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International Journal of Pharmaceutical

Biological and Chemical Sciences

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS)

| OCT-DEC 2013 | VOLUME 2 | ISSUE 4 | 13-19 www.ijpbcs.net or www.ijpbcs.com

Research Article

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EFFECTS OF TRAMADOL ON SERUM BONE ALKALINE PHOSPHATASE,

BONE MORPHOGENETIC PROTEIN 2 AND HEALING OF

DENTAL IMPLANT IN A SHEEP MODEL

Fayhaa AM Al – Mashhadane1, Ghada Abdul-Rhman Taqa

2, Tahani A. AL-Sandook

3

1,2Assistant professor in pharmacology, University of Mosul /College of Dentistry, Iraq

3Professor in pharmacology, University of Mosul /College of Dentistry, Iraq

*Corresponding Author Email: [email protected]

INTRODUCTION

Several categories of prescription medications, when

taken long-term, can cause reduced bone density and

strength. Analgesic drugs are very commonly prescribed

for long-term patients, such as those suffering from

arthritis or chronic back pain and They could have such

effect on bone(1).Analgesics are also one of the drugs that

are widely used in dental surgery and their potential

effect on bone metabolism is worth studying.

Since the mid 1970s the effects of NSAIDs on bone

metabolism and bone healing have been studied (2). In

1976, significant reduced fracture healing was reported

in rats treated with indomethacin .In the same year , a

64-years old man with an ankle fracture / luxation was

purposefully treated with indomethacin to prevent callus

formation in anticipation of surgery .(3)

Opioids, an alternative type of analgesic drugs, have

been shown to be associated with a decreased bone

mineral density .An increased risk of fracture have been

also reported with use of opioids ,although a significant

differences may exist between different types of opioids

.(4)

The most common opioid analgesics used are: morphine,

codeine, Pethidine, heroin and methadone. Tramadol is a

ABSTRACT:

Aim of the study: To examine the effect of tramadol on serum bone alkaline phosphatase (BALP) , Bone

morphogenetic protein 2 (BMP2) and healing of dental implant in sheep model. Methods: Six apparently healthy

mature male sheep aged 10-12 months and body weight of 25 ± 2kg from local market were included in the study. The

animals were randomized into two groups of 3 animals: Group 1 not treated by any drug (control), Group 2 were treated

by tramadol hydrochloride (Mepha LLC;Switzerland) in dose of 5mg/kg/day for 12 weeks before surgery and additional

6 weeks after surgery of dental implant. Blood samples(10 mL) from each sheep was drawn via jugular vein puncture

before the drug administration and at the 10 ,20,30,40,50,60,70,80 days of treatment for analysis of BALP , BMP2 by

using ELISA kitS from MyBiocource company (USA) . At the end of 12th week of the study ,two animal were selected

from each of the groups mentioned above ,received general anesthesia inducted by intramuscular injection of a mixture

containing (10mg/kg) ketamine with (2mg/kg) xylazine as sedative , analgesic and muscle relaxant solution .Local

anesthesia ,2% lidocaine HCL with epinephrine 1:80,000 local anesthetic agent was administered by infiltration at the

surgical site. For the selected animals, four implants/sheep were performed. Titanium Tixos Implants (LEADER

ITALIA) were used in this study. Animals with dental implants were sacrificed after 6 weeks from day of implants

installation .The tibiae with dental implants from each animal were dissected ,freed from soft tissue .Tibial bones with

implants were subjected to assessment of implant removal torque using torque meter device(LEADER, ITALY).

Results: Data analysis by using Independent Samples Test showed that there were no significant differences between

the mean level of serum BALP and BMP2 in control group (8.195±1.622 ng/ml , 97.079±20.886 ng/ml) compared to

treatment group(7.535±2.588 pg/ml , 103.626±20.597 pg/ml ) respectively , at p-value ˂ 0.05 . On clinical observation

,the sites of implants installation were covered by sound apparently healthy bone for tramadol group ,while for

control group the sites of implants showed healing with sound bone but without covering the implants .Data analysis by

using Independent Samples Test showed that there were significant differences between the mean level of implant

removal torque readings between control and treatment groups (22.50±2.88 Ncm , 55.00 ± 4.082 Ncm ) respectively at

p – value = 0.000. Conclusion: Tramadol ,a serotonin reuptake inhibitor, can be a useful analgesic drug to be prescribed

as for dental implant surgery .

KEYWORDS: Prescription medications, Analgesics, dental surgery

mailto:[email protected]

* Fayhaa AM Al – Mashhadane et al; Effects of tramadol on serum bone alkaline phosphatase , bone morphogenetic protein 2.

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | OCT-DEC 2013 | VOLUME 2 | ISSUE 4 | 13-19| www.ijpbcs.net

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synthetic analgesic of opioid class with the chemical

formula of (C16H25NO2).It is a centrally acting analgesic

whose mechanism of action is predominantly based on

blockade of serotonin reuptake. Also it has been found to

inhibit norepinephrine transporter function. Because it is

only partially antagonized by naloxone, it is believed to be only a weak μ-receptor agonist. (5)

The internal structure of bone is described in terms of

quality or density which reflects a number of

biomechanical properties, such as strength and modulus

of elasticity. In dental implant surgery, the density of

bone is a determining factor in treatment plan , implant

design , surgical approach ,healing time.(6) Gap healing

and implant fixation could be affected by quality and

quantity of bone in the local environment(7),which can be

evaluated by measuring biochemical bone markers like

BALP and BMP2. Osteoblasts control mineralization

and osteogenesis by regulating the passage of calcium and phosphate ions across their surface membranes. The

latter contain alkaline phosphatase, which is used to

generate phosphate ions from organic phosphates. So,

bone alkaline phosphatase (BALP) contributes to

mineralization (8). The level of BALP in serum indicates

the metabolic status of osteoblasts .It is level in serum

provides information useful in the evaluation and

treatment of patients with bone diseases, also its level

increase rapidly in response to anabolic therapy. The loss

and deterioration of bone tissues is caused by a net

imbalance in bone remodeling and this can be estimated by measuring bone mineral density, but this estimation is

unable to provide direct information on the micro

architectural deterioration of bone which is associated

with deeper resorption sites .BALP can reflect this

underlying remodeling process in a highly specific and

sensitive way. Another bone formation marker is Bone

morphogenetic protein 2 (BMP-2), a potent osteo

inductive cytokine from the TGF-β super family that

triggers the development of stem cells into osteoblasts(9).

BMP2 may be useful for enhancing bone growth into

gaps around cementless implants (10). Analgesics are

widely prescribed for dental implant patients ,not only for postoperative pain relief but also for other systemic

diseases like muscular and skeletal disorders .Given

their frequency of use, likely cross –correlation of

analgesics with implant failure could indicate that they

cannot used safely for pain relief during the early

postoperative period and a possible increased risk of

implant failure if performed for patient after chronic use

of such drugs(11).Although the effects of analgesics in

maxillofacial surgery have not been adequately

investigated , their negative effect on bone healing has

been confirmed in orthopaedics .(12-14)

Aim of this study was to examine the effect of tramadol

as an analgesic drug on serum BALP, BMP2 and healing

of dental implant in sheep model.

MATERIALS AND METHODS This study was carried out in the department of Basic

Sience ,College of Dentistry, University of Mosul .It was

conducted at the time interval between November /2012

till March/ 2013 .The study protocol was approved by

scientific committee /department of basic science

/college, of Dentistry/University of Mosul.

Six apparently healthy mature male sheep of 10-12

months old and body weight of 25 ± 2kg purchased from

local market were included in the study. During the

entire period of the study, the animals were permanently housed indoors in animal house of College of

Dentistry/University of Mosul .They were kept in group

housing under photoperiod cycle of light: from 6:00 to

18:00 h and dark: from 18:00 to 6:00 at temperature 20 ±

2Cº .This protocol was used in view of published reports

of seasonal variation in bone remodeling and circadian

rhythm variation of the biochemical markers of bone

turnover. The animals were fed twice daily with

standardized diet with tap water. Each sheep was

subjected to a clinical examination, also they were

examined daily by veterinarian until slaughtering .The

animals were randomized into two groups of 3 animals: Group 1 not treated by any drug, Group 2 were treated

by tramadol hydrochloride (Mepha LLC; Switzerland) in

dose of 5mg/kg/day (15)

for 12 weeks before surgery and

additional 6 weeks after surgery.

Blood sampling

All animals were blood sampled before the drug

administration and at the 10 ,20,30,40,50,60,70,80 days

of treatment .The animals were prevented from feeding

12 hr before blood sampling ,then 10 ml of blood were

drawn via jugular vein puncture between 10:00 - 11:00

am for the analysis of biochemical parameters , placed into plain tubes ,kept at room temperature for 30 minutes

,centrifuged for 15 minutes at 3000 rpm, then the

separated serum was removed by micropipette and

transferred to 6 eppendroff tubes ,stored at -20°C till

analysis.

Determination of Bone Alkaline Phosphatase and

bone morphogenetic protein 2:

BALP ELISA kit and BMP2 ELISA kit from

MyBiocource Company (USA) used for the

quantitative determination of Sheep BAP and BMP2

ASSAY PROCEDURE The desired numbers of coated wells in the holder have

been secured then 100 µL of Standards or Samples were

added to the appropriate well in the antibody pre-coated

Microtiter Plate. 100 µL of Blank Solution (pH 7.0-7.2)

was added in the blank control well, then 50 µL of

Conjugate was added to each well (NOT blank control

well), Mixed well and covered then incubated for 1 hour

at 37°C. After that the micro titer plate washed by using

Automated Washing five times with diluted wash solution (400 µL/well/wash) using an auto washer and

dried. A 50 µL Substrate A and 50 µL Substrate B were

added to each well including blank control well,

subsequently, covered and incubated for 10-15 minutes

at 20-25°C, followed by addition of 50µL of Stop

Solution to each well including blank control well.

Mixed well. Finally, the Optical Density (O.D.) at 450

nm was determined by using a microplate reader of

ELISA device immediately.

Induction of Anesthesia

At the end of 12th week of the study, two animal were selected from each of the groups mentioned above .At



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the day of operation the selected animals were checked

by veterinarian for their general statement and health,

then received general anesthesia inducted by

intramuscular injection of a mixture containing

(10mg/kg) ketamine hydrochloride general anesthetic

agent and (2mg/kg) xylazine sedative, analgesic and muscle relaxant solution. Complete anesthesia was

obtained within 10 minutes .Local anesthesia, 2%

lidocaine HCL with epinephrine 1:80,000 local

anesthetic agent was administered by infiltration at the

surgical site prior the incision for hemostasis.

Surgical procedure

For the selected animals, four implants/sheep were

performed. Titanium Tixos Implants (LEADER

ITALIA) were used in this study.

The operation sites were shaved ,washed and disinfected

with 10% povidon iodine .A skin incision was made

along the longitudinal axis of both posterior tibial bones at the external surface, fascia of each bone was dissected

and a full – thickness flap was reflected to expose a

segment of 10 cm of each tibia under aseptic condition

.After exposure and reflection of soft tissue , the

preparation of implant was performed following

manufactureʼs recommendations given in the manual of

implant system Implant beds were prepared at a distance

of 20 mm apart , using rotating hand piece with speed of

1900 rpm under copious irrigation with sterile 0.9 %

physiological saline with back and forth motion to

minimize the risk of bone overheating .Thereafter, dental implants were carefully installed and fixed manually

using implant driver with ratchet till the implant bodies

were submerged in bone .

At completion of implant placement ,the flap was gently

reapproximated and primary wound closure was

performed in layers, periosteium with periosteium using

3-0 synthetic , braided , absorbable polyglycolic acid

surgical suture and skin with skin using 3-0 non

resorbable black silk suture which was to be removed

10 days postoperatively .Finally ,the wounds were

dressed for 24 hours then dressing was removed and

implants were left to heal in submerged fashion. Immediately post-operatively antibiotic was

administered intramuscularly with (10 mg/kg) of

oxytetracycline, then the same dose was repeated every

12 hour for three times.

Along the period of implant healing which extended to 6

weeks ,the animals were received periodical veterinary

care with no evidence of serious adverse local or

systemic complications observed throughout the period

of implant healing .Animals with dental implants were

sacrificed according to the Islamic regulation after 6

weeks from day of implants installation. The tibiae with dental implants from each animal were

dissected, freed from soft tissue. A small head round bur

mounted on a straight surgical hand piece under copious

normal saline irrigation was used to expose the implant

sites in tramadol group .So , all implant sites were be

ready for measurement of measurement of stability.

Implant torque measurement

Tibial bones with implants were subjected to assessment

of implant removal torque using Torque Meter Device

(LEADER, ITALY); this torque control device enables

the setting of the ratchet to any value between 10 and 60

Ncm. The setting precision is absolutely valid because it

is exclusively based on the force of gravity.

RESULTS Standard descriptive statistic of BALP for each study

time in tramadol group was shown in Table (1).

Dental Implant

Healing over the site of surgery was uneventful

throughout the study period for all implants .The sheep

was in good health at the time of sacrifice. On clinical

observation, the sites of implants installation were

covered by sound apparently healthy bone for tramadol

group, while for control group the sites of implants showed healing with sound bone but without covering

the implants .There were no signs of infection in both

groups. After 6 weeks from the date of surgery, implant

stability was measured by using torque meter device.

Data analysis by using Independent Samples Test

showed that there were significant differences between

the mean level of implant removal torque readings

between control and treatment groups (22.50±2.88,

55.00 ± 4.082) respectively at p – value = 0.000.Table

(4)

DISCUSSION In the present study effects of Tramadol on BALP,

BMP2 and healing of dental implant were examined.

The unique mechanism of action in tramadol and its

alternative metabolic pathways can lead to this

hypothesis that it does not interfere with bone

metabolism. In this study no significant changes of

means of serum levels of both BALP and BMP2 was

found between control and treatment groups (Table 3)

.Since that BALP and BMP2 reflect underlying bone remodeling process in a highly specific and sensitive

way and their activities demonstrating rapid and

extensive osteogenic differentiation (16), This result

indicated that tramadol have neutral effect on bone

remodeling process.

Bone metabolism and remodeling is a phenomenon

which occurs frequently in response to external stimuli

as well as biochemical changes. Osteoblasts and

osteoclasts are two main cells that carry out this process.

(17-19).

Tramadol is known as atypical opioid in that it has some

distinguishing features other than general characteristics

of opioids (20). Most of the effects of drugs in opioid

class are mediated via receptors. These receptors are G-

protein- coupled receptors with subtypes known as

kappa, delta, mµ. These receptors are present in central

nervous system as well as peripheral sensory neurons (21).

Unlike other opioid drugs Tramadol has 2 unique

mechanism of action: First:Tramadol has low affinity for

mu receptors and even lower affinity for delta and kappa

receptors. Second:Tramadol is a serotonin releasing agent and also norepinephrine- reuptake inhibitor.

Tramadol plays its analgesic role by inhibiting the

reuptake of monamines through mu receptors. (22)



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Tramadol possess the distinctive feature of acting via

non-opioid ways. This mechanism of action is the reason

why the analgesic effects of tramadol is not fully

antagonized by mu receptor antagonists (naloxone) a

feature that is not seen in other opioids (23). In a study on

rats treatment with morphine showed a significant biochemical and histological osteoporotic changes while

treatment with tramadol leads to non-significant

osteoporotic effect. (24)

In regard to dental implant ,significant differences was

found in the implant removal torque readings between

control and treatment groups .Treatment group showed

enhanced bone formation around dental implant that need greater removal torque compared to control

group.(Table 4)

Table (1): Descriptive Statistics of BALP for Tramadol group during study times

study times

N Mean Std.

Deviation

Std. Error Minimum Maximum

Time 1 3 5.103 .6590 .3800 4.418 5.734

Time 2 3 7.726 .6370 .3670 6.999 8.185

Time 3 3 12.735 4.508 2.603 9.768 17.923

Time 4 3 9.293 1.213 .7000 7.964 10.342

Time 5 3 7.413 2.360 1.362 5.887 10.132

Time 6 3 7.333 1.846 1.065 5.756 9.363

Time 7 3 6.411 .3830 .2210 6.103 6.840 Time 8 3 5.007 1.704 .9830 3.939 6.972

Time 9 3 5.389 .8280 .4780 4.685 6.303 Time 1: blood sample before the drug administration

Time 2: blood sample 10 days after the drug administration

Time 3: blood sample 20 days after the drug administration Time 4: blood sample 30 days after the drug administration Time 5: blood sample 40 days after the drug administration Time 6: blood sample 50 days after the drug administration Time 7: blood sample 60 days after the drug administration Time 8: blood sample 70 days after the drug administration Time 9: blood sample 80 days after the drug administration

Table (2): Descriptive Statistics of BMP2 for Tramadol group during study times

Study times

N Mean Std.

Deviation

Std. Error Minimum Maximum

Time 1

Time 2

Time 3

Time 4

Time 5

Time 6

Time 7 Time 8

Time 9

3 87.680 9.574 5.527 78.839 97.849

3 110.377 14.023 8.096 96.945 124.926

3 126.530 29.948 17.291 92.985 150.581

3 110.222 22.097 12.757 85.030 126.328

3 113.751 27.572 15.9190 84.899 139.836

3 115.361 15.058 8.694 103.625 132.340

3 111.888 12.975 7.491 99.993 125.725 3 94.959 13.698 7.908 83.086 109.946

3 94.058 9.695 5.597 83.471 102.505

Table (3): Independent Samples Test comparison between control and treatment groups for the mean level of

serum BALP and BMP2

Bone markers Study groups No. mean ±SD t-value df p-value

BALP control 9 8.195 1.622 -0.648 16 0.526

treatment 9 7.535 2.588

BMP2 control 9 97.079 20.886 0.669 16 0.513

treatment 9 103.626 20.597



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Table (4): Independent Samples Test comparison between control and treatment groups

for the mean level of implant removal torque readings

J. Cell Biol. Vol. 191 No. 1 7–13, 2010

Figure 1. The different effects of gut- and brain-derived serotonin on the osteoblast. The free circulating form of gut-

derived serotonin directly signals to the osteoblast by binding to the Htr1b receptor. This binding inhibits the

phosphorylation of CREB by PKA, leading to decreased expression of Cyclin (Cyc) genes and decreased osteoblast

proliferation. As a result, bone formation is slowed down. In contrast, serotonergic neurons of the dorsal raphe (DR)

signal to VMH neurons via the Htr2c receptor to inhibit the synthesis of epinephrine and thereby decrease sympathetic

tone. This decrease is relayed in osteoblasts by decreased signaling via the 2 adrenergic receptor (Adr 2), which

negatively controls osteoblast proliferation via a molecular clock gene/cyclinD1 (Cyc D1) cascade and positively

regulates bone resorption via activation of a PKA/ATF4-dependent pathway, leading to increased synthesis of Rankl,

an activator of osteoclast differentiation and function. The inhibition of sympathetic activity by brain derived serotonin thus results in increased formation and decreased resorption. Solid lines, direct actions; broken lines, indirect

mechanisms.

This neutral effect of tramadol on bone formation and

healing of dental implant was predictable given the

distinctive features of this drug and its mechanism of

action comparing to other drugs of its class and it is

corresponded to the results from other study that carried

out to investigate effects of tramadol on orthodontic

tooth movement in rats, it was found that this drug does

not interfere with the process of bone remodeling and

tooth movement in rat and it does not affect osteoclastic

activity and bone resorption(25) .In other study on rats

Study

groups

No. mean ±SD t-

value

df p-

value

control 4 22.50 2.887

13.00

6

0.000

treatment 4 55.00 4.082



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treated with morphine showed a significant biochemical

and histological osteoporotic changes while those

treated with tramadol showed non-significant

osteoporotic effect(26). Considering the fact that most of

the pharmacological effects of tramadol are associated

with inhibition of reuptake of serotonine and norepinephrine and acting via opioid receptors is not as

key factor as it is in other opioids, it is necessary to

understand the effect of serotonin on bone biology.

The serotonin molecule has some remarkable properties

.It is synthesized by two different genes at two different

sites, and, surprisingly, plays antagonistic functions on

bone mass accrual at these two sites. When produced

peripherally, serotonin acts as a hormone to inhibit bone

formation. In contrast, when produced in the brain,

serotonin acts as a neurotransmitter to exert a positive

and dominant effect on bone mass accrual by enhancing

bone formation and limiting bone resorption(27,28).This dissociation is explained by the fact that serotonin cannot

cross the blood–brain barrier; hence, altering its levels

peripherally does not influence its central concentration,

or vice versa(29). In other words, serotonin central and

peripheral functions may be completely dissociated..

Serotonin is synthesized by two distinct genes.

Peripherally Tph1, involved in the synthesis of serotonin

in the gut and centrally Tph2, involved in the synthesis

of serotonin in the brain (26). (Figure 3)

Pharmacologic, genetic expression, and cell culture

studies subsequently confirmed that Tph1 and gut-derived serotonin synthesis were potent regulators of

bone formation (26, 28, 29) .In contrast with the Tph1, the

severe low bone mass phenotype observed in the absence

of Tph2 results from an effect on both arms of bone

remodeling: it is secondary to a decrease in bone

formation parameters as well as to an increase in bone

resorption parameters (30). Further analysis of the

molecular basis of this phenotype revealed that both

these effects are mediated by an increase in sympathetic

tone (26, 30).

So, the central effect of tramadol and it is mechanism of

action which is predominantly based on blockade of serotonin reuptake (31,32) could explain it is neutral effect

on bone remodeling and bone healing. The present study

suggests further research to investigate the healing of

dental implant in patients receiving different doses and

durations of tramadol treatment.

CONCLUSION In summary, this study does provide support for a

possible role for serotonin in regulating bone density and structure. It also reveals that tramadol, a serotonin

reuptake inhibitor, can be useful to be prescribed as

analgesic drug for dental implant surgery.

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*Corresponding author address:

Fayhaa AM Al – Mashhadane Assistant professor in pharmacology,

University of Mosul /College of Dentistry, Iraq

fayhaa am al , tahani a. al-sandookijpbcs.net/ijpbcsadmin/upload/ijpbcs_5282ded8de30c.pdfbone...

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