Chenguang Li

Cervical Intervertebral Disc Degeneration Induced by Unbalanced Dynamic and Static Forces: A Novel in Vivo Rat Model

Study Design. Establishment of a novel in vivo animal model of cervical spondylosis. Objective. To investigate apoptotic, degenerative, and inflammatory changes occurring in the cervical intervertebral discs of rats. Summary of Background Data. Cervical degeneration occurs as the result of imbalance of both static and dynamic spinal stabilizers. The disc degeneration that occurs is characterized by increased local inflammation and increased apoptosis of intervertebral disc cells. Methods. By excising the paraspinal musculature and posterior cervical spinal ligaments of rats, both static and dynamic cervical stabilizers were disrupted. The resultant biomechanical imbalance resulted in biochemical and histologic changes, which were characterized by light microscopy, electron microscopy, immunostaining, enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization. Results. Histologic analysis showed characteristic degenerative changes of the intervertebral discs and vertebral endplates following surgery. Ultrastructural examination revealed apoptotic changes, which were verified by immunostaining. Instability also resulted in significant up-regulation of inflammatory factors, as shown by enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization. Conclusions. By creating static and dynamic posterior instability of the cervical spine, this novel model of cervical spondylosis results in rapid intervertebral disc degeneration characterized by increased apoptosis and local inflammation, such as that seen clinically.

Prolonged upright posture induces degenerative changes in intervertebral discs in rat lumbar spine.

Study Design. Both forelimbs of rats were amputated, and these rats were kept in the custom-made cages that kept the rats in prolonged upright posture. Pathologic changes were observed in the lumbar spine at different time points after the surgery. Objective. To investigate the effect of upright posture on intervertebral discs of rat lumbar spine. Summary of Background Data. Previous studies have shown that increased axial forces on the spine can decrease the height of the intervertebral disc, but there are no data to indicate whether or not long-term and repeated assumption of the upright posture could result in degenerative changes. Methods. The forelimbs of 30 rats were amputated when they were 1-month old. These rats were kept in the custom-made cages and were forced to stand upright on their hind-limbs and tails to obtain water and food. Normal rats of the same ages kept in regular cages were used as control. The rats were killed at 5, 7, and 9 months after the surgery, and the intervertebral discs samples of lumbar spine were harvested for histologic and immunohistochemical studies. Total RNA isolated from these samples was used for real-time PCR of type II collagen (Col2&agr;1), type X collagen (Col10&agr;1), matrix metalloproteinase-13 (MMP-13), aggrecan, and disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). Results. Histologic analysis showed degenerative changes of the intervertebral discs after surgery such as disordered collagen structure of endplate cartilage, fragmentation of annulus fibrosus, and decreased height of disc. Immunostaining revealed decreased protein level of type II collagen and increased protein expression of type X collagen. Real-time PCR showed upregulated expression of MMP 13, ADAMTS-5, and Col10&agr;1 mRNA and downregulated mRNA expression of Col2&agr;1 and aggrecan. Conclusion. Long-term and repeated assumption of the upright stance accelerates disc degeneration in rats.

A continuous observation of the degenerative process in the intervertebral disc of Smad3 gene knock-out mice.

Study Design. Pathologic changes were observed in the spine of small mother against decapentaplegic (Smad) 3−/− mice at different time points. Objective. To observe the degeneration of the intervertebral disc (IVD) in Smad3 gene knock-out mice with growth. Summary of Background Data. Smad3 gene knock-out (Smad3−/−) mice displays phenotypes similar to human osteoarthritis. Despite the similarities between IVD cartilage endplate and the articular cartilage, there has been relatively little interest in exploring the possibility that IVD degeneration might be driven by the deficiency of Smad3. Methods. The Smad3−/− mice were killed at the 10th, 30th, and 60th day after their birth and the IVD samples of spine were harvested for histologic and immunohistochemical studies. Total RNA isolated from these samples were used for real-time PCR analysis of type II collagen (Col2&agr;1), type X collagen (Col10&agr;1), aggrecan, and transforming growth factor-&bgr;1 (TGF-&bgr;1). Results. Compared with the wild-type mice, Smad3−/− mice appeared significantly smaller in size. Radiograph showed that the spine of Smad3−/− mice is malformation and kyphosis. Histologic analysis revealed the declined height of cartilage endplate, decreased proteoglycan and collagen content in disc of Smad3−/− mice. With growth, especially of the 30- and 60-day old Smad3−/− mice, the protein positive staining of type II collagen, aggrecan, and TGF-&bgr;1 in the disc decreased, while that of type X collagen increased. And the analysis of real-time PCR showed that the mRNA expression of Col2&agr;1, aggrecan, and TGF-&bgr;1 decreased, while that of Col10&agr;1 increased. Conclusion. Smad3 gene knock-out mice develop IVD degeneration with growth.

Mechanism of resveratrol on the promotion of induced pluripotent stem cells

OBJECTIVE To investigate the effects of resveratrol (RV) in reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) and the related mechanism. METHODS Primary MEFs were isolated from E13.5 embryos and used within three passages. Retroviruses expressing Sox2 and Oct4 were produced by transfecting GP2-293t cells with recombinant plasmids (MSCV)-Sox2 and MSCV-Oct4. Supernatants containing retroviruses were obtained after 48-hour transfection and MEFs were then infected. Different concentrations (0, 5, 10 and 20 μmol/L) of RV were added to embryonic stem cell (ESC) medium to culture MEFs 48 h post-infection. iPSC clones emerged and were further cultured. Expression of pluripotent markers of iPSCs was identified by cell immunofluorescence and reverse transcription-polymerase chain reaction. Both cytotoxicity and cell proliferation were assayed by Western blot analysis after RV was added into ESC medium. The ultrastructure change of mitochondria was observed by electron microscopy. RESULTS More than 2.9-fold and 1.3-fold increases in colony number were observed by treatment with RV at 5 and 10 μmol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 μmol/L RV. The proliferation effect on MEFs or MEFs infected by two factors Sox2/Oct4 (2 factors-MEFs, 2F-MEFs) was investigated after RV treatment. At 20 μmol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 μmol/L treatments. Clones were selected from the 10 μmol/L RV-treated group and cultured. Green fluorescent protein expression from one typical clone was silenced one month later which expressed ESC-associated marker genes Gdf3, Nanog, Ecat1, Fgf4 and Foxd3. Electron transmission microscope showed obvious cavitations in mitochondria. The expression of hypoxia-inducible factor-1α was up-regulated when 2F-MEFs were treated with RV compared to the control group. CONCLUSION RV improved the efficiency of reprogramming 2F-MEFs into iPSCs at low and moderate concentrations (5 and 10 μmol/L). The effect of 10 μmol/L RV on reprogramming was much greater than that of 5 μmol/L RV. However, high concentration of RV (20 μmol/L) led to more severe cavitations in mitochondria and caused cytotoxic effects. Taken together, these findings suggest that RV mimics hypoxia in cells and promotes reprogramming at a low concentration.

Protective Effect of Ligustrazine on Lumbar Intervertebral Disc Degeneration of Rats Induced by Prolonged Upright Posture

Most chronic low back pain is the result of degeneration of the lumbar intervertebral disc. Ligustrazine, an alkaloid from Chuanxiong, reportedly is able to relieve pain, suppress inflammation, and treat osteoarthritis and it has the protective effect on cartilage and chondrocytes. Therefore, we asked whether ligustrazine could reduce intervertebral disc degeneration. To determine the effect of ligustrazine on disc degeneration, we applied a rat model. The intervertebral disc degeneration of the rats was induced by prolonged upright posture. We found that pretreatment with ligustrazine for 1 month recovered the structural distortion of the degenerative disc; inhibited the expression of type X collagen, matrix metalloproteinase (MMP)-13, and MMP3; upregulated type II collagen; and decreased IL-1β, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression. In conclusion, ligustrazine is a promising agent for treating lumbar intervertebral disc degeneration disease.

Prolonged upright posture induces calcified hypertrophy in the cartilage end plate in rat lumbar spine.

Study Design. Both forelimbs of rats were amputated and these rats were kept in the custom-made cages to keep prolonged and repeated upright posture. Changes of bone were observed in the lumbar vertebrae at three different time points after the surgery. Objective. To investigate the effect of prolonged and repeated upright posture on the cartilage end plate of rat lumbar vertebrae. Summary of Background Data. Previous studies show calcified hypertrophy is related to mechanical stress, but there are no clear evidences to indicate whether or not long-term and repeated assumption of the upright posture could result in calcified hypertrophy in cartilage end plate of rat lumbar spine. Methods. The forelimbs of 30 rats were amputated when they were 1 month old. These rats were kept in the custom-made cages and were forced to stand upright on their hind-limbs and tails to obtain water and food. Normal rats of the same ages kept in regular cages were used as control. The rats were killed at 5, 7, and 9 months after the surgery and lumbar vertebrae samples were harvested for micro-CT, histologic, and immunohistochemical studies. Total RNA isolated from these samples were used for real-time RT-PCR of type X collagen (Col10&agr;1), vascular endothelial growth factor (VEGF), and transforming growth factor &bgr;1 (TGF-&bgr;1). Results. Micro-CT showed increased inner part of cartilage end plate. Histologic revealed peripheral hypertrophy of disc after the surgery. Immunostaining and real-time RT-PCR showed increased protein and mRNA expression of type X collagen, VEGF, and TGF-&bgr;1. Conclusion. Prolonged upright posture induces cartilage end plate calcification and hypertrophy in rat lumbar spine.

Prolonged and repeated upright posture promotes bone formation in rat lumbar vertebrae.

Study Design. Both forelimbs of rats were amputated and these rats were kept in the custom-made cages to keep prolonged and repeated upright posture. Changes of bone were observed in the lumbar vertebrae at three different time points after the surgery. Objective. To investigate the effect of prolonged and repeated upright posture on bone formation of rat lumbar vertebrae. Summary of Background Data. Previous studies show long-term and repeated load-induced bone formation, but there are no clear evidences to indicate whether or not long-term and repeated assumption of the upright posture could result in bone formation at rat lumbar vertebrae. Methods. The forelimbs of 30 rats were amputated when they were 1 month old. These rats were kept in the custom-made cages and were forced to stand upright on their hindlimbs and tails to obtain water and food. Normal rats of the same ages kept in regular cages were used as control. The rats were sacrificed at 5, 7, and 9 months after the surgery and lumbar vertebrae samples were harvested for micro-computed tomographic (CT), histological, and immunohistochemical studies. Total RNA isolated from these samples were used for real-time polymerase chain reaction of type I collagen (Col1&agr;2), type X collagen (Col10&agr;1), transforming growth factor-&bgr;1 (TGF-&bgr;1) and its related nuclear transcript factor runt-related transcription factor 2 (Runx2), as well as the biomarker of angiogenesis and vascular invasion, which is also a prerequisite for endochondral bone formation: vascular endothelial growth factor (VEGF). Results. Micro-CT and histological studies showed increased trabecular bone density and increased osteoblast quantities of lumbar vertebrae after surgery. Immunostaining revealed increased protein expression of type I collagen, type X collagen, TGF-&bgr;, and VEGF. Real-time polymerase chain reaction showed upregulated expression of Col1&agr;2, Col10&agr;1, VEGF, TGF-&bgr;1, and Runx2 mRNA. Conclusion. Upright posture induces bone acquisition in the rats’ lumbar spine, primarily through the mode of the endochondral ossification, which is associated with increased loading, activated VEGF, and TGF-&bgr;1 signaling.

Bioavailable 25(OH)D but Not Total 25(OH)D Is an Independent Determinant for Bone Mineral Density in Chinese Postmenopausal Women

Total 25(OH)D levels were determined to assess bone health in elderly populations; however, the bioavailability of 25(OH)D is regulated by the albumin and vitamin D binding protein (DBP) levels and DBP variations. Whether bioavailable 25(OH)D level is a superior biomarker for vitamin D than total 25(OH)D level regarding the BMD and the bone metabolism were not yet fully understood. With a community based cross-sectional study of 967 postmenopausal women, we found that the variant rs7041, but not rs4588, of DBP was significantly associated with the blood DBP level, which was positively correlated with the total 25(OH)D level but negatively associated with bioavailable 25(OH)D levels. Both total and bioavailable 25(OH)D levels were significantly correlated with the BMD value in postmenopausal women; however, only the bioavailable 25(OH)D level was an independent determinant of the BMD values when adjusted for age, body mass index and bone turnover biomarkers (OST and β-CTX). The bioavailable and total 25(OH)D were negatively correlated with bone formation biomarkers (OST, PINP and ALP) and PTH levels, while they were positively correlated with osteoprotegerin (OPG) level; however, the bone resorption biomarker (β-CTX) was not correlated with the 25(OH)D levels. An increment of PTH level, along with reduced bioavailable 25(OH)D levels, was evident when the bioavailable 25(OH)D level was < 5 ng/mL, which may be the optimal cutpoint for sufficient vitamin D in Chinese elderly women. The blood calcium, magnesium, ALP, TSH, FGF23, and phosphorus levels were not correlated with the total or the bioavailable 25(OH)D levels. These results suggested that high bioavailable 25(OH)D levels were correlated with reduced bone turnover processes and were a biomarker superior to total 25(OH)D for vitamin D in assessing the risks of bone-related diseases. The results indicate that the bioavailable 25(OH)D level should be determined in assessing the bone health.