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 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.
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 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.
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 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 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 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 investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro.MethodsThe nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO2, 20%O2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO2, 1%O2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.ResultsHE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A (P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B (P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D (P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B (P<0.05).ConclusionHypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
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.