Anti-Brucella activity of Caryopteris mongolica Bunge root extract against Brucella melitensis infection in mice

Tsevelmaa N
Dariimaa D
Batkhuu J
Anti-Brucella activity of Caryopteris mongolica Bunge root extract against Brucella melitensis infection in mice
10.1186/s12906-018-2220-yR E S E A R C H A R T I C L E Open Access
Background: The current treatment for human brucellosis requires a combination of antibiotics for long periods of time, and the reported incidence and prevalence of the disease vary widely in nomadic livestock of Mongolia. The objective of the present study was to evaluate the in vivo antibacterial activity of the C. mongolica root extract against B. melitensis.Methods:In this study, we used of 6 groups of mice (n = 5). Five groups of BALB/c mice were inoculated intraperitoneally with the M16 strain of B. melintensis, as follows: (i) one group was used for pretreatment monitoring; (ii) the control group was administered 2% Tween 80 and was used as the non-treatment group; and the other three groups were treated with one oral gavage per day for 21 days with (iii) doxycycline (2 mg/day), (iv) doxycycline (1 mg/day) with root extract (20 mg/day), and (v) C. mongolica root extract (20 mg/day). The one group that was kept non-infected was used as a healthy control group.Results: This study demonstrated that daily treatment with doxycycline alone and in combination with C. mongolica root extract significantly reduced splenic infection at the end of treatment. However, the spleen index of both the doxycycline-treated and the combination-treated groups of mice decreased by approximately 50% compared to that of the healthy control mouse group. Treatment with the C. mongolica root extract resulted in a 1.47log reduction in splenic infection compared to the non-treatment group, and the spleen index of the C. mongolica-treated group of mice was the same as that of the normal mouse group. In all treatment groups, neutrophil phagocytic activity significantly decreased, and all treatment groups demonstrated splenic regeneration.Conclusions:The present study showed that the C. mongolica root extract may be useful in the treatment of brucellosis patients, in combination with doxycycline or other antibiotics, to reduce the toxicity of high-dosage antibiotics, to prevent the development of antibiotic resistance and to prevent Brucella infection.

Background

Caryopteris mongolica Bunge (Lamiaceae) grows in areas such as Khentii, Khangai, Mongol Daguur, Mongol Altai, and Dornod Govi in Mongolia and some areas of northern China. C. mongolica has long been used as a traditional medicine in Mongolia to alleviate aches, oedema, and rheumatism [1]. We have reported the phytochemical analysis of root extract of Caryopteris mongolica. We isolated of three new compounds and five known diterpene derivatives, namely, demethylcryptojaponol, incanone, 6α-hydroxydemethylcryptojaponol, cyrtophyllone B, and 14-deoxycoleon U, from the root extract of Caryopteris mongolica, used HPLC, MS and NMR techniques. Among these isolates, a new compound of abietane diterpene derivatives showed strong antibacterial activity [2]. Brucella is a gram-negative, aerobic, coccobacillary, non-motile microorganism [3]. In addition, Brucella species are facultative intracellular pathogens that localize predominantly in the cells and organs of the mononuclear phagocytic system, such as macrophages in the liver and spleen [4]. Brucellosis mainly affects domestic animals, in which Brucella colonize the reticulendothelial system and genital organs, leading to abortion, stillbirth, orchitis, epididymitis, and infertility and resulting in significant economic losses. The illness continues to be one of the most widely distributed zoonoses and is transmissible to humans [5,6]. Human brucellosis is usually caused by Brucella melintensis and has a wide spectrum of clinical symptoms, including irregular fever, sweating, arthralgia, myalgia, headache and weakness [7]. The reported incidence and prevalence of the disease vary widely in nomadic livestock in Mongolia. Selenge T., and others reported that 20.2%, or one in five, herders are infected with brucellosis [8]. The current treatment for human brucellosis requires a combination of antibiotics for long periods of time because antibiotics either lose their antimicrobial activity in the intracellular environment or do not persist long enough to produce a therapeutic effect [9]. World Health Organization guidelines recommend a 6-week course of doxycycline plus rifampin [10]. More recent recommendations also propose the use of doxycycline for 6 weeks with the aminoglycoside streptomycin for 2 to 3 weeks or with gentamicin for 1 week [11]. Combination therapies are more effective than single-agent therapies; however, new therapies are necessary due to the difficulties of patient adherence to the treatment itself along with the side effects of combination therapy and the dangers of antibiotic resistance [12]. Herbal treatments are gentle, inexpensive and effective in controlling the disease. On the basis of the in vitro screening results, the C. mongolica root extract had high anti-Brucella activity against B. melintensis (a concentration of 50 μg/disc produced an inhibition zone of 15 mm). To identify the anti-Brucella activity of the crude extract of C. mongolica root, we used the disc diffusion method. B. melitensis was grown in tryptone soy broth media for 72 h, and 100 μl (10 6 CFU/ml) was spread over the surface of tryptone soy agar medium in Petri dishes with diffusion discs treated with the root extract. Doxycycline-treated discs (a concentration of 10 μg/disc produced an inhibition zone of 35 mm) were used as a positive control. In other studies, plants such as Scrophularia deserti [13], Origanum syriacum, Thymus syriacus [14], Prunus mahaleb seeds [15], Satureja hortensis [16], Oliveria decumbens [17], Teucrium polium [18], and Moringa oleifera [19] have been examined for in vitro activity against Brucella. However, studies have not reported the efficacy of these medicinal plants for the in vivo treatment of brucellosis. The aim of this study was to evaluate the in vivo antibacterial activity of the C. mongolica root extract against B. melitensis.

Methods

Bacterial strain and culture conditions

Tryptone soy agar (TSA), Brucella agar, and tryptone soy broth (TSB) were purchased from Biolab, Hungary. Brucella melintensis 16 M (ATCC23456, biotype 1), a smooth virulent strain, was used in this study. The experiment was performed with fresh bacteria incubated on TSA plates at 37°C until the exponential growth phase. The bacterial clones were then counted and diluted with sterile saline solution.

Plant material

The roots of Caryopteris mongolica were collected in August 2014 from the Bayanchandmani soum of Tov Province. The plant was identified by Dr. Ch. Sanchir of the Institute of Botany at the Mongolian Academy of Sciences. A voucher specimen has been deposited in the herbarium at the National University of Mongolia (No. 20140803).

The C. mongolica root was extracted with acetone:H 2 O (8:2), evaporated, and air dried. For the in vivo study, the dry extract was dissolved in Tween 80 (polyoxyethylene [20] sorbitan monooleate), which was purchased from Sigma. We confirmed the acute toxicity of the C. mongolica root extract in accordance with Guideline 423 of the Globallу Harmonised System. The solution of dried root extract in Tween 80 was administered by oral gavage at concentrations of 1000, 3000, 5000, 8000, and 15,000 mg/kg to 3 mice per concentration. The results of this experiment showed that C. mongolica root would be classified as non-toxic (data not shown).

Animals

Female BALB/c mice of 12-15 weeks of age (weight 25-28 g) were supplied by the Research Institute of Veterinary Medicine. The mice were randomly placed into 6 groups (n = 5 mice/group), housed in transparent cages and fed a normal laboratory diet of nutrient pellets with access to drinking water. The mice were housed in a temperature-controlled facility (23 ± 5°C) with a 12 h light-dark cycle. All experimental protocols were approved by the Animal Care and Ethics Committee of the Institute of Veterinary Medicine of Mongolia.

Inoculation

The mice were infected with a single intraperitoneal dose of 1.7 × 10 5 CFU/mouse Brucella melintensis in 0.1 ml of sterile 0.9% saline. The one group that was kept noninfected was used as a healthy control group. On the 15th day after infection, a group of mice was selected to have their spleens aseptically removed and examined for bacterial infection.

Treatment

The control group (ii) was kept untreated (administered 2% Tween80 solution) and was sacrificed at 38 days postinfection. The other groups were administered a daily oral gavage for 21 days, as follows: (iii) antibiotic only, (iv) antibiotic and extract, and (v) extract only. The respective doses for each mouse in these groups were as follows: 2 mg doxycycline in 0.1 ml (80 mg/kg/day), 1 mg doxycycline + 20 mg extract in a total of 0.3 ml, and 20 mg extract in 0.25 ml (800 mg/kg/day) at intervals of 24 h. Doxycycline was purchased from Sigma Aldrich for use as an antibacterial treatment.

Bacterial counts in spleen

At the end of the treatment period (38 days postinfection), we first collected blood samples from the tail vein of the mice in all groups and then aseptically removed the spleens and placed them inside pre-weighted sterile plastic Whirl-Pak Bags (Nasco) for weighing. The mice were anaesthetized with isoflurane delivered by an RC 2 -rodent Circuit Controller (VetEquip, California, USA) and euthanized in a CO 2 chamber. To the sterile plastic bag containing each spleen, we added a solution of 9 parts sterile saline and then homogenized the tissue by hand. After the samples were homogenized, 10-fold serial dilutions of the homogenate were performed with a sterile saline solution; 100 μl of the dilutions was then inoculated onto TSA plates. The inoculum was distributed with a sterile spatula until fully dispersed on the agar surface. Two plates per sample were inoculated. The plates were incubated at 37°C for 3 days. The splenic bacterial loads were calculated by multiplying the CFU by the dilution factor. The spleen index is a comparison of the spleen weight with the body weight. The bacterial counting experiments were conducted in accordance with the protocol described by Elias B et al. [20].

Haemotological and histological analysis

The blood samples were collected from the tail vein of anaesthetized mice into heparinized tubes and were analysed with a blood analyser (Diff/poch-100ivd Sysmex, Japan). Additionally, neutrophil phagocytic activity was examined by the nitroblue tetrazolium (NBT) reduction test (Sigma Aldrich procedure number 840). For the histological assay, the spleens were fixed in a 10% formalin solution for 24 h; washed in flowing water; dehydrated in ethanol, xylene, and paraffin solutions; and then embedded in paraffin. The paraffin blocks were sliced with a Yamato Konki microtome and stained with haematoxylineosin (HE). Finally, the slides were analysed with a Nikon E-600 microscope.

Statistical analysis

One-way ANOVA followed by the Newman-Keuls multiple comparison test was used to compare the results between the samples using GraphPad Prism 5 (P ≤ 0.05 or 0.01).

Results

Comparison of infection-related physical and splenic changes in treatment groups

We first examined splenic infection on the 15th day postinfection. The spleen index of the pretreatment (postinfection day 15) monitoring group (i) was increased threefold (Table 1 and Fig. 1b) compared to that of the normal mouse group.

In addition, the administered Brucella inoculum produced a baseline splenic infection of 5.46 log CFU/0.1 g spleen (Table 1 and Fig. 1b). This result showed that B. melitensis infection was successful in the mice. We started treatment with antibiotic, C. mongolica root extract or a combination of the two 15 days after inoculation to allow the establishment of chronic infection.

Within the first 10 days of treatment, the mice in the groups that were administered antibiotic and the combination of antibiotic and root extract displayed physical signs of weakness, slower body movement, and ruffled fur. Furthermore, 2 mice in the group treated with only antibiotic died before the end of treatment. However, these signs associated with ill health were not evident in the group that was administered the root extract treatment.

At the end of treatment, we examined splenic infection in the non-treatment (day 38 post-infection) control group (ii) and observed that the bacteria were gradually eliminated compared to the pretreatment group. The mean splenic bacterial count in the non-treatment group was 4.5 log CFU (Table 1, Fig. 1c). Thus, the treatment efficacies in the treated groups were compared to the nontreatment group by the splenic bacterial count.

In comparison with no treatment, daily treatment with doxycycline alone (iii) (2.05 log CFU) and with the combination of doxycycline and C. mongolica root extract (iv) (2.1 log CFU) significantly reduced splenic infection at the end of treatment. However, the spleen index of both the doxycycline-treated and the combination-treated mouse groups decreased by approximately 50% compared to that of the normal mouse group (Table 1, Fig. 1a, d, e). The C. mongolica root extract-treated group (v) demonstrated a 1.47 log CFU reduction in splenic bacterial count compared with the non-treatment group, for which the spleen index was the same as that of the normal mouse group (vi) (Table 1, Fig. 1a, f). We included 5 mice in each group; however, we used 3 mice from the antibiotic treatment group for the spleen index calculation. Furthermore, we used 3 mice from each group except the healthy control group for the splenic CFU counting experiment.

Comparison of haematological changes in treatment groups

We also examined the haematological parameters of B. melitensis-infected BALB/c mice that were treated with C. mongolica root extract, antibiotic or a combination of both. The white blood cell (WBC) count of the mice in the pretreatment and non-treatment groups, compared to that of the normal mice, increased twofold. On the other hand, the WBC count decreased onefold in all post-treatment groups compared to the WBC count of the non-treatment group. However, the counts of other cell types (LYM, OTHR, EO) showed no significant reduction in any post-treatment group compared to those of the non-treatment group. Neutrophil phagocytic activity increased about threefold in the non-treatment group (at 38 days post-infection) compared to the normal group; in contrast, it decreased significantly in all treatment groups compared to the control group. In the normal mice, the neutrophil phagocytic activity was 13%, and it increased to 38% in the non-treatment group. Neutrophil phagocytic activity decreased to 14.5, 18.5 and 21% in the groups administered antibiotic only, combination therapy and extract only, respectively ( Table 2). We included 5 mice in each group; however, we used 3 mice from the antibiotic treatment group for blood analysis.

Comparison of histological changes in treatment groups

The splenic histological study showed that lymphocyte necrosis occurred around the white pulp and increased the amount of red pulp, lymphocytes at different stages of development, macrophages and neutrophils in the nontreatment group compared with other groups (Fig. 2a, b). On the other hand, the mouse groups that were administered the combination of antibiotic plus root extract and root extract only exhibited a decrease in red pulp and neutrophils, as well as regeneration of areas of splenic necrosis (Fig. 2c, d). Interestingly, in the combination-treated group, haemosiderin pigmentation at the interface of the red and white pulp can be observed (Fig. 2c). We used of Statistical analysis: *P < 0.05 and **P < 0.01 comparing the non-infected and non-treatment groups

Discussion

The efficacy of different antibiotics for the treatment of human brucellosis has also been evaluated in mouse models [20][21][22]. Sasha    group. Interestingly, in the doxycycline-treated and combination-treated groups, spleen sizes were dramatically reduced (a decrease of approximately 1.5 times) compared to the spleen sizes of the normal mice. The group treated only with C. mongolica root extract displayed more modest results in the ability to kill Brucella compared to the other groups; however, the spleen sizes of the mice in this group were the most similar to the spleen sizes of normal mice. Additionally, in the combination-treated group, haemosiderin pigmentation at the interface of the red and white pulp can be observed.

Haemosiderin is a golden-brown, granular pigment derived from the breakdown of red blood cells and is present within the cytoplasm of macrophages. Background haemosiderin levels reflect the normal removal of effete red blood cells by the spleen, but the haemosiderin level can increase in cases of chemically induced haemolytic anaemia or methaemoglobinaemia [24]. Rare side effects of doxycycline include haemolytic anaemia, thrombocytopaenia, eosinophilia, and neutropaenia [25][26][27]. Several studies have demonstrated that histological examinations reveal characteristic aminoglycoside-induced renal alterations such as tubular lipidosis and tubulonephrosis foci in mice that have received a gentamicin solution either alone or in combination with doxycycline [28,29].

We predicted that a significant effect on spleen size resulting from the strong antibiotic treatment could cause anaemia to occur. Anaemia may lead the mice in the antibiotic-treated or combination-treated groups to exhibit physical signs of discomfort, such as motor weakness and ruffled fur. Root extract has been seen as a gentler treatment alternative, with a therapeutic effect that has yet to produce abnormal symptoms such as a reduction in spleen size. Previous histological studies note that the white pulp/red pulp ratio in the normal spleen is close to 1/1, while the ratio in the brucellosis-infected spleen is close to 1/4 [30]. Brucella bacteria are removed from the local tissue lymphocytes and transferred into the general circulation, from whence they are disseminated throughout the body, with a characteristic tropism for the cells and organs of the mononuclear phagocytic system, such as macrophages in the liver and spleen [31]. After two weeks of infection, concomitant with splenic swelling, granulomas and giant cells increase in this organ; a trend that continues until the middle of the chronic steady phase. During the 3rd week post-infection, the apparent depletion of lymphocytes in the white pulp reaches its maximum. Then, after the 4th week, lymphoid hyperplasia and extra medullary haematopoiesis gradually increase, with the appearance of several mitotic figures and the multifocal accumulation of macrophages that surround and sometimes cover the periarteriolar lymphoid sheaths. Therefore, the infiltration of macrophages and neutrophils increases their number to relatively large proportions.

This increase is proportional to the swelling and infiltration of blood and phagocytic cells in the spleen [32]. Our study noted an increase in red pulp, smeared splenic cells and a high activity of neutrophil phagocytosis in the mice in the non-treatment group compared to those in the treatment groups. Mice in the C. mongolica root extract-treated group demonstrated regeneration of areas of splenic necrosis and a normal ratio of white to red pulp. Neutrophil phagocytic activity increases in the presence of bacterial infection, whereas the effectiveness of antibiotic therapy may be indicated by a reduction in the percentage of neutrophils positive for phagocytosis. The mean baseline neutrophil phagocytic activity was 9.6% in normal dogs, and a two-to fourfold increase was demonstrated in endotoxin-stimulated dogs [33,34].

The haematological and histological results clearly showed that the C. mongolica root extract is effective for curbing inflammatory processes in B. melitensis-infected mice.

The antibiotics currently used for the treatment of human brucellosis are capable of acting efficiently under acidic conditions because they have to penetrate macrophages. Celli Jet al. indicated that Brucella organisms integrate with components of the early endosomal pathway and reside in acidified phagosomes but not in lysosomes [35]. Interestingly, gentamicin modified into hydrophobic and non-capsulated forms enhanced the intracellular killing of Brucella, thereby decreasing intracellular infection [36]. Doxycycline, like other tetracycline antibiotics, is bacteriostatic activity. Doxycycline works by preventing bacteria from reproducing through the inhibition of protein synthesis. The lipophilic properties of doxycycline may account its ability to inhibit some bacterial strains that are more hydrophilic agents [37]. Our previous study demonstrated of three new compounds and five known diterpene derivatives from the root extract of C. mongolica. Among these isolates, a new compound of abietane diterpene derivatives showed strong antibacterial activity [2]. The diterpenoids can act on multiple biochemical targets of the bacteria and it has been suggested that the activity of these compounds results from their ability to cross or damage microbial cell membranes due to their amphiphilic property [38,39]. Urzua et al. suggested that promote the efficient antibacterial effect displayed by diterpenoids include a lipophilic decalin ring system, capable of insertion into a lipophilic region, and one strategically positioned hydrogen-bond-donor group (HBD; hydrophilic group), capable of interactions with the membrane phosphorylated groups. These hydrogen-bondinteractions between the phosphorylated groups in the membrane and the HBD in the diterpene are very important for the antibacterial activity. The totarol diterpene inhibits NADH-CoQ in the bacterial electron transport chain [40]. Horminone, an abietane diterpene quinone inhibiting bacterial protein synthesis, according bind to ribosomal RNA phosphate groups [41], ferruginol potentiates activity by acting as an efflux pump inhibitor [42]. The quinone diterpenoids of C.mongolica root may obtainable penetrate into the lipophilic cell membrane and bind to phosphate groups on macromolecules due to this, degrading of the cell membrane or inhibiting of proteins synthesis.

The recently study validated of pharmacokinetics of three diterpenes (carnosic acid, carnosol, rosmanol) after oral administration in rats of different doses (0.25, 0.82, 2.45 g/kg) Rosemarine officinalis.L extracts. They suggested that diterpenes are rapidly absorb, slowly eliminate, the values of maximum plasma concentration (C max ), the time to reach the maximum concentrations (t max ) directly depend on dosage. In the elimination phase, a double-peak phenomenon appeared on the mean plasma concentrationtime profiles. The probably reason may be caused by redistribution and enterohepatic circulation after the analytes were excreted into the gastrointestinal tract through the bile [43]. Our previous study demonstrated that diterpenes are main components which antibacterial active in C.mongolica root extract. Also, R.officinalis and C.mongolica are both belong in Lamiaceae family. Therefore pharmacokinetic properties of C.mongolica extract may similar with the suggested study. Further, its necessary of action mechanism and pharmacokinetic study for new diterpenoids from C.mongolica root. Also, we have to more study about synergistic or additive effect of C.mongolica extract with other anti-brucella agents.

This study clearly demonstrated that crude extracts from C. mongolica root have good efficacy against in vivo B. melitensis infection and "mild" or "low toxicity" effective than antibiotic treatment.

Conclusions

This study demonstrated that daily treatment with doxycycline both alone and in combination with C. mongolica root extract significantly reduced splenic infection at the end of the treatment. However, the spleen index of both the doxycycline-treated and the combination-treated mouse groups decreased by approximately 50% compared to that of the normal mice group. The C. mongolica root extracttreated group demonstrated a 1.47 log CFU reduction in splenic infection compared to the non-treatment group and a spleen index that was similar to that of the normal mouse group. In all treatment groups, the activity of neutrophil phagocytosis significantly decreased, and splenic regeneration was observed. The present study has concluded that C. mongolica root extract may be useful in the treatment of brucellosis patients, in combination with doxycycline or other antibiotics, to avoid the toxicity of high dosage antibiotics and prevent the development of antibiotic resistance. Furthermore, the root extract can possibly be used to prevent Brucella infection.

Fig. 1
1Splenic enlargement in a Brucella-infected mouse compared with a normal mouse and countable Brucella colonies in 0.1 g spleen homogenate from mice in the following groups inoculated on TSA plates. a health control mouse spleen, b pretreatment, c non-treatment, d doxycycline only, e combination of antibiotic and root extract, f C. mongolica root extract only 2 mice from the healthy control, non-treatment, combination and only C. mongolica root extract treatment groups for the histological experiment.

Fig. 2
2Splenic section obtained after administration in mice from the following groups: a normal, b non-treatment, c combination of antibiotic and root extract, d C. mongolica root extract only. a -white pulp, b -red pulp, c -lymphocytes in different stages of development, d -haemosiderin pigmentation, ↗ -lymphocyte necrosis. Haematoxylin and eosin stain, magnification 400× and 40×

Table 1
1Antibacterial efficacies of C. mongolica root extract, antibiotic, and the combination of both treatments in B. melitensisinfected BALB/c miceTreatment group
Spleen index (mean ± SD)
Spleen index reduction
Log CFU in 0.1 g spleen (mean ± SD)
Log reduction

Non infected
0.3 ± 0.2
Control
-

Pretreatment
1.09 ± 0.4*
↑0.79
5.46 ± 0.15

Non treatment
0.96 ± 0.2*
↑0.66
4.5 ± 0.1
Control

Doxycycline
0.15 ± 0.01*
↓0.15
2.05 ± 0.07**
2.45

Combination treatment
0.19 ± 0.07*
↓0.11
2.1 ± 0.2**
2.4

C.mongolica root extract
0.32 ± 0.05
↑0.02
3.03 ± 0.15
1.47

Table 2
2Haematological parameters of B. melitensis-infected BALB/c mice treated with C. mongolica root extract, antibiotic or the combination of bothTreatment group
Content of Blood cells (in μl)

WBC (×10 3 )
LYM (%)
OTHR (%)
EO (%)
Neutrophil phagacytosis activity (%)

Non infected
3.05 ± 0.2
73.4 ± 8.5
22.2 ± 4
1.4 ± 0.28
13 ± 2.8

Pretreatment
6.4 ± 0.7
59.7 ± 4.2
38.03 ± 5.6
3.6 ± 0.75
49 ± 6.5

Non treatment
5.7 ± 0.1
63.8 ± 9.02
34.9 ± 8.9
1.26 ± 0.7
38 ± 2

Doxycycline
4.7 ± 0.01*
60.05 ± 0.3
37.5 ± 0.3
2.4 ± 0.1
14.5 ± 3.5*

Combination treatment
4.3 ± 0.5*
61.8 ± 8.4
32.7 ± 5.5
1.93 ± 0.4
18.5 ± 3.5*

C.mongolica root extract
4.2 ± 0.9*
63.4 ± 3.6
34.4 ± 3.3
2.1 ± 0.9
21 ± 2.5*

Statistical analysis: *P < 0.05 compared to the non-treatment group

AcknowledgementsThe authors thank Dr. Galindev. B and other colleagues (Core laboratory of the Mongolian National University of Medical Sciences) for their helpful discussions and Altankhuu. B and Enerel. E for their technical assistance.FundingThis study was supported by the Innovation Project (IN-09/2014) of the Science and Technology Foundation of Mongolia.Availability of data and materialsAll data generated and/or analysed during the current study are available from the corresponding author upon reasonable request.Authors' contributions TN, NB, and DD performed the experiments and data analysis. OO and BJ supervised the experimental work. TN designed the study and wrote the manuscript. All authors read and approved the final manuscript.Ethics approvalThis study followed international animal care and welfare guidelines for handling mice and designing experimental protocols and was approved by the Animal Care and Ethics Committee of the Institute of Veterinary Medicine of Mongolia.Competing interestsThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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The effect of hyperlipidemia on bone graft regeneration of peri-implantal created defects in rabbits

Mehmet Bugrul Tekin
Hulya Toker
The effect of hyperlipidemia on bone graft regeneration of peri-implantal created defects in rabbits
10.1186/s40729-019-0170-xR E S E A R C HPeri-implantHyperlipidemiaBone regenerationImplant stability
Aim: It is reported that hyperlipidemia affects quality and density of bone and adversely affects wound healing. The effect of hyperlipidemia on implant osseointegration and peri-implant defect regeneration has not been fully explained. The purpose of this study was to examine the effects of hyperlipidemia on the healing potential of the materials used for peri-implant bone regeneration and implant stability. Materials and methods: Twelve male, New Zealand rabbits were used in this study. Half of the rabbits were fed a 2% cholesterol diet for 8 weeks to induce hypercholesterolemia. Peri-implant defects (7 mm diameter) were created in the tibias of rabbits and placed implants (3.3 mm in diameter). This study was conducted as a split-mouth design. Animals were randomly divided into two groups: (1) hypercholesterol+autogenous graft group and hypercholesterol+xenograft group (n = 6), and (2) autogenous graft and xenograft groups as controls (n = 6). At 8 weeks after surgery, the rabbits were euthanized. During implant surgery and at 8 weeks, implant stability was measured with resonance frequency analysis (RFA values). Bone-to-implant contact (BIC) was analyzed via histomorphometric analysis. Results: Hyperlipidemic groups showed significantly lower BIC values than those of the control groups at 8 weeks (p < 0.05). According to baseline RFA readings, there was no significant difference between control and hyperlipidemic groups (p ˃ 0.05). The hypercholesterol+autogenous graft group had significantly lower RFA readings and BIC values than the hypercholesterol+xenograft group at 8 weeks (p < 0.05). Conclusion: Within the limitations of this study, it was found that hyperlipidemia may negatively affect the implant stability especially in the autogenous group and also, may decrease peri-implant bone regeneration. However, further studies are necessary to confirm these results more.

Summary

These findings suggest that hyperlipidemia reduced bone graft regeneration and decreased implant stability in experimental peri-implant defects in rabbits.

Dental implant survival is mainly dependent on successful osseointegration following placement. Any alteration of this biological process may adversely affect the success rate. Also, the long-term prognosis is adversely affected by inadequate bone volume at implant sites. There are several risk factors that were defined as implant failure. One of the risks of implant's failure depends on the systemic health of the subject (such as diabetes mellitus, osteoporosis, smoking) Recently, some authors suggested that there is a relationship between hypercholesterolemia and dental implant osseointegration [1,2].

Hyperlipidemia is a state with an abnormal lipid profile, which is characterized by elevated blood concentrations of triglycerides, elevated levels of total cholesterol and LDL, and decreased levels of HDL cholesterol [3]. Hyperlipidemia is associated with several diseases such as atherosclerosis and osteoporosis. The National Health and Nutrition Examination Survey (NHANES III) reported that 63% of osteoporotic patients have hyperlipidemia [4]. Furthermore, in the USA, among the approximately 300,000 implants placed due to fractures every year, 5-10% exhibit impaired healing or osseointegration [5,6]. The main mechanisms of the relationship between hyperlipidemia and bone tissue metabolism are the involved aspects of some metabolic changes, including lower bone mineral density, increase in the number of osteoclasts, and the inhibition of osteoblastic activity. However, several investigators suggested that lipidlowering drugs, such as statins, had beneficial effects on bone metabolism and also favorable effects on statins observed on osteogenesis around implants [7].

Bone augmentation procedures stimulate or facilitate the growth of new bone into the defected site. Although autologous or autogenous bone grafts are the ideal graft material for use in periodontal and implant surgeries, its use is restricted by the limitation of graft volume available, donor site morbidity, and prolongation of the operation. Thus, various alternative materials have been used including allografts and xenografts [8][9][10]. Xenografts are bone grafts that are derived from a donor of different species and are accepted as an osteoconductive material [11]. It was reported that xenograft subsequently replaced newly formed bone and bone apposition around titanium implants [12]. Furthermore, Hockers et al. found that deproteinized bone mineral and autogenic bone grafts appeared to be equally well integrated into the regenerating bone around implants [13].

To the best of our knowledge, peri-implant bone graft healing in the presence of hyperlipidemia has not been studied extensively yet. However, there is only one study that has investigated the effects of hyperlipidemia on implant osseointegration in mice via implant push-in test and micro-CT analysis. In that study, Keuroghlian [1] found that high-fat diet-fed mice had significantly increased implant loss as well as decreased formation and strength of the bone-to-implant interface. Therefore, based on these unfavorable aspects of hyperlipidemia, the two hypotheses that will be tested is that high levels of cholesterol can impair bone graft regeneration around a peri-implantal defect and can decrease implant stability. Therefore, the goal of this study was to investigate the histomorphometric evaluation of xenograft and autogenous bone graft healing in high-fat diet-induced animals with peri-implant bone defects.

Materials and methods

Animals

Prior to the study, the protocol was approved by the Animal Ethics Committee of Cumhuriyet University Faculty of Medicine under protocol number 65202830/130. Twelve New Zealand male rabbits (6-months old), with a mean weight of 3-3.2 kg, were included in this study.

The animals were maintained in individual cages at 21°C with 12-h day/night cycles and free to access to food and fresh water.

Induction of hyperlipidemia

Hyperlipidemia was induced on half of the animals by a 2% high-lipid diet including 95% pure cholesterol extract (Acros Organics, Geel, Belgium). Animals started on the diet 8 weeks prior to implant placement. Hyperlipidemic diet increased the total cholesterol levels fivefold at 8 weeks (Table 1), then experimental protocols were conducted. Also, blood samples were collected for the analyses of triglyceride, total cholesterol [14], and fraction (HDL and LDL) at the beginning, 4 weeks, and 8 weeks during the experiment.

This study was conducted as a split-mouth design. After the hyperlipidemia induction, two implants were placed in every animal and the peri-implant defect that was restored had different treatment protocols, and the study groups were the following:

Hypercholesterol+autogenous graft (HPL+AG) group and hypercholesterol+xenograft (HPL+XG) group (n = 6), Autogenous graft (AG) and xenograft (XG) groups as controls (n = 6).

Surgical procedures

All animals were anesthetized with an intramuscular injection of ketamine (40 mg/kg, Eczacibasi Ilac Sanayi, Istanbul, Turkey) and xylazine (10 mg/kg, Bioveta a.s., Komenskeho, Chezh Republic). The surgical site was disinfected with iodine solution and shaved. A 2-cm incision was made for rising full-thickness flap in the tibia. Circumferential defect (7-mm wide and 4-mm depth) was created using trephine bur (MIS Implant Technologies, Sholomi, Israel) with a low-speed handpiece under continuous irrigation with sterile saline at both sides of the tibia. İmplant osteotomy was performed at 800 rpm and the submerged type implants (Ø3.3 mm × 8 mm, RBM surface, İmplance, Trabzon, Turkey) were placed. Circumferential defects were grafted with either AG or XG. Bovine bone (Bio-Oss, Geistlich Pharma North America, NJ, USA) with a form of granules measuring 0.25-1 mm in size was used in this study. Furthermore, the bone obtained during the defect formation was crushed in the bone crusher (Schwert, Seitingen/Oberflacht, Germany) and then implanted into defects as an autogenous graft. The wound was sutured and postoperative antibiotic (ceftriaxone, intramuscularly (i.m.) 30 mg/kg, 3 days 1 × 1) and analgesic (carprofen, i.m. 4 mg/kg, 3 days, 1 × 1) were administered to animals to prevent postoperative infection and pain.

The animals were euthanized (by an overdose of 3% pentobarbital sodium) at 8 weeks after implant placement.

Resonance frequency analysis measurement

The RFA was measured soon after insertion of the implant (baseline) and after 8 weeks before removal of bone blocks from tibiae, using a resonance frequency analysis device (Osstell Mentor; Integration Diagnostics AB, Göteborg, Sweden). A Smartpeg (Integration Diagnostics AB) was screwed into each implant and tightened to approximately 5 N-cm. The transducer probe was aimed at the small magnet at the top of the Smartpeg at a distance of 2 or 3 mm and held stable during the pulsing until the instrument beeped and displayed the RFA value. The measurements were taken twice in the buccolingual direction and twice in the mesiodistal direction. The mean of the four measurements was recorded as the RFA values.

Histomorphometric analysis

Bone block sections including implants were removed and the samples were fixed in 4% neutral buffered formalin for 24 h then consecutively dehydrated using alcohol and embedded with resin (Tecnovit 7200 VLC, Heraus Kulzer GmbH, Wehrheim, Germany). The resin blocks were polymerized and sectioned in a mesiodistal plane using a cutting-grinding unit (Exakt 300 CL, Apparatbau, Norderstedt, Hamburg, Germany) in 300-μm thickness. After, the thickness of sections was carefully reduced to 40 μm using a micro-grinding system (Exakt 400 CS, Apparatbau, Norderstedt, Hamburg, Germany). Following this, the samples were stained with toluidine blue.

The histomorphometric analysis was performed by a single examiner (BT) who was masked from the samples' identities. The images of the sections in all groups were captured by a digital camera connected to a light microscope (Olympus® CX41, Tokyo, Japan) and loaded to a computer. The image analysis software (Analysıs LS Research, Version 5.0, Olympus Soft Imaging Solutions) was used for the measurements. Bone-to-implant contact (BIC) was measured as the amount of direct contact between the bone and the implant surface. Also, the amount of BIC was recorded as micrometers.

Statistical analysis

Statistical analyses were performed with SPSS, version 22 (IBM Corporation, New York, USA). Kolmogorov-Smirnov test was performed to test the normality of the data distribution. Mann-Whitney U test was used to compare groups. Wilcoxon test was performed in the intragroup analysis. The data were presented as a mean ± standard deviation and p < 0.05 was considered statistically significant.

Results

The animals tolerated the surgical treatment during the experiment well. However, one animal from the hyperlipidemic group and one animal from nonhyperlipidemic group died from an infection. Also, at the end of the study, the clinical examination performed prior to sacrifice disclosed that all implants were stable and that the surrounding mucosae were clinically noninflamed.

The means of the biochemical analyses were presented in Table 2. During the study period, no significant differences were found in lipid biomarkers (TG, LDL, HDL, TC) and these levels were also higher in hyperlipidemic groups than those of the non-hyperlipidemic group (p < 0.05).

In Fig. 1 were shown the mean value of BIC of all groups. After 8 weeks, BIC value was greater in the control groups compared to their respective hyperlipidemic groups as shown in Fig. 2 (p < 0.05). Also, there was a significantly higher BIC value in the HPL+XG group than those in the HPL+AG group (p < 0.05). However, there was no significant difference in BIC value between the AG and XG groups (p > 0.05). RFA was measured at the time of implant placement and after 8 weeks (Fig. 3). According to intragroup comparisons, there was a significant difference in RFA readings between baseline and 8 weeks (p < 0.05). In all groups, RFA readings of implants were lower at baseline and were higher at 8 weeks. Also, no significant differences were found in RFA readings between AG and XG groups at 8 weeks (70.8 and 72.6, respectively) (p ˃ 0.05). However, in HPL+AG group, RFA readings were lower than those of the HPL+XG group at 8 weeks (p < 0.05). RFA readings of HPL+XG group were not different compared to XG group (p ˃ 0.05).

Discussion

Although bone regeneration is an efficient process in physiological conditions, many factors such as inflammation, hormonal changes, and also, elevated serum lipid levels associated with the impaired or delayed healing process. In the present study, an initial objective of the research was to identify the effects of hyperlipidemia on bone graft regeneration around peri-implant defects in rabbits. To the best of our knowledge, this is the first study to verify the inhibition of bone graft regeneration and implant stability in peri-implantal defect under hyperlipidemic conditions. And also, we demonstrated that the amount of bone-to-implant contact and implant stability improved in HPL+XG group compared to HPL +AG group.

A wound-healing situation that is unaffected by external factors such as microbial colonization, plaque accumulation is a prerequisite for evaluating potential regenerative or augmentation treatment modalities. Therefore, in our study, the tibia of a rabbit was selected for extraoral defect model. Rabbits are commonly used for screening implant materials prior to testing in a larger animal model because they are easy to handle, and due to the size of their tibia, allowing the creation of a large peri-implant defect and the installation of conventional implants in the proximal region of the tibia [15].

In literature, different experimental protocols have been used to induce hyperlipidemia and no available threshold to confirm hyperlipidemia such as diabetic models that blood glucose levels were greater than 300 mg/dl. Earlier studies were used to induce hyperlipidemia by a different concentration or times such as a 1% cholesterol diet for 4 weeks [16], 1.5% cholesterol diet for 12 weeks [17], and 0.5% cholesterol diet for 12 weeks [18]. Also, in a recent study [19], rabbits were fed a 2% cholesterol diet for 8 weeks, as we used in our study. After 8-week feeding, we observed a fivefold increase in total cholesterol and triglyceride levels in rabbits. Furthermore, these levels lasted during the study period as shown in Table 2.

Graft resorption called biodegradation should take place at a time appropriate to the activation of new bone formation [20]. Some of the studies showed that the xenograft was slowly resorbed or was encapsulated [20][21][22]. In another study that was investigated histologically the simultaneous placement of endosseous implants into the sinus cavity and the surgical elevation of the sinus floor including filling the cavity with different grafting materials, they found that hydroxyapatite or natural bovine bone mineral demonstrated newly formed bone with direct contact at the implant surface [12]. Similarly, our results showed that xenograft particles were almost replaced by new bone. But, bone-to-implant contact was decreased in the hyperlipidemic group. However, there is no clinical data available in which a clinical outcome obtained following regenerative therapy of hyperlipidemic patient appears to be more unfavorable than those of the non-hyperlipidemic patients. Only one retrospective study suggested that high total serum cholesterol levels tend to increase graft failure rates whilst it did not influence implant failures. In that study, only autologous and/ or deproteinized bovine bone was used for all cases, as we used in this study [2].

Hyperlipidemia increases the risk for the generation of lipid oxidation products. Pirih's animal study found that hyperlipidemia induces secondary hyperparathyroidism and impaired bone regeneration and mechanical strength through actions of oxidized lipids which accumulate in the subendothelial spaces of vasculature and bone [23]. Also, in a recent study, the authors evaluated the effects of hyperlipidemia on implant osseointegration in mice via micro-CT analysis [1]. They reported that HF diet-fed mice had significantly decreased bone formation and bone-to-implant contact. Interestingly, in our study, the HPL+AG group showed less BIC compared to the HPL+XG group, probably by upregulating osteoclastogenesis and suppressing mineralization. But keeping in mind that small sample size may affect these results and therefore it must be confirmed in future studies using a larger sample size.

Osseointegration and bone healing are similar processes as they both involve similar cells, hormones, and systems. Therefore, drugs or systemic diseases that affect bone healing can also decrease osseointegration [24]. In addition, the quality and quantity of the bone surrounding the implants are a critical factor to the long-term prognosis. There are several methods that have been purposed to evaluate implant osseointegration, such as the cutting torque resistance analysis and the resonance frequency analysis [25]. RFA analysis aims at providing an objective and non-invasive measurement, and also the RFA has been extensively used in experimental and clinical research [26]. However, Balshi et al. reported that RFA measurements were associated with the bone density, the location of placement, and the gender [27]. Furthermore, in a study that investigated the correlations between the RFA analysis and peri-implant bone levels in surgically created dehiscence defects and circumferential defects, they found that RFA readings were correlated to circumferential and wide-dehiscence defects but not for narrow-dehiscence defects. According to the authors, the size of surrounding bone defects was highly associated with RFA readings. Huang et al. suggested that the highest resonance frequency value was found when the implant was placed into the type I surrounding bone and in contrast, the resonance frequency of the implant with type IV bone quality was found almost fourfold less than that found in the type I model [28]. In our study, baseline RFA readings were decreased in all groups due to circumferential intra-bony defects (class 1-e, according to defect classifications of Schwarz et al.) and the wide bone marrow of the tibial bone and no significant differences among the groups [29].

In reviewing the literature, only one study has explored the effect of hyperlipidemia on implant stability in mice [1]. In that study, the most important finding was that high-fat diet-fed mice had increased implant loss and the high-fat diet group required a lower load to break the bone-to-implant contact at 4 and 8 weeks according to push-in test results. Also, in that study, there was no peri-implantal defect and all implants were inserted pristine bone of mice. However, our results showed that RFA readings were decreased in HPL+AG groups compared to controls, and RFA readings in the HPL+XG group were better results than that in the HPL +AG group. These results must be confirmed in a larger sample size because of a low sample size is the main limitation of this study.

In conclusion, within the limitations of this animal study, these findings support the hypothesis that hyperlipidemia impaired bone graft regeneration especially, autogenous graft healing with peri-implant defects and also decreased implant stability. Thus, according to our results, we confirmed the adverse effects of hyperlipidemia on implant osseointegration and peri-implant defect regeneration. However, future studies needed to investigate the mechanism of action of hyperlipidemia on graft materials in selecting appropriate graft materials for hyperlipidemic patients.

Fig. 1
1Mean BIC value of study groups at 8 weeks. *p < 0.05 versus HPL+AG and HPL+XG group, # p < 0.05 versus HPL+XG group (n of each group is 5)Fig. 2Histologic views of the study groups at 8 weeks (toluidine blue, × 10). a AG group, b XG group, c HPL+AG group, d HPL+XG group (NB new bone, BM bone marrow, red arrow indicates boneto-implant contact, black arrow indicates residual xenograft particles)

Fig. 3
3Mean and standard deviation of RFA readings in study groups at baseline and 8 weeks. a p < 0.05 versus baseline, b p < 0.05 versus HPL+AG group, c p < 0.05 versus AG group (n of each group is 5)

Table 1
1Biochemical parameters of study groups during hyperlipidemia induction prior to implant placement protocol (mean ± SD)Total cholesterol Triglyceride HDL
LDL

AG
Baseline 28 ± 1.1
22 ± 1.5
20 ± 2
14 ± 1.5

4 weeks 27 ± 1.6
21 ± 1.6
19 ± 1.4 15 ± 2

8 weeks 31 ± 1.9
25 ± 2
20 ± 1.4 18 ± 1.9

XG
Baseline 25 ± 1.4
20 ± 1.6
18 ± 1.9 17 ± 1.5

4 weeks 30 ± 1.5
24 ± 1.5
18 ± 1.5 20 ± 1.6

8 weeks 29 ± 1.8
19 ± 1.1
20 ± 1.4 21 ± 1.4

HPL+AG Baseline 25 ± 2.5
23 ± 2.2
18 ± 1.2 15 ± 1.3

4 weeks 61 ± 2.1
55 ± 1.5
38 ± 1.7 34 ± 2.6

8 weeks 101 ± 2
90 ± 1.5
39 ± 1.5 38 ± 2.3

HPL+XG Baseline 22 ± 1.8
18 ± 2
17 ± 2
16 ± 1.9

4 weeks 70 ± 2.2
53 ± 1.3
36 ± 2.1 37 ± 2

8 weeks 110 ± 1.6
94 ± 1.4
38 ± 1.6 41 ± 1.5

Table 2
2Biochemical parameters of study groups during experiment (mean ± SD)Total cholesterol Triglyceride HDL
LDL

AG
Baseline 28 ± 2.4
22 ± 1.5
20 ± 2
14 ± 1.4

4 weeks 27 ± 1.1
21 ± 2
19 ± 1.2 15 ± 2

8 weeks 31 ± 1.9
25 ± 1.6
20 ± 1.5 18 ± 1.8

XG
Baseline 25 ± 1.5
20 ± 1.5
18 ± 1.9 17 ± 1.3

4 weeks 30 ± 1.6
24 ± 1.7
18 ± 1.4 20 ± 1.4

8 weeks 29 ± 1.5
19 ± 2.1
20 ± 1.5 21 ± 1.7

HPL+AG Baseline 101 ± 2.5
90 ± 2.2
39 ± 1.5 38 ± 1.3

4 weeks 105 ± 1.5
98 ± 1.2
40 ± 1.4 41 ± 2.7

8 weeks 110 ± 1.4
100 ± 1.8
41 ± 1.7 54 ± 1.7

HPL+XG Baseline 110 ± 2
94 ± 3.1
38 ± 2.1 41 ± 1.9

4 weeks 113 ± 2.2
96 ± 2.2
40 ± 2
43 ± 2

8 weeks 114 ± 1.5
101 ± 2.1
39 ± 1.4 47 ± 1.6

AcknowledgementsNot applicableFunding This study was supported by the Cumhuriyet University Unit of Scientific Research Projects (project number DIS-171).Availability of data and materialsNot applicableAuthors' contributionsMBT performed the experimental protocol and histomorphometric analysis and was a major contributor in writing the manuscript. HT analyzed and interpreted the data. Both authors read and approved the final manuscript.Ethics approval and consent to participateConsent for publication Not applicableCompeting interests Mehmet Bugrul Tekin and Hulya Toker declare that they have no competing interests.
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