation enzymes matrix metalloproteinase and ADAMTS increases, whereas expression of ECM synthesis–related aggrecan and COL2A1 decreases. In addition, the Wnt signaling pathway is reportedly involved in the process of IDD. Bu-Shen-Huo-Xue-Fang (BSHXF), a Chinese traditional medicine formula that contains six Chinese traditional medicinal herbs, is widely used in the treatment of IDD. Herein, we obtained the serum containing BSHXF from BSHXF-fed rat and demonstrated that the BSHXF promoted NP cell proliferation and ECM synthesis through the Wnt signaling pathway. By using DIANA online tools and luciferase reporter gene assays, we confirmed that miR-483-3p and miR-23c regulated CTNNB1 and GSK3B, respectively, through direct targeting, thereby affecting the effect of BSHXF on NP cell proliferation and ECM synthesis through the Wnt signaling pathway. Taken together, we demonstrated the function and mechanism of BSHXF in regulating NP cell proliferation and ECM remodeling through the Wnt signaling pathway during IDD.     

Dynamic compression and co-culture with nucleus pulposus cells promotes proliferation and differentiation of adipose-derived mesenchymal stem cells

Adipose-derived stem cells (ASCs) are a set of multi potent stem cells potentially used in cartilage tissue engineering. We hypothesized that the effect of dynamic compression and co-culture with nucleus pulposus cells (NPCs) promotes ASC proliferation and chondrogenic differentiation. A controlled dynamic compression loading device was utilized to stimulate ASCs obtained from Sprague Dawley (SD) rats and identified by flow cytometry. The proliferation index was measured by carboxyfluorescein succinimidyl ester (CFSE) staining. Dynamic compression, as well as co-culture enhanced chondrogenic differentiation of ASCs as indicated by the expression of SOX-9, type-II collagen and aggrecan, which were measured by real-time PCR and Western blot. In our study, we found dynamic compression promoted the proliferation of ASCs and induced its differentiation into NP-like cells. Combination of dynamic compression and co-culture showed an additive effect on NP-like cell differentiation.     

Stiffness of photocrosslinked RGD-alginate gels regulates adipose progenitor cell behavior

Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long-term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell-microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD-alginate to investigate the adipogenic and pro-angiogenic potential of 3T3-L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca(2+) concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self-renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications.     

Serum‐free,chemically defined medium with TGF‐β3 enhances functional properties of nucleus pulposus cell‐laden carboxymethylcellulose hydrogel constructs

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum‐containing medium formulation was compared to a chemically defined, serum‐free medium to determine the effect on matrix elaboration and functional properties of NP cell‐laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor‐beta 3 (TGF‐β₃). Glycosaminoglycan (GAG) content increased in both TGF‐β₃‐treated groups and was highest for treated, serum‐free constructs (9.46 ± 1.51 µg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum‐containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum‐free constructs. Type II collagen content was greater in both serum‐free groups and highest in treated, serum‐free constructs. The equilibrium Young's modulus was highest in serum‐free samples supplemented with TGF‐β₃ (18.54 ± 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 ± 0.46 vs. 19.94 ± 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum‐free, TGF‐β₃ supplemented medium, indicating the importance of medium formulation in NP construct development. Biotechnol. Bioeng. 2010; 105: 384–395. © 2009 Wiley Periodicals, Inc.