The Effects Of Alendronate On The Lumbar Intervertebral Disc Degeneration In Ovariectomized Rats

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 ratsObjective: 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 densityThe BMD values of L3-6vertebral bodies in the OVX group weresignificantly decreased, when compared with the Sham group (P<0.05).2Bone histomorphometryIn 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 scoresIn 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 ratsObjective: 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 densityThe 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 measurementsQuantification 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 bodyCompared 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 scoresIn 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 heightThe 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 endplateBoth 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).7ImmunohistochemistryThe 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 PCRThe 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 ratsObjective: 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 densityThe 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 measurementsQuantification 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 scoresIn 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).4ImmunohistochemistryThe 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.