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 compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
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.
Objective To research the biological feature of intervertebral disc nucleus pulposus cells (NPCs) by observing cell morphous, phenotype and ultramicrostructure. Methods The NPCs from 2-week-old healthy rabbit werecultured in DMEM/F12 medium with 15% FBS. The cell biological features were observed by inverted phase contrast microscope, l ight microscope, electron microscope, cell vital ity assay, cell growth curve and cells staining after harvest and during the periods of culturing the primary, the 1st passage and 2nd passage. Results The results of inverted phase contrast microscope showed that the primary passage adhered at 5 days, grew exponentially at 6-8 days, and were subcultured after covering the bottom at 17 days. The phenotype of the NPCs changed from polygon to long fusiform with passage increased; the vital ity assay showed that there was about 95%-97%, 98%-100%, 100% and 75%-80% NPCs survived just after isolation from intervertebral disc, during the period of culturing the primary, the 1st passage and the 2nd passage, respectively. The toluidine blue staining of the NPCs was bly positive, and HE staining showed clear cell nucleus and cytoplasm. The I collagen immunohistochemical staining showed negative results in the 1st passage, but II collagen immunohistochemical staining and safranin O staining showed positive results. However, the I collagen immunohistochemical staining showed positive result in the 2nd passage, and II collagen immunohistochemical staining and safranin O staining showed weakly positive results. The cell growth curve showed the same as the growth course of cell cultured in vitro. The results of TEM showed that there were many glycogen particles and less chondriosomes in the primary passage. With the increased passage, the glycogen particles decreased and the chondriosomes increased, and cell organ became swell. Conclusion This study clarifies the biological feature of NPCs in vitro, providing the experimental basis for the seed cell research of the nuclues pulposus tissue.
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.
ObjectiveTo investigate the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in microenvironment of premature senescence of nucleus pulposus cells (NPCs) so as to lay a foundation for the repair of intervertebral disc degeneration by BMSCs transplantation.
MethodsHuman degenerative nucleus pulposus and normal bone marrow were collected, and then NPCs and BMSCs were isolated, cultured, and identified. The 3rd passage BMSCs and the 1st passage NPCs with premature senescence were co-cultured without contact in the Transwell culture system. NPCs to BMSCs ratio was 75%:25% (group A), 50%:50% (group B), and 0:100% (group C). The morphological changes of BMSCs were observed by inverted phase contrast microscopy and transmission electron microscopy. At 3 and 6 days after co-culture, cell counting kit 8 was used to detect cell viability, flow cytometry was used to observe the cell cycle and detect DNA metabolism after BrdU labeling. Cell senescence was also evaluated by detecting senescence associated β-galactosidase (SA-β-gal) activity.
ResultsThe typical morphology of cell senescence was seen in groups A and B, especially in group A. At 3 and 6 days after co-culture, the cell survival rate of group A was significantly lower than that of group B (P<0.05). At 3 days after co-culture, the proportion of cells in G1 phase in group A was significantly higher than that in groups B and C (P<0.05), the proportion of cells in S phase in group A was significantly lower than that in groups B and C (P<0.05). At 6 days, the proportion of cells in G1 phase in group A was about 81.0%, and the proportion of cells in S phase and G2 phase decreased, showing significant difference when compared with groups B and C (P<0.05); the proportion of cells in G1 phase in group B was about 74.4%, showing significant difference when compared with group C (P<0.05). BrdU content in group A was significantly lower than that in groups B and C at 3 and 6 days after co-culture (P<0.05), but no significant difference was found between groups B and C at 3 days (P>0.05); Brdu content in group B was also significantly reduced when compared with group C (P<0.05) at 6 days. At 6 days, SA-β-gal activity was significantly increased in groups A and B, and significant difference was shown in SA-β-gal positive cell number between groups (P <0.05).
ConclusionPremature senescence of NPCs can down-regulate the proliferation capacity of co-cultured BMSCs by the paracrine effect. The greater proportion of NPCs with premature senescence is, the earlier senescence of BMSCs will be induced.
ObjectiveTo investigate the expression of p16INK4a in nucleus pulposus (NP) and to clarify its relationship with intervertebral disc degeneration so as to provide evidence for biological repair of intervertebral disc.
MethodsThe NP specimens were obtained from 17 patients with intervertebral disc degeneration undergoing discectomy, who aged 40-50 years (mean, 45.4 years). Based on the preoperative MRI, there were 10 cases of grade Ⅲ degeneration, and 7 cases of grade IV degeneration. Cell senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-gal) activity. Senescence marker (p16INK4a) and disc degeneration markers [A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5), Aggrecan, and Sryrelated HMG box transcri ption factor 9 (Sox-9)] were determined in the NP specimens with immunohistochemistry and Western blot. The correlation between ADAMTS 5 and p16INK4a was analyzed.
ResultsClustered distribution of green SA-β-gal-positive cells was seen in the NP with grade Ⅲ and IV degeneration. A few single round SA-β-gal-positive NP cells (NPCs) wrapped by the layered extracellular matrix were also seen in the NP with grade Ⅲ degeneration. It was difficult to see single distribution of NPCs in the NP with grade IV degeneration. The percentage of SA-β-gal-positive cells was 22.7%±5.4% and 37.1%±7.6% in the NP with grade Ⅲ and IV degeneration respectively, showing significant difference (t=-9.666, P=0.000). The percentages of p16INK4a-positive and ADAMTS 5-positive NPCs in the NP with grade IV degeneration were significantly higher than those with grade Ⅲ degeneration (P<0.05). The percentages of Aggrecan-positive and Sox-9-positive NPCs in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The protein expressions of Aggrecan and Sox-9 in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The NP with grade IV degeneration showed significantly higher protein expressions of p16INK4a and ADAMTS 5 (P<0.05). Importantly, there was a good correlation between p16INK4a and ADAMTS 5 protein expressions (r=0.908, P=0.000).
ConclusionPremature senescent NPCs increase in the NP with the advancing disc degeneration. The expression of p16INK4a and its association with degeneration grades suggest that the p16INK4a may play a significant role in the pathogenesis of intervertebral disc degeneration.
Objective To introduce the research of cell transplantation for treating intervertebral disc degeneration. Methods The original articles in recent years about cell transplantation for treating intervertebral disc degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. Results Transplantation of intevertebraldisc-derived cells or BMSCs by pure cell transplantation or combined with collagen scaffold into intervertebral disc couldexpress nucleus pulposus-l ike phenotype. All the cells transplanted into intervertebral disc could increase extracellular matrix synthesis and rel ieve or even inhibit further intervertebral disc degeneration. Conclusion Cell transplantation for treating intervertebral disc degeneration may be a promising approach.
ObjectiveTo research the biological characteristics of different generations of rabbit nucleus pulposus cells (NPCs) that were cultured with natural culture and subculture method.MethodsThe thoracolumbar segments of New Zealand white rabbits (6-8 weeks old and weighing 1.5-2.5 kg) were obtained and nucleus pulposus were isolated from disc regions. And NPCs were harvested by enzymatic digestion from nucleus pulposus. Primary NPCs were counted as P0 generation. Then, NPCs were passaged by trypsin and counted as P1, P2, P3 with a totle of 4 generations. P0 to P3 generations NPCs were separately examined by observation of cell morphology and proliferation time, detection of apoptosis rates of cells by flow cytometry, and detection of hypoxia-inducible factor 1α (HIF-1α), matrix metalloproteinases 2 (MMP-2), Aggrecan, and collagen type Ⅱ proteins by immunofluorescence and Western blot.ResultsThe morphology of NPCs transformed from triangular or polygonal in P0 generation to spindle in P3 generation; the characteristic vacuolated cells gradually disappeared; and the cell volume and cell proliferation time increased. The cell apoptosis rates were 5.47%±0.91%, 13.77%±2.42%, 33.46%±1.82%, and 38.76%±1.50% from P0 to P3 generations, with the increase of culture time, and there were significant differences between 4 generations (P<0.05). Immunofluorescence staining showed that with the increase of cells generation, the fluorescence intensity of HIF-1α, collagen type Ⅱ, and Aggrecan decreased, and the fluorescence intensity of MMP-2 increased. Western blot results showed that the relative expression of HIF-1α protein was high in P0 generation, the P1 generation has a rising trend, and then gradually decreased; the differences between generations were significant (P<0.05). The relative expression of collagen type Ⅱ protein decreased from P0 to P3 generations and there were significant differences between generations (P<0.05). The relative expression of Aggrecan protein decreased from P0 to P2 generations and there were significant differences between generations (P<0.05); but no significant difference was found between P2 and P3 generations (P>0.05). The relative expression of MMP-2 protein increased significantly in P3 generation; except that the difference between P0 and P2 generations was not significant (P>0.05), the significant differences were found between the other generations (P<0.05).ConclusionRabbit NPCs degeneration model was successfully established by the natural culture and subculture method. Transforming of NPCs morphology, increasing of cell apoptosis rates, decreasing of anabolism, and increasing of catabolism were presented in NPCs degeneration model.
Objective
To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere.
Methods
Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression.
Results
The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05).
Conclusion
Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.
