阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的作用及其机制的研究
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摘要
腰椎间盘退变是导致下腰部疼痛的主要病因之一，下腰痛给患者带来巨大痛苦的同时，给家庭及社会带来沉重的经济负担。一些临床及流行病学研究认为，骨质疏松与脊柱退行性疾病或椎间盘退变呈负相关性，但最近的一项关于绝经期前后妇女椎体骨密度与腰椎间盘退变相关性的研究显示，腰椎骨密度与腰椎间盘退变呈正相关性，跟骨及桡骨的骨密度同样与椎间盘退变密切相关。此外，在前期研究中发现卵巢切除大鼠腰椎间盘退变与椎体骨量丢失密切相关，提示对骨质疏松的治疗可能会对椎间盘退变进程发挥一定的作用。骨质疏松与椎间盘退变关系仍存在争议，因此两者之间的关系还有待进一步研究。
研究显示，椎间盘退变与脊柱结构完整有关，包括邻近的结构如椎体及终板。椎体及终板结构的破坏将导致异常力学负荷作用于椎间盘。对绝经期前后妇女骨密度变化的研究结果显示，骨密度与椎间盘的高度存在一定的相关性，说明椎体的结构特性与椎间盘退变程度密切相关，并且组织结构的改变同样会影响到其它结构进而出现改变。骨密度降低所引起的椎体骨折可导致椎间盘高度降低，并加速椎间盘的退变进程。软骨终板不仅是维持椎间盘正常功能的重要结构，同时还是髓核获取营养的重要途径。Nachemson等人发现，软骨终板钙化会导致软骨终板增厚，软骨终板内软骨层变薄，从而影响营养物质通过软骨终板向椎间盘的扩散，最终导致椎间盘早期出现退行性变。
雌激素缺乏可引起绝经后妇女终板出现退行性改变，进而最终导致椎间盘发生退行性变。雌激素替代治疗被建议用于骨质疏松相关的椎间盘退变的治疗。然而，患者因雌激素治疗尚存在一些不良反应而拒绝用药，因此，当前迫切需要一种新的药物用于骨质疏松相关的椎间盘退变的治疗，如二膦酸盐类药物：阿仑膦酸钠、唑来膦酸钠等。阿仑膦酸钠作为一种潜在的二膦酸盐类药物，已广泛用于绝经后妇女骨质疏松的治疗。阿仑膦酸钠可提高患者骨密度，抑制骨转换率，降低骨折发生率。最近，Neogi等人的研究显示，阿仑膦酸钠可以抑制绝经后妇女椎间隙变窄，提示二膦酸盐类药物对椎间盘退变具有一定作用。阿仑膦酸钠对椎间盘退变的作用机制尚不清楚，因此本研究拟通过影像学、组织形态计量学、分子生物学等方法，探讨阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的作用及其相关作用机制，为临床治疗骨质疏松相关的腰椎间盘退变提供理论依据及临床用药指导。
第一部分卵巢切除大鼠椎体骨量丢失与腰椎间盘退变关系的实验研究
目的：探讨卵巢切除大鼠椎体骨量丢失与大鼠腰椎间盘退变的关系。
方法：20只3月龄雌性Sprague-Dawley（SD）大鼠随机分为两组：假手术组（Sham）及卵巢切除组（OVX）。Sham组大鼠进行假手术，仅暴露卵巢组织但不切除。OVX组大鼠经卵巢切除术切除双侧卵巢组织，卵巢切除术后6个月，采用过量失血方法处死各组大鼠并收集L3-6节段及其椎间盘组织。椎体骨密度检测及椎体骨组织形态计量学检测用以评价大鼠椎体骨量及椎体微观结构的变化。对椎间盘进行van Gieson染色进行椎间盘组织学观察并进行组织学评分。
结果：
1骨密度检测结果
OVX组大鼠L3-6椎体BMD显著低于Sham组（P<0.05）。
2椎体骨组织形态计量学检测结果
OVX组大鼠L4椎体骨小梁相对体积（BV/TV），骨小梁厚度（Tb.Th）及骨小梁数量（Tb.N）显著低于Sham组（P<0.05），而骨小梁分离度（Tb.Sp），荧光周长百分率（%L.Pm），矿化沉积率（MAR）及骨形成率（BFR/BV）显著高于Sham组（P<0.05）。
3组织形态学观察及组织学评分结果
OVX组大鼠椎间盘髓核内脊索细胞大量减少，软骨样细胞大量增殖，并可见粘液样变性。软骨终板可见大量骨样组织及髓腔生成。OVX组椎间盘组织学评分显著高于Sham组（P<0.05）。
第二部分阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的预防作用的实验研究
目的：本研究旨在应用骨质疏松大鼠模型探讨预防性应用阿仑膦酸钠对腰椎间盘退变的作用。
方法：30只3月龄雌性Sprague-Dawley（SD）大鼠随机等分为三组：（1）假手术组（Sham）；（2）卵巢切除+盐水组（OVX+V）；（3）卵巢切除+阿仑膦酸钠组（OVX+ALN）。Sham组大鼠进行假手术，仅暴露卵巢组织但不切除。OVX+V组及OVX+ALN组大鼠经卵巢切除术切除双侧卵巢组织后，OVX+ALN组大鼠给予阿仑膦酸钠颈后皮下注射，剂量为15μg/kg，一周两次。OVX+V组大鼠给予等体积的生理盐水作为对照。卵巢切除术后6个月，采用过量失血方法处死各组大鼠并收集L3-6节段椎体及其间盘组织。椎体骨密度检测、micro-CT检测及椎体生物力学检测用以评价大鼠腰椎椎体骨组织特性及微观结构的变化。对椎间盘进行van Gieson染色进行椎间盘组织学观察并进行组织学评分。同时对各组大鼠椎间盘高度以及软骨终板厚度的变化进行分析比较。免疫组织化学染色及real-time PCR用于检测各组大鼠椎间盘中aggrecan、I型胶原、II型胶原、MMP-1、MMP-3及MMP-13表达的变化。
结果：
1椎体骨密度检测结果
OVX+V组大鼠L3-4及L5-6椎体BMD显著低于Sham组（P<0.05）。OVX+ALN组大鼠L3-4及L5-6椎体BMD显著高于OVX+V组（P<0.05）
2Micro-CT检测结果
OVX+V组BV/TV、Tb.N显著低于Sham组，而Tb.Sp及SMI显著高于Sham组（P<0.05）。OVX+ALN组椎体BV/TV、Tb.N显著高于OVX+V组，Tb.Sp及SMI显著低于OVX+V组（P<0.05）。而Tb.Th在三组间无统计学差异（P>0.05）。
3椎体生物力学检测结果
OVX+V组椎体极限负荷（maximum load），屈服应力（yield stress），极限应力（maximum stress）及弹性模量（elastic modulus）均显著性低于Sham组（P<0.05）。OVX+ALN组椎体极限负荷，屈服应力，极限应力及弹性模量均显著高于OVX+V组（P<0.05）。
4组织形态学观察及组织学评分结果
OVX+V组椎间盘退变明显，而OVX+ALN组椎间盘髓核内仅出现少量的软骨样细胞，髓核未见粘液样变性，在纤维环与髓核交接处可见纤维软骨增殖现象。软骨终板内可见大量异常骨样组织及髓腔生成。而OVX+ALN组椎间盘退变程度较轻，仅在髓核内出现少量的软骨样细胞增殖，以及在髓核与纤维环交界处出现一些软骨细胞增殖，髓核内未出现粘液样变性。并且在软骨终板中段未发现有异常骨样组织生成。OVX+V组L5-6椎间盘组织学评分显著高于Sham组，而OVX+ALN组组织学评分显著低于OVX+V组（P<0.05）。
5椎间盘高度检测结果
OVX+V组L5-6椎间盘高度显著低于Sham组（P<0.05），OVX+ALN组L5-6椎间盘高度显著高于OVX+V组（P<0.05），OVX+ALN组与Sham组L5-6椎间盘高度无显著性差异（P>0.05）。
6软骨终板厚度及软骨终板内骨样组织面积检测结果
OVX+V组软骨终板厚度及软骨终板内骨样组织与软骨终板面积比值均显著高于Sham组（P<0.05），OVX+ALN组大鼠软骨终板厚度显著低于OVX+V组（P<0.05），且软骨终板内骨样组织与软骨终板面积比值显著低于OVX+V组（P<0.05）。
7椎间盘免疫组织化学检测结果
OVX+V组髓核内，aggrecan及II型胶原染色强度显著低于Sham组。然而，OVX+ALN组aggrecan及II型胶原染色强度显著高于OVX+V组。OVX+V组髓核内MMP-1、MMP-3及MMP-13染色强度显著高于Sham组，而OVX+ALN组髓核内MMP-1、MMP-3及MMP-13染色强度显著低于OVX+V组。
OVX+V组纤维环中MMP-1、MMP-3及MMP-13表达增高，其IOD值显著高于Sham组（P<0.05）。但OVX+ALN组纤维环中MMP-1、MMP-3和MMP-13的表达显著降低，其IOD值显著低于OVX+V组（P<0.05）。
OVX+V组纤维环中I型胶原表达增高及Ⅱ型胶原表达降低，其IOD值显著高于或低于Sham组（P<0.05）。而OVX+ALN组纤维环中I型胶原表达降低及Ⅱ型胶原表达增强，其IOD值显著低于或高于OVX+V组（P<0.05）。OVX+V组软骨终板区域可见X型胶原免疫组织化学染色程度强于Sham组，而OVX+ALN组X型胶原染色强度低于OVX+V组。
8Real-time PCR检测结果
与Sham组相比，OVX+V组Col2α1mRNA表达降低（P>0.05），而aggrecan及Col1α1mRNA表达显著增高（P<0.05）。而OVX+ALN组aggrecan及Col2α1mRNA显著高于OVX+V组（P<0.05），Col1α1mRNA表达显著低于OVX+V组（P<0.05）。OVX+V组MMP-1、MMP-3及MMP-13mRNA表达显著高于Sham组（P<0.05）。而阿仑膦酸钠可有效抑制由卵巢切除引起的MMP-1、MMP-3及MMP-13mRNA表达的增高（P<0.05）。
第三部分阿仑膦酸钠治疗卵巢切除大鼠腰椎间盘退变作用效果的实验研究
目的：探讨阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的治疗作用。
方法：30只3月龄雌性SD大鼠随机分为三组：（1）假手术组（Sham）；（2）卵巢切除+盐水治疗组（OVX+V）；（3）卵巢切除+阿仑膦酸钠治疗组（OVX+ALN）。OVX+V组及OVX+ALN组大鼠行双侧卵巢切除术切除双侧卵巢组织，Sham组大鼠仅暴露卵巢组织但不切除。OVX+ALN组大鼠于卵巢切除术3个月后给予阿仑膦酸钠颈后皮下注射，剂量为15μg/kg，一周两次。OVX+V组大鼠给予等体积的生理盐水作为对照。卵巢切除术后6个月，过量失血处死各组大鼠并收集L3-6椎体标本。椎体骨密度检测及Micro-CT检测用以评价大鼠腰椎椎体骨骼特性及微观结构的变化。应用van Gieson染色进行椎间盘组织学检测及组织学评分。免疫组织化学染色用于检测各组大鼠椎间盘中aggrecan、I型胶原、II型胶原、MMP-1及MMP-13表达的变化。
结果：
1椎体BMD检测结果
OVX+V组大鼠L3-4及L5-6椎体BMD显著低于Sham组（P<0.05），而OVX+ALN组大鼠L3-4及L5-6椎体BMD显著高于OVX+V组（P<0.05）。
2Micro-CT检测结果
OVX+V组BV/TV、Tb.N显著低于Sham组，而Tb.Sp显著高于Sham组（P<0.05）。而OVX+ALN组椎体BV/TV、Tb.Th显著高于OVX+V组，Tb.Sp显著低于OVX+V组（P<0.05）。
3椎间盘组织形态学观察及评分结果
OVX+V组髓核内，脊索细胞数量显著减少，并出现大量软骨样细胞增殖，髓核内可见粘液样变性。在纤维环与髓核的过渡区出现大量体积较小的软骨细胞增殖。软骨终板内异常钙化现象较为明显，并且在软骨终板的中段出现大量的异位骨样组织及髓腔。OVX+ALN组髓核内脊索细胞数量未出现显著性减少，髓核中出现一定量的软骨样细胞，并出现轻度的黏液样变性。在纤维环与髓核交接处可见纤维软骨细胞增殖现象。
OVX+V组椎间盘组织学评分显著高于Sham组（P<0.05），而OVX+ALN组椎间盘组织学评分显著低于OVX+V组（P<0.05），但Sham组与OVX+ALN组椎间盘组织学评分无显著性差异（P>0.05）。4免疫组织化学染色结果
OVX+V组髓核内aggrecan及II型胶原表达较Sham组显著降低，MMP-1及MMP-13表达增强，而OVX+ALN组aggrecan及II型胶原表达高于OVX+V组，并且MMP-1及MMP-13表达弱于OVX+V组。OVX+V组纤维环中MMP-1及MMP-13表达增高，其IOD值显著高于Sham组（P<0.05）。但OVX+ALN组纤维环中MMP-1和MMP-13的表达显著降低，其IOD值显著低于OVX+V组（P<0.05）。
OVX+V组纤维环中Ⅱ型胶原表达降低，而I型胶原表达增高，其IOD值分别显著低于或高于Sham组（P<0.05）。而OVX+ALN组纤维环中Ⅱ型胶原表达增强，I型胶原表达减弱，其IOD值显著高于或低于OVX+V组（P<0.05）。
OVX+V组软骨终板内X型胶原表达显著高于Sham组，而OVX+ALN组软骨终板内X型胶原表达较OVX+V组显著降低。
结论：
1卵巢切除大鼠椎体发生骨质疏松同时可合并出现腰椎间盘的退行性改变。
2阿仑膦酸钠可有效延缓卵巢切除大鼠腰椎间盘退变进程。其具体作用机制一方面可能与维持椎体及软骨终板结构的完整性及功能有关，另一方面与调控细胞外基质代谢，保护椎间盘的功能有关。
3阿仑膦酸钠是一种防治与骨质疏松相关的腰椎间盘退变的潜在性药物。
Lumbar intervertebral disc degeneration (LVD) is the leading cause oflow back pain and other disc disorders, which are responsible for enormoushuman suffering, high health care costs, and significant socioeconomic losses.Although some clinical and epidemiological studies observed thatosteoporosis was inversely related to spinal degeneration diseases orintervertebral disc degeneration, a recent correlation study of BMD and LVDin premenopausal and postmenopausal women showed that the BMD of notonly the lumbar vertebrae but also the calcaneus and radius were associatedwith LVD. In addition, in a previous study, we found a strong associationbetween osteopenia and disc degeneration in ovariectomized (OVX) rats,suggesting that measures to reduce osteoporotic changes might have inhibitoryeffects on disc degeneration. These variant consequences indicate that thecontraversial relationship between osteoporosis and LVD remains to be furtherclarified.
Increasing evidence indicates that disc degeneration is associated with thedisruption of an intact spinal structure, including adjacent structures, such asthe vertebral body and endplate, which might result in imbalanced mechanicalloading on the disc. BMD assays in both pre-and postmenopausal womenrevealed a correlation between BMD and disc height, suggesting that thevertebral structural propoties are closely related to the degree of discdegeneration, and the milieu influencing one structure may similarly influenceothers. Vertebral fractures caused by low BMD can result in decreased discheight and exacerbate disc degeneration. The endplate is not only anotheressential structure in maintaining the integrity and physiological function ofthe avascular intervertebral disc, but also acts as a gateway for nutrient supplyto the nucleus. Nachemson found that calcification of the endplate may impede nutrient diffusion into disc, leading to thinner cartilage, but increasedthickness of the endplate, and consequently premature disc degeneration.Estrogen deficiency also might influence the severity of disc degeneration inpostmenopausal females, by negatively affecting endplate quality andinducing endplate degeneration. Estrogen replacement therapy is suggested asan alternative treatment for disc degeneration related to osteoporosis. However,many patients refuse estrogens for various reasons, which drove us to seeknew drug targets for the treatment of osteoporosis related disc degeneration,such as bisphosphonates, including alendronate (ALN) and zoledronate. ALN,a potent bisphosphonate, has widely been used as a first line drug in thetreatment of osteoporosis in postmenopausal women, where it can increasebone mineral density (BMD), suppress bone turnover and reduce the incidenceof fractures. Most recently, a study by Neogi et al. showed that ALN mightlead to a reduction in disc space narrowing in postmenopausal women,suggesting a novel role for bisphosphonate in altering the pathological processof disc degeneration. We hypothesize that ALN may be an alternative drugtreatment for lumbar disc degeneration related to osteoporosis, although theunderlying mechanisms by which ALN impacts the process of discdegeneration remain unclear. In the present study, we intends to investigate theeffect of alendronate on lumbar intervertebral disc in OVX rats by radiology,histomorphometry, molecular biology, in order to provide clinical guidelinesfor treatment of osteoporosis related intervertebral disc degeneration in clinic.
Part I Lumbar intervertebral disc degeneration can induced byosteoporosis in ovariectomized rats
Objective: To investigate the relationship between osteoporosis andlumbar intervertebral disc degeneration in ovariectomized rats．
Methods：Twenty female Sprague-Dawley rats aged3months underwenteither sham-operation (sham)(N=10) or bilateral ovariectomy (OVX)(N=10).After animals were sacrificed at6months post-OVX, the L3-6spinalsegments were harvested. Bone mineral density (BMD) and histomorphometryanalysis were performed to evaluate the bone mass and microstructural changes in the lumbar vertebral bodies. Histological analysis with van Giesonstain and the histological score were used to identify the characteristics of thedegenerative discs.
Results:
1Bone mineral density
The BMD values of L3-6vertebral bodies in the OVX group weresignificantly decreased, when compared with the Sham group (P<0.05).
2Bone histomorphometry
In the OVX group, the value in bone volume fracture (BV/TV),trabecular bone thickness (Tb.Th), and trabecular number (Tb.N) weresignificantly decreased, and trabecular separation (Tb.Sp), percent labeledperimeter (%L.Pm), bone formation rate (BFR/BV) and mineral appositionrate (MAR) were significantly increased when compared with Sham group(P<0.05).
3Histological findings and histological scores
In the OVX+V group, the discs showed degenerative changes, where thenucleus pulposus comprised relatively few, clustered, doublets ofchondrocyte-like cells. Mucoid degeneration could be seen eroding thenucleus pulposus. Bony tissues became more obvious in the cartilage endplate.The histological score of the discs in the OVX group was significantly higherthan the Sham group (P<0.05).
Part II The effect of alendronate sodium on degenerated intervertebraldisc tissue in ovariectomized rats
Objective: This study aims to investigate the effects of ALN on lumbarintervertebral disc degeneration related to osteoporosis using anovariectomized (OVX) rat model.
Methods: Thirty female Sprague-Dawley rats aged3months wererandomly divided into three groups (with10rats each) as follows: the Shamgroup underwent sham surgery; the OVX+ALN group had twice-a-weeksubcutaneous injections of ALN (15μg/kg) for6months. The OVX+V groupreceived an equivalent volume of saline solution as placebo post-OVX. After animals were sacrificed at6months post-OVX, the L3-6spinal segments wereharvested. Bone mineral density (BMD), micro-CT analysis andbiomechanical testing were performed to evaluate the bone quality andmicrostructural changes in the lumbar vertebral bodies. Histological analysiswith van Gieson stain and the histological score were used to identify thecharacteristics of the degenerative discs. The disc height and the thickness ofthe cartilage endplate were measured and compared. Immunohistochemistryand real-time PCR measurements for aggrecan, type I collagen, type IIcollagen, and matrix metalloprotease (MMP)-1, MMP-3and MMP-13expressions on the disc were performed to assess the underlying molecularsignaling changes in matrix metabolism during intervertebral discdegeneration.
Results:
1Bone mineral density
The BMD values of L3-4and L5-6vertebral bodies in the OVX+V groupwere significantly decreased, when compared with the Sham group (P<0.05).However, the BMD values of L3-4and L5-6vertebral bodies in theOVX+ALN group were significantly increased, when compared with theOVX+V group (P<0.05)
2Micro-CT measurements
Quantification of three-dimensional trabecular structures revealed thatBV/TV and Tb.N in the OVX+V group was significantly decreased comparedwith the Sham group (P<0.05), and Tb.Sp and SMI was higher in the OVX+Vgroup than in the Sham group (P<0.05)(Table4). The BV/TV and Tb.N inALN group are significantly higher than their counterparts in OVX+V group(P<0.05). Meanwhile, the Tb.Sp and SMI was markedly lower in theOVX+ALN group than in the OVX+V group (P<0.05). There was nosignificant difference in Tb.Th among the three groups (P>0.05).
3Mechanical testing for the lumbar vertebral body
Compared with the Sham group, the values of maximum load, yieldstress, maximum stress and elastic modulus were significantly decreased in the OVX+V group (P<0.05). However, the values of maximum load, yield stress,maximum stress and elastic modulus were significantly higher in theOVX+ALN group than in the OVX+V group at6months post-surgery(P<0.05), suggesting that ALN could effectively maintain the biomechanicalstrength of the vertebrae.
4Histological findings and histological scores
In the OVX+V group, the discs showed degenerative changes, where thenucleus pulposus comprised relatively few, clustered, doublets ofchondrocyte-like cells. Mucoid degeneration could be seen eroding thenucleus pulposus, with clefts forming within them. An increased number ofsmall chondrocytes appeared in the inner layer of the annulus fibrosus, whichwas present in the form of fibers invading the nucleus pulposus.. Bony tissuesbecame more obvious in the deep zone of cartilage endplate. In contrast, thehistological morphology in the OVX+ALN group didn’t show obviouschanges, there was no significant difference in the OVX+ALN group in thenumber of the notochordal cells, where only a few of chondrocyte-like cellsappeared in the nucleus pulposus, and some small chondrocytes were observedin the inner layer of the annulus fibrosus. Mucoid degeneration was notobserved in most samples in the OVX+ALN group and there were no bonytissues formed in the middle cartilage endplate. The histological score of thediscs in the OVX+V group was significantly higher than the Sham group(P<0.05); however, the histological score of the discs in the OVX+ALN groupwas markedly lower than the OVX+V group (P<0.05).
5Disc height
The disc height in the OVX+V group was significantly decreasedcompared with the Sham group (P<0.05). The disc height in the OVX+ALNgroup was significantly higher than the OVX+V group (P<0.05), but there wasno significant difference between the OVX+ALN and Sham groups.
6Thickness of the cartilage endplate
Both the cartilage endplate thickness and the bony tissue area within thecartilage endplate, especially in the middle cartilage endplate, were markedly increased in the OVX+V group when compared with the Sham group(P<0.05). The thickness of cartilage endplate and the bony tissues area ofcartilage endplate in the OVX+ALN group were both significantly decreasedwhen compared with the OVX+V group (P<0.05).
7Immunohistochemistry
The density of aggrecan and type II collagen staining in the OVX+Vgroup markedly decreased compared with the Sham group. However, muchstronger immunostaining was observed for aggrecan and type II collagen inthe OVX+ALN group compared with the OVX+V group. Strongerimmunostaining for MMP-1, MMP-3and MMP-13could be observed in thechondrocyte-like cells in the OVX+V group. In the OVX+ALN group,immunostaining for MMP-1, MMP-3and MMP-13were much weaker than inthe OVX+V group.
The IOD value of MMP-1, MMP-3, MMP-13positive cells in theannulus fibrosus was significantly higher in the OVX+V group than that in theSham group (P<0.05). The IOD values of type II and type I collagens weresignificantly lower and higher, respectively, in the OVX+V group comparedwith the Sham group (P<0.05). However, the IOD values of MMP-1, MMP-3and MMP-13were significantly lower in the OVX+ALN group comparedwith the OVX+V group (P<0.05), and the IOD values for type II and type Icollagens were significantly higher and lower, respectively (P<0.05). Type Xcollagen was observed in the cartilage endplate, with stronger positive stainingin the chondrocytes of the OVX+V group than in the Sham group, but weakerin the OVX+ALN group compared with the OVX+V group.
8Real-time PCR
The expression of Col2α1mRNA was decreased (P>0.05), and theexpressions of aggrecan and Col1α1mRNAs were significantly increased inthe OVX+V group compared with the Sham group (P<0.05). The expressionsof aggrecan and Col2α1mRNAs were significantly increased in theOVX+ALN group compared with the OVX+V group (P<0.05), and theexpression of Col1α1was significantly lower than in the OVX+V group (P<0.05).
The expressions of MMP-1, MMP-3and MMP-13mRNAs weresignificantly higher in the OVX+V group than in the Sham group at6monthspost-surgery (P<0.05). ALN treatment significantly depressed theOVX-induced up-regulation of MMP-1, MMP-3and MMP-13mRNAexpressions (P<0.05).
Part III The therapeutic effect of alendronate on degeneratedintervertebral disc tissue in ovariectomized rats
Objective: The aim of the present study is to investigate the therapeuticeffect of alendronate on degenerated intervertebral disc tissue inovariectomized rats.
Methods: Thirty female Sprague-Dawley rats aged3months wererandomly divided into three groups (with10rats each) as follows: the Shamgroup underwent sham surgery; the OVX+ALN group had twice-a-weeksubcutaneous injections of ALN (15μg/kg) at3months post-OVX. TheOVX+V group received an equivalent volume of saline solution as placebopost-OVX. After animals were sacrificed at6months post-OVX, the L3-6spinal segments were harvested. Bone mineral density (BMD) and micro-CTanalysis were performed to evaluate the bone quality and microstructuralchanges in the lumbar vertebral bodies. Histological analysis with van Giesonstain and the histological score were used to identify the characteristics of thedegenerative discs. Immunohistochemistry for aggrecan, type I collagen, typeII collagen, and matrix metalloprotease (MMP)-1and MMP-13expressions onthe disc were performed to assess the underlying molecular signaling changesin matrix metabolism during intervertebral disc degeneration.
Results:
1Bone mineral density
The BMD values of L3-4and L5-6vertebral bodies in the OVX+V groupwere significantly decreased, when compared with the Sham group (P<0.05).However, the BMD values of L3-4and L5-6vertebral bodies in theOVX+ALN group were significantly increased, when compared with the OVX+V group(P<0.05)
2Micro-CT measurements
Quantification of three-dimensional trabecular structures revealed thatBV/TV and Tb.N in the OVX+V group was significantly decreased comparedwith the Sham group (P<0.05), and Tb.Sp was higher in the OVX+V groupthan in the Sham group (P<0.05). The BV/TV and Tb.Th in OVX+ALN groupare significantly higher than their counterparts in OVX+V group (P<0.05).Meanwhile, the Tb.Sp was markedly lower in the OVX+ALN group than inthe OVX+V group (P<0.05).
3Histological findings and histological scores
In the OVX+V group, the discs showed degenerative changes, where thenucleus pulposus comprised relatively few, clustered, doublets ofchondrocyte-like cells. Mucoid degeneration could be seen eroding thenucleus pulposus, with clefts forming within them. An increased number ofsmall chondrocytes appeared in the inner layer of the annulus fibrosus, whichwas present in the form of fibers invading the nucleus pulposus. Bony tissuesbecame more obvious in the deep zone of cartilage endplate. In contrast, thehistological morphology in the OVX+ALN group didn’t show obviouschanges, there was no significant difference in the OVX+ALN group in thenumber of the notochordal cells, where only a few of chondrocyte-like cellsappeared in the nucleus pulposus, and some small chondrocytes were observedin the inner layer of the annulus fibrosus. Mucoid degeneration was notobserved in most samples in the OVX+ALN group and there were no bonytissues formed in the middle cartilage endplate. The histological score of thediscs in the OVX+V group was significantly higher than the Sham group(P<0.05); however, the histological score of the discs in the OVX+ALN groupwas markedly lower than the OVX+V group (P<0.05).
4Immunohistochemistry
The density of aggrecan and type II collagen staining in the OVX+Vgroup markedly decreased compared with the Sham group. However, muchstronger immunostaining was observed for aggrecan and type II collagen in
the OVX+ALN group compared with the OVX+V group. Strongerimmunostaining for MMP-1and MMP-13could be observed in thechondrocyte-like cells in the OVX+V group. In the OVX+ALN group,immunostaining for MMP-1and MMP-13were much weaker than in theOVX+V group.
The IOD value of MMP-1, MMP-13positive cells in the annulusfibrosus was significantly higher in the OVX+V group than that in the Shamgroup (P<0.05). The IOD values of type II and type I collagens weresignificantly lower and higher, respectively, in the OVX+V group comparedwith the Sham group (P<0.05). However, the IOD values of MMP-1andMMP-13were significantly lower in the OVX+ALN group compared with theOVX+V group (P<0.05), and the IOD values for type II and type I collagenswere significantly higher and lower, respectively (P<0.05).
Conclusions:
1Lumbar intervertebral disc can induced by osteoporosis inovariectomized rats.
2ALN can retard the progression of lumbar intervertebral discdegeneration in OVX rats. The underlying mechanisms might be related topreservation of the structural integrity and function of the adjacent structures,including the vertebrae and endplates, which further links with modulations inextracellular matrix metabolism to protect the disc from degeneration.
3ALN might be a promising drug agent for preventing and curing lumbarintervertebral disc degeneration related to osteoporosis.
研究显示，椎间盘退变与脊柱结构完整有关，包括邻近的结构如椎体及终板。椎体及终板结构的破坏将导致异常力学负荷作用于椎间盘。对绝经期前后妇女骨密度变化的研究结果显示，骨密度与椎间盘的高度存在一定的相关性，说明椎体的结构特性与椎间盘退变程度密切相关，并且组织结构的改变同样会影响到其它结构进而出现改变。骨密度降低所引起的椎体骨折可导致椎间盘高度降低，并加速椎间盘的退变进程。软骨终板不仅是维持椎间盘正常功能的重要结构，同时还是髓核获取营养的重要途径。Nachemson等人发现，软骨终板钙化会导致软骨终板增厚，软骨终板内软骨层变薄，从而影响营养物质通过软骨终板向椎间盘的扩散，最终导致椎间盘早期出现退行性变。
雌激素缺乏可引起绝经后妇女终板出现退行性改变，进而最终导致椎间盘发生退行性变。雌激素替代治疗被建议用于骨质疏松相关的椎间盘退变的治疗。然而，患者因雌激素治疗尚存在一些不良反应而拒绝用药，因此，当前迫切需要一种新的药物用于骨质疏松相关的椎间盘退变的治疗，如二膦酸盐类药物：阿仑膦酸钠、唑来膦酸钠等。阿仑膦酸钠作为一种潜在的二膦酸盐类药物，已广泛用于绝经后妇女骨质疏松的治疗。阿仑膦酸钠可提高患者骨密度，抑制骨转换率，降低骨折发生率。最近，Neogi等人的研究显示，阿仑膦酸钠可以抑制绝经后妇女椎间隙变窄，提示二膦酸盐类药物对椎间盘退变具有一定作用。阿仑膦酸钠对椎间盘退变的作用机制尚不清楚，因此本研究拟通过影像学、组织形态计量学、分子生物学等方法，探讨阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的作用及其相关作用机制，为临床治疗骨质疏松相关的腰椎间盘退变提供理论依据及临床用药指导。
第一部分卵巢切除大鼠椎体骨量丢失与腰椎间盘退变关系的实验研究
目的：探讨卵巢切除大鼠椎体骨量丢失与大鼠腰椎间盘退变的关系。
方法：20只3月龄雌性Sprague-Dawley（SD）大鼠随机分为两组：假手术组（Sham）及卵巢切除组（OVX）。Sham组大鼠进行假手术，仅暴露卵巢组织但不切除。OVX组大鼠经卵巢切除术切除双侧卵巢组织，卵巢切除术后6个月，采用过量失血方法处死各组大鼠并收集L3-6节段及其椎间盘组织。椎体骨密度检测及椎体骨组织形态计量学检测用以评价大鼠椎体骨量及椎体微观结构的变化。对椎间盘进行van Gieson染色进行椎间盘组织学观察并进行组织学评分。
结果：
1骨密度检测结果
OVX组大鼠L3-6椎体BMD显著低于Sham组（P<0.05）。
2椎体骨组织形态计量学检测结果
OVX组大鼠L4椎体骨小梁相对体积（BV/TV），骨小梁厚度（Tb.Th）及骨小梁数量（Tb.N）显著低于Sham组（P<0.05），而骨小梁分离度（Tb.Sp），荧光周长百分率（%L.Pm），矿化沉积率（MAR）及骨形成率（BFR/BV）显著高于Sham组（P<0.05）。
3组织形态学观察及组织学评分结果
OVX组大鼠椎间盘髓核内脊索细胞大量减少，软骨样细胞大量增殖，并可见粘液样变性。软骨终板可见大量骨样组织及髓腔生成。OVX组椎间盘组织学评分显著高于Sham组（P<0.05）。
第二部分阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的预防作用的实验研究
目的：本研究旨在应用骨质疏松大鼠模型探讨预防性应用阿仑膦酸钠对腰椎间盘退变的作用。
方法：30只3月龄雌性Sprague-Dawley（SD）大鼠随机等分为三组：（1）假手术组（Sham）；（2）卵巢切除+盐水组（OVX+V）；（3）卵巢切除+阿仑膦酸钠组（OVX+ALN）。Sham组大鼠进行假手术，仅暴露卵巢组织但不切除。OVX+V组及OVX+ALN组大鼠经卵巢切除术切除双侧卵巢组织后，OVX+ALN组大鼠给予阿仑膦酸钠颈后皮下注射，剂量为15μg/kg，一周两次。OVX+V组大鼠给予等体积的生理盐水作为对照。卵巢切除术后6个月，采用过量失血方法处死各组大鼠并收集L3-6节段椎体及其间盘组织。椎体骨密度检测、micro-CT检测及椎体生物力学检测用以评价大鼠腰椎椎体骨组织特性及微观结构的变化。对椎间盘进行van Gieson染色进行椎间盘组织学观察并进行组织学评分。同时对各组大鼠椎间盘高度以及软骨终板厚度的变化进行分析比较。免疫组织化学染色及real-time PCR用于检测各组大鼠椎间盘中aggrecan、I型胶原、II型胶原、MMP-1、MMP-3及MMP-13表达的变化。
结果：
1椎体骨密度检测结果
OVX+V组大鼠L3-4及L5-6椎体BMD显著低于Sham组（P<0.05）。OVX+ALN组大鼠L3-4及L5-6椎体BMD显著高于OVX+V组（P<0.05）
2Micro-CT检测结果
OVX+V组BV/TV、Tb.N显著低于Sham组，而Tb.Sp及SMI显著高于Sham组（P<0.05）。OVX+ALN组椎体BV/TV、Tb.N显著高于OVX+V组，Tb.Sp及SMI显著低于OVX+V组（P<0.05）。而Tb.Th在三组间无统计学差异（P>0.05）。
3椎体生物力学检测结果
OVX+V组椎体极限负荷（maximum load），屈服应力（yield stress），极限应力（maximum stress）及弹性模量（elastic modulus）均显著性低于Sham组（P<0.05）。OVX+ALN组椎体极限负荷，屈服应力，极限应力及弹性模量均显著高于OVX+V组（P<0.05）。
4组织形态学观察及组织学评分结果
OVX+V组椎间盘退变明显，而OVX+ALN组椎间盘髓核内仅出现少量的软骨样细胞，髓核未见粘液样变性，在纤维环与髓核交接处可见纤维软骨增殖现象。软骨终板内可见大量异常骨样组织及髓腔生成。而OVX+ALN组椎间盘退变程度较轻，仅在髓核内出现少量的软骨样细胞增殖，以及在髓核与纤维环交界处出现一些软骨细胞增殖，髓核内未出现粘液样变性。并且在软骨终板中段未发现有异常骨样组织生成。OVX+V组L5-6椎间盘组织学评分显著高于Sham组，而OVX+ALN组组织学评分显著低于OVX+V组（P<0.05）。
5椎间盘高度检测结果
OVX+V组L5-6椎间盘高度显著低于Sham组（P<0.05），OVX+ALN组L5-6椎间盘高度显著高于OVX+V组（P<0.05），OVX+ALN组与Sham组L5-6椎间盘高度无显著性差异（P>0.05）。
6软骨终板厚度及软骨终板内骨样组织面积检测结果
OVX+V组软骨终板厚度及软骨终板内骨样组织与软骨终板面积比值均显著高于Sham组（P<0.05），OVX+ALN组大鼠软骨终板厚度显著低于OVX+V组（P<0.05），且软骨终板内骨样组织与软骨终板面积比值显著低于OVX+V组（P<0.05）。
7椎间盘免疫组织化学检测结果
OVX+V组髓核内，aggrecan及II型胶原染色强度显著低于Sham组。然而，OVX+ALN组aggrecan及II型胶原染色强度显著高于OVX+V组。OVX+V组髓核内MMP-1、MMP-3及MMP-13染色强度显著高于Sham组，而OVX+ALN组髓核内MMP-1、MMP-3及MMP-13染色强度显著低于OVX+V组。
OVX+V组纤维环中MMP-1、MMP-3及MMP-13表达增高，其IOD值显著高于Sham组（P<0.05）。但OVX+ALN组纤维环中MMP-1、MMP-3和MMP-13的表达显著降低，其IOD值显著低于OVX+V组（P<0.05）。
OVX+V组纤维环中I型胶原表达增高及Ⅱ型胶原表达降低，其IOD值显著高于或低于Sham组（P<0.05）。而OVX+ALN组纤维环中I型胶原表达降低及Ⅱ型胶原表达增强，其IOD值显著低于或高于OVX+V组（P<0.05）。OVX+V组软骨终板区域可见X型胶原免疫组织化学染色程度强于Sham组，而OVX+ALN组X型胶原染色强度低于OVX+V组。
8Real-time PCR检测结果
与Sham组相比，OVX+V组Col2α1mRNA表达降低（P>0.05），而aggrecan及Col1α1mRNA表达显著增高（P<0.05）。而OVX+ALN组aggrecan及Col2α1mRNA显著高于OVX+V组（P<0.05），Col1α1mRNA表达显著低于OVX+V组（P<0.05）。OVX+V组MMP-1、MMP-3及MMP-13mRNA表达显著高于Sham组（P<0.05）。而阿仑膦酸钠可有效抑制由卵巢切除引起的MMP-1、MMP-3及MMP-13mRNA表达的增高（P<0.05）。
第三部分阿仑膦酸钠治疗卵巢切除大鼠腰椎间盘退变作用效果的实验研究
目的：探讨阿仑膦酸钠对卵巢切除大鼠腰椎间盘退变的治疗作用。
方法：30只3月龄雌性SD大鼠随机分为三组：（1）假手术组（Sham）；（2）卵巢切除+盐水治疗组（OVX+V）；（3）卵巢切除+阿仑膦酸钠治疗组（OVX+ALN）。OVX+V组及OVX+ALN组大鼠行双侧卵巢切除术切除双侧卵巢组织，Sham组大鼠仅暴露卵巢组织但不切除。OVX+ALN组大鼠于卵巢切除术3个月后给予阿仑膦酸钠颈后皮下注射，剂量为15μg/kg，一周两次。OVX+V组大鼠给予等体积的生理盐水作为对照。卵巢切除术后6个月，过量失血处死各组大鼠并收集L3-6椎体标本。椎体骨密度检测及Micro-CT检测用以评价大鼠腰椎椎体骨骼特性及微观结构的变化。应用van Gieson染色进行椎间盘组织学检测及组织学评分。免疫组织化学染色用于检测各组大鼠椎间盘中aggrecan、I型胶原、II型胶原、MMP-1及MMP-13表达的变化。
结果：
1椎体BMD检测结果
OVX+V组大鼠L3-4及L5-6椎体BMD显著低于Sham组（P<0.05），而OVX+ALN组大鼠L3-4及L5-6椎体BMD显著高于OVX+V组（P<0.05）。
2Micro-CT检测结果
OVX+V组BV/TV、Tb.N显著低于Sham组，而Tb.Sp显著高于Sham组（P<0.05）。而OVX+ALN组椎体BV/TV、Tb.Th显著高于OVX+V组，Tb.Sp显著低于OVX+V组（P<0.05）。
3椎间盘组织形态学观察及评分结果
OVX+V组髓核内，脊索细胞数量显著减少，并出现大量软骨样细胞增殖，髓核内可见粘液样变性。在纤维环与髓核的过渡区出现大量体积较小的软骨细胞增殖。软骨终板内异常钙化现象较为明显，并且在软骨终板的中段出现大量的异位骨样组织及髓腔。OVX+ALN组髓核内脊索细胞数量未出现显著性减少，髓核中出现一定量的软骨样细胞，并出现轻度的黏液样变性。在纤维环与髓核交接处可见纤维软骨细胞增殖现象。
OVX+V组椎间盘组织学评分显著高于Sham组（P<0.05），而OVX+ALN组椎间盘组织学评分显著低于OVX+V组（P<0.05），但Sham组与OVX+ALN组椎间盘组织学评分无显著性差异（P>0.05）。4免疫组织化学染色结果
OVX+V组髓核内aggrecan及II型胶原表达较Sham组显著降低，MMP-1及MMP-13表达增强，而OVX+ALN组aggrecan及II型胶原表达高于OVX+V组，并且MMP-1及MMP-13表达弱于OVX+V组。OVX+V组纤维环中MMP-1及MMP-13表达增高，其IOD值显著高于Sham组（P<0.05）。但OVX+ALN组纤维环中MMP-1和MMP-13的表达显著降低，其IOD值显著低于OVX+V组（P<0.05）。
OVX+V组纤维环中Ⅱ型胶原表达降低，而I型胶原表达增高，其IOD值分别显著低于或高于Sham组（P<0.05）。而OVX+ALN组纤维环中Ⅱ型胶原表达增强，I型胶原表达减弱，其IOD值显著高于或低于OVX+V组（P<0.05）。
OVX+V组软骨终板内X型胶原表达显著高于Sham组，而OVX+ALN组软骨终板内X型胶原表达较OVX+V组显著降低。
结论：
1卵巢切除大鼠椎体发生骨质疏松同时可合并出现腰椎间盘的退行性改变。
2阿仑膦酸钠可有效延缓卵巢切除大鼠腰椎间盘退变进程。其具体作用机制一方面可能与维持椎体及软骨终板结构的完整性及功能有关，另一方面与调控细胞外基质代谢，保护椎间盘的功能有关。
3阿仑膦酸钠是一种防治与骨质疏松相关的腰椎间盘退变的潜在性药物。
Lumbar intervertebral disc degeneration (LVD) is the leading cause oflow back pain and other disc disorders, which are responsible for enormoushuman suffering, high health care costs, and significant socioeconomic losses.Although some clinical and epidemiological studies observed thatosteoporosis was inversely related to spinal degeneration diseases orintervertebral disc degeneration, a recent correlation study of BMD and LVDin premenopausal and postmenopausal women showed that the BMD of notonly the lumbar vertebrae but also the calcaneus and radius were associatedwith LVD. In addition, in a previous study, we found a strong associationbetween osteopenia and disc degeneration in ovariectomized (OVX) rats,suggesting that measures to reduce osteoporotic changes might have inhibitoryeffects on disc degeneration. These variant consequences indicate that thecontraversial relationship between osteoporosis and LVD remains to be furtherclarified.
Increasing evidence indicates that disc degeneration is associated with thedisruption of an intact spinal structure, including adjacent structures, such asthe vertebral body and endplate, which might result in imbalanced mechanicalloading on the disc. BMD assays in both pre-and postmenopausal womenrevealed a correlation between BMD and disc height, suggesting that thevertebral structural propoties are closely related to the degree of discdegeneration, and the milieu influencing one structure may similarly influenceothers. Vertebral fractures caused by low BMD can result in decreased discheight and exacerbate disc degeneration. The endplate is not only anotheressential structure in maintaining the integrity and physiological function ofthe avascular intervertebral disc, but also acts as a gateway for nutrient supplyto the nucleus. Nachemson found that calcification of the endplate may impede nutrient diffusion into disc, leading to thinner cartilage, but increasedthickness of the endplate, and consequently premature disc degeneration.Estrogen deficiency also might influence the severity of disc degeneration inpostmenopausal females, by negatively affecting endplate quality andinducing endplate degeneration. Estrogen replacement therapy is suggested asan alternative treatment for disc degeneration related to osteoporosis. However,many patients refuse estrogens for various reasons, which drove us to seeknew drug targets for the treatment of osteoporosis related disc degeneration,such as bisphosphonates, including alendronate (ALN) and zoledronate. ALN,a potent bisphosphonate, has widely been used as a first line drug in thetreatment of osteoporosis in postmenopausal women, where it can increasebone mineral density (BMD), suppress bone turnover and reduce the incidenceof fractures. Most recently, a study by Neogi et al. showed that ALN mightlead to a reduction in disc space narrowing in postmenopausal women,suggesting a novel role for bisphosphonate in altering the pathological processof disc degeneration. We hypothesize that ALN may be an alternative drugtreatment for lumbar disc degeneration related to osteoporosis, although theunderlying mechanisms by which ALN impacts the process of discdegeneration remain unclear. In the present study, we intends to investigate theeffect of alendronate on lumbar intervertebral disc in OVX rats by radiology,histomorphometry, molecular biology, in order to provide clinical guidelinesfor treatment of osteoporosis related intervertebral disc degeneration in clinic.
Part I Lumbar intervertebral disc degeneration can induced byosteoporosis in ovariectomized rats
Objective: To investigate the relationship between osteoporosis andlumbar intervertebral disc degeneration in ovariectomized rats．
Methods：Twenty female Sprague-Dawley rats aged3months underwenteither sham-operation (sham)(N=10) or bilateral ovariectomy (OVX)(N=10).After animals were sacrificed at6months post-OVX, the L3-6spinalsegments were harvested. Bone mineral density (BMD) and histomorphometryanalysis were performed to evaluate the bone mass and microstructural changes in the lumbar vertebral bodies. Histological analysis with van Giesonstain and the histological score were used to identify the characteristics of thedegenerative discs.
Results:
1Bone mineral density
The BMD values of L3-6vertebral bodies in the OVX group weresignificantly decreased, when compared with the Sham group (P<0.05).
2Bone histomorphometry
In the OVX group, the value in bone volume fracture (BV/TV),trabecular bone thickness (Tb.Th), and trabecular number (Tb.N) weresignificantly decreased, and trabecular separation (Tb.Sp), percent labeledperimeter (%L.Pm), bone formation rate (BFR/BV) and mineral appositionrate (MAR) were significantly increased when compared with Sham group(P<0.05).
3Histological findings and histological scores
In the OVX+V group, the discs showed degenerative changes, where thenucleus pulposus comprised relatively few, clustered, doublets ofchondrocyte-like cells. Mucoid degeneration could be seen eroding thenucleus pulposus. Bony tissues became more obvious in the cartilage endplate.The histological score of the discs in the OVX group was significantly higherthan the Sham group (P<0.05).
Part II The effect of alendronate sodium on degenerated intervertebraldisc tissue in ovariectomized rats
Objective: This study aims to investigate the effects of ALN on lumbarintervertebral disc degeneration related to osteoporosis using anovariectomized (OVX) rat model.
Methods: Thirty female Sprague-Dawley rats aged3months wererandomly divided into three groups (with10rats each) as follows: the Shamgroup underwent sham surgery; the OVX+ALN group had twice-a-weeksubcutaneous injections of ALN (15μg/kg) for6months. The OVX+V groupreceived an equivalent volume of saline solution as placebo post-OVX. After animals were sacrificed at6months post-OVX, the L3-6spinal segments wereharvested. Bone mineral density (BMD), micro-CT analysis andbiomechanical testing were performed to evaluate the bone quality andmicrostructural changes in the lumbar vertebral bodies. Histological analysiswith van Gieson stain and the histological score were used to identify thecharacteristics of the degenerative discs. The disc height and the thickness ofthe cartilage endplate were measured and compared. Immunohistochemistryand real-time PCR measurements for aggrecan, type I collagen, type IIcollagen, and matrix metalloprotease (MMP)-1, MMP-3and MMP-13expressions on the disc were performed to assess the underlying molecularsignaling changes in matrix metabolism during intervertebral discdegeneration.
Results:
1Bone mineral density
The BMD values of L3-4and L5-6vertebral bodies in the OVX+V groupwere significantly decreased, when compared with the Sham group (P<0.05).However, the BMD values of L3-4and L5-6vertebral bodies in theOVX+ALN group were significantly increased, when compared with theOVX+V group (P<0.05)
2Micro-CT measurements
Quantification of three-dimensional trabecular structures revealed thatBV/TV and Tb.N in the OVX+V group was significantly decreased comparedwith the Sham group (P<0.05), and Tb.Sp and SMI was higher in the OVX+Vgroup than in the Sham group (P<0.05)(Table4). The BV/TV and Tb.N inALN group are significantly higher than their counterparts in OVX+V group(P<0.05). Meanwhile, the Tb.Sp and SMI was markedly lower in theOVX+ALN group than in the OVX+V group (P<0.05). There was nosignificant difference in Tb.Th among the three groups (P>0.05).
3Mechanical testing for the lumbar vertebral body
Compared with the Sham group, the values of maximum load, yieldstress, maximum stress and elastic modulus were significantly decreased in the OVX+V group (P<0.05). However, the values of maximum load, yield stress,maximum stress and elastic modulus were significantly higher in theOVX+ALN group than in the OVX+V group at6months post-surgery(P<0.05), suggesting that ALN could effectively maintain the biomechanicalstrength of the vertebrae.
4Histological findings and histological scores
In the OVX+V group, the discs showed degenerative changes, where thenucleus pulposus comprised relatively few, clustered, doublets ofchondrocyte-like cells. Mucoid degeneration could be seen eroding thenucleus pulposus, with clefts forming within them. An increased number ofsmall chondrocytes appeared in the inner layer of the annulus fibrosus, whichwas present in the form of fibers invading the nucleus pulposus.. Bony tissuesbecame more obvious in the deep zone of cartilage endplate. In contrast, thehistological morphology in the OVX+ALN group didn’t show obviouschanges, there was no significant difference in the OVX+ALN group in thenumber of the notochordal cells, where only a few of chondrocyte-like cellsappeared in the nucleus pulposus, and some small chondrocytes were observedin the inner layer of the annulus fibrosus. Mucoid degeneration was notobserved in most samples in the OVX+ALN group and there were no bonytissues formed in the middle cartilage endplate. The histological score of thediscs in the OVX+V group was significantly higher than the Sham group(P<0.05); however, the histological score of the discs in the OVX+ALN groupwas markedly lower than the OVX+V group (P<0.05).
5Disc height
The disc height in the OVX+V group was significantly decreasedcompared with the Sham group (P<0.05). The disc height in the OVX+ALNgroup was significantly higher than the OVX+V group (P<0.05), but there wasno significant difference between the OVX+ALN and Sham groups.
6Thickness of the cartilage endplate
Both the cartilage endplate thickness and the bony tissue area within thecartilage endplate, especially in the middle cartilage endplate, were markedly increased in the OVX+V group when compared with the Sham group(P<0.05). The thickness of cartilage endplate and the bony tissues area ofcartilage endplate in the OVX+ALN group were both significantly decreasedwhen compared with the OVX+V group (P<0.05).
7Immunohistochemistry
The density of aggrecan and type II collagen staining in the OVX+Vgroup markedly decreased compared with the Sham group. However, muchstronger immunostaining was observed for aggrecan and type II collagen inthe OVX+ALN group compared with the OVX+V group. Strongerimmunostaining for MMP-1, MMP-3and MMP-13could be observed in thechondrocyte-like cells in the OVX+V group. In the OVX+ALN group,immunostaining for MMP-1, MMP-3and MMP-13were much weaker than inthe OVX+V group.
The IOD value of MMP-1, MMP-3, MMP-13positive cells in theannulus fibrosus was significantly higher in the OVX+V group than that in theSham group (P<0.05). The IOD values of type II and type I collagens weresignificantly lower and higher, respectively, in the OVX+V group comparedwith the Sham group (P<0.05). However, the IOD values of MMP-1, MMP-3and MMP-13were significantly lower in the OVX+ALN group comparedwith the OVX+V group (P<0.05), and the IOD values for type II and type Icollagens were significantly higher and lower, respectively (P<0.05). Type Xcollagen was observed in the cartilage endplate, with stronger positive stainingin the chondrocytes of the OVX+V group than in the Sham group, but weakerin the OVX+ALN group compared with the OVX+V group.
8Real-time PCR
The expression of Col2α1mRNA was decreased (P>0.05), and theexpressions of aggrecan and Col1α1mRNAs were significantly increased inthe OVX+V group compared with the Sham group (P<0.05). The expressionsof aggrecan and Col2α1mRNAs were significantly increased in theOVX+ALN group compared with the OVX+V group (P<0.05), and theexpression of Col1α1was significantly lower than in the OVX+V group (P<0.05).
The expressions of MMP-1, MMP-3and MMP-13mRNAs weresignificantly higher in the OVX+V group than in the Sham group at6monthspost-surgery (P<0.05). ALN treatment significantly depressed theOVX-induced up-regulation of MMP-1, MMP-3and MMP-13mRNAexpressions (P<0.05).
Part III The therapeutic effect of alendronate on degeneratedintervertebral disc tissue in ovariectomized rats
Objective: The aim of the present study is to investigate the therapeuticeffect of alendronate on degenerated intervertebral disc tissue inovariectomized rats.
Methods: Thirty female Sprague-Dawley rats aged3months wererandomly divided into three groups (with10rats each) as follows: the Shamgroup underwent sham surgery; the OVX+ALN group had twice-a-weeksubcutaneous injections of ALN (15μg/kg) at3months post-OVX. TheOVX+V group received an equivalent volume of saline solution as placebopost-OVX. After animals were sacrificed at6months post-OVX, the L3-6spinal segments were harvested. Bone mineral density (BMD) and micro-CTanalysis were performed to evaluate the bone quality and microstructuralchanges in the lumbar vertebral bodies. Histological analysis with van Giesonstain and the histological score were used to identify the characteristics of thedegenerative discs. Immunohistochemistry for aggrecan, type I collagen, typeII collagen, and matrix metalloprotease (MMP)-1and MMP-13expressions onthe disc were performed to assess the underlying molecular signaling changesin matrix metabolism during intervertebral disc degeneration.
Results:
1Bone mineral density
The BMD values of L3-4and L5-6vertebral bodies in the OVX+V groupwere significantly decreased, when compared with the Sham group (P<0.05).However, the BMD values of L3-4and L5-6vertebral bodies in theOVX+ALN group were significantly increased, when compared with the OVX+V group(P<0.05)
2Micro-CT measurements
Quantification of three-dimensional trabecular structures revealed thatBV/TV and Tb.N in the OVX+V group was significantly decreased comparedwith the Sham group (P<0.05), and Tb.Sp was higher in the OVX+V groupthan in the Sham group (P<0.05). The BV/TV and Tb.Th in OVX+ALN groupare significantly higher than their counterparts in OVX+V group (P<0.05).Meanwhile, the Tb.Sp was markedly lower in the OVX+ALN group than inthe OVX+V group (P<0.05).
3Histological findings and histological scores
In the OVX+V group, the discs showed degenerative changes, where thenucleus pulposus comprised relatively few, clustered, doublets ofchondrocyte-like cells. Mucoid degeneration could be seen eroding thenucleus pulposus, with clefts forming within them. An increased number ofsmall chondrocytes appeared in the inner layer of the annulus fibrosus, whichwas present in the form of fibers invading the nucleus pulposus. Bony tissuesbecame more obvious in the deep zone of cartilage endplate. In contrast, thehistological morphology in the OVX+ALN group didn’t show obviouschanges, there was no significant difference in the OVX+ALN group in thenumber of the notochordal cells, where only a few of chondrocyte-like cellsappeared in the nucleus pulposus, and some small chondrocytes were observedin the inner layer of the annulus fibrosus. Mucoid degeneration was notobserved in most samples in the OVX+ALN group and there were no bonytissues formed in the middle cartilage endplate. The histological score of thediscs in the OVX+V group was significantly higher than the Sham group(P<0.05); however, the histological score of the discs in the OVX+ALN groupwas markedly lower than the OVX+V group (P<0.05).
4Immunohistochemistry
The density of aggrecan and type II collagen staining in the OVX+Vgroup markedly decreased compared with the Sham group. However, muchstronger immunostaining was observed for aggrecan and type II collagen in
the OVX+ALN group compared with the OVX+V group. Strongerimmunostaining for MMP-1and MMP-13could be observed in thechondrocyte-like cells in the OVX+V group. In the OVX+ALN group,immunostaining for MMP-1and MMP-13were much weaker than in theOVX+V group.
The IOD value of MMP-1, MMP-13positive cells in the annulusfibrosus was significantly higher in the OVX+V group than that in the Shamgroup (P<0.05). The IOD values of type II and type I collagens weresignificantly lower and higher, respectively, in the OVX+V group comparedwith the Sham group (P<0.05). However, the IOD values of MMP-1andMMP-13were significantly lower in the OVX+ALN group compared with theOVX+V group (P<0.05), and the IOD values for type II and type I collagenswere significantly higher and lower, respectively (P<0.05).
Conclusions:
1Lumbar intervertebral disc can induced by osteoporosis inovariectomized rats.
2ALN can retard the progression of lumbar intervertebral discdegeneration in OVX rats. The underlying mechanisms might be related topreservation of the structural integrity and function of the adjacent structures,including the vertebrae and endplates, which further links with modulations inextracellular matrix metabolism to protect the disc from degeneration.
3ALN might be a promising drug agent for preventing and curing lumbarintervertebral disc degeneration related to osteoporosis.
引文
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