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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 14  |  Issue : 1  |  Page : 17-24

Histological and immunohistochemical evaluation to the effects of orally ingested atorvastatin on the alveolar bone of white albino rats


1 Department of Oral Biology, Faculty of Dentistry, Minia University, Minia, Egypt
2 Department of Oral Biology, Faculty of Dentistry, Damanhour University, Damanhur, Egypt

Date of Web Publication14-Mar-2017

Correspondence Address:
Saher S Mohammed
33, El-Zamalek Street, Abu-Qurqas, El-Minya
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_58_16

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  Abstract 

Introduction: Statins have been used as prospective agents in the management of some bone diseases as osteoporosis. Reviews of medical studies have shown increased bone mass in patients on long-term treatment with statins.
Objective: The objective of this study was to evaluate histologically and immunohistochemically effects of ingestion of atorvastatin on the cortical plates of alveolar bone of white albino rats in normal intact periodontium.
Materials and methods: Fifteen males of adult albino rats at the age 6–8 weeks, weighting 150–200 g were used in the study. They were divided into two main groups, control group and two experimental groups. Rats in experimental group I received 20 mg/kg body weight atorvastatin by gastric tube for 6 weeks. Experimental group II rats, received 40 mg/kg body weight atorvastatin for the same period. Specimens were fixed using formal saline solution, demineralized using EDTA solution then processed to be stained using hematoxylin and eosin stain for general histological examination and osteopontin for immunohistochemical study.
Results: Histological results revealed increased alveolar bone turnover in both experimental groups. In experimental group I, osteoclasts appeared in their Howships' lacunae resorbing old bone and reversal lines were found to separate between the newly formed bone and the old bone. In experimental group II, filling cones of the newly formed bone were clearly detected. Immunohistochemical results showed positive immunoreactive cement lines to osteopontin in both experimental groups. Experimental group II appeared with weak positive immunoreactions in the matrix of the newly formed bone.
Conclusion: It has been concluded that atorvastatin increased alveolar bone remodeling in dose dependent manner. The effect was clearly detected in the cortical plates of the alveolar bone in the form of cutting cones lined by osteoclasts and filling cones with central capillaries.

Keywords: alveolar bone, atorvastatin, cement lines, osteopontin


How to cite this article:
Mohammed SS, Shredah MT. Histological and immunohistochemical evaluation to the effects of orally ingested atorvastatin on the alveolar bone of white albino rats. Tanta Dent J 2017;14:17-24

How to cite this URL:
Mohammed SS, Shredah MT. Histological and immunohistochemical evaluation to the effects of orally ingested atorvastatin on the alveolar bone of white albino rats. Tanta Dent J [serial online] 2017 [cited 2021 Oct 19];14:17-24. Available from: http://www.tmj.eg.net/text.asp?2017/14/1/17/202059


  Introduction Top


Statins are among the most widely prescribed pharmaceutical agents that effectively lower serum cholesterol levels. They are used for hypercholesterolemia and atherosclerosis prevention and treatment [1]. Several studies suggest that statins may have other effects beyond lipid reducing function. They have shown anabolic effect on bone tissue [2].

Between various statins, atorvastatin stands out because of its lipophilicity, which is closely linked to its pleiotropic effects. Moreover, it was found to have few adverse effects and better cost-effectiveness relationship, compared with other statins. As a result, it is widely used in clinical practice [3].

Statins target hepatocytes and inhibit 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA reductase) which is the enzyme that converts HMG-CoA into mevalonic acid. The later is a cholesterol precursor. Statins alter the structure of the enzyme when they bind to its active site. This prevents HMG-CoA reductase from attaining a functional structure. The change in conformation at the active site makes these drugs very effective and specific [4].

Atorvastatin is rapidly absorbed following administration [5]. Its half-life is ˜14 h [6]. It is metabolized by hepatic cytochrome P450 enzymes [7].

Considering the effects of orally administered different doses of simvastatin and atorvastatin on the skeletal bone of normal female rats, it was found that small statins' doses decreased bone mineral density. Meanwhile, high-dose simvastatin was found to increase bone formation and resorption while its low dose decreased bone formation and increased bone resorption [8].

The effect of statin therapy duration on bone turnover markers was investigated in dyslipidemic patients. It was reported that statins showed marked influence on osteocalcin and serum receptor activator of nuclear factor κ-B ligand (RANKL), resulting in bone metabolism improvement in patients with longer duration of statin therapy [9].

In-vitro studies proved that statins enhanced bone morphogenic protein 2 (BMP-2) mRNA expression in cultured mouse cells resulting in enhanced bone formation in murine calvaria. It was also reported that simvastatin and lovastatin augmented bone formation when injected subcutaneously over the murine calvaria. In addition, it was stated that the statin-mediated activation of the BMP-2 promoter can be abolished by the addition of mevalonate, the downstream metabolite of HMG-CoA reductase. These reported results strongly suggested that bone deposition is a result of HMG-CoA reductase inhibition and lipophilicity of statins is important in producing pleiotropic effects such as bone formation [2].

Bone is a metabolically active tissue in which the pattern of organization of its mineral and organic components determines its successful mechanical function. Bone turnover is controlled by definite agents that regulate bone formation and bone resorption, which are the two major processes of bone remodeling. Disturbances in these mechanisms can lead to either bone resorption or bone deposition [10].

In normal periodontium, bone deposition and resorption are produced by resident cells in the periodontal ligament. Members of the tumor necrosis factor ligand and tumor necrosis factor receptor superfamilies, regulate osteoclastic differentiation process through cell-to-cell interactions. The RANKL is expressed on periosteal osteoblasts and fibroblasts. RANKL is expressed on osteoblasts as a membrane-bound protein. It interacts with its corresponding receptor, RANK, on osteoclast precursors resulting in their activation to osteoclasts. The effects of RANKL are blocked by its soluble decoy receptor osteoprotegerin (OPG), thus inhibiting osteoclasts differentiation. When the concentrations of OPG are high in relation to RANKL expression, OPG binds RANKL, inhibiting the RANK – RANKL interaction and preventing osteoclasts differentiation to enhance bone deposition. In contrast, when the levels of OPG are low relative to the levels of RANKL, RANKL binds RANK on osteoclast precursors and bone resorption proceeds [11],[12].

Osteopontin (OPN) is a glycoprotein that was first detected in osteoblasts. It is also known as bone sialoprotein I which is a multifunctional protein that is highly expressed in bone. The prefix of the word 'osteo' indicates that the protein is expressed in bone while the suffix 'pontin' is taken from the Latin word 'pons' denoting a bridge that identifies OPN's role as a linking protein. The chemical structure of OPN is single chain polypeptide composed of about 300 amino acids with nearly 30 attached carbohydrate residues. The protein is rich in acidic residues that are either aspartic or glutamic acid [13].

Although many studies were made regarding the effect of different statins on skeletal bone of the body, few studies are available concerning its influence on the alveolar bone. So, this work aimed to evaluate the effect of atorvastatin on the alveolar bone of intact albino rats using histological and immunohistochemical studies.


  Materials and Methods Top


Fifteen male Sprague-Dawley albino rats at the age 6–8 weeks, weighting 150–200 g, specific-pathogens free were used in this study. Each animal group was housed in separate clean cage at Faculty of Dentistry, Minia University. This work was approved by the Ethical Committee and followed the guidelines of Animal Welfare Organization [14].

Reagents

Atorvastatin (Lipitor) 40 mg tablets were obtained from Pfizer (Ad Doqi, Giza Governorate, Egypt). The tablets were ground and then the different doses 20 and 40 mg/kg were calculated. Each dose was freshly dissolved in distilled water before the oral intake.

Experimental design

Animals were randomly divided into two main groups as following:

Control group

The control group included five rats received distilled water by gastric tube to control the influence of stress.

Experimental groups

  1. Experimental group I (20 mg/kg atorvastatin treated group): this group included five rats. Rats were received 20 mg/kg/day of atorvastatin dissolved in distilled water by a gastric tube for 6 weeks
  2. Experimental group II (40 mg/kg atorvastatin treated group): this group included five rats. Rats were received 40 mg/kg/day of atorvastatin dissolved in distilled water by a gastric tube for 6 weeks.


Histological procedures

Rats from all groups were killed after 6 weeks by decapitation under light halothane anesthesia. Alveolar bone samples were obtained from the mandible for tissue preparation and fixed in 10% formal saline for 48 h. The specimens were demineralized using a 10% EDTA solution at pH 7.8 in a microwave oven [15],[16].

The demineralized specimens were washed by tap water and prepared for histological study by dehydration using ascending grades of alcohol (50, 70, and 90% then in absolute alcohol). Specimens were then cleared from alcohol with xylene (clearing agent) followed by infiltration with paraffin in constant temperature oven (60°C) for 2–3 h. As the specimens were completely infiltrated, they were removed and placed in the center of the box of melted paraffin, the bottom of which was the surface of cutting. The box containing the paraffin embedded specimen was then immersed in cool water to harden. By the use of microtome, serial sections were done from the paraffin blocks. The cutting was done to obtain sections with 4–6 μm in thickness. Suitable lengths of the paraffin ribbon were mounted on the prepared microscope slides. The slides were placed on a constant temperature drying table at about 37–42°C [17].

For immunohistochemical staining with OPN, sections were deparafinized and rehydrated through xylene and serial dilutions of ethyl alcohol to distilled water. Then, they were incubated in Antigen Retrieval Citra (BioGenex, San Ramon, California, USA) at 95°C for 15 min. Sections were washed in PBS at pH 7.4; and with 0.02% Triton X-100 PBS twice for 3 min each. All incubations were done in a humidity chamber at room temperature and followed by 2, 3 min washes in PBS. To label sections with OPN, the specimens were incubated with levamisol (Vector Laboratories; Burlingame, California, USA) for 30 min to block endogenous alkaline phosphatase activity. Polyclonal primary antibody to OPN (OP-199) was diluted in PBS with 1% normal goat serum, for 90 min then was applied at a concentration of 74 μg/ml. All sections were incubated with biotinylated anti-goat immunoglobulin G for 20 min. Vectastain ABC-AP (Vector Laboratories) was prepared and applied for 30 min. Finally, color was developed using NBT/BCIP (Dako, Carpinteria, California, USA). The sections were dehydrated in ethyl alcohol and xylene and mounted using permanent medium [18],[19].

Measuring thickness of alveolar bone

Image analysis for alveolar bone thickness was done using Image J22 software. Standard measuring frame per five photomicrographs for each group using a magnification ×400 by light microscopy were transferred to the monitored screen [20].

Steps of measuring alveolar bone thickness:

  1. Straight line tool selection was used to make a line selection that corresponds to alveolar bone thickness
  2. The unit of length measurement was determined which was centimeter
  3. The distance in pixels field was automatically filled in based on the length of the line selection.


Statistical analysis

The results of measuring alveolar bone thickness were summarized as mean and SD. The significance of the results was assessed by determining the probability factor (P), where P value less than or equal to 0.05 is considered statistically significant. Calculating was made using statistical package for the social sciences, version 15 (SPSS Inc., Chicago, Illinois, USA).


  Results Top


Histological study of the alveolar bone using hematoxylin and eosin

The control group

Longitudinal sections in the cortical plates of alveolar bone in rats of the control group revealed normal compact bone architecture. The cortical plates appeared to be composed of several osteons with centrally located haversian canals ([Figure 1]).
Figure 1: A Photomicrograph of transverse section in the cortical plate of alveolar bone of control group showing: a - cortical plate with osteons having centrally located haversian canals b - periodontal ligament c - cellular cementum d - dentin (H and E, ×200).

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Experimental group I (20 mg/kg atorvastatin treated group)

Longitudinal sections of cortical plates in this group showed some areas subjected to bone remodeling. Reversal lines appeared to separate between the old bone and the small amount of the newly formed bone by the uninucleated osteoblasts ([Figure 2]).
Figure 2: Photomicrograph of longitudinal section in cortical plate of alveolar bone in experimental group I showing: a - dentin b - periodontal ligament c - old bone with its resting lines d -osteoclasts e -cement or reversal line (H and E, ×200).

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In transverse sections, multinucleated osteoclasts were clearly detected in their Howship's lacunae in the leading edge of the cutting cones to resorb old bone ([Figure 3]).
Figure 3: Photomicrograph of transverse section in cortical plate of alveolar bone in experimental group I showing: a - osteoclasts in Howshipæs lacunae b - old bone c - periodontal ligament d - cementum e - dentin (H and E, ×400).

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Experimental group II (40 mg/kg atorvastatin treated group)

Transverse sections in the cortical plates of the alveolar bone in this group showed massive areas of cortical bone remodeling. Filling cones in cross sections appeared with central capillaries and osteoblasts that deposited new bone in concentric lamellae. Cement lines appeared scalloped separating between old bone resorbed by osteoclasts and new bone formed osteoblasts ([Figure 4],[Figure 5],[Figure 6]).
Figure 4: Photomicrograph of transverse section in the cortical plate of alveolar bone in experimental group II showing: a - remodeled compact bone b - dentin c - periodontal ligament d - cementum (H and E, ×200).

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Figure 5: Photomicrograph of transverse section in cortical plate of alveolar bone in experimental group II showing: a - filling cones b - new bone c - cement line (H and E, ×400).

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Figure 6: Photomicrograph of transverse section in cortical plate of alveolar bone in experimental group II showing: a - filling cones with central capillaries b - osteoclsats in Howships lacunae c - periodontal ligament d - cementum e-dentin (H and E, ×400).

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Immunohistochemical results using osteopontin

The positive immunoreactivity for OPN appeared in the form of brown coloration of the cement lines separating between the new and old bone and brown coloration in the matrix of the new bone.

The control group

Transverse sections of cortical plates in rats' specimens of control group appeared with no detectable immunostained cement lines or bone matrix ([Figure 7]). The staining was really specific as we tested its specificity by immunohistochemical control methods [21].
Figure 7: A photomicrograph of transverse section in cortical plate of alveolar bone in the control group showing no detectable immunostained bone matrix (Osteopontin, ×400).

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Experimental group I (20 mg/kg atorvastatin treated group)

Longitudinal sections of the cortical plates of alveolar bone in 20 mg/kg body weight treated rats revealed positively immunoreactive cement lines for OPN ([Figure 8]).
Figure 8: A photomicrograph of longitudinal section in the cortical plate of alveolar bone in experimental group I showing: a - negatively immunostained dentin b - negatively immunostained periodontal ligament c - positively immunostained cement line (Osteopontin, ×400).

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Experimental group II (40 mg/kg atorvastatin treated group)

Transverse sections of rats' specimens in this experimental group showed weak immunoreactive areas in the matrix of the newly formed bone together with moderately immunoreactive cement lines for OPN ([Figure 9]).
Figure 9: photomicrograph of transverse section in the cortical plate of alveolar bone in experimental group II showing: a - negatively immunostained matrix in old bone b - weakly immunostained matrix of the new bone c - positively immunoreactive cement lines (Osteopontin, ×400).

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Image analysis results

On measuring the alveolar bone thickness in the control and experimental groups, significant increase in the thickness of alveolar bone from the control group to the experimental group I was recorded. Meanwhile, P value and mean ± SD results revealed insignificant increase in the alveolar bone thickness from the experimental group I to the experimental group II ([Table 1] and [Figure 10]).
Table 1: Comparison between the studied groups regarding thickness of the alveolar bone

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Figure 10: A histogram showing comparison between the studied groups regarding alveolar bone thickness.

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  Discussion Top


Statins, are originally developed to treat hypercholesterolemia. They are inhibitors of the enzyme 3-hydroxy-3-methylglutaryl which is the rate-limiting enzyme of the mevalonate pathway. Statin's effects are pleiotropic because the inhibited mevalonate pathway is responsible for the synthesis of many other important biochemical molecules. In particular, statins can greatly affect the process of bone turnover and regeneration as they target important cell types, including mesenchymal stem cells, osteoblasts, endothelial cells, and osteoclasts. Statins have also been shown to have anti-inflammatory properties suggesting a useful role in the process of bone regeneration as inflammation can interfere with normal bone healing. As a result, the use of statins for bone therapy is a promising and growing area of research [22].

Several studies have investigated the effects of statins administration on various skeletal bones of the body. In particular, there is no histopathological information about the effect of systemic administered statins on the alveolar bone of intact periodontium in rats. In the present work we evaluated the effect of two different doses of atorvastatin on the cortical plates of the alveolar bone in pathogens free rats without induction of any inflammatory processes. All of the statins that are currently available target the liver and decrease cholesterol biosynthesis, but they are poorly distributed to bone. Atorvastatin is one of the most recent synthetic potent statins that might get past the liver and reach the bone [10]. Consequently, atorvaststin was chosen for this study.

In the current study, different structural changes were detected in the cortical plates of the alveolar bone by light microscope in rats receiving 20 and 40 mg/kg body weight of atorvastatin. Using hematoxlyin and eosin, increased bone remodeling was easily detected in the two experimental groups. Scalloped cement or reversal lines were clearly found separating between old resorbed and newly deposited bone in both groups. In experimental group II, the leading edge of the cutting cone appeared to be lined with multinucleated osteoclasts that resorbed the old bone. Filling cones were identified behind with central capillaries and osteoblasts lining that deposited bone in concentric lamellae.

Regarding new bone formation after statins administration it was stated that statins could enhance osteogenesis by inhibiting the synthesis of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Statins were found to inhibit HMG-CoA reductase, and thus prevent the synthesis of mevalonate. FPP and GGPP are the downstream products of mevalonate. As a result, statins could reduce the synthesis of cellular FPP and GGPP [23].

Atorvastatin showed a protective effect in the affected periodontium by ligature-induced periodontitis. It was recorded that atorvastatin inhibited the inflammatory process and increased the anabolic activity of the alveolar bone [24].

Concerning with alveolar bone remodeling, the effect of administration of 20 mg of atorvastatin per day was studied on biochemical bone turnover markers. The study was done using bone specific alkaline phosphatase as bone formation marker and C-telopeptide as bone resorption marker in postmenopausal women for 8 weeks. It was proved that atorvastatin might influence bone metabolism resulting in protective effects mainly in older women. However, the results failed to show a significant effect of atorvastatin on bone metabolism in the younger women of age less than 63 years old [25].

In addition, in-vitro studies supported our findings regarding alveolar bone remodeling. OPG and RANKL production in cultured resting human gingival fibroblasts were evaluated in the presence of atorvastatin. Atorvastatin was found to increase resting fibroblasts RANKL production at low concentrations insignificantly. It was also recorded that atorvastatin significantly increased the RANKL/OPG ratios at higher concentrations under noninflammatory conditions. Consequently, it was proved that atorvastatin might influence the production of RANKL and OPG by human gingival fibroblasts to enhance bone catabolism and remodeling without induction of inflammatory processes [26].

In contrast to our findings concerned with differentiation of osteoclasts, the levels of OPG and RANKL mRNA were measured using semiquantitative reverse transcription-PCR in mouse bone cell cultures. Statins were found to increase OPG mRNA expression and decrease RANKL mRNA expression [27].

The effect of simvastatin on osteoclasts using tartrate-resistant acid phosphatase staining was examined. It was recorded that RANKL induced differentiation of osteoclasts was blocked after treatment with simvastatin in an earlier stage and at a higher concentration [28].

Osteoclasts were reported to move along compact bone during the process of bone remodeling creating a resorption channel (cutting cone) which is characterized by the scalloped array of the resorption lacunae (Howship's lacunae). The preosteoblasts migrate onto the roughened surface of the resorbed old bone to deposit a thin coating of noncollagenous matrix proteins layer. This layer was found to be composed mainly of OPN that acts as cohesive mineralized layer between old and new bone. These findings might explain our results showing immunoreactive reversal lines for OPN in both experimental groups [29].

OPN was reported to be produced by osteoblasts when they form bone matrix. Consequently, it was proved to accumulate in the mineralized matrix binding strongly to hydroxyapatite and possibly explaining its presence in the bone matrix of the newly formed bone [30].

Our immunohistochemical findings failed to detect positive immunoreaction in osteoblast or osteoclast cells. These results came in contrast to other studies showed that OPN was expressed by both types of cells that are implicated in bone remodeling. Osteoclast derived OPN was also found to inhibit the formation of hydroxyapatite during normal bone mineralization [31].

Our image analysis results revealed significant increase in the alveolar bone thickness from the control group to experimental group I. The control group recorded a mean value of 1.4 ± 0.1 while experimental group I recorded 3.03 ± 0.7 mean value. On the other hand, experimental group II showed insignificant increase in the alveolar bone thickness when compared with experimental group I. Experimental group II recorded 3.5 ± 0.4 mean value.

The increase in the alveolar bone thickness in both experimental groups of our study was proved to be dose dependent using image analysis. Statins were recorded to be effective activators of BMP-2 and powerful stimulators of osteoblastic differentiation [32].


  Conclusion Top


It has been concluded that atorvastatin increased alveolar bone remodeling. This effect was proved to be dose dependent. The effect had been clearly shown in the cortical plates of the alveolar bone in the form of cutting edges with osteoclasts and filling cones with central capillaries and osteoblasts.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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