The Effect And Its Mechanism Of Alendronate On Disc Degeneration Adjacent To A Lumbar Fusion In Ovariectomized Rats

Part one Experimental study on disc degeneration adjacent to a lumbar fusion in ovariectomized ratsObjective:To investigate the characteristics of disc degeneration adjacent to a lumbar fusion in ovariectomized rats. And to investigate the incidence and pathological features of adjacent-segment disc degeneration as well as the effect of osteoporosis on the adjacent-segment disc degeneration.Methods:40 female SD rats were include in this study. The rats underwent either a bilateral ovariectomy or sham operation. Four weeks later, all but ten rats underwent PLF consisting of an intertransverse process fusion using an autologous-iliac-bone graft with spinous-process wire fixation at L4-5. And randomly divided into 4 groups: Sham group, OVX group, PLF group, OVX+PLF group. Animals were sacrificed 12 weeks after PLF surgery. We compared X-ray imaging and imaging score before sacrificed. After the rats were killed, the L3-6 segment discs were removed, dissected of muscle, and assessed palpated manually. Next, L5-6 of adjacent-segment disc were fixed in formalin for histology, histomorphometric and immunohistochemical analysis at adjacent intervertebral disc of L5-6 segments and scored.Results:1 Fusion evaluationThe imaging score of PLF group was 3.00±0.92, the imaging score of OVX+PLF group was 2.15±0.88. The radiography fusion scores were markedly lower in the OVX+PLF group than in the PLF group(P<0.05).The manual palpation test revealed no detectable movement at the level of fusion in any of the rats that underwent PLF surgery.2 Adjacent-segment disc degeneration evaluationThere was a marked loss of notochord cells in the OVX, PLF, and OVX+PLF groups. A portion of these cells had been replaced by chondrocyte-like cells distributed in clusters, and the surrounding matrix displayed mucinous degeneration. There was various degrees of derangement in the annulus fibrosus. The endplate appeared to have large areas of varying degrees of calcification and contained many vascular buds. The OVX, PLF, and OVX+PLF groups showed significantly higher scores compared with the Sham group(P<0.05). These degenerative changes were most obvious in the OVX+PLF group.Conclusion:PLF surgery using spinous process wire fixation can lead to adjacent-segment disc degeneration. Osteoporosis exacerbates the adjacent-segment disc degeneration induced by posterolateral lumbar fusion in an ovariectomized rat model. The pathological process of ASDD is extensive, limiting its study. The methods used in our study provides a good model for the pathological process of elaboration of adjacent-segment disc degeneration and drug therapy efficacy.Part two The protential effects of alendronate on disc degeneration adjacent to a lumbar fusion in ovariectomized ratsObjective:The aim of this part is to determined the effect of alendronate on the disc degeneration adjacent to a lumbar fusion in ovariectomized rats.Methods:50 female SD rats were include in this study. The rats underwent either a bilateral ovariectomy or sham operation. Four weeks later, all but ten rats underwent PLF consisting of an intertransverse process fusion using an autologous-iliac-bone graft with spinous-process wire fixation at L4-5. Animals were subcutaneously administered vehicle(V) or ALN(Sigma-Aldrich, MO, USA) 70 μg/kg/week once a week for 12 weeks. All PLF animals were divided into five groups(n=10) as follows: Sham+V, OVX+V, PLF+V, OVX+PLF+V, and OVX+PLF+ALN group. The animals were euthanized 12 weeks after the PLF surgery. Blood samples were collected from abdominal aorta and stored at-80 °C for analysis the serum bone metabolism biomarkers. The L3-6 segment discs were removed, dissected of muscle, and assessed palpated manually. Then, separate the bilateral L4-5 intertransverse fusion site and L5-6 intervertebral disc, which decalcified, embedded, sectioned to dyeing in turn. Fusion site was stained with HE followed by the histological score, while adjacent-segment intervertebral disc was stained with VG followed by the histological score. The height of the intervertebral disc endplate, endplate thickness and endplate calcification area ratio were measured based on the VG staining images.Results:1 Fusion evaluationQualitative histological analysis of fusion mass showed well-developed bone neogenesis in the OVX+PLF+PTH group compared with OVX+PLF+V group. In the OVX+PLF+PTH group, cross-linked trabecular bone surrounded richly by bone marrow were observed. In contrast, there were cartilaginous and fibrosis-like tissue in the PLF+V group and OVX+PLF+V group, both exists sequestrum. The histological score were: PLF+V 3.60±1.00; OVX+PLF+V 3.21±1.21; OVX+PLF+ALN 5.34±1.40. The average Emery grade obtained for the OVX+PLF+PTH group was significantly greater than the mean scores recorded in the OVX+PLF+V group(P<0.05).For manual palpation, the steel wire of all PLF-surgery rats remained intact and connected to the spinous process. There was no detectable movement at the levels of fusion on manual palpation in all PLF-surgery rats.2 Adjacent-segment disc degeneration evaluationIn the Sham group, the adjacent-segment intervertebral discs appeared normal. Cartilage endplates were hyaline cartilage composed of chondrocytes. The nucleus pulpous is full of bulging gel with abundant notochord cells while collagen bundles within the lamellae are parallel with arranged regular. In contrast, disc degeneration was observed in the OVX+V, PLF+V and OVX+PLF+V groups. Calcification tissue was prevalent in the cartilage endplate. The size of notochord cells were reduced and some were replaced by chondrocytes-like cells in the nucleus pulpous. Loss of organized parallel arrangement of the fibres were also observed in the annulus fibrosus. The effect from OVX was evident when comparing PLF+V group to the OVX+PLF+V group, and mucoid degeneration was more prevalent in the nucleus pulpous in the OVX+PLF+V group. In contrast, the histomorphology in the OVX+PLF+ALN group is much better than that in the OVX+PLF+V group. As shown in OVX+PLF+ALN group, reduced chondrocyte-like cells and the size of mucoid degeneration in the nucleus pulposus while fibres arranged more neatly in the annulus fibrosus were observed compared with the OVX+PLF+V group. Calcification tissue rarely appeared in the middle cartilage endplate in the OVX+PLF+V group.The histological score confirmed these changes, the OVX+V, PLF+V, and OVX+PLF+V groups showed significantly higher scores compared with the Sham+V group(P<0.05), and the OVX+PLF+ALN group had markedly lower scores than that in the OVX+PLF+V group(P<0.05).3 Morphological ParametersDisc height was significantly lower in the OVX+V, PLF+V, and OVX+PLF+V groups than the Sham+V group(P<0.05). The endplate thickness and relative area of endplate calcification were significantly higher in the OVX+PLF+V group compared with the Sham+V group(P<0.05). The OVX+PLF+ALN group showed a marked increase in disc height(P<0.05), whereas the endplate thickness and relative area of endplate calcification were decreased compared with those in the OVX+PLF+V group(P<0.05).4 Serum bone metabolism markersCompared to Sham+V group, the serum OC levels was significantly decreased while the serum CTX-I levels was significantly increased in the OVX+V and OVX+PLF+V groups(P<0.05). In contrast, the serum OC levels were significantly higher, but the serum CTX-I levels were significantly lower in the OVX+PLF+ALN group than that in the OVX+PLF+V group(P<0.05).Conclusion:Alendronate is a novel pharmacotherapeutic strategy that effectively suppresses the progression of adjacent-segment disc degeneration without producing obvious adverse effects on the lumbar posterolateral fusion in ovariectomized rats. Based on the considerable discrepancies between species, further comprehensive investigations should be conducted prior to the clinical application of ALN. Part three The mechanism of alendronate on disc degeneration adjacent to a lumbar fusion in ovariectomized ratsObjective:Alendronate effectively suppresses the progression of adjacent-segment disc degeneration in ovariectomized rats, and assumed as a potential drug for the relief of intervertebral disc degeneration. However, its regulatory mechanism has not yet been explained. In this part, we used an osteoporotic adjacent-segment disc degeneration rat model, to observe the effect of ALN on intervertebral disc degeneration adjacent to a lumbar fusion in ovariectomized rats and to explore its mechanism of protective effect.Methods:50 female SD rats were include in this study. The rats underwent either a bilateral ovariectomy or sham operation. Four weeks later, all but ten rats underwent PLF consisting of an intertransverse process fusion using an autologous-iliac-bone graft with spinous-process wire fixation at L4-5. Animals were subcutaneously administered vehicle(V) or ALN(Sigma-Aldrich, MO, USA) 70 μg/kg/week once a week for 12 weeks. All PLF animals were divided into five groups(n=10) as follows: Sham+V, OVX+V, PLF+V, OVX+PLF+V, and OVX+PLF+ALN group. The animals were euthanized 12 weeks after the PLF surgery. We compared X-ray imaging and imaging score before sacrificed. After the rats were killed, the L3-6 segment discs were removed, dissected of muscle, and assessed palpated manually.. Then, separate the bilateral L4-5 intertransverse fusion site and L5-6 intervertebral disc, which decalcified, embedded, sectioned to dyeing in turn. Fusion site was stained with HE followed by the histological score, while adjacent-segment intervertebral disc was stained with VG followed by the histological score.The MVD and and vascular buds relative area analysis were measured based on the VG staining images. Immunohistochemistry and real-time PCR for the detection of aggrecan, I collagen, type II collagen, MMP-13 changes and ADAMTS-4 expression in the disc. The six degrees motion in L5-6 intervertebral disc was detected by 3-D motion testing system.Results:1 Fusion evaluationThe steel wire of all PLF-surgery rats remained intact and connected to the spinous process. There was no detectable movement at the levels of fusion on manual palpation in all PLF-surgery rats.2 Bone mineral density and micro-CT parameters of the L6 lumbar vertebraeThe BMD of the L6 vertebrae in the OVX+V group and OVX+PLF+V group were significantly decreased compared with the Sham group. There are no statistical difference between OVX+V and OVX+PLF+V group. As a function of ALN treatment, the significantly difference was found between OVX+PLF+ALN and OVX+PLF+V group.Compared to Sham group, BV/TV, TB.Th and Tb.N values were significantly decreased but Tb.Sp and SMI values were significantly increased in the OVX+V group and OVX+PLF+V group(P<0.05). BV/TV, TB.Th and Tb.N values were significantly increased and Tb.Sp and SMI values were significantly decreased in the OVX+PLF+ALN group compared with the OVX+PLF+V group(P<0.05).3 Adjacent-segment disc degeneration evaluation and scoringDisc degeneration was observed in the OVX+V, PLF+V and OVX+PLF+V groups. Calcification tissue was prevalent in the cartilage endplate. The size of notochord cells were reduced and some were replaced by chondrocytes-like cells in the nucleus pulpous. Loss of organized parallel arrangement of the fibres were also observed in the annulus fibrosus. The effect from OVX was evident when comparing PLF+V group to the OVX+PLF+V group, and mucoid degeneration was more prevalent in the nucleus pulpous in the OVX+PLF+V group. In contrast, the histomorphology in the OVX+PLF+ALN group is much better than that in the OVX+PLF+V group. As shown in OVX+PLF+ALN group, reduced chondrocyte-like cells and the size of mucoid degeneration in the nucleus pulposus while fibres arranged more neatly in the annulus fibrosus were observed compared with the OVX+PLF+V group. Calcification tissue rarely appeared in the middle cartilage endplate in the OVX+PLF+V group.The histological score confirmed these changes, the OVX+V, PLF+V, and OVX+PLF+V groups showed significantly higher scores compared with the Sham+V group(P<0.05), and the OVX+PLF+ALN group had markedly lower scores than that in the OVX+PLF+V group(P<0.05).4 Morphological parameters resultsMVD and vascular buds relative area in the OVX+V, PLF+V and OVX+PLF+V groups were significantly decreased compared with Sham+V group(P<0.05). The effect from OVX was evident when comparing PLF+V group to the OVX+PLF+V group. However, the MVD and vascular buds relative area in the OVX+PLF+ALN group was significantly higher than that in the OVX+PLF+V group(P<0.05).5 Immunohistochemistry AnalysisChondrocyte-like cells metaplasia at the nucleus pulposus and dearrangement of annulus fibrosus were observed in the OVX+V, PLF+V and OVX+PLF+V groups, accompanied by stronger immunostainings of type I collagen, MMP-13 and ADAMTS-4 and weaker immunostainings of Aggrecan and type II Collagen were observed in matrix compared with Sham+V group. The extent of the ASDD was significantly increased by addition of OVX. However, OVX+PLF+ALN group showed less chondrocyte-like cells and more neatly arranged annulus fibrosus, with stronger immunostainings of Aggrecan and type II collagen as well as weaker immunostainings of type I collagen, MMP-13 and ADAMTS-4 in matrix than that in the OVX+PLF+V group, as comfirmed by analysis of IOD value.6 Real-time PCR analysisThe OVX+V, PLF+V, and OVX+PLF+V groups showed lower Col2α1 and Aggrecan m RNA levels and higher Col1α1, MMP-13 and ADAMTS-4 m RNA levels compared with the Sham+V group(P<0.05). Col2α1 and Aggrecan m RNA expression was significantly increased in the OVX+PLF+ALN group compared with the OVX+PLF+V group(P<0.05). Additionally, Col1α1, MMP-13 and ADAMTS-4 expression was lower in the OVX+PLF+ALN group than the OVX+PLF+V group(P<0.05).7 3D motion testing resultsIn loading of flexion, lateral bending and rotation, the range of motion was significantly reduced in the PLF+V, OVX+PLF+V and OVX+PLF+ALN group compared with Sham+V group(P<0.05). No difference between the groups.Conclusion:1 Osteoporosis exacerbates the djacent-segment disc degeneration induced by posterolateral lumbar fusion in an ovariectomized rat model, suggested that osteoporosis is a risk of adjacent segment disc degeneration factors after spinal fusion.2 This study showed that ALN attenuates PLF-induced ASDD progression in an osteoporotic rat model by maintaining the structural integrity and function of the adjacent vertebrae and endplates and preventing metabolic changes in the extracellular matrix of the adjacent disc.3 ALN can be considered as a potential drug can alleviate the disc degeneration adjacent to a lumbar fusion in osteoporosis condition.