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 Table of Contents  
Year : 2015  |  Volume : 5  |  Issue : 1  |  Page : 2-9

Histomorphometric and histological evaluations of the simvastatin effect on alveolar bone loss induced by cyclosporine A in rats

1 Department of Oral Biology, Faculty of Dentistry, Medical Research Institute, Alexandria University, Alexandria, Egypt
2 Department of Histochemistry and Cell Biology, Medical Research Institute, Alexandria University, Alexandria, Egypt

Date of Web Publication26-Aug-2015

Correspondence Address:
Samir Hagar
Department of Oral Biology, Faculty of Dentistry, Alexandria University, Alexandria
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2229-6360.163640

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Background: Cyclosporin-A- has been used as an immunosuppressant to prevent the rejection of organ transplants. However, alveolar bone loss is an important negative side-effect of this drug. Simvastatin, a hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, is known to inhibit cholesterol biosynthesis. It has advanced effects on bone formation in vivo and in vitro. So,we evaluated the histological and histomorphometric analysis of osteoblast and osteoclast cells after administration of simvastatin in cyclosporin-A-associated alveolar bone loss in rats.
Aim of the Study: To evaluate the effect of simvastatin and cyclosporin -A- on Alveolar bone by investigating the histological and histomorphometric results of osteoblast and osteoclast cells.
Materials and Methods: 24 adult male rats will be divided into 3 groups: Group I: Control group; 4 rats, Group II: cyclosporine -A- group; 10 rats (10 mg/kg) subcutaneous injection, Group III: cyclosporine -A-/simvastatin group; 10 rats, simvastatin will be taken orally daily (20mg/kg/day). Two rats from the control group and 5 rats from each of the studied experimental groups (group II & III) were sacrificed on days 15 and 30 consecutively using Histological and Histomorphometric investigations.
Results: Histological results revealed higher bone volume and osteoblast cells, and decreased number of osteoclast cells in Simvastatin group than in CsA group. The same results was statistically significant in Histomorphometric results of both osteoblast and osteoclast cells counts. In Histomorphometrical analysis showed a significant increase of osteoblast cells in Simvastatin group than CsA group, and significant decrease of osteoclast cells in Simvastatin group than CsA group.
Conclusion: We can conclude that Simvastatin counteract the adverse effect of CsA induced alveolar bone loss that induced new bone formation.

Keywords: Cyclosporin A; bone loss; simvastatin

How to cite this article:
Hagar S, Sahar S, Mona Y, Nancy M. Histomorphometric and histological evaluations of the simvastatin effect on alveolar bone loss induced by cyclosporine A in rats. Indian J Multidiscip Dent 2015;5:2-9

How to cite this URL:
Hagar S, Sahar S, Mona Y, Nancy M. Histomorphometric and histological evaluations of the simvastatin effect on alveolar bone loss induced by cyclosporine A in rats. Indian J Multidiscip Dent [serial online] 2015 [cited 2021 Jun 22];5:2-9. Available from: https://www.ijmdent.com/text.asp?2015/5/1/2/163640

  Introduction Top

It is generally accepted that the single most important factor in the development of alveolar bone loss is periodontitis, which is derived from bacterial infection coincident to subgingival plaque. [1],[2],[3] Osteoporosis is defined as "a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. [4]" In osteoporosis, disruption of the resorption and formation processes in the bone remodeling cycle results in net bone loss and therefore, in a lower bone mass and increased risk of fracture. [5] Osteoporosis is a significant adverse reaction in transplant recipients .

Recently, an increasing number of immunosuppressive programs use glucocorticoid-free regimens, but other immunosuppressants, such as calcineurin inhibitors (e.g., cyclosporine A [CsA]), are also associated with the pathogenesis of transplantation-related osteoporosis. [6],[7],[8] Patients undergoing solid organ transplantation are long-term users of cyclosporine to prevent allograft rejection. Glucocorticoids are one of the immunosuppressive regimens after transplantation. It has been established that even low doses of glucocorticoids are accompanied with significant bone loss. Glucocorticoids induce apoptosis of osteoblasts and osteocytes and decrease replication and differentiation of osteoblasts. They also inhibit expression of genes for type I collagen, osteocalcin, transforming growth factor-β (TGF-β), and receptor activator for nuclear factor-K β κB-ligand (RANKL). [9]

Cyclosporine is now widely used in patients with immune-based disease. CsA is a cyclical polypeptide of 11 amino acids, one of which is unique to the cyclosporins. First isolated as an antifungal agent, it has been shown to have marked immunomodulatory properties. These properties have meant that the drug can be used as an immunosuppressant to prevent the rejection of transplants, both of organs (kidney, liver, and pancreas) and bone marrows, [10] as well as used to manage several illnesses, including psoriasis. [11]

CsA suppresses the immune response by inhibiting evolutionary conserved signal transduction pathways. CsA binds to their intracellular receptors, immunophilins, creating composite surfaces that block the activity of specific targets. For CsA/cyclophilin, the target is calcineurin. Inhibition of the action of calcineurin results in a complete block in the translocation of the nuclear factor of activated T-cells (NF-AT), resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin (IL-4) and CD40 ligand as well as those necessary for T-cell proliferation such as IL-2. [16]

Some of the immunosuppressive effects of CsA have been attributed to the ability to induce the production of the potent immunosuppressive cytokine TGF-ββ. TGF-ββ is a powerful immunosuppressive molecule considered to be at least 10,000 times more potent than CsA. [17] The main disadvantages of CsA are nephrotoxicity, hypertension, neurotoxicity, hepatoxicity, hyperlipidemia, anorexia, nausea, vomiting, paresthesia, gingival hyperplasia, and tremor. Long-term treatment with CsA-associated with many side-effects including hyperlipidemia and an increased risk of atherosclerosis. CsA demonstrates complex effects on lipoprotein metabolism and bile acid production and affects endothelial cells, smooth muscle cells, and macrophages, all of which are critical to the atherosclerotic process. [18]

One of the proposed side-effects of cyclosporine is bone loss after long-term use. Although it is a controversial topic, it was recently shown that cyclosporine causes an alveolar bone loss in rats. [19] Increased osteoclasia and decreased bone formation were also observed in periodontal sites in the alveolar bones of cyclosporine-treated rats. [20] Cyclosporine inhibits osteoblast differentiation, osteocalcin production, and collagen synthesis, reducing the bone replaced in each remodeling cycle, [21],[22] Some have proposed that the role of T-lymphocytes via RANKL is essential for triggering cyclosporine associated bone loss; however, the exact mechanism for this action is still obscure. [23]

Gingival overgrowth (GO) is one of the most common side-effects of CsA. [24] However, a much lesser known oral complication is severe lip enlargement. [25],[26] CsA may interfere with the wound healing following dental extractions by inhibition of the activity of matrix metalloproteinases 2 and 9. [27] A widely used group of drugs is that of statins like simvastatin, atorvastatin, cerivastatin, etc. They act on the mevalonate pathway, albeit at a different level. [28] Simvastatin is a butanoic acid with the empirical formula C25H38O5. They are competitive inhibitors of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR). [29] Since cholesterol is the main product of the mevalonate pathway statins are used orally to treat hypercholesterolaemia and hyperlipidemia. The safety profile of statins is well documented. [30] Inhibition of HMG-CoAR prevents the production of cholesterol (hence the effective use of statins for the treatment of hypercholesterolemia), but also prevents the synthesis of isoprenoid lipids necessary for the prenylation of small guanosine triphosphatases (GTPases), critical signaling molecules that require the addition of an isoprenoid lipid tail to direct them to cell membranes. [31] This diminished signaling by GTPases may affect cytokine expression and coupled with the inhibition of the mevalonate pathway, may result in inhibition of osteoclast differentiation or activation. [32],[33] Simvastatin, a synthetic statin has a number of pleiotropic effects as well. In addition to its anti-resorptive actions, it has been found to exert anabolic effects on bone. [28]

Inhibition of bone resorption

Inhibition of the enzyme HMG-CoAR and the subsequent blockade of the mevalonate pathway is probably the most important mechanism of inhibition of bone resorption by simvastatin. Upregulation of bone formation: Local stimulation of bone morphogenetic protein 2 (BMP-2), a major bone growth regulatory factor, can lead to new bone formation. Mundy et al. [34] identified that lovastatin and simvastatin, mevastatin, and fluvastatin increased gene expression for BMP-2 in osteoblasts. Additionally, it has been observed that statins like simvastatin, atorvastatin, and cerivastatin markedly enhance gene expression for vascular endothelial growth factor (VEGF) in MC3T3-E1 cells (preosteoblastic murine cells). VEGF is involved in the process of endochondral bone formation and stimulates osteoblastic differentiation leading to new bone formation. [35]

  Materials and Methods Top

Experimental animals

Twenty-five adult male rats weighing 90-100 g will be used in this study. These animals will be obtained from the Institute of Medical Research, Alexandria University. Animals will be housed in specially designed wire mesh bottom cages, three animals per cage. The animals will be supplied a regular diet throughout the whole experimental period which will last for 30 days.

The animals will be divided into three groups:

Group I: Control (consisting of 5 rats) will be injected with 1 ml saline

Group II: CsA - group (consisting of 10 rats), which will be treated with CsA (Novartis PharmaAG, Stein, Isvicre, Turkey), subcutaneous daily injection (10 mg/kg body weight, once a day) [34]

Group III: CsA/simvastatin group (consisting of 10 rats), this group will be treated with both CsA - and Simvastatin (Global Pharma ,6 October, Egypt), oral daily doses (once a day) of simvastatin at 20.0 mg/kg. [35],[36]

From each group, 5 rats will be sacrificed after 15 and 30 days consecutively after the commencement of the daily treatments.

Histological examination

The hemimandibles of each rat in all groups will be carefully removed, dissected and fixed in 10% formalin for 48 h. Decalcification will be carried out in trichloroacetic acid (8%) till complete decalcification then dehydrated in ascending concentrations of ethyl alcohol, infiltrated with xylene and embedded in paraffin wax. Serial sections of 5 μm thickness will be cut by microtome and subsequently stained with hematoxylin and eosin for general examination and trichrome stain for collagen. [37]

Histomorphometric analysis

The morphometric analysis will be carried out in the alveolar bone of hematoxylin and eosin-stained section. Images will be viewed and recorded using the same microscope and software. The image of each section of all groups will be captured using ×10 objective lens (Barr = 200 μm) with numerical aperture of a high resolution (16 bit digital camera, 1280 × 1024 pixel) for counting osteoclast and osteoblast cells.

Alveolar bone will be measured using the image analysis system by measuring: Osteoblast count (N.Ob/mm 2 ) and osteoclast count (N.Oc/mm 2 ) values indicate the number of osteoblasts and osteoclasts per definite surface area of bone surface (BS), respectively; [38] the mean of this measurement will be calculated.

  Results Top

Histological findings

Histological findings in Group IA (control, 15 days) the alvear bone showing normal osteoblast cells arranged on the bone surface with minimal amount of reversal lines indicating normal bone remodeling, normal bone thickness, normal osteocytes, actual bone marrow spaces [Figure 1].
Figure 1: Decalcified section of alveolar bone of Group IA: (control, 15 days) (H and E, ×400)

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In Group IIA (CsA, 15 days) the alveolar process showed areas of reabsorption. The reversal lines indicating high rate of bone turn over as result of bone resorption. Osteoblasts morphologically similar to the control group could be found in a parallel and adjacent position as related to the trabeculae and in other areas of bone tabeculae showed lesser osteoblast cells [Figure 2].
Figure 2: Decalcified section of alveolar bone of Group IIA: (cyclosporine A, 15 days) (H and E, ×400)

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But in Group IIIA (CsA and Simvastatin) revealed restoration of the normal alveolar bone trabeculae thickness, normal osteoblast cells arranged on bone surface, reversal lines with new bone formation are noticed as a result of simvastatin therapy, and large osteocytes in the newly formed bone indicating the role of osteocyte in bone mineralization [Figure 3].
Figure 3: Decalcified sectIon of alveolar bone of Group IIIA: (cyclosporine A and Simvastatin, 15 days) (H and E, ×400)

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The results of the second interval (30 days), In Group IB (control, 30 days) showed normal alveolar bone trabeculae with normal osteoblast cells resting on osteoid tissue, normal osteocyte lacunae with normal nuclei, and reversal lines appeared indicating normal bone turn over [Figure 4].
Figure 4: Decalcified section of alveolar bone of Group IB (control, 30 days) (H and E, ×400)

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On the other hand, group IIB (cyclosporine A, 30 days) Showed loss of the bone tissue with signs of disorganization and replacement by medular and fibrous tissue. In addition, a higher concentration of osteoclasts coupled with depressions in the bone tissue could be also noted, indicating sever bone resorbtion as result of continous cyclosporine A administration, and notice few osteocytes with pyknotic nuclei [Figure 5].
Figure 5: Decalcified section of alveolar bone of Group IIB (cyclosporine A, 30 days) (H and E, ×400)

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As a results of Simvastatin administration in group III B showed increase in the bone volume and architecture. Several reversal lines appeared indicating new bone formation, and osteocytes appeared with relatively similar to that in the control group [Figure 6].
Figure 6: Decalcified section of alveolar bone of Group IIIB (cyclosporine A and simvastatin, 30 days) (H and E, ×400)

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Histomorphometric results

The histomorphometric findings show the comparison between the number of osteoblast and osteoclast cells in different studied groups at the different interval of times. It was found that there was a statistical difference between the different studied groups at the different interval of times.

Statistical analysis

Data were fed to the computer and analyzed using IBM Statistical Package for Social Sciences (SPSS) software package version 20.0. Quantitative data were described using range (minimum and maximum), mean, standard and median.

The statistical test used as follow: (1) Mean and standard deviation of each category. (2) Mann-Whitney test was used for comparison between unpaired signed ranks test. The 5% was chosen as the cut-off level of significance.

From this [Table 1],[Table 2],[Table 3] and [Table 4] we concluded that there was statistical significant difference in osteoblasts number between studied Group IIA (CsA 10 mg/kg/day) and Group IIIA (CsA 10 mg/kg/day and simvastatin 20 mg/kg/day), Also statistical significant difference in osteocytes number between that both groups.
Table 1: Comparison between the different studied groups according to osteoblast

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Table 2: Comparison between the different studied groups according to osteoblast

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Table 3: Comparison between the different studied groups according to osteoclast

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Table 4: Comparison between the different studied groups according to osteoclast

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  Discussion Top

In the present study, an established and well-characterized animal model were used to evaluating, firstly, the role of simvastatin in the prevention of CsA-induced alveolar bone loss in the absence of inflammation and secondly, estimate the expression of BMP-2 after simvastatin therapy indicating bone formation. In this study, histological observations confirmed that administration of immunosuppressive doses of CaA for 30 days caused alveolar bone loss, in agreement with previous reports. [39] In addition, we showed that administration of simvastatin counteracted the deleterious effects of CsA on bone turnover in the absence of inflammation. These results are compatible with those reported by Ohno et al. [40] who showed that treatment with cerivastatin (a synthetic statin) improves CsA-induced high-turnover osteopenia in transplanted bone, mainly through the inhibition of bone resorption. The Histological observations were confirmed by histomorphometrical analysis, and we can thus suggest that cyclosporin A at a dose of 10 mg/(kg body weight day) leads to the decrease in BV/TV seen during CsA treatment was the result of a lower trabecular number (Tb.N) and thickness (Tb.Th) and higher trabecular separation. [41]

Our results also show that the decrease in BV/TV resulting from cyclosporine a treatment was associated with a significant decrease in the number of osteoblasts (N.Ob/BS) and an increase in osteoclasts (N.Oc/BS), which was significantly affected by simvastatin in alveolar bone tissues.

Cyclosporine inhibits osteoblast differentiation, osteocalcin production, and collagen synthesis, reducing the bone replaced in each remodeling cycle. [42] Some have proposed that the role of T-lymphocytes via RANKL is essential for triggering cyclosporine-associated bone loss; however, the exact mechanism for this action is still obscure. It has been demonstrated that CsA inhibits osteoclast formation by inhibiting calcineurin and subsequently, the expression of the NF-ATc1, a transcription factor involved in osteoclast differentiation and function. [43] It is interesting that the immunosuppressive drug, CsA, produced increases in IL-1β β and PGE2 protein and inducible nitric oxide synthase mRNA in noninflamed tissues, and a similar regulation of these genes has also been observed in experimentally induced inflammation. These cytokines are considered to play a role in bone resorption, probably through the stimulation of osteoclast proliferation and differentiation, leading to bone loss.

A previous study by Wondimu et al. [44] showed that CsA enhanced PGE2 formation in human gingival fibroblasts. According to this author, these findings indicate that the potentiation of PGE2 formation induced by CsA is due to an increased level and/or activity of phospholipase A2, the enzyme responsible for the release of arachidonic acid from phospholipids in the cell membrane. Thus, it can be suggested that CsA may also affect the production of the metabolites of the lipoxygenase pathway, which can also stimulate osteoclastic bone resorption. Also, cyclooxygenase enzymes appear to be potential mediators involved in the pathogenesis of cyclosporine-induced GO. [45]

As shown in our histomorphometric analysis that in CsA treated group, the number of osteoblast significantly decrease. Also, Nassar et al. observed in three studies the same results in CsA-treated groups, suggesting a modest negative effect of CsA on bone formation. [46] As shown in our histomorphic results that Simvastatin increase the number of osteoblast cells that is followed by bone formation. In the fact that simvastatin enhanced the proliferation and osteoblastic differentiation of human periodontal ligament (HPDL) cells via inhibition of the mevalonate pathway [47] that result in more bone formation.

Another cause of increased bone formation by simvastatin was stated by Mundy et al., [34] who showed that simvastatin can induce the expression of BMP-2, a member of the transforming growth factor superfamily and a key regulator of bone morphogenesis. Statins can also stimulate the expression of bone anabolic factors, such as VEGF, and promote osteoblast differentiation and mineralization in MC3T3 cells. [35],{50} Also, simvastatin stimulated signal transduction of TGF-β β depends on increasing dose. So, it induced not only BMPs but also TGF-β1 in HPDL cells. These TGF-ββ1and BMP-2 inductions were caused by the inhibition of cholesterol biosynthetic pathway.

Previous studies have demonstrated that statins inhibit the release of IL-1, IL-6 and TNF-a by upregulating the nuclear receptors, peroxisome proliferator-activated receptor (PPAR-a and PPAR-c; 22), which may represent a direct antagonist effect to CsA. The histological analysis in our study showed that many osteocytes were incorporated into the low-mineralized bone in the simvastatin-treated rats. Recently, the function of osteocytes in bone metabolism has been discussed, in which nucleobindin, a calcium-binding proteins, modulates bone matrix maturation by localizing in osteocytes and osteoblasts. {51} It has also been reported that sclerostin is secreted from osteocytes and inhibits the mineral maturation of compact bone. {52} Thus, as osteocytes have an important role in the maturation of the bone matrix, low-mineralized bone induced by simvastatin may have the potency to form mature alveolar bone.

From the histological findings, it is interesting that not only alveolar bone but also periodontal ligament and connective tissue, recovered in the simvastatin-treated group. However, the effect of simvastatin on cementum formation was not clear in this study.

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Conflicts of interest

There are no conflicts of interest.[48]

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4]

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Amany A. Rabea
Archives of Oral Biology. 2020; : 104887
[Pubmed] | [DOI]


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