Sulfated Undaria pinnatifida Polysaccharide Promotes Endocytosis of Nano-Calcium Oxalate Dihydrate by Repairing Subcellular Organelles in HK-2 Cells.

The clinical manifestation of primary hyperoxaluria includes hyperoxaluria and recurrent urinary calculi. In this study, an oxidative damage model was constructed based on oxalate damage to the human renal proximal tubular epithelial cells (HK-2), and a comparative study was carried out on four different sulfated levels of Undaria pinnatifida polysaccharides (UPP0, UPP1, UPP2, and UPP3 with sulfate group [-OSO3-] contents of 1.59%, 6.03%, 20.83%, and 36.39%, respectively) on the repair of oxidatively damaged HK-2 cells. The results showed that after repair by UPPs, cell viability was enhanced, healing ability was improved, the intracellular superoxide dismutase level and mitochondrial membrane potential were increased, malondialdehyde, reactive oxygen species, and intracellular Ca2+ levels were reduced, cellular autophagy was reduced; lysosomal integrity was improved, and cytoskeleton and cell morphology were restored. The ability of repaired cells to endocytose nano-calcium oxalate dihydrate crystals (nano-COD) was enhanced. The activity of UPPs was closely related to their -OSO3- content. A too high or too low -OSO3- content was not conducive to polysaccharide activity, and only UPP2 exhibited the best cell repair ability and strongest ability to promote the cell endocytosis of crystals. UPP2 may be used as a potential agent to inhibit CaOx crystal deposition caused by high oxalate concentration.

Sequencing and bioinformatics analysis of miRNA from rat endplate chondrogenic exosomes.

Exosomes have a key role in various diseases, such as arthritis, heart disease and respiratory disease. Exosomes from various sources have also been indicated to improve intervertebral disc degeneration. However, the role of endplate chondrogenic exosomes in intervertebral disc degeneration has remained largely elusive. The aim of the present study was to compare exosomal microRNA (miRNA) expression patterns in endplate chondrocytes before and after degeneration, and their potential roles in the pathogenesis of intervertebral disc degeneration (IVDD). Endplate chondrocytes were extracted from rats and cultured to obtain pre- and post-degeneration chondrocytes. Exosomes were obtained from the chondrocytes by centrifugation. The two groups of exosomes were subjected to small RNA sequencing, miRNA identification, novel miRNA prediction, quantitative analysis of miRNA expression and differentially expressed (DE) miRNA screening, in addition to miRNA target gene (TG) prediction and TG functional annotation and enrichment analysis. The percentage of miRNAs isolated from the exosomes before and after degeneration was found to differ. A total of 58 DE miRNAs were analyzed, the expression levels of which were significantly different post-degeneration compared with pre-degeneration. Cell experiments were also performed, in which the exosomes were co-cultured with nucleus pulposus (NP) cells. The results indicated that the chondrocyte-derived exosomes were taken up by the NP cells and influenced the expression of aggrecan and collagen 1A and 2A, suggesting that they may inhibit IVDD via their action on NP cells. The specific miRNAs present in exosomes during IVDD may be used to develop new targets for the treatment and diagnosis of this condition. DE exosomal miRNAs derived from endplate cartilage pre- and post-degeneration may be associated with the risk of IVDD and could help to distinguish patients with IVDD. Furthermore, the expression of certain miRNAs may be associated with disease progression, which may contribute to understanding the pathophysiology of IVDD from an epigenetic perspective.

[Effects of oral and intravenous tranexamic acid on perioperative blood loss after lumbar spinal canal decompression and fusion].

OBJECTIVE: To explore the effects of different administration methods of tranexamic acid(TXA) on the perioperative blood loss, hidden blood loss, transfusion rate and adverse reactions in lumbar spinal decompression and fusion. METHODS: Sixty patients who received lumbar spinal canal decompression and fusion from July 2019 to July 2020 were enrolled and divided into observation group and control group, with 30 cases in each group. The observation group was given 2 g TXA orally at 2 hours before operation, control group was given 1 g TXA for 5-10 min before skin incision and 6 hours after operation intravenously. The intraoperative blood loss, postoperative drainage, total blood loss, hidden blood loss, drainage tube removal time, blood transfusion rate, venous thrombosis rate, adverse event rate were recorded respectively. The changes of hemoglobin(Hb) and hematocrit (HCT) were observed before operation and 1, 3 days after operation. RESULTS: Hb and HCT at 1 and 3 days after operation were significantly improved compared with those before operation(P<0.01). However, there was no significant difference between the groups(P>0.05). There were no significant difference in amount of blood loss, postoperative drainage, total blood loss, intraoperative blood loss, hidden blood loss, postoperative drainage time, and blood transfusion rate between two groups (P>0.05). There were no venous thrombosis and adverse events occurred in both groups. CONCLUSION: During the perioperative period of lumbar spinal decompression and fusion, oral TXA and intravenous TXA have the same effect in reducing perioperative blood loss and are safe and reliable. It is recommended that oral TXA be used to save medical costs and convenience.

Interaction between nanometer calcium oxalate and renal epithelial cells repaired with carboxymethylated polysaccharides.

OBJECTIVE: Injury of renal tubular epithelial cells (HK-2) is an important cause of kidney stone formation. In this article, the repairing effect of polysaccharide (PCP0) extracted from the traditional Chinese medicine Poria cocos and its carboxymethylated derivatives on damaged HK-2 cells was studied, and the differences in adhesion and endocytosis of the cells to nanometer calcium oxalate monohydrate (COM) before and after repair were explored. METHODS: Sodium oxalate (2.8 mmol/L) was used to damage HK-2 cells to establish a damage model, and then Poria cocos polysaccharides (PCPs) with different carboxyl (COOH) contents were used to repair the damaged cells. The changes in the biochemical indicators of the cells before and after the repair and the changes in the ability to adhere to and internalize nano-COM were detected. RESULTS: The natural PCPs (PCP0, COOH content = 2.56%) were carboxymethylated, and three carboxylated modified Poria cocos with 7.48% (PCP1), 12.07% (PCP2), and 17.18% (PCP3) COOH contents were obtained. PCPs could repair the damaged HK-2 cells, and the cell viability was enhanced after repair. The cell morphology was gradually repaired, the proliferation and healing rate were increased. The ROS production was reduced, and the polarity of the mitochondrial membrane potential was restored. The level of intracellular Ca2+ ions decreased, and the autophagy response was weakened. CONCLUSION: The cells repaired by PCPs inhibited the adhesion to nano-COM and simultaneously promoted the endocytosis of nano-COM. The endocytic crystals mainly accumulated in the lysosome. Inhibiting adhesion and increasing endocytosis could reduce the nucleation, growth, and aggregation of cell surface crystals, thereby inhibiting the formation of kidney stones. With the increase of COOH content in PCPs, its ability to repair damaged cells, inhibit crystal adhesion, and promote crystal endocytosis all increased, that is, PCP3 with the highest COOH content showed the best ability to inhibit stone formation.

Sulfated Undaria pinnatifida polysaccharide inhibits the formation of kidney stones by inhibiting HK-2 cell damage and reducing the adhesion of nano­calcium oxalate crystals.

OBJECTIVE: The formation of kidney stone is closely related to cell injury and crystal adhesion. METHOD: The sulfur trioxide-pyridine method was used to sulfate raw Undaria pinnatifida polysaccharide (UPP) with a molecular weight (Mw) of 8.33 kDa. Four polysaccharides with the sulfate group (-OSO3-) contents of 1.59% (UPP0), 6.03% (UPP1), 20.83% (UPP2), and 36.39% (UPP3) were obtained. The antioxidant activity of the four UPPs, the difference in oxidative damage inflicted by nano-CaOx monohydrate (nano-COM) on human proximal tubular epithelial (HK-2) cells before and after protection by UPPs, and the inhibitory effect on nano-COM adhesion were explored. RESULTS: Structural characterization showed that sulfation was successful. As the -OSO3- content in the UPPs was increased, the antioxidant activity and capability of the UPPs to regulate the growth of calcium oxalate (CaOx) crystals gradually increased. The damage caused by nano-COM crystals to HK-2 cells under protection by UPPs was weakened. This effect enhanced cell viability, enabled the maintenance of good cell morphology, reduced reactive oxygen species (ROS) levels, and inhibited the decrease in mitochondrial membrane potential, as well as decreased the eversion of phosphatidylserine (PS) and the expression of the adhesion proteins osteopontin (OPN), heat shock protein (HSP 90), and Annexin A1 (ANXA1). The adhesion of nano-COM to HK-2 cells was inhibited under the protection by UPPs. CONCLUSION: UPP3 with the highest content of -OSO3- presented the best antioxidant activity and crystal regulation ability, while UPP2 with the second highest -OSO3- content showed optimal cell protection ability and crystal adhesion inhibition ability. The biological activity of UPPs was regulated by Mw and -OSO3- content. UPP2 with moderate -OSO3- content may become a potential drug for preventing CaOx stones.

Antioxidant activity of sulfated Porphyra yezoensis polysaccharides and their regulating effect on calcium oxalate crystal growth.

The nucleation, growth and aggregation of calcium oxalate (CaOx) crystals and the oxidative damage of renal tubular epithelial cells are the key factors to induce kidney stones. In this study, degraded Porphyra yezoensis polysaccharide (PYP0) with 14.14% sulfate group (-OSO3-) content was modified via the sulfur trioxide-pyridine method to obtain three kinds of sulfated P. yezoensis polysaccharides (PYPs), namely, PYPS1, PYPS2, and PYPS3, with -OSO3- group contents of 17.11%, 20.28%, and 27.14% respectively. Fourier transform infrared spectroscopy, 1H NMR, and 13C NMR analyses showed that the -OSO3- groups replaced the hydroxyl groups at the C2, C4, and C6 positions on (1 â†’ 3)-linked ß-D-galactose, the basic structural skeleton unit of PYP0. The antioxidant activity of the PYPSs increased after sulfation, and their scavenging capacity for OH and DPPH free radicals was enhanced with the increase in their -OSO3- group content. Calcium oxalate (CaOx) crystal growth experiments showed that sulfated PYPs promoted the conversion of the thermodynamically stable and sharp CaOx monohydrate (COM) crystals into the thermodynamically unstable and round CaOx dihydrate crystals. With the increase in the -OSO3- group content of the polysaccharides, the concentration of soluble Ca2+ ions in the supernatant increased and the amount of CaOx precipitate decreased. PYPs were nontoxic to human kidney proximal tubular epithelial cells (HK-2) and could protect HK-2 from oxidative damage caused by nano-COM and reduce the level of reactive oxygen species in cells. PYPS3, which had the highest degree of sulfation, had the best protective capability. The results of this work showed that sulfation improved the biological activity of PYPs. This study could provide inspiration for the development of new drugs for the prevention and treatment of kidney stones.

Effects of Selenized Astragalus Polysaccharide on the Adhesion and Endocytosis of Nanocalcium Oxalate Dihydrate after the Repair of Damaged HK-2 Cells.

An oxidative damage model of human proximal renal epithelial cells (HK-2) was established using oxalate damage. The repair effects of Astragalus polysaccharide (APS) and selenized APS (Se-APS) on damaged HK-2 cells were investigated. Differences in the adhesion and endocytosis of HK-2 cells to calcium oxalate dihydrate crystals with a size of approximately 100 nm before and after APS and Se-APS repair were also explored. The results showed that after being repaired by APS and Se-APS, HK-2 cells exhibited increased cell viability, restored cell morphology, reduced reactive oxygen species level, increased mitochondrial membrane potential, reduced phosphatidylserine eversion, and osteopontin expression. Moreover, the amount of adherent crystals on the cell surface decreased, but the amount of endocytic crystals increased. At the same concentration, Se-APS exhibited better repair effects on the damaged HK-2 cells than APS. All these findings revealed that Se-APS may be a potential drug candidate for inhibiting the formation of kidney stones.

Degraded Porphyra yezoensis polysaccharide protects HK-2 cells and reduces nano-COM crystal toxicity, adhesion and endocytosis.

We studied the protection of degraded Porphyra yezoensis polysaccharide (PYP) on human proximal tubular epithelial cells (HK-2) from cytotoxicity of nano-calcium oxalate monohydrate (COM) crystal, and the regulation of adhesion and endocytosis of the COM crystal. Four degraded fractions, namely, PYP1, PYP2, PYP3, and PYP4, were successfully obtained, with molecular weights (Mws) of 576.2, 49.5, 12.6, and 4.02 kDa, respectively. PYP protection reduced the crystal toxicity, prevented the destruction of cell morphology and cytoskeleton, inhibited the production of reactive oxygen species and the decline of lysosomal integrity, and reduced the expression of osteopontin and transmembrane protein (CD44). PYPi inhibited the adhesion and endocytosis of HK-2 cells by nano-COM. Endocytic COM crystals were accumulated in the lysosomes. With decreasing molecular weight, the ability of PYP to reduce cell damage and inhibit cell adhesion and endocytosis increased. PYP4, which has the smallest molecular weight, weaker intramolecular hydrogen bonds and more reducing groups, showed the best biological activity. PYPi can reduce the oxidative damage of the crystal to the cell, inhibit the adhesion and endocytosis of the crystal, and reduce the risk of kidney stone formation. Therefore, PYP, especially PYP4, has potential for use as a green drug to inhibit the formation and recurrence of calcium oxalate stones.

[One-stage transpedicular debridement, posterior internal fixation and fusion for the treatment of senile thoracolumbar tuberculosis].

OBJECTIVE: To evaluate the clinical outcomes of one-stage transpedicular debridement, posterior internal fixation, RBK mixed streptomycin filled bone grafting for the treatment of elderly patients with thoracolumbar tuberculosis. METHODS: The clinical data of 20 elderly patients with thoracolumbar tuberculosis underwent one stage transpedicular debridement, posterior internal fixation, OSTEOSET RBK mixed streptomycin-filled bone grafting from September 2006 to July 2017 were retrospectively analyzed. There were 12 males and 8 females, aged from 62 to 83 years with an average of (72.4±6.9) years old. Visual analogue scale (VAS), Oswestry Disability Index (ODI)were used to evaluate the pain and spinal function. The kyphosis angle (Cobb angle) of the lesion segment and the bone growth of the lesion area were observed by the X-ray films. RESULTS: All the operations were successful, the operation time was (160.9±23.8) min, and the intraoperative blood loss was (317.9± 112.7) ml. The incisions were healed by first intention, and no sinus and incision were delayed. Spinal tuberculosis was completely cured, Frankel grade has one or more improvements. The VAS score decreased from (7.50±1.15) points before surgery to (1.70±1.39) points at 12 months after surgery (P<0.05). The ODI score decreased from preoperative (92.50±1.17)% to (12.80±0.89)% at the final follow up (P<0.05). The sagittal Cobb angle of the lesion segment decreased from preoperative (24.2±1.6)° to (8.3±0.7)°at 12 months after surgery(P<0.05), the kyphosis deformity was significantly corrected. In all cases, bone fusion was achieved in bone graft area, without bone nonunion and device fracture complications. CONCLUSION: One-stage transpedicular debridement, posterior internal fixation, RBK mixed streptomycin filled bone grafting is suitable for thoracolumbar tuberculosis patients with good general condition and less vertebral destruction.

Preoperative plasma leptin levels predict delirium in elderly patients after hip fracture surgery.

Leptin is considered to be a modulator of the immune response. Hypoleptinemia increases the risk for Alzheimer's disease and vascular dementia. The present study aimed to investigate the ability of plasma leptin level to predict delirium in elderly patients after hip fracture surgery. Postoperative delirium (pod) was evaluated using the Confusion Assessment Method. Prolonged postoperative delirium (ppod) was defined as delirium lasting more than 4 weeks. Plasma leptin levels of 186 elderly patients and 186 elderly controls were measured by an enzyme-linked immunosorbent assay. Plasma leptin level was substantially lower in patients than in controls (4.6±2.2ng/ml vs. 7.5±1.8ng/ml, P<0.001). It was identified as an independent predictor for pod [odds ratio, 0.385; 95% confidence interval (CI), 0.286-0.517; P<0.001] and ppod (odds ratio, 0.283; 95% CI, 0.152-0.527; P<0.001) using a multivariate analysis, and had high area under receiver operating characteristic curve for pod [area under curve (AUC), 0.850; 95% CI, 0.790-0.898] and ppod (AUC, 0.890; 95% CI, 0.836-0.931). The predictive value of leptin was markedly bigger than that of age for pod (AUC, 0.705; 95% CI, 0.634-0.770; P=0.002) and ppod (AUC, 0.713; 95% CI, 0.642-0.777; P=0.019). In a combined logistic-regression model, leptin improved the AUC of age to 0.890 (95% CI, 0.836-0.931) (P<0.001) for pod and 0.910 (95% CI, 0.860-0.947) (P=0.005) for ppod. Thus, preoperative plasma leptin level may be a useful, complementary tool to predict delirium and also prolonged delirium in elderly patients after hip fracture surgery.

Effect of pcDNA3.1- vascular endothelial growth factor 165 recombined vector on vascular buds in rabbit vertebral cartilage endplate.

BACKGROUND: This study aimed to investigate the effect of pcDNA3.1-vascular endothelial growth factor (VEGF)165 vector on vertebral cartilage endplate vascular buds and intervertebral discs. METHODS: Rabbits were randomly assigned to the control and experimental groups with 10 in each. In the experimental group, we anesthetized the rabbits and exposed the front vertebral body. Using the mark of the longitudinal ossature of the front vertebral body of the lumbar vertebrae, we advanced a needle at the central point of the front fourth and fifth lumbar intervertebral discs and injected 20 µl pcDNA3.1-VEGF165. Similarly, in the control group, we injected 20 µl pcDNA3.1. At 4 and 8 weeks post-injection, we examined the changes of the vertebral cartilage endplate using X-ray radiograph, histology, and scanning electron microscopy. RESULTS: The vertebral cartilage endplate calcification and degeneration in the experimental group were less than those in the control group at 8 weeks post-operation. The average number and diameter of vascular buds obviously increased in the experimental group at 4 and 8 weeks post-operation. The number of vascular buds and the diameter in the region of the inner annulus increased when compared to those in the area near the nucleus pulposus. CONCLUSIONS: The pcDNA3.1-VEGF165 plasmid can increase the average number and diameter of vascular buds and decelerate intervertebral disc degeneration.

[Effects of vascular endothelial growth factor vector on vascular buds of vertebral cartilaginous endplate in rabbits].

OBJECTIVE: To directly inject recombinant pcDNA3.1-VEGF165 plasmid into degeneration intervertebral disc and explore its effects on vascular buds of vertebral cartilage endplate and intervertebral disc in rabbits. METHODS: Rabbits were randomly assigned into the experimental and control groups (n = 10 each). For the experimental group, the animals were anesthetized and the front vertebral body exposed. With the longitudinal ossature of front vertebral body of lumbar vertebrae as a mark, a needle was inserted at the central point of the front fourth and fifth lumbar intervertebral disc and 20 µl pcDNA3.1-VEGF165 injected. For the control group, 20 µl pcDNA3.1 was injected. At Weeks 4 and 8 post-injection, the changes of vertebral cartilage endplate were monitored by radiograph, histology and scanning electron microscopy. RESULTS: The vertebral cartilage endplate calcification and degeneration in the experimental group were less pronounced than that in the control group at Week 8 post-operation. The average number and diameter of vascular buds obviously increased in the experimental group at Weeks 4 and 8 post-operation. The number and diameter of vascular buds in the region of inner annulus increased compared with those in the area near nucleus pulposus. CONCLUSION: The pcDNA3.1-VEGF165 plasmid may promote the vascular buds of vertebral cartilage endplate by increasing their average number and diameter and arresting the intervertebral disc degeneration.

Modified unilateral transpedicular percutaneous vertebroplasty for treatment of osteoporotic vertebral compression fractures.

OBJECTIVE: To comparatively assess the clinical outcome of modified unilateral percutaneous vertebroplasty for the treatment of osteoporotic vertebral compression fractures. METHODS: The clinical outcome and incidence of cement extrusion in a consecutive group of 70 patients at our institution between December 2005 and December 2008 was retrospectively reviewed. Thirty-five patients were randomly distributed to modified percutaneous vertebroplasty (Group A) and 35 to traditional percutaneous vertebroplasty (Group B). A visual analog scale (VAS) was used on the first post-operative day and 1 year later to assess the severity of pain before and after vertebroplasty. The incidence of cement extrusion on CT scan was also compared between the two groups. RESULTS: The treatment was successful in all seventy patients. The incidence of cement extrusion was 14.29% (5/35 patients) in group A, and 37.12% (13/35 patients) in group B, this difference being statistically significant (P < 0.05). No patients had serious complications. Complete pain relief was achieved in 50 patients, and significant relief in the other 20 (20/70 patients). There was no statistically significant difference between Groups A and B. CONCLUSION: Modified percutaneous vertebroplasty enhances the accuracy of cement injection into the center of the vertebral body, increasing the safety of the procedure with no increase in cost. It is a safer and more easily performed technique for treating patients with osteoporotic vertebral compression fractures than traditional percutaneous vertebroplasty.

[Altered expression of Ank protein in vertebral end plate].

OBJECTIVE: To observe the expression change of ANK protein in normal and degenerative vertebral endplate chondrocytes and explore the correlation between ANK gene expression and intervertebral disc degeneration. METHODS: Cartilaginous endplates of 45 patients were divided into experiment group (28 with cervical spondylotic myelopathy including 17 males and 11 females) and control group (17 with fracture or dislocation of cervical spine including 10 males and 7 females). The MRI examinations revealed that all the endplate in control group were grade 0 and grade I-III in experiment group according to Miller's classification. Pathological examination demonstrated that intervertebral discs were grade I in control group and grade III-V in experiment group according to Thompson's classification. The morphological appearances and calcification of cartilaginous endplates were observed by HE and Von kossa staining. Immunohistochemical SABC staining, RT-PCR and Western blot were used to detect the ANK mRNA and protein expression in chondrocytes. RESULTS: More calcium deposit could be observed in the experiment group than in the control group by Von kossa staining. ANKH protein was found positive in cell membrane of chondrocytes. Compared with chondrocytes of control group, the expression of ANK mRNA in experiment group markedly decreased (P < 0.05). And the level of ANK protein expression decreased too (P < 0.05). CONCLUSION: Following the degeneration of cartilaginous endplate, the intervertebral disc degeneration worsened and the expression level of ANK decreased in vertebral endplate chondrocytes. Also calcification is positively correlated with the degree of intervertebral disc degeneration. Modulating the expression of ANK in endplate chondrocytes may be a new approach to treat the degeneration of intervertebral disc.

[Finite element analysis of screw in percutaneous axial lumbosacral interbody fusion].

OBJECTIVE: To establish a 3D finite element model of L5/S1 motion segment with percutaneous axial lumbosacral interbody fusion (AxiaLIF) screw and conduct a preliminary analysis of biomechanical stress. METHODS: The titanium screw was implanted in L5 and S1 vertebral body. Solid model was established by CAD software according to the actual dimensions of screw. Then computer graphics were obtained from iges format and imported into the finite element analysis software to establish the finite element model. With the aid of Mechanical Virtual Human of China, a 3D finite element model of L5/S1 motion segment with AxiaLIF screw was established. Vertical compression, torque moment and bending moment were loaded respectively on the upper surface of L5 vertebrae to simulate the load stress in human body. Stress distribution of screw was obtained. RESULTS: Generally stress values were relatively low. Peak stresses of screw under three loading conditions were 175.334 Mpa, 183.765 Mpa and 146.237 Mpa respectively. Stress value was relatively high at the central part of interbody fusion. And all the highest values were localized at the first thread below the central part. Result of comparison between three conditions: torque load was the highest, followed by vertical load and lateral bending. CONCLUSION: Percutaneous axial lumbosacral screw can meet the normal loading conditions. Further clinical applications are recommended.

Finite element analysis of the screw in percutaneous axial lumbosacral interbody fusion.

OBJECTIVE: To establish a three-dimensional finite element model of the L(5)/S(1) motion segment with percutaneous axial lumbosacral screw and analyze the biomechanical stress on the screw. METHODS: With the help of related software and the Mechanical Virtual Human of China, a three-dimensional finite element model was established. Three different loading conditions on the screw were analyzed with this model. RESULTS: Peak stresses on the screw under three loading conditions were 175.334 MPa, 183.765 MPa and 146.237 MPa, respectively. Generally, stress values were relatively low. The stress values were relatively high at the point of interbody fusion and middle part of the screw, all the highest values being localized to the upper and lower threads closest to the middle part. Comparison among the three conditions showed that torque load was the greatest, followed by vertical load, with lateral bending being the least. CONCLUSION: Percutaneous axial lumbosacral screw easily meets normal loading conditions and may be an effective method for lumbosacral fusion.

[Establishment of an in vitro model of natural degeneration of lumbar endplate chondrocytes and expression of Sox9 therein].

OBJECTIVE: To establish an in vitro model of natural degeneration of lumbar endplate chondrocytes and explore the role and the expression change of Sox9 gene, an important gene in the differentiation and maturation of chondrocyte, in the process of natural degeneration of endplate chondrocytes. METHODS: The lumbar vertebrae of 35 SD rats were taken out to obtain the endplates. Endplate chondrocytes were isolated by enzyme digestion and cultured so as to establish an in vitro natural degeneration model of chondrocytes. The morphological appearances and biological characteristics of the chondrocytes of different generations were observed by HE staining, immunocytochemical staining and toluidine blue et cetera; RT-PCR was used to detect the mRNA expression of Sox9 gene and type II collagen in differential generations. RESULTS: The lumbar cartilaginous endplate chondrocytes of rat expressed collagen II, and it's phenotype and biological characteristics were similar to those of articular cartilage cells. When the cells were passaged to the forth or fifth generations they were fusiform and their proliferative speed decreased. Compared with the primary generation chondrocytes, the expression of Sox9 mRNA in the forth and fifth generation chondrocytes was markedly decreased (P < 0.05). And the mRNA expression level of type II collagen, regulated by Sox9 gene, decreased too (P < 0.05). The mRNA expression of Sox9 was positively correlated with the mRNA expression of type II collagen (r = 0.912, P < 0.05). CONCLUSION: A model of natural degeneration of lumbar endplate chondrocytes has been established successfully, thus providing a good cytological basis for the study of degeneration of lumbar endplate. Sox9 gene may play a role in the process of natural degeneration of endplate chondrocytes.

[Correlation between chondrocyte apoptosis of vertebral cartilage endplate and degeneration of intervertebral disc].

OBJECTIVE: To investigate the correlation between chondrocyte apoptosis of cartilage endplate and intervertebral disc degeneration. METHODS: Forty adult New Zealand rabbits were randomized into 2 equal groups: experimental group, undergoing exposure of the upper and lower margins of vertebral bodies C4 and C5, lower ARFIN OF c3, and upper margin of C6, and wadding of bone cement to the depth of posterior margin to interrupt the nutrition supply to the vertebral endplate, thus establishing the models of intervertebral disc degeneration, and control group, only undergoing exposure of the cervical vertebrae. Four and 8 weeks later ten rabbits from each group were sacrificed to obtain specimens of cartilage endplate and tissues of intervertebral disc. TUNEL approach was used to detect the apoptosis of chondrocytes in the vertebral cartilage endplate and immunohistochemistry was used to determine the expression of type II collagen from the intervertebral disc tissues. RESULTS: Four weeks after the operation the number of apoptotic chondrocytes of the cartilage endplate in the experimental group was 9.9 +/- 0.88/HP, significantly higher than that of the control group (9.7 +/- 0.94/HP, P < 0.05). Eight weeks after the operation the number of apoptotic chondrocytes of the cartilage endplate in the experimental group was 12.4 +/- 0.71/HP, significantly higher than that of the control group (11.7 +/- 0.91/HP, P < 0.05). Four and 8 weeks after the operation, the numbers of type II collagen positive cells of the experiment group were 27.7 +/- 1.52/HP and 19.5 +/- 1.57/HP respectively, both significantly lower than those of the control group (29.6 +/- 1.54/HP and 20.6 +/- 1.40/HP respectively, both P < 0.05). Correlation analysis showed that vertebral cartilage endplate chondrocyte apoptosis was highly negatively correlated with the expression of type II collagen operation 4 and 8 weeks after the operation with the coefficient values of 0.86 and 0.82 respectively (both P < 0.05). CONCLUSION: Chondrocyte apoptosis in vertebral cartilage endplate may result in the decreased expression of type II collagen in intervertebral discs and lead to the degeneration of intervertebral discs.