Objective To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor α (TNF-α). Methods The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-α, and DMEM/ F12 medium containing 10% PBS, 100 ng/ mL TNF-α, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-α group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry. Results Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-α group had no expression. The cell apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 34.24% ± 4.60%, 6.59% ± 1.03%, and 0.40% ± 0.15%, respectively. The early apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 22.16% ± 2.69%, 5.03% ± 0.96%, and 0.49% ± 0.05%, respectively; the late cell apoptosis rates were 13.96% ± 4.86%, 10.68% ± 3.42%, and 0.29% ± 0.06%, respectively. Compared with TNF-α group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P lt; 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P lt; 0.05). Conclusion Ad-hIGF-1 could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α.
Objective To review the progress of the mechanisms of Wnt/β-catenin and nuclear factor-kappa B (NF-кB) pathways in the process of the intervertebral disc degeneration. Methods The related literature about the mechanisms of Wnt/β-catenin and NF-кB pathways in the process of the intervertebral disc degeneration was reviewed, analyzed, and summarized. Results Wnt/β-catenin and NF-кB pathways are both activated in the process of the intervertebral disc degeneration, and exist interaction. However, the specific mechanisms and interactive mediums of Wnt/β-catenin and NF-кB pathways in the process of the intervertebral disc degeneration are still unclear. Conclusion The mechanisms of Wnt/β-catenin and NF-кB pathways in the process of the intervertebral disc degeneration have to be studied deeply.
Objective To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology. Methods Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology. Results Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology. Conclusion The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
Objective To review the research progress of the seed cells, scaffolds, growth factors, and the prospects for clinical application of the intervertebral disc regeneration. Methods The recent literature concerning the regeneration strategies and tissue engineering for treatment of degenerative intervertebral disc disease was extensively reviewed and summarized. Results Seed cells based on mesenchymal stem cells (MSCs) and multiple-designed biomimetic scaffolds are the hot topic in the field of intervertebral disc regeneration. It needs to be further investigated how to effectively combine the interactions of seed cells, scaffolds, and growth factors and to play their regulation function. Conclusion The biological regeneration of intervertebral disc would have a very broad prospects for clinical application in future.
Objective To summarize the research situation of stem cells transplantation for intervertebral disc (IVD) degeneration. Methods The original articles about stem cells transplantation for repair of IVD degeneration were extensively reviewed; the clinical applications, the mechanisms, and related factors to influence repair effect were analyzed; and obstacles in stem cells transplantation for repair of IVD degeneration. Results Autogenic stem cells transplantation can repair IVD degeneration and effectively relieve the symptoms of low back and leg pain. Stem cells can differentiate into disc chondrocytes in the disc microenvironment, increase the production of various growth factors, and exert a trophic effect on disc cells. It is also evident that the transplanted stem cells can potentially protect disc cells from apoptosis and maintain an immune-privileged state in the IVD. Multiple factors such as tissue origin of stem cells, methods to pre-modulate the seeds, choice of injectable scaffolds, and even the severity of degeneration are closely related to the repair effects. To get a more efficient stem cell therapy, future researches are challenged to modulate the migration and distribution of stem cells in the IVD, avoid flow back, and better understand their ability to restore stemness properties within the degenerative disc niche. Conclusion Stem cells transplantation is proven to be a promising biological approach for repair of IVD degeneration.
Objective To investigate the expression and significance of growth-associated protein 43 (GAP-43) in the dorsal root ganglion (DRG) and intervertebral disc in the rat model of intervertebral disc inflammation. Methods A total of 103 adult male Sprague Dawley rats (weighing, 200-250 g) were randomly divided into the experimental group (n=48), the control group (n=48), and the blank control group (n=7). Fluoro-gold (F-G) as tracer was injected into the L5, 6 intervertebral disc of 3 groups; after 7 days of F-G injection, complete Freund’s adjuvant (50 µL) and the same volume of saline were injected in the experimental group (to prepare the model of intervertebral disc inflammation) and the control group, respectively, and the blank control group had no further treatment. After 1, 3, 7, and 14 days, T13-L6 DRG and L5, 6 intervertebral disc of experimental group and control group were harvested to detect the GAP-43 by using fluorescent immunohistochemistry, in situ hybridization, and RT-PCR. The DRG and intervertebral disc of blank control group were also harvested after 8 days of F-G injection. Results Fluorescent immunohistochemistry results showed that the number of F-G-labeled GAP-43 immunoreaction (GAP-43-IR) cells of the DRGs in the experimental group was significantly higher than that in the control group (P lt; 0.05) at 3 days, and no significant difference was found at the other time points (P gt; 0.05). There was no significant difference in the cross-sectional area of F-G-labeled GAP-43-IR cells between the experimental group and the control group at each time point (P gt; 0.05). The co-expression of GAP-43 with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding glycoprotein exhibited that the expression of CGRP was 91.4% ± 7.4% in the control group and was 87.6% ± 7.8% in the experimental group, showing no significant difference between 2 groups (P gt; 0.05). There was no IB4-binding glycoprotein expression in GAP-43-IR cells of the DRGs in 2 groups. The expressions of GAP-43, CGRP, and IB4-positive nerve fibers in the intervertebral disc exhibited that the GAP-43-IR nerve fibers in the experimental group were significantly more than that in the control group (P lt; 0.05), but no significant difference was found in the expression of CGRP between 2 groups (P gt; 0.05); and there was no IB4-binding glycoprotein expression in GAP-43-IR nerve fibers of the intervertebral disc in 2 group. In situ hybridization and RT-PCR detection showed that the positive expression cells ratio of GAP-43 mRNA and the level of GAP- 43 mRNA were significantly higher in the experimental group than in the control group at 1 day (P lt; 0.05), and no significant difference was found at the other time points (P gt; 0.05). Conclusion Intradiscal inflammatory environment may induce the expression of GAP-43, and potentially promote the nerve fiber ingrowth of rat.
Objective To summarize the role of cellular senescence and senescent secretary phenotype in the intervertebral disc (IVD) degeneration. Methods Relevant articles that discussed the roles of cellular senescence in the IVD degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. The senescent phenomenon during IVD degeneration, senescent secretary phenotype of the disc cells, senescent pathways within the IVD microenvironment, as well as the anti-senescent approaches for IVD regeneration were systematically reviewed. Results During aging and degeneration, IVD cells gradually and/or prematurely undergo senescence by activating p53-p21-retinoblastoma (RB) or p16INK4A-RB senescent pathways. The accumulation of senescent cells not only decreases the self-renewal ability of IVD, but also deteriorates the disc microenvironment by producing more inflammatory cytokines and matrix degrading enzymes. More specific senescent biomarkers are required to fully understand the phenotype change of senescent disc cells during IVD degeneration. Molecular analysis of the senescent disc cells and their intracellular signaling pathways are needed to get a safer and more efficient anti-senescence strategy for IVD regeneration. Conclusion Cellular senescence is an important mechanism by which IVD cells decrease viability and degenerate biological behaviors, which provide a new thinking to understand the pathogenesis of IVD degeneration.
Objective To research the transfer of adenovirus human bone morphogenetic protein 4 (Ad-hBMP-4) to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion (aged, 27-50 years). The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc cells at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 × 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR (P lt; 0.05), and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot (P lt; 0.05). There were significant differences between 3 days and 6 days in experimental group (P lt; 0.05). Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions. hBMP-4 may play an important role in the repair process during early disc degeneration.
Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.
Objective The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells. Methods The primary NP cells were harvested from male SpragueDawley rats (8-10 weeks old); the hypoxia inducible factor 1α (HIF-1α), HIF-1β, matrix metalloproteinase 2 (MMP-2), andcollagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-β-galactosidase (SA-β-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells prol iferation. Results Immunocytochemical staining showed that NP cells expressed HIF-1α, HIF-1β, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-β-gal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P lt; 0.001). And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (Plt; 0.001). The flow cytometry showed that the G1 phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P lt; 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P lt; 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA. Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.