The material properties and volume proportion of the fibers as well as the cross-sectional area proportion of nucleus pulposus vary greatly in different studies. The effect of these factors on the mechanical behavior of intervertebral discs (IVDs) are uncertain. The IVDs finite element models with different parameters were created to investigate the pressure, height, rotation, stress, and strain of the IVDs under loads: pure compression, rotation after compression or axial moment after compression. The results showed that the material properties of fibers had great impact on the mechanical behavior of IVDs, especially on the rotation angle. When the fiber volume ratio was small, its changes had a significant impact on the rotation angle of the IVDs. The area proportions of nucleus pulposus had relatively little effect on the mechanical behavior of IVDs. The IVDs rotation should be observed when validating the model. By adjusting the elastic modulus or volume ratio of fibers within a reasonable range, a model that could simulate the mechanical behavior of normal IVDs could be obtained. It was reasonable to make the area proportion of nucleus pulposus within 25%–50% for the IVDs finite element model. This study provides guidance and reference for finite element modeling of the IVDs and the investigation of the IVDs degeneration mechanism.
ObjectiveTo investigate the influence of ISOBAR TTL dynamic internal fixation system on degeneration of adjacent intervertebral disc by MRI measurement of lumbar nucleus pulposus volume in treating lumbar degenerative disease after operation. MethodsBetween March 2010 and October 2011, 34 patients with lumbar intervertebral disc herniation (23 cases of paracentral type and 11 cases of lateral type) underwent operation with ISOBAR TTL dynamic internal fixation system for fixation of single segment, and the clinical data were analyzed retrospectively. There were 20 males and 14 females, aged 39-62 years (mean, 47.5 years). The disease duration was 6-18 months (mean, 14 months). Involved segments included L4, 5 in 21 cases and L5, S1 in 13 cases. The X-ray films and MRI images were taken at 6, 12, 18, 24, 36, and 48 months after surgery. Based on X-ray films, the height of intervertebral space was measured using angle bisectrix method. The nucleus pulposus volume was measured based on the MRI scan. The postoperative change of nucleus pulposus volume and intervertebral disc height were used to evaluate the influence of ISOBAR TTL system on degeneration of adjacent intervertebral disc nucleus pulposus. ResultsThirty patients were followed up 48 months. The height of intervertebral space showed no significant difference between at pre-and post-operation (P>0.05). The nucleus pulposus volume increased after operation, showing no significant difference at 6, 12, and 18 months when compared with preoperative value (P>0.05), but significant difference was found at 24, 36, and 48 months when compared with preoperative value (P < 0.05). The height of nucleus pulposus increased after operation but the width was decreased; the values showed no significant difference at 6, 12, and 18 months when compared with preoperative ones, but showed significant difference at 24, 36, and 48 months when compared with preoperative ones (P < 0.05). The diameter of nucleus pulposus at 18, 24, 36, and 48 months after operation was significantly langer than that at preoperation (P < 0.05). ConclusionISOBAR TTL dynamic internal fixation system can prevent or delay the degeneration of intervertebral discs.
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.
Objective To detect the cell density, apoptotic rate, and the expressions of BNIP3 in nucleus pulposus of degenerative intervertebral disc of rabbits, so as to further understand the mechanism of intervertebral disc degeneration. Methods Thirty male New Zealand white rabbits, aging 3 months and weighing (2.3 ± 0.2) kg, were divided into sham operation group (control group, n=10) and intervertebral disc degeneration model group (experimental group, n=20). Interbertebral disc degeneration models were establ ished by puncture of L3,4, L4,5, and L5,6 intervertebral discs in the experimental group; intervertebral discs were exposed only and then sutured in the control group. The degree of intervertebral disc degeneration was evaluated according to Pfirrmann classification by MRI at 4 and 8 weeks after establ ishing models. Apototic cells were determined by TUNEL and histological methods, and the immunohistochemical staining was performed to detect the expressions of BNIP3 in nucleus pulposus of intervertebral disc. Results MRI examination showed that the signal intensity decreased gradually at 4 and 8 weeks in the experimental group. There wassignificant difference in the degree of intervertebral disc degeneration between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The histological observation and TUNEL test showed that high density of nucleus pulposus cells and only a few apoptotic cells were observed in the control group; at 4 and 8 weeks, the density of nucleus pulposus cells decreased gradually with more apoptotic cells in the experimental group. There were significant differences in the nucleus pulposus cell density and positive rate of TUNEL staining between 2 groups, and between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The expression of BNIP3 of nucleus pulposus was negative in the control group; however, in the experimental group, the positive expression rates of BNIP3 of nucleus pulposus (the gray values) were 13.45% ± 1.16% and 32.00% ± 1.82% (194.32 ± 4.65 and 117.54 ± 2.11) at 4 and 8 weeks respectively, showing significant differences (P lt; 0.05). Conclusion The decrease of cell density in nucleus pulposus is involved in the development of intervertebral disc degeneration. Cell apoptosis is one of reasons in the decrease of nucleus pulposus cell; BNIP3 is involved in nucleus pulposus cell apoptosis in the degenerative intervertebral disc.
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 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 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.
Objective To explore a practical method of culturing discs organ system by observing the changes of the nucleus pulposus after the whole intervertebral discs (including cartilage end-plate, nucleus pulposus, and annulus fibrous)were cultivated. Methods A total of 335 intervertebral discs were taken out completely from 60 healthy SD rats (about150 g) aged 5-6 weeks of clear grade and rinsed by high osmotic sal ine solution containing heparin, then put to the culture plate after being divided into 5 groups randomly. The whole intervertebral discs were cultured with high osmotic (410 mOsmol/ kg) culture medium and changed the medium once every day, then the cell viabil ity (n=15), HE staining (n=15), Safranin O staining (n=15), and immunohistochemistry staining (n=2) were observed at 0, 3, 7, 14, and 21 days; RT-PCR result (n=5) was observed at 0, 3, 7, and 14 days. Results The cell viabil ity was not changed significantly within 14 days (P gt; 0.05) and was significantly lower at 21 days than at other time points (P lt; 0.01). The immunohistochemistry staining results for collagen type II were positive in nucleus pulposus cells at every time point. HE staining showed that the tissue integrity and morphology of the whole intervertebral discs were not changed within 14 days. Safranin O staining showed no significant difference in the matrix grey scale within 14 days (P gt; 0.05) and significant differences between 21 days and 0-14 days (P lt; 0.05). RT-PCR results showed that the mRNA expression of collagen type I increased with time, but the expressions of collagen type II, aggrecan, and decorin decreased, showing significant differences in the mRNA expressions of the matrix protein at each time point (P lt; 0.05). Conclusion High osmotic sal ine solution containing heparin could be used to cultivate the whole intervertebral discs, it is an ideal model for futher studies on physiology and pathology of intervertebral discs.
Objective To determine whether the transforminggrowth factor β1 (TGF-β1) is a key regulatory molecule required for an increase or a balance of extracellular matrix (ECM) and DNA synthesis in the goat passaged nucleus pulposus (NP) cells. Methods The NP cells isolated from the goat intervertebral discs were cultured in vitro for a serial of passages and transfected with the replicationincompetent adenoviral vectors carrying the human TGF-β1 (hTGF-β1) or lacZ genes. Then, they were cultured in monolayer or alginate bead 3dimensional (3-D) systems for 10 days.The changes in the production and the molecular components of ECM that occurredin the NP cells transfected with Ad/hTGF-β1 or the controls were evaluated by Westernblot and absorbance of glycosaminoglycan (GAG)-Alcian Blue complexes. Differences of DNA synthesis in the variant cells and culture systems were assessed by fluorometric analysis of the DNA content. ResultsA quantitation in the variant culture systems indicated that in monolayers the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher cell viability and more DNA synthesis(P＜0.05); however, in the alginate 3-D culture system, the NP cells transfected with Ad/hTGF-β1 did not have any significant difference from the controls(P＞0.05). The Western blotting analysis ofthe protein sample isolated from the variant cells for TGF-β1, type Ⅱ collagen, and Aggrecan expression indicated that in the monolayers and alginate 3-D culture systems the NP cells at Passage 3 transfected with Ad/hTGF-β1 revealed much higher protein levels than the controls(P＜0.05)； whereas the type Ⅰcollagen content was much lower than the controls (P＜0.05), but a significatly increased ratio of type Ⅱ/type Ⅰ collagen was found in both of the cell culture systems(P＜0.05). The GAG quantification also showed a positive result in both the cell culture systems and the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher GAG content than the controls(P＜0.05). Conclusion To a greaterextent, hTGF-β1 can play a key role in maintaining the phenotype of the NP cells and can still have an effect of the phenotypic modulation after a serial of the cell passages. The NP cells that are genetically manipulated to express hTGF-β1 have a promising effect on the restoration of the intervertebral disc defects. The NP cells transfected with Ad/hTGF-β1 cultured in the 3-D alginate bead systems can show a nearly native phenotype.
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.