Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.
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 isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to valuate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell prol iferation and phenotype. Methods The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) andco-cultured with notochord cells (group B) (1 ∶ 1), and cell prol iferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. Results The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 μm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 μm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P gt; 0.05). The co-cultured cells (group B) increased in cell prol iferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell prol iferation in group B was more than that in group A after 4-day culture (P lt; 0.05). The cocultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. Conclusion The notochord cells are conducive for the prol iferation and phenotypekeeping of the chondroid cells and may play a key role in preventing degeneration of the disc.
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 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 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.
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.
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.
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.
【Abstract】 Objective To detect the expression of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3)in cell death induced by nutrition deprivation in nucleus pulposus cells so as to further understand the mechanism of deathin nucleus pulposus cells. Methods Two adult Sprague Dawley rats, male or female, weighing 150-200 g, were involvedin this experiment. The cells isolated from rat caudal disc were cultured under the condition of L-DMEM culture media,10%FBS, and 21%O2 (control group) and under the condition of DMEM-free glucose culture media, no serum, and 1% O2(experimental group). The expressions of BNIP3 gene and protein were detected by real-time fluorescent quantitative PCR,immunofluorescence staining, and Western blot. The cell apoptosis rate and mitochondrial membrane potential were measuredby flow cytometry at 24, 48, and 72 hours after culture. Results The expression of BNIP3 decreased in the control group;the expressions of BNIP3 showed an increasing tendency with time in the experimental group, and BNIP3 combined withmitochondria. Significant differences were observed in the expressions of BNIP3 gene and protein between 2 groups at the othertime (P lt; 0.05) except that no significant difference was observed in the expression of BNIP3 gene at 24 hours (P gt; 0.05). Thecell apoptosis rate and mitochondrial membrane potential were significantly lower in the experimental group than those in thecontrol group (P lt; 0.05). Conclusion Upregulation of BNIP3 and translocation to mitochondria may be involved in nucleuspulposus cell death in nutrition deprivation.