Human Nucleus Pulposus Cell Line Research Articles

Effects of Fluid Shear Stress on Human Intervertebral Disc Nucleus Pulposus Cells Based on Label-Free Quantitative Proteomics.

Objective To explore the possible mechanism of fluid shear stress on human nucleus pulposus cells based on label-free proteomics technology. Methods The human nucleus pulposus cell line was purchased and subcultured in vitro. The Flexcell STR-4000 multiflow field cell fluid shear stress loading culture system was used to apply continuous laminar fluid shear stress (12 dyne/cm2, 45 mins) to the monolayer adherent cells. Those without mechanical loading were used as the control group, and those subjected to fluid shear loading were used as the experimental group. Differential protein expression was identified using mass spectrometry identification technology, and bioinformatics analysis was performed using Gene Ontology GO (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes KEGG (Kyoto Encyclopedia of Genes and Genomes). Results The proteomics results of the experimental group and the control group showed that the total number of mass spectra was 638653, the number of matched mass spectra was 170110, the total number of identified peptides was 32050, the specific peptide was 30564, and the total number of identified proteins was 4745. Comparing the two groups, 47 proteins were significantly differentially expressed, namely, 25 upregulated proteins and 22 downregulated proteins. Bioinformatics analysis showed that significantly different proteins were mainly manifested in cellular process, biological regulation, metabolic process, binding, catalytic activity, cellular components (cell part), organelle part (organelle part), and other molecular biological functions. Conclusion Using proteomics technology to screen human nucleus pulposus cells after fluid shear stress loading, the differential protein expression provides a basis for further exploration of the mechanism of mechanical factors on nucleus pulposus.

Mechanical loading mediates human nucleus pulposus cell viability and extracellular matrix metabolism by activating of NF-κB.

Lower back pain is one of the most frequent complaints in US orthopedic outpatient departments. Intervertebral disc degeneration (IDD) is an important cause of lower back pain. Previous studies have found that mechanical loading was associated with IDD, but the underlying mechanism remains unclear. In the present study, a human nucleus pulposus cell line was used to establish an in vitro mechanical loading model. Mechanical loading, western blot analysis, quantitative PCR, ELISA, cell viability assay and IHC staining were used in the current study. It was found that a short loading time of 4 h followed by a long period of rest (20 h) exerted protective effects against matrix degradation in nucleus pulposus cells, whilst a longer loading time of 20 h followed by a shorter period of rest (4 h) resulted in cell apoptosis and extracellular matrix (ECM) degradation. Excessive mechanical loading may induce ECM degradation by activation of the NF-κB signaling pathway. Taken together, these findings demonstrated that whilst moderate mechanical loading exerted beneficial effects on nucleus pulposus cells, excessive mechanical loading inhibited human nucleus pulposus cell viability and promoted ECM degradation by activating NF-κB.

Role of miR-589-3p in human lumbar disc degeneration and its potential mechanism.

The present study aimed to investigate the role of miR-589-3p in lumbar disc degeneration (LDD) and to explore the underlying mechanisms. Nucleus pulposus (NP) cells were stimulated with lipopolysaccharide (LPS) to simulate an in vitro model of intervertebral disc degeneration. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of microRNA (miR)-589-3p in the NP cells, and the results demonstrated that the increased expression of miR-589-3p in LPS stimulated NP cells compared with the control. To further investigate the role of miR-589-3p in LDD, a human NP cell line with high/low miR-589-3p expression was generated using miR-589-3p mimics/inhibitors. In addition, a human NP cell inflammation model was conducted by LPS (10 µM) treatment. Western blot analysis and RT-qPCR were performed for detection of associated genes and proteins. Protein levels of pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were evaluated by ELISA. Flow cytometry was used for cell apoptosis determination. Furthermore, Targetscan was used to predict potential targets of miR-589-3p, and a dual luciferase reporter assay was used to verify the prediction. The findings verified that miR-589-3p was significantly upregulated in LDD. In vitro, miR-589-3p mimics/inhibitors significantly increased/reduced the production of TNF-α, IL-1β and IL-6 in LPS stimulated NP cells. Furthermore, miR-589-3p mimics/inhibitors significantly promoted/inhibited LPS stimulated NP cell apoptosis. MiR-589-3p mimics/inhibitors significantly repressed/enhanced type II collagen and aggrecan expression in LPS stimulated NP cells. In addition, it was demonstrated that mothers against decapentaplegic homolog (Smad) 4 was a direct target gene of miR-589-3p, and was negatively regulated by miR-589-3p in NP cells. In conclusion, miR-589-3p may function as a promoter in LDD development via the regulation of Smad4.

Inhibition of miR‑27a suppresses the inflammatory response via the p38/MAPK pathway in intervertebral disc cells

The current study aimed to investigate the role of miR-27a in intervertebral disc degeneration (IDD) and to examine the underlying mechanisms. Quantitative polymerase chain reaction (qPCR) was performed to detect the expression level of miR-27a in the nucleus pulposus (NP) tissues of patients with IDD, and the results revealed an increasing expression of miR-27a in IDD compared with the control. To further investigate the role of miR-27a in IDD, a stable human NP cell line with low miR-27a expression was generated by transfecting cells with a lentiviral antigomiR-27a inhibitor. In addition, a human NP cell inflammation model was established by lipopolysaccharide (LPS; 10 µM) stimulation. The miR-27a expression in NP cells was determined by qPCR, while the expression of its target proteins; p-p38 and nuclear factor (NF-κB) was measured by western blot analysis. Furthermore, the mRNA and protein expression levels of proinflammatory factors, including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α), were also evaluated by qPCR and ELISA, respectively. The current results confirmed that miR-27a was significantly upregulated in IDD. In vitro, downregulation of miR-27a in LPS-stimulated NP cells by transfection with the miR-27a inhibitor resulted in suppression of p-p38 and NF-κB expression levels. Furthermore, the production of the proinflammatory factors IL-1β, IL-6 and TNF-α was significantly reduced in LPS-stimulated NP cells with downregulated miR-27a. In conclusion, miR-27a may function as a promoter in IDD development, while inhibition of miR-27a may suppress proinflammatory factors released by intervertebral disc cells by regulating the p38/mitogen-activated protein kinase (MAPK) signaling pathway.

Collagen type II is downregulated in the degenerative nucleus pulposus and contributes to the degeneration and apoptosis of human nucleus pulposus cells.

Degenerative disc disease (DDD) is a common degenerative condition initiated mainly within the nucleus pulposus (NP). To date, the etiopathogenesis of DDD remains unclear, and because no effective therapeutic strategies are available to target its pathological processes, DDD is still treated with symptomatic interventions that are far from adequate. Collagen type II is one of the major matrix components of the NP, and is considered to be essential to NP homeostasis. However, the specific mechanisms by which collagen type II influences NP cells remain unknown. In the present study, collagen type II expression was detected using immunohistochemistry analysis and quantitative polymerase chain reaction, and it was demonstrated to be significantly downregulated in NP tissues from patients with DDD compared with nondegenerative controls. To further explore the mechanism in vitro, interleukin (IL)-1β stimulation was used to induce degeneration of a human NP cell line. IL-1β stimulation upregulated both the mRNA and protein levels of the catabolic markers matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), while it downregulated the anabolic makers aggrecan and collagen type II. However, addition of purified collagen type II prevented this IL-1β-induced metabolic disturbance of the NP cells. Furthermore, IL-1β stimulation significantly promoted apoptosis in NP cells, while collagen type II treatment decreased the apoptotic rate and the protein levels of cleaved caspase-3. In conclusion, collagen type II exhibited protective effects in suppressing NP cell degeneration through its anticatabolic, proanabolic and antiapoptotic effects, suggesting that it may be a promising therapeutic agent for the prevention and treatment of DDD.

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.

Overexpression of hTERT extends replicative capacity of human nucleus pulposus cells, and protects against serum starvation‐induced apoptosis and cell cycle arrest

The nucleus pulposus (NP) cells are chondrocyte-like cells that are required for the resistance of compressive loads through the synthesis of collagen fibrils and proteoglycan aggrecans, and the generation of a hydrostatic swelling pressure, and thus play an important role in the intervertebral disc. Here, we report the production and characterization of an immortalized human NP cell line from normal NP cells using stable transfection of recombinant human telomerase reverse transcriptase (hTERT) gene. The hTERT-transfected NP cells exhibited morphological characteristics typical of native cells. When compared with the first generation of normal NP cells, the hTERT-transfected NP cells grew faster and had an increased level of IGF-1 and TGF-β gene expression. They were successfully passaged over 20 generations without significant change in the levels of type II collagen and proteoglycan aggrecan expression. In addition, they showed resistance to serum starvation-induced apoptosis, G1 cell cycle arrest, and gene expression of p53, CCNE1, Fas, and Caspase 3. Moreover, histology revealed that no tumorigenicity of NP cells over expressing hTERT was observed after they were implanted in nude mice. Taken together, an immortalized human NP cell line was established, which has an extended lifespan, retains phenotypic features similar to primary parent NP cells, and should provide a suitable model for studying the biology of NP cells.

Low-intensity pulsed ultrasound stimulates cell proliferation, proteoglycan synthesis and expression of growth factor-related genes in human nucleus pulposus cell line

Low-intensity pulsed ultrasound (LIPUS) stimulation has been shown to effect differentiation and activation of human chondrocytes. A study involving stimulation of rabbit disc cells with LIPUS revealed upregulation of cell proliferation and proteoglycan (PG) synthesis. However, the effect of LIPUS on human nucleus pulposus cells has not been investigated. In the present study, therefore, we investigated whether LIPUS stimulation of a human nucleus pulposus cell line (HNPSV-1) exerted a positive effect on cellular activity. HNPSV-1 cells were encapsulated in 1.2% sodium alginate solution at 1x10(5) cells/ml and cultured at 10 beads/well in 6-well plates. The cells were stimulated for 20 min each day using a LIPUS generator, and the effects of LIPUS were evaluated by measuring DNA and PG synthesis. Furthermore, mRNA expression was analyzed by cDNA microarray using total RNA extracted from the cultured cells. Our study revealed no significant difference in cell proliferation between the control and the ultrasound treated groups. However, PG production was significantly upregulated in HNPSV cells stimulated at intensities of 15, 30, 60, and 120 mW/cm(2) compared with the control. The results of cDNA array showed that LIPUS significantly stimulated the gene expression of growth factors and their receptors (BMP2, FGF7, TGFbetaR1 EGFRF1, VEGF). These findings suggest that LIPUS stimulation upregulates PG production in human nucleus pulposus cells by the enhancement of several matrix-related genes including growth factor-related genes. Safe and non-invasive stimulation using LIPUS may be a useful treatment for delaying the progression of disc degeneration.

Feasibility of Using a Human Nucleus Pulposus Cell Line as a Cell Source in Cell Transplantation Therapy for Intervertebral Disc Degeneration

Assessment of the potential use of an immortalized human nucleus pulposus cell line as an alternative cell source in cell transplantation therapy for intervertebral disc degeneration. To evaluate the effect of transplanting the human nucleus pulposus cell line into a disc degeneration model in rabbits and to define whether it is capable of becoming an alternative cell source for cell transplantation therapy for disc degeneration. Interest in cell transplantation therapy for disc degeneration has been growing for several years, and a range of different cell types have been examined as possible donor cells. In addition, the establishment of a novel cell line that possesses some of the major characteristics of a normal human nucleus pulposus cells has been reported. Human nucleus pulposus cell line was established, and cells were transplanted into a rabbit disc degeneration model. At 4, 8, and 24 weeks after transplantation, inhibition of intervertebral disc degeneration was assessed by examining the disc height, macroscopic appearance, histologic findings, and immunohistochemistry. In addition, aggrecan, versican, and Type II collagen gene expression in the nucleus pulposus were measured semiquantitatively at the mRNA level. Furthermore, the survival of transplanted cells was examined using immunohistochemistry for Simian Virus 40 T antigen, and the presence of graft-versus-host reaction was assessed by immunohistochemistry for CD4 and CD58. The disc height was significantly greater in the transplanted group than in the degenerative group's disc from 4 weeks' posttransplantation. Macroscopically, the nucleus pulposus was absent and there was loss of disc height in the degenerative group at 24 weeks after transplantation, whereas the nucleus pulposus was preserved in the transplanted group. Histologic examination showed that the structure of the inner anulus fibrosus was significantly preserved in the transplanted group, and the boundary between the nucleus and anulus could be clearly visualized. Expression of mRNAs of the nucleus pulposus matrix, aggrecan, and Type II collagen was significantly greater in the transplanted group than in the degenerative group. This indicates that transplantation of human nucleus pulposus cell line helped to preserve the matrix of the nucleus pulposus. Thus, transplantation of a human nucleus pulposus cell line was shown to delay disc degeneration in this rabbit model. The human nucleus pulposus cell line may become an alternative cell source for cell transplantation therapy of intervertebral disc degeneration.

Atelocollagen for culture of human nucleus pulposus cells forming nucleus pulposus-like tissue in vitro: Influence on the proliferation and proteoglycan production of HNPSV-1 cells

Nucleus pulposus (NP) is responsible for maintaining function and structure of the disc. Scaffolds to culture disc cells three-dimensionally are emphasized in recent reports on development of a new method for treating disc degeneration using cell transplantation and tissue engineering. Among artificial scaffolds and cell carrying materials, Atelocollagen is a collagen gel that has an advantage in safety issues over others. However, to date there has been no study that investigated culture of human nucleus pulposus cells in Atelocollagen. To investigate whether Atelocollagen could be used as a culture scaffold and if it has any effect on cell proliferation and proteoglycan (PG) production, as well as to find the optimal commercially available Atelocollagen for NP cell transplantation and tissue engineering, we cultured human NP cell line HNPSV-1, in three different Atelocollagen and compared with alginate. Furthermore, NP-like tissues were generated using these cells and different Atellocollagen solutions. Results showed that both DNA synthesis and content is significantly greater when cultured in Atelocollagen than in alginate. On the other hand, proteoglycan synthesis and accumulation was significantly greater in alginate compared with the 0.3% Atelocollagen scaffolds; with 3% Atelocollagen, however, results were similar. NP-like tissue generated by Atelocollagen showed good water and proteoglycan preservation. The current study demonstrates that the use of Atelocollagen as an in vitro culture scaffold for three-dimensional culture of human NP cell lines is indeed feasible and moreover, Atelocollagen possesses the potential to become a candidate scaffold for cell transplantation or tissue engineering for the treatment of intervertebral disc degeneration.

New strategies for disc repair: novel preclinical trials

Degeneration of lumbar intervertebral discs is a major cause of low back complaints, an irreversible occurrence with no currently available treatment. Furthermore, various surgical procedures can accelerate disc degeneration. On the other hand, recent experimental studies on disc cells have demonstrated an important role for the nucleus pulposus in preserving overall disc structure. The author's group has already found that nucleus pulposus cells activated annulus fibrous cells, and reinsertion of nucleus pulposus cells slowed further disc degeneration. We have designed three subsequent studies that were designed to examine further possibilities for clinical transplantation: (1) activation of nucleus pulposus cells by mesenchymal stem cells; (2) focus on the multilineage differentiation potential of mesenchymal stem cells as an alternative cell source for cell transplantation therapy of disc degeneration; (3) the possibility of a human nucleus pulposus cell line as a cell source for cell transplantation therapy. Activation of nucleus pulposus could be achieved by co-culture with autogenous mesenchymal stem cells allowed to have direct cellular interaction. This would be a useful clinical cell source. Induction of nucleus pulposus cells by autogenous mesenchymal stem cells also would be an important subject for a clinical trial. Clinical application of the cells derived from a human nucleus pulposus cell line is an important project to be undertaken in the near future.

Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector: establishment of a novel cell line for the study of human nucleus pulposus cells.

Establishment and characterization of a de novo cell line derived from human nucleus pulposus cells using a recombinant simian virus 40 (SV40) adenovirus vector. To assess the feasibility of human nucleus pulposus cell line procurement and to evaluate the character of the resultant outcome to better understand the nature of human nucleus pulposus cells. Despite recent advances in disc cell biologic research, the fundamental nature of nucleus pulposus cells, especially in the context of human cell lines, is still not well understood. Therefore, a broad-based analysis of these cells is of significant necessity. Because of the limited amount of existing human cells, establishment of an immortal cell line would greatly facilitate resource supply. After release of informed consent, tissue samples of nucleus pulposus were obtained from the lumbar intervertebral disc of a 19-year-old man undergoing anterior fusion for burst fracture. Samples with no apparent damage were selected and digested enzymatically for primary culture and then were infected with recombinant SV40 adenovirus vector (Ad/SV40). The infected cells were maintained in culture for more than 40 population doublings, after which they were considered immortalized. Next, confirmation of expression of T antigen was performed and resultant immortalized cell lines were designated and classified as human nucleus pulposus cell line derived from Ad/SV40 infection-1 (HNPSV-1). HNPSV-1 cells were characterized and compared with their mother cells under two designated culture conditions: monolayer and three-dimensional. Morphologic and immunocytochemical analyses were performed at various intervals. Cell proliferation, DNA synthesis, proteoglycan synthesis, gene expression profiling, and karyotypic analyses were also performed. Moreover, HNPSV-1 cells were injected into rabbit discs to assess the presence of tumorigenesis. Recombinant SV40 adenovirus vector infected nucleus pulposus cells with relatively high efficiency (90%> at multiplicity of infection 100). HNPSV-1 demonstrated marked prolongation of cell life with continuous cell doublings for over 5 months (60-100 cell population doublings). Despite significant increase in cell proliferation and DNA synthesis when compared with its mother cells, resultant cell lines expressed strikingly similar cell morphology and functional characteristics. Atypical karyotypes were noted; however, no apparent tumorigenesis was seen in rabbit discs 24 weeks after injection of HNPSV-1. HNPSV-1 was successfully established using recombinant SV40 adenovirus vector. Results showed that human nucleus pulposus cells are capable of immortalization with maintenance of original cell characteristics. It is anticipated that these cells will be useful for in vitro studies of the biologic nature of human nucleus pulposus cells.