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.
ObjectiveTo investigate the feasibility to culture rabbit annulus fibrosus cells on the KLD-12 polypeptide nanofiber gel so as to search for the seed cells and the scaffolds for tissue engineering. MethodsThe rabbit annulus fibrosus cells were isolated with pancreatin and cultured; the cells at passage 3 were seeded on the KLD-12 polypeptide nanofiber gel to prepare the KLD-12 polypeptide/annulus fibrosus cells gel. The cell morphology change was observed by inverted microscope. The cell counting kit 8 (CCK-8) was used to detect the cell proliferation, and Calcein-AM/propidium iodide (PI) fluorescent staining to observe the cell vitality. The alcian blue method was used to measure the glycosaminoglycan (GAG) content, immunofluorescence technique to observe the collagen type II level, and real-time fluorescence quantitative PCR (RT-qPCR) to measure the mRNA expressions of Aggrecan and collagen type II. ResultsThe cells on the scaffolds grew well, showing round shape on the scaffolds and spindle or fusiform shape at the edge of the scaffold. The cell proliferation exhibited increasing trend with time, and it was significantly higher at 14 days than the other time points (P < 0.05), and on KLD-12 polypeptide nanofiber gel than on blank gel (P < 0.05). The ratios of living cells were 89.32%±8.58% at 5 days and 97.81%±1.09% at 14 days, showing no significant difference (t=-1.962, P=0.097). The GAG content gradually increased with culture time, reached the peak at 8 days, and then gradually decreased; the GAG content at 5, 8, and 11 days was significantly higher than that at 2 and 14 days (P < 0.05). The level of collagen type II was normal. The mRNA expressions of collagen type II and Aggrecan could be measured at 5 and 14 days; the relative expression levels of collagen type II and Aggrecan mRNA were significantly higher at 14 days than 5 days (P < 0.05). ConclusionThe rabbit annulus fibrosus cells on KLD-12 polypeptide nanofiber gel are able to grow well and to produce extracellular matrix, so KLD-12 polypeptide nanofiber gel has the potential to serve as a scaffold for the treatment 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 investigate the effect of local injection of curcumin-loaded mesoporous silica nanoparticles (Cur@MSN) on the repair and treatment of degenerative intervertebral disc tissue in rats, and provide a new strategy for the treatment of intervertebral disc degeneration. Methods Mesoporous silica nanoparticles (MSN) and Cur@MSN were prepared according to the method reported in the literature. Rat nucleus pulposus cells were co-cultured with curcumin and Cur@MSN, respectively, and the growth status and activity of cells in normal environment and inflammatory environment (adding lipopolysaccharide) were observed respectively. Twelve 8-week-old SD rats were randomly divided into 4 groups, including normal group, degeneration group, curcumin group, and Cur@MSN group, with 3 rats in each group. Acupuncture degeneration model was established in coccygeal intervertebral discs (Co7-8, Co8-9) of rats, and corresponding intervention were injected. Imaging, gross pathology, and histological examination were performed after 4 weeks, respectively, to observe the tissue structure and pathological changes of intervertebral discs. Results Under scanning electron microscope, Cur@MSN was spherical in shape, with groove-like pores on its surface. Particle size analysis showed that the particle size of MSN was concentrated in 120-160 nm, and that of Cur@MSN was distributed in 130-170 nm. Zeta potential analysis showed that the average Zeta potential of MSN, curcumin, and Cur@MSN was ?12.5, ?22.5 and ?13.5 mV, respectively. The entrapment efficiency of Cur@MSN was 20.4%, and the drug loading rate was 0.2%. Curcumin released by Cur@MSN in 12 h accounted for about 60% of the total drug dose, and curcumin released in 28 h accounted for about 70%. In cell experiment, there was no significant difference in cell proliferation absorbance among the groups in normal environment (P>0.05), but the cell proliferation absorbance in the Cur@MSN group on the 3rd and 5th day in inflammatory environment was significantly higher than that in the control group and the curcumin group (P<0.01). The percentage of disc height index and the Pfirrmann grade of the Cur@MSN group were better than those of the degeneration group and the curcumin group (P<0.01). The histological score of the Cur@MSN group was lower than that of the degeneration group and the curcumin group (P<0.01). Conclusions Cur@MSN can delay the degeneration process of rat coccygeal intervertebral disc, and has certain repair and treatment effects on its degenerated intervertebral disc. Among them, curcumin can delay the degeneration of intervertebral disc by inhibiting inflammation, and the loading of MSN is helpful for curcumin to exert its biological effects.
Objective To research the transfer of adenovirus human bone morphogenetic protein 4 (Ad-hBMP-4) to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion (aged, 27-50 years). The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc cells at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 × 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR (P lt; 0.05), and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot (P lt; 0.05). There were significant differences between 3 days and 6 days in experimental group (P lt; 0.05). Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions. hBMP-4 may play an important role in the repair process during early 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.
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 review the research advances in animal models of human disc degeneration. Methods The relative articles in recent years were extensively reviewed. Studies both at home and abroad were analyzed and classified. The advantages and disadvantages of each method were compared. Results Studies were classified as either experimentally induced models or spontaneous models. The induced models were subdivided as mechanical (alteration of forces on the normal disc), structural (injury or chemical alteration) and genetically induced models. Spontaneous models included those animals that naturally developed degenerative disc disease. Conclusion Animal model of intervertebral disc degeneration is an important path for revealing the pathogenesis of human disc degeneration, and play an important role in testing novel interventions. With recent advances in the relevance of animal models and humans, it has a great prospect in study of human disc degeneration.
ObjectiveTo review the research progress of endogenous repair strategy (ERS) in intervertebral disc (IVD).MethodsThe domestic and foreign literature related to ERS in IVD in recent years was reviewed, and its characteristics, status, and prospect in the future were summarized.ResultsThe key of ERS in IVD is to improve the vitality of stem/progenitor cells in IVD or promote its migration from stem cell Niche to the tissue that need to repair. These stem/progenitor cells in IVD are derived from nucleus pulposus, annulus fibrosus, and cartilaginous endplate, showing similar biological characteristics to mesenchymal stem cells including the expression of the specific stem/progenitor cell surface markers and gene, and also the capacity of multiple differentiations potential. However, the development, senescence, and degeneration of IVD have consumed these stem/progenitor cells, and the harsh internal microenvironment further impair their biological characteristics, which leads to the failure of endogenous repair in IVD. At present, relevant research mainly focuses on improving the biological characteristics of endogenous stem/progenitor cells, directly supplementing endogenous stem/progenitor cells, biomaterials and small molecule compounds to stimulate the endogenous repair in IVD, so as to improve the effect of endogenous repair.ConclusionAt present, ERS has gotten some achievements in the treatment of IVD degeneration, but its related studies are still in the pre-clinical stage. So further studies regarding ERS should be carried out in the future, especially in vivo experiments and clinical transformation.
Objective To summarize the research situation of stem cells transplantation for intervertebral disc (IVD) degeneration. Methods The original articles about stem cells transplantation for repair of IVD degeneration were extensively reviewed; the clinical applications, the mechanisms, and related factors to influence repair effect were analyzed; and obstacles in stem cells transplantation for repair of IVD degeneration. Results Autogenic stem cells transplantation can repair IVD degeneration and effectively relieve the symptoms of low back and leg pain. Stem cells can differentiate into disc chondrocytes in the disc microenvironment, increase the production of various growth factors, and exert a trophic effect on disc cells. It is also evident that the transplanted stem cells can potentially protect disc cells from apoptosis and maintain an immune-privileged state in the IVD. Multiple factors such as tissue origin of stem cells, methods to pre-modulate the seeds, choice of injectable scaffolds, and even the severity of degeneration are closely related to the repair effects. To get a more efficient stem cell therapy, future researches are challenged to modulate the migration and distribution of stem cells in the IVD, avoid flow back, and better understand their ability to restore stemness properties within the degenerative disc niche. Conclusion Stem cells transplantation is proven to be a promising biological approach for repair of IVD degeneration.