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.
Objective Toreview theresearch progress of nucleus pulposus cells phenot ypic markers. Methods The domestic and international l iterature about nucleus pulposus cells phenotypic markers was reviewed extensively and summarized. Results Due to different biomechanical properties，nucleus pulposus cells and articular chondrocytes have differences in morphology and extracellular components such as the ratio of aggrecan to collagen type II α1. Nucleus pulposus cells can be identified by surface marker (CD24), gene markers (hypoxia inducible factor 1α, glucosetransporter protein 1, matrix metalloproteinase 2, vascular endothel ial growth factor A, etc), and various markers (keratin 19 and glypican 3，paired box 1, forkhead box F1 and integrin-binding sialoprotein, etc). Conclusion Nucleus pulposus cells and articular chondrocytes have different phenotypic markers, but nucleus pulposus cells are still lack of specific markers.
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.
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 The biological treatment of intervertebral disc degeneration becomes a research hotspot in recentyears. It is necessary to find an effective approach to induce bone marrow mesenchymal stem cells (BMSCs) differentiate to disc cells which could make appl ication of cell transplantation as a treatment of intervertebral disc degeneration. To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-l ike cells. Methods The eukaryotic expression vector of pcDNA3.1IE-SOX9Flag was constructed. Rabbit BMSCs were isolated and cultured from one-month-old New Zealand white rabbits and were induced into osteogenetic cells in the osteogenesis supplement medium; and the cell surface markers were detected by flow cytometry. The cells at the 3rd passage were randomly divided into 3 groups: in transfected group, the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag; in negative control group, the cells were transfected with plasmid pcDNA3.1; and in blank control group, the cells were treated with the media without recombinant plasmid. After selected by G418 for 7 days, the cells were harvested and RT-PCR was employed to assay SOX9 mRNA and collagen type II gene (Col2al) mRNA expressions in BMSCs. The expression of SOX9 protein was assayed by Western blot and collagen type II expression was also observed by immunohistochemical staining. Results The SOX9 eukaryotic expression vector was constructed successfully. The BMSCs after 5 days of osteogenetic induction were positive for the alkal ine phosphatase staining. What was more, CD44 expression was positive but CD34 and CD45 expressions were negative. The transfection efficiency was 34.32% ± 1.75% at 72 hours after transfection. After 2 weeks of transfection, BMSCs turned to polygonal and ell iptical. And the cell prol iferation was gradually slow which was similar to the growth characteristic of nucleus pulposus cells. RT-PCR identification showed that SOX9 mRNA and Col2al mRNA expressions were positive in transfected group, and were negative in 2 control groups. Western blot detection showed that SOX9 protein expressed in transfected group but did not express in the control groups. At 2 weeks after transfection, the result of the immunohistochemicalstaining for collagen type II was positive in transfected group. Conclusion The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rabbit BMSCs, the transfected BMSCs can differentiate into nucleus pulposus-l ike cells, which lays a theoretical foundation for treatment of intervertebral disc degeneration with BMSCs transplantation.
Objective Bone marrow mesenchymal stem cells (BMSCs) transplantation can potentially regenerate the degenerated intervertebral disc, with the underlying regenerating mechanism remaining largely unknown. To investigate the potential of human BMSCs protecting nucleus pulposus cells (NPCs) from oxidative stress-induced apoptosis in a coculturesystem, and to illustrate the possible mechanisms of BMSCs transplantation for intervertebral disc regeneration. Methods BMSCs collected by density gradient centrifugation in Percoll solution were cultured and sub-cultured till passage 3, and the surface molecules of CD34, CD45, and CD13 were identified. NPCs were isolated by collagenase digestion and the chondrocyte l ike phenotype was confirmed by morphologic observation after HE staining, inverted phase contrast microscope, proteoglycan, and collagen type II expression after toluidine blue and immunocytochemistry staining. The 3rd passage BMSCs and the 1st passage NPCs were divided into four groups: group A, NPCs (1 × 106 cells) were cultured alone without apoptosis inducing (negative control); group B, NPCs (1 × 106 cells) were co-cultured with BMSCs (1 × 106 cells) with apoptosis inducing; group C, NPCs (1 × 106 cells) were co-cultured with BMSCs (3 × 105 cells) with apoptosis inducing; group D, NPCs (1 × 106 cells) were cultured alone with apoptosis inducing (positive control). After 3 or 7 days of culture or co-culture, the NPCs in groups B, C, and D were exposed to 0.1 mmol hydrogen peroxide for 20 minutes to induce apoptosis. With DAPI staining cellular nucleus, Annexin-V/propidium iodide staining cellular membrane for flow cytometry analysis, the apoptosis of NPCs in each group was studied both qual itatively and quantitatively. Besides, the changes in Bax/Bcl-2 gene transcription and Caspase-3 protein content, were analyzed with semi-quantitative RT-PCR and Western blot. Results BMSCs were successfully isolated and CD34-, CD45-, and CD13+ were demonstrated; after isolated from degenerated intervertebral discs and sub-cultured, the spindle-shaped 1st passage NPCs maintained chondrocyte phenotype with the constructive expressions of proteoglycan and collagen type II in cytoplasm. DAPI staining showed the nucleus shrinkage of apoptosis NPCs. Co-cultured with BMSCs for 3 days and 7 days, the apoptosis rates of NPCs in groups B (29.26% ± 8.90% and 18.03% ± 2.25%) and C (37.10% ± 3.28% and 13.93% ± 1.25%) were lower than that in group D (54.90% ± 5.97% and 26.97% ± 3.10%), but higher than that of groupA (15.67% ± 1.74% and 8.87% ± 0.15%); all showing significant differences (P lt; 0.05). Besides, semi-quantitative RT-PCR showed Bcl-2 gene transcription up-regulated (P lt; 0.05) and no significant change of Bax (P gt; 0.05); Western blot result showed that the Caspase-3 protein expression of groups B and C was lower than that of group D, and was higher than that of group A; all showing significant differences (P lt; 0.05). Conclusion In a co-culture system without direct cellular interactions, the oxidative stress-induced apoptosis of human NPCs was amel iorated by BMSCs. The enhanced anti-apoptosis abil ity of NPCs preconditioned by co-culturing with BMSCs might come from the decreased Bax/Bcl-2 gene transcription ratio.
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 verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor β1 (hTGF-β1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring. Methods Rabbit models of disc degeneration were established by injecting the 25 μL fibronectin fragment (Fn-f, 1 mmol/ L), 4 weeks later,saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n=8). Group A received the 25 μL rAAV2-hTGF-β1 (1 × 1012 vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-β1 was performed on the sl ices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection. Results Immunohistochemical staining showed that extensive and intense positive immunohisochemical staining for hTGF-β1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P lt; 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks. Conclusion The discs injected with rAAV2-hTGF-β1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
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.
