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 explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
Objective
To summarize the research progress of microRNA (miRNA) and its non-viral vector in intervertebral disc degeneration (IDD) and to investigate the potential of non-viral vector delivery of miRNA in clinical application.
Methods
The related literature about the role of miRNA in IDD and its non-viral delivery system was reviewed and analyzed.
Results
MiRNA can regulate the related gene expression level and further participate in the pathophysiologic process in degenerated intervertebral disc, miRNA delivered by various non-viral vectors has obtained an ideal effect in some diseases.
Conclusion
MiRNA plays a great role in the cellular and molecular mechanisms of IDD, as a safe and effective strategy for gene therapy, non-viral vector provides new possibilities for IDD treated with miRNA.
Objective To investigate the effect of local injection of curcumin-loaded mesoporous silica nanoparticles (Cur@MSN) on the repair and treatment of degenerative intervertebral disc tissue in rats, and provide a new strategy for the treatment of intervertebral disc degeneration. Methods Mesoporous silica nanoparticles (MSN) and Cur@MSN were prepared according to the method reported in the literature. Rat nucleus pulposus cells were co-cultured with curcumin and Cur@MSN, respectively, and the growth status and activity of cells in normal environment and inflammatory environment (adding lipopolysaccharide) were observed respectively. Twelve 8-week-old SD rats were randomly divided into 4 groups, including normal group, degeneration group, curcumin group, and Cur@MSN group, with 3 rats in each group. Acupuncture degeneration model was established in coccygeal intervertebral discs (Co7-8, Co8-9) of rats, and corresponding intervention were injected. Imaging, gross pathology, and histological examination were performed after 4 weeks, respectively, to observe the tissue structure and pathological changes of intervertebral discs. Results Under scanning electron microscope, Cur@MSN was spherical in shape, with groove-like pores on its surface. Particle size analysis showed that the particle size of MSN was concentrated in 120-160 nm, and that of Cur@MSN was distributed in 130-170 nm. Zeta potential analysis showed that the average Zeta potential of MSN, curcumin, and Cur@MSN was ?12.5, ?22.5 and ?13.5 mV, respectively. The entrapment efficiency of Cur@MSN was 20.4%, and the drug loading rate was 0.2%. Curcumin released by Cur@MSN in 12 h accounted for about 60% of the total drug dose, and curcumin released in 28 h accounted for about 70%. In cell experiment, there was no significant difference in cell proliferation absorbance among the groups in normal environment (P>0.05), but the cell proliferation absorbance in the Cur@MSN group on the 3rd and 5th day in inflammatory environment was significantly higher than that in the control group and the curcumin group (P<0.01). The percentage of disc height index and the Pfirrmann grade of the Cur@MSN group were better than those of the degeneration group and the curcumin group (P<0.01). The histological score of the Cur@MSN group was lower than that of the degeneration group and the curcumin group (P<0.01). Conclusions Cur@MSN can delay the degeneration process of rat coccygeal intervertebral disc, and has certain repair and treatment effects on its degenerated intervertebral disc. Among them, curcumin can delay the degeneration of intervertebral disc by inhibiting inflammation, and the loading of MSN is helpful for curcumin to exert its biological effects.
Objective To review the research advances in animal models of human disc degeneration. Methods The relative articles in recent years were extensively reviewed. Studies both at home and abroad were analyzed and classified. The advantages and disadvantages of each method were compared. Results Studies were classified as either experimentally induced models or spontaneous models. The induced models were subdivided as mechanical (alteration of forces on the normal disc), structural (injury or chemical alteration) and genetically induced models. Spontaneous models included those animals that naturally developed degenerative disc disease. Conclusion Animal model of intervertebral disc degeneration is an important path for revealing the pathogenesis of human disc degeneration, and play an important role in testing novel interventions. With recent advances in the relevance of animal models and humans, it has a great prospect in study of human disc degeneration.
ObjectiveTo comprehensively analyze the relationship between microRNAs and intervertebral disc degeneration at home and abroad.
MethodsThe literature about the relationship between microRNAs and intervertebral disc degeneration was reviewed and analyzed.
ResultsMicroRNA can lead to intervertebral disc degeneration by regulating the gene expression, thus influencing the cell's apoptosis and proliferation, increasing of the production of inflammatory mediator and protease, which play important roles in intervertebral disc degeneration.
ConclusionMicroRNA is a research focus in the field of intervertebral disc degeneration. Further research of the relationship between microRNAs and intervertebral disc degeneration will help to identify the pathogenesis of intervertebral disc degeneration and furnish the new ideal for the diagnosis and treatment of intervertebral disc degeneration.
Objective To introduce the research of nucleus pulposus cells for treating intervertebral disc degeneration. Methods The original articles in recent years about nucleus pulposus cells for treating intervertebral disc degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. Results Nucleus pulposus cells are not only simply a remnant of embryonic notochordal cells, but have also an important influence on the well-being of the whole disc. The biological treatment strategies aim to regenerate the disc by either trying to improve the micro-enviroment within the disc or to increase the popoulation of the nucleus pulposus, which includes transplanting mesenchymal stem cellsto differentiate into nucleus-l ike cells in the degenerated intervertebral disc. Conclusion Nucleus pulposus cells or ucleus pulposus l ike cells based cell transplantation methods prove to be a promising and real istic approach for the intervertebral disc regeneration.
ObjectiveTo explore the effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α) and serum deprivation.
MethodsThe NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups:Vit C group (group A), TNF-α group (group B), and serum deprivation group (group C). Group A was reassigned to A1 subgroup (basic medium), A2 subgroup (100 μg/mL Vit C), and A3 subgroup (200 μg/mL Vit C). Group B was reassigned to B0 subgroup (control group), B1 subgroup (100 ng/mL TNF-α), B2 subgroup (100 μg/mL Vit C+100 ng/mL TNF-α), and B3 subgroup (200 μg/mL Vit C+100 ng/mL TNF-α). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2%FBS+100 μg/mL Vit C), and C3 subgroup (2% FBS+200 μg/mL Vit C). After C1 subgroup (2% FBS), C2 subgroup (2%FBS+100 μg/mL Vit C), and C3 subgroup (2% FBS+200 μg/mL Vit C). After application of 100 μg/mL or 200 μg/mL Vit C for 24 hours, NP cells were stimulated by TNF-α and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type Ⅰ, collagen type Ⅱ, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR.
ResultsGroup A:Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with A1 subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type Ⅰ, collagen type Ⅱ, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B:TNF-α significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C:2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05).
ConclusionVit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 μg/mL Vit C may delay the apoptosis induced by TNF-α and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.
Objective To summarize recent research progress in hydrogel-based growth factors for treatment of intervertebral disc degeneration (IDD). Methods The relevant literature on hydrogel-based growth factors for IDD treatment at home and abroad was extensively reviewed, and their advantages and therapeutic effects in repairing IDD were analyzed and summarized. Results Hydrogels exhibit high hydration, biocompatibility, and biodegradability, enabling targeted delivery and sustained release of growth factors such as growth differentiation factors and transforming growth factors. This facilitates enhanced efficacy in promoting cell proliferation, extracellular matrix synthesis, and reducing inflammatory responses. Consequently, hydrogels demonstrate broad application prospects in the repair of IDD. ConclusionResearch on hydrogel-based growth factors for treating IDD demonstrates advantages such as avoiding disc damage caused by repeated injections and controlling growth factor release concentrations. However, drawbacks include the limited variety of loaded growth factors and the need to verify the long-term stability and biocompatibility of hydrogels. Therefore, further research is required on aspects such as the types of loaded growth factors and the long-term stability and biocompatibility of hydrogels to establish an experimental foundation for their clinical application.
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.
