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Study Design: Neovascularization and expression of inflammatory cytokines were examined in Osteoprotegerin (Opg) knockout (KO) mice that show intervertebral disc (IVD) degeneration., Objective: The aim of this study was to clarify the pathological changes in lumbar IVD degeneration in Opg KO mice., Summary of Background Data: Osteoporosis is a controversial risk factor for IVD degeneration. Deletion of Opg resulted in IVD degeneration in mice. Neovascularization and inflammatory cytokines are key factors in IVD degeneration., Methods: Opg KO mice and their wild-type (WT) littermates were euthanized. Lumbar IVDs were harvested. Safranin O/Fast Green staining was performed to examine the pathological changes. Microcomputed tomographic (micro-CT) analysis was performed to determine the structural changes at the junction of lumbar IVD cartilage and vertebrae. Tartrate-resistant acid phosphatase (TRAP) staining was performed to evaluate osteoclast formation. Protein expression of vascular endothelial growth factor A (VEGF-A), CD31, VE-cadherin, CD 34, interleukin-1β (IL-1β), and tumor necrosis factors α (TNF-α) were analyzed by immunohistochemistry (IHC) assays. Gene expressions of IL-1β, IL-6, and TNF-α were analyzed by real-time polymerase chain reaction (RT-PCR)., Results: In 12-week-old Opg KO mice, new bone was formed in the endplate cartilage of lumbar IVDs and this became more obvious in 24-week-old Opg KO mice. Three-dimensional (3D) μCT reconstruction analyses showed that the edges of the L4 and L5 vertebrae were rugged with bone marrow cavities in it. Protein expression of VEGF-A, CD31, VE-cadherin, and CD34 was increased in the endplate and growth plate of lumbar IVDs of Opg KO mice. Gene expression of IL-1β, IL-6, and TNF-α as well as protein expression of IL-1β and TNF-α were highly expressed in the lumbar IVDs of Opg KO mice., Conclusion: Deletion of Opg leads to increased neovascularization and expression of inflammatory cytokines in the lumbar disc in Opg KO mice, which may play important roles in IVD degeneration., Level of Evidence: N/A.

Objective: To observe effects of removing arms and ovarian on lumbar intervertebral disc and vertebral bone mineral density (BMD) by establishing rat model of lumbar intervetebral disc degeneration (IDD) with kidney deficiency, and to explore internal mechanism of disc degeneration, relationship between disc degeneration and osteoporosis., Methods: Thirty Sprague-Dawley female rats aged one month were randomly divided into control group, lumbar IDD group and lumbar IDD with kidney deficiency group (combined group), 10 rats in each group. Lumbar IDD group removed double arms, lumbar IDD with kidney deficiency group removed double arms after 3 months, both ovaries were removed. Vertebral bone mineral density were observed by Micro-CT scan; morphological changes were tested by safranine O-fast green staining; II, X collagen protein expression in the intervertebral disc were obsevered by immunohistochemistry; extracellular matrix gene expression were obsevered by real-time polymerase chain reaction (RT-PCR), in order to evaluate the effects of removed of forelimbs and double ovarian on degeneration and vertebral bone mineral density of intervertebral disc., Results: Micro-CT scan showed osteoporosis in kidney deficiency group was obviously worse than other two groups; safranine O-fast green staining showed that intervertebral space became narrowed, intervertebral disc tissue degenerated obviously, chondral palte was underdeveloped in kidney deficiency group; immunohistochemistry showed that X collagen expression increased, type II collagen expression decreased in kidney deficiency group; RT-PCR showed that type II collagen expression in lumbar IDD group and kidney deficiency group was lower than control group, and had statistical meaning among three groups (P=0.000, P=0.000); Age 1 in lumbar IDD group and kidney deficiency group was lower than control group, and had statistical meaning among three groups (P=0.000, P= 0.000); while type X collagen expression was higher than control group, but no significant meaning; MMP-13 in lumbar IDD group and kidney deficiency group was higher than control group, with significant meaning compared among three groups (P= 0.000, P=0.000); aggrecanase-2 in lumbar IDD group and kidney deficiency group was higher than control group, with significant meaning compared among three groups (P=0.006, P=0.008)., Conclusion: Rats model of lumbar disc degeneration established by removed forelimbs and ovariectomized can occure "bone like"--osteoporosis, which is similar with clinical kidney lumbar disc degeneration in tissue morphology, molecular cell biology expression.

Objective: To investigate the effects of serum containing Yiqi Huayu Bushen Recipe, a compound Chinese herbal medicine, on expressions of aggrecan and type X collagen mRNAs of the degenerated intervertebral disc cells in patients with cervical spondylotic myelopathy., Methods: Six inpatients diagnosed as cervical spondylotic myelopathy and undergoing cervical decompression and fusion surgery to remove the degenerated disc tissue were enrolled in this study. Primary intervertebral disc cells were isolated using small tissue mass method. Serum containing Yiqi Huayu Bushen recipe were obtained after SD rats were fed with Yiqi Huayu Bushen recipe for 3 days. MTT method was used to detect proliferation of the first-passage cells isolated from degenerated intervertebral discs and growth curve was drawn. In the following tests, the first-passage cells were cultured with the sera containing drugs and sodium chloride (NaCl), respectively. Proliferation of intervertebral disc cells was detected by MTT method and expressions of aggrecan and type X collagen mRNAs were detected by real-time polymerase chain reaction., Results: The growth curve of the first-passage cells of the degenerated intervertebral discs showed that the time of cell proliferation peak was the sixth day. Compared with the same concentrations of NaCl solution, serum containing Yiqi Huayu Bushen Recipe at 5%, 10% and 20% promoted cell proliferation and expression of aggrecan mRNA and decreased expression of type X collagen mRNA (P<0.01). 10% serum containing Yiqi Huayu Bushen recipe was more effective than 5% and 20%., Conclusion: Yiqi Huayu Bushen Recipe inhibits degeneration of the intervertebral disc cells by regulating collagen and proteoglycan expressions.

Study Design: An in vivo study of the cervical intervertebral discs (IVDs) response to upright posture was performed using an amputated bipedal rat model., Objective: To investigate the effects of upright posture on IVDs of rat cervical spine., Summary of Background Data: The distinct arrangement of human neck muscle from that of cat and rhesus indicated that in the evolution process, upright posture might have affected cervical spine of human ancestors. However, the effects of upright posture on cervical spine have not been assessed., Methods: Forty-one-month-old rats were randomly divided into 5-month-control, 5-month-surgery, 7-month-control, and 7-month surgery group (n = 10 per group). Both forelimbs of 2 surgery group rats were amputated, and those rats were then induced to be upright in the custom-made cages. Two control group rats were kept in regular cages. These rats were respectively killed at the fifth and seventh month after surgery and the IVD samples of lumbar spine were harvested for histologic and immunohistochemical studies. Total RNA isolated from these samples were used for real-time polymerase chain reaction of type II collagen (Col2a1), type X collagen, matrix metalloproteinase 13 (MMP-13), MMP-3, aggre-can, and aggrecanase-2 (ADAMTS-5)., Results: Upright posture affects histologic changes of the cervical IVDs such as fissures of anulus fibrosus and decreased height of disc, decreased protein level of Col2a1 at nucleus pulposus and anulus fibrosus, up-regulated MMP-13, MMP-3, ADAMTS-5, and type X collagen mRNA expression, and downregulated mRNA expression of Col2a1 and aggrecan., Conclusion: Upright stance accelerates cervical disc degeneration in rats.