ObjectiveTo investigate the feasibility of establishing intervertebral disc degeneration (IDD) model by using minimally invasive acupuncture and rotary-cutting. MethodsForty New Zealand white rabbits [male or female, (2.9±0.3) kg in weight] were randomly divided into control group (n=20) and experimental group (n=20). No treatment was done in the control group; percutaneous puncture was performed on L4, 5 and L5, 6 intervertebral disc by using 18G needle under C-arm X-ray monitoring for rotary-cutting of nucleus pulposus to promote degeneration of the disc in the experimental group. At 4, 8, 12, and 16 weeks after operation, general observation and MRI observation were done, and intervertebral disc degeneration was accessed based on Pfirrmann grade; the specimens were harvested for Masson staining and Safranine O staining. ResultsThe nucleus pulposus showed dark colors and reduced elasticity in the experimental group when compared with the control group. T2-weighted MRI images indicated that the disc signal intensity of control group had no obvious change at early stage, and weakened slightly at late stage; disc signal intensity of the experimental group decreased with time. According to Pfirrmann grade for disc degeneration, disc degeneration degree was significantly aggravated with time in 2 groups (P < 0.05); degeneration was significantly more severe in the experimental group than the control group at the other time points (P < 0.05) except 4 weeks (P > 0.05). Masson staining results showed that irregular arrangement of annulus with integrate structure was observed in the control group with time; the annulus of the experimental group arranged in disorder, or even disc fibrous circle rupture appeared with time. Safranin O staining showed that the nucleus pulposus cells reduced significantly in the experimental group, but did not in the control group. ConclusionMinimally invasive acupuncture and rotary-cutting could successfully establish the IDD model in rabbits.
Objective To introduce the research of mesenchymal stemcells(MSCs) transplantation for treating intervertebral disc degeneration. Methods The recent original articles about the MSCs transplantation for treating intervertebral disc degeneration were extensively reviewed. Results Transplanted MSCs in intervertebral disc can express chrondcyte-like phenotype in certain conditions, increase matrix synthesis and release intervertebral disc degeneration. Conclusion MSCs transplantation for treating intervertebral disc degeneration may be a future approach.
Objective To review the mechanism of extracellular vesicles (EVs) in treating intervertebral disc degeneration (IVDD). Methods The literature about EVs was reviewed and the biological characteristics and mechanism of EVs in the treatment of IVDD were summarized. Results EVs are a kind of nano-sized vesicles with a double-layered lipid membrane structure secreted by many types of cells. EVs contain many bioactive molecules and participate in the exchange of information between cells, thus they play important roles in inflammation, oxidative stress, senescence, apoptosis, and autophagy. Moreover, EVs are found to slow down the process of IVDD by delaying the pathological progression of the nucleus pulposus, cartilage endplates, and annulus fibrosus. Conclusion EVs is expected to become a new strategy for the treatment of IVDD, but the specific mechanism remains to be further studied.
Objective Degenerative lumbar scol iosis and spinal stenosis are more common in elderly patients. Because of many factors, treatment choices are more complex. To investigate the step treatment strategy of degenerative lumbarscol iosis and spinal stenosis. Methods Between January 2005 and December 2009, 117 patients with degenerative lumbar scol iosis and spinal stenosis were treated with step treatment methods, including conservative therapy (43 cases), posterior decompression alone (18 cases), posterior short segment fusion (1-2 segments, 41 cases), and posterior long segment fusion ( ≥ 3 segments, 15 cases). Step treatment options were made according to patient’s will, the medical compl ications, the degree of the symptoms of low back and lower extremity pain, the size of three-dimensional lumbar scol iosis kyphosis rotating deformity, lumbar spine stabil ity (lateral sl ip, degenerative spondylolysis), and the overall balance of the spine. The visual analogue scale (VAS) score of low back and lower extremity pain, Oswestry disabil ity index (ODI), lumbar lordosis angle, and scol iosis Cobb angle were measured and compared before and after treatments. Results Seventy-two cases were followed up more than 12 months, and there was no death or internal fixation failure in all patients. Of them, 19 patients underwent conservative treatment; the mean follow-up period was 19.3 months (range, 1-5 years); no symptom deterioration was observed; VAS score of low back and lower extremity and ODI were significantly decreased at last follow-up (P lt; 0.05); and lordosis angle was decreased and scol iosis Cobb angle was increased, but there was no significant difference (P gt; 0.05). Twelve cases underwentposterior decompression alone; the average follow-up was 36 months (range, 1-5 years); VAS score of lower extremity and ODI were significantly decreased at last follow-up (P lt; 0.05); and scol iosis Cobb angle was increased and lordosis angle was decreased, but there was no significant difference (P gt; 0.05). Thirty-one patients underwent posterior short segment fusion; the mean follow-up period was 21.3 months (range, 1-3 years); postoperative hematoma, poor wound heal ing, cerebrospinal fluid leakage, and superficial infection occurred in 1 case, respectively, and were cured after symptomatic treatment; VAS score of low back and lower extremity and ODI were significantly decreased (P lt; 0.05); and postoperative lumbar scol iosis Cobb angle and lordosis angle were significantly improved at last follow-up (P lt; 0.05). Ten patients underwent posterior long segment fusion; the mean follow-up period was 17.1 months (range, 1-3 years); postoperative symptoms worsened in 1 case and was cured after physical therapy and drug treatment for 3 months, and deep infection occurred in 1 case and was cured after debridement and continuous irrigation drainage; VAS score and ODI were significantly decreased (P lt; 0.05); and postoperative scol iosis Cobb angle and lordosis angle were improved significantly at last follow-up (P lt; 0.05). Conclusion The treatment of degenerative lumbar scol iosis and spinal stenosis should be individual and step. Surgery treatment should be rely on decompression while deformity correction subsidiary. Accurate judgment of the responsible segment of symptoms, scol iosis and lordosis can prevent the operation expansion and increase safety of surgery with active control bleeding.
Low back pain caused by intervertebral disc degeneration is a common clinical chronic disease. The regenerative ability of intervertebral disc tissue is extremely poor. Meanwhile, current treating methods can not fundamentally solve such problems. With the increasing awareness of the mechanism of disc degeneration and the rapid development of the fields of cellular and molecular biology, gene and materials engineering, using stem cells and tissue engineering technology to slow down or reverse the progress of disc degeneration may become possible. The author reviewed the application of stem cells for treating degenerative discs from present researching status and concepts for the future in the combination of researches reported both at home and abroad.
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 compare the effectiveness and radiological changes of posterior decompression combined with Coflex interspinous dynamic reconstruction or lumbar 360° fusion for degenerative lumbar spinal disorders at L4, 5. MethodsBetween October 2008 and November 2010, a comparative study was carried out on patients with degenerative lumbar spinal disorders at L4, 5. In group A, 29 patients underwent posterior decompression combined with Coflex interspinous dynamic reconstruction; there were 20 males and 9 females with an average age of 45.1 years (range, 21-67 years); and the disease duration was 2 months to 4 years. In group B, 31 patients underwent posterior decompression combined with lumbar 360° fusion treatment; there were 16 males and 15 females with an average age of 56.2 years (range, 32-86 years); and the disease duration was 3 months to 6 years. Except the age, there was no significant difference in gender, disease duration, and etiology etc. between 2 groups (P gt; 0.05). The results were assessed by Japanese Orthopaedic Association (JOA), visual analogue scale (VAS) scores, and Oswestry disabil ity index (ODI). The range of motion (ROM) and intervertebral height of affected and adjacent segments, and the ROM of lumbar were measured before operation and last follow-up. Results Significant differences were found in the operative time and blood loss between 2 groups (P lt; 0.05). Intraoperative dural tear occurred in 1 case of group B, spinal canal venous plexus hemorrhage in 1 case of group B, and postoperative cerebrospinal fluid leakage in 2 cases of group A and B respectively, showing no significant difference (χ2=0.119, P =0.731). The follow-up was 12-21 months in group A and was 12-23 months in group B. At the last follow-up, the JOA, VAS scores, and ODI of groups A and B were significantly improvedwhen compared with the preoperative values (P lt; 0.05). The VAS score of group A was significantly higher than that of group B (P lt; 0.05). There was no significant difference in the intervertebral height of L4, 5 and L5, S1 of groups A and B between pre- and post-operation (P gt; 0.05). In group B, the intervertebral height of L3, 4 was significantly reduced (P lt; 0.05) compared with the preoperative one. There was no significant difference in the ROM of L5, S1 and ROM of lumbar in groups A and B between preand post-operation (P gt; 0.05). At last follow-up, the ROM of L4, 5 was significantly reduced in group A (P lt; 0.05), and the ROM of L3, 4 was significantly increased in group B (P lt; 0.05). Except significant differences in the intervertebral height and ROM of L3, 4 between 2 groups (P lt; 0.05), no significant difference was found in other parameters (P gt; 0.05). Conclusion Posterior decompression combined with Coflex interspinous dynamic reconstruction has the same effectiveness as lumbar 360° fusion in treating degenerative lumbar spinal disorders at L4, 5, but the former has a protective effect on the adjacent segments of fusion and is recommended for initial treatment of young adults and the elderly and frail patients with recurrent.
Objective Polylactic acid (PLA) patch has proper steric configuration, sufficient mechanic strength, and flexibil ity, to investigate the blocking effect on the intra-discal inflammation after annulus puncture sticked by medical glue so as to seal the pinhole left after annulus puncture. Methods Twenty healthy New Zealand white rabbits (weighing 2.0-2.5 kg) were randomly divided into 4 groups (n=5): groups A, B, C, and D. In group A, the rabbits underwent exposure of intervertebral disc and transverse process at L2-7 as a control; in group B, the rabbits received annulus puncture at L2-7 with an 18-gauge needle; and in groups C and D, the pinholes were sealed respectively with a PLA patch sticked with medical gel and medical gel alone after annulus puncture at L2-7. General condition of rabbits was observed after operation. The intervertebral disc tissue was harvested 1 week after operation. The tissue structure was observed by HE and Masson staining. And the expressions of inflammatory factors l ike interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOs) were detected with immunohistochemistry and ELISA. Results All the animals survived till the end of the experiment. In group A at 1 week, the nucleus pulposus tissue had normal structure. In group B at 1 week, leak of nucleus pulposus from the pinhole and sl ight adhesion to the adjacent tissue could be seen, and the nucleus pulposus tissue had significant degenerative change by histological observation. In groups C and D, clots of coagulated medical gel and extensive adhesion to the adjacent tissue could be seen; histological observation suggested that the nucleus pulposus tissue of group C had similar histology manifestation to that of group A; while group D had similar histology manifestation to group B with obviously-decreased cells and disorder of matrix. ELISA test showed remarkably elevated expression level of inflammatory factors including IL-1β, TNF-α, and iNOs in groups B and D when compared with groups A and C, showing significant differences (P lt; 0.05), and similar expression level were observed in groups A and C, groups B and D (P gt; 0.05). Conclusion The PLA patch sticked with medical gel is effective in blocking the intra-discal inflammation 1 week after annulus puncture.
ObjectiveTo observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP.MethodsHuman degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot.ResultsHE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group (P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group (t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A (P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant (P<0.05).ConclusionBoth IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.
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.