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 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.
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 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 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.
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 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.
ObjectiveTo investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits.MethodsTwenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L3, 4, L4, 5, and L5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot.ResultsThe comprehensive score of biological response in each group after injection was significantly lower than that before injection (P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups (P<0.05), and there was no significant difference among other groups (P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection (P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection (P<0.05). There was no significant difference between other groups (P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks (P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks (P<0.05), and there was no significant difference between other groups (P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group (P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group (P<0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group (P<0.05). There was no significant difference in the expression of p65 protein between groups (P>0.05).ConclusionZinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.