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 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 summarize the research progress of microRNA (miRNA) and its non-viral vector in intervertebral disc degeneration (IDD) and to investigate the potential of non-viral vector delivery of miRNA in clinical application.
Methods
The related literature about the role of miRNA in IDD and its non-viral delivery system was reviewed and analyzed.
Results
MiRNA can regulate the related gene expression level and further participate in the pathophysiologic process in degenerated intervertebral disc, miRNA delivered by various non-viral vectors has obtained an ideal effect in some diseases.
Conclusion
MiRNA plays a great role in the cellular and molecular mechanisms of IDD, as a safe and effective strategy for gene therapy, non-viral vector provides new possibilities for IDD treated with miRNA.
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.
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.
To review the advance in the experimental studies and evaluate the potential therapeutic appl ication of the growth differentiation factor 5(GDF-5) and osteogenic protein 1 (OP-1) in intervertebral disc degeneration.Methods Relevant l iterature at home and abroad publ ished in recent years was searched and analyzedcomprehensively. Results The growth factor was one of the most potential proteins in curing the intervertebral discdegeneration. In vitro, exogenous GDF-5 or OP-1 increased the deoxyribonucleic acid and proteoglycan contents ofboth nucleus pulposus and annlus fibrosis cells types significantly. GDF-5 at 200 ng/mL or OP-1 significantly stimulatedproteoglycan synthesis and collagen synthesis. In vivo, the injection of GDF-5(100 μg) or OP-1(100 μg in 10 μL 5% lactose) resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores had statistical significance. Conclusion A single injection of GDF-5 or OP-1 has a reparative capacity on intervertebral discs, presumably based on its effect to stimulate matrix metabol ism of intervertebral disc cells and enhance extracellular matrix production. A single injection of exogenous GDF-5 or OP-1 in the degenerated disc shows a good prospect.
Objective To investigate the therapeutic effect of BMSCs- chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its cl inical appl ication. Methods Two mill il iter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by 5-BrdU and mixed with chitosan hydrogel to prepare BMSCs- chitosan hydrogel complex. Six rabbitswere selected to establ ish the model of intervertebral disc degeneration and randomized into 3 groups (n=2 per group): control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 μL of autogenous BMSCs- chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 μL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU label ing detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted. Results Cell label ing detection showed that autogenous BMSCs survived and prol iferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O stainning notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was l ighter than that of other two groups, with a indistinct structure.Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the ransplantation and the degeneration group was 223.84 ± 3.93, 221.03 ± 3.53 and 172.50 ± 3.13, respectively, indicating there was no significant difference between the control and the transplantation group (P gt; 0.05), but a significant difference between the control and transplantation groups and the degeneration group (P lt; 0.05). Conclusion The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the cl inical appl ication of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.
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.
ObjectiveTo review the research status on the molecular basis of intervertebral disc degeneration and the repairing effect of platelet-rich plasma.
MethodsThe related literature about the molecular basis of intervertebral disc degeneration and the repairing effect of platelet-rich plasma was reviewed, analyzed, and summarized.
ResultsThe molecular basis of intervertebral disc degeneration includes genetic influences, cell senescence, decreased matrix production, increased degradative enzyme production, proinflammatory cytokine expression, apoptosis, and neural ingrowth. Platelet-rich plasma can release a series of growth factors to promote intervertebral disc cells proliferation, differentiation, and extracellular matrix synthesis. It can also inhibit proinflammatory effect and apoptosis.
ConclusionAlthough the prospect of using platelet-rich plasma to repair intervertebral disc degeneration is encouraging, further studies are still needed.
ObjectiveTo review the biomaterial and clinical prospects of annulus fibrosus tissue engineering.
MethodsThe recent literature concerning annulus fibrosus tissue engineering, including cell source, bioactive molecules, and biomaterial was extensively reviewed and summarized.
ResultsMesenchymal stem cells (MSCs) is an ideal seed cells. When annulus fibrosus cells and MSCs in the ratio of 2:1 are cultured, it shows the closest mRNA expression levels of annulus fibrosus-related markers. Bioactive molecules can be divided into 4 types:growth factors, morphogens, catabolic enzyme inhibitors, and intracellular regulators. They play an active role in promoting the synthesis of extracellular matrix, and maintaining intervertebral disc homeostasis and a balance between anabolic- and catabolic process in the disc. Based on the source, biological materials can be divided into natural materials, synthetic materials, and composite materials. The mechanical properties of the annulus fibrosus is an important basis for material design. Up to now, none of these scaffold materials is accepted as the most suitable one. The selection of scaffold materials is still to be further studied. The development of novel composite biomaterials is a trend.
ConclusionThe annulus fibrosus tissue engineering for the anulus fibrosus regeneration and repair will bring very broad prospects for clinical application in future.
