94
NICOTINE EFFECTS ON THE NUMBER OF OSTEOCLAST AND
95 INTRODUCTION
Dental implant is a material that implanted in the jawbone and support restorations that resemble a teeth. Currently, the material of dental implant is commonly made of Titanium (Ti). Dental implants as a replacement for lost teeth have been commonly used in the last two decades. Even with the increase in life expectancy, dental implant users also increased. Since 2005, more than 1 million dental implants have been installed each year 1.
The success of dental implants depends on comfort, aesthetics and function as much as possible natural tooth. Dental implants are said to be functionally successful in case of osseointegration, resulting in union between the implant. Osseointegration occurs when during the formation and healing process, the bone does not get interference and it is highly dependent on cellular mechanisms. Healing process depends on osteoclast and osteoblast cells. Osteoblasts are cells involved in bone formation, is located on the bone surface and derived from progenitor cells. In the active state, osteoblasts synthesize matrix, cuboidal- shaped, and the inactive state shaped flat and often referred to as bone lining cells.
Osteoclasts play a role in bone resorption and during this process, hydrogen ions are formed from carbonic anhydrase into the plasma membrane to dissolve the bone matrix.
Various factors that influence the formation of bone and bone healing include systemic, genetic, molecular and pharmacologic factors. Other factors are smoking, alcoholism, and certain drugs (bisphosphonates), age and gender 2,3,4.
Research on the effect of smoking on dental implant has been previously done.
Examination to know osseointegration is to calculate the percentage of new bone into the thread. Bone density is calculated by looking at the number of osteoblasts in the thread.
Results count histometric this study stated that osseointegration in smokers was lower than in nonsmokers 5.
In this study, researchers will explore the nicotine effect on osteoclast and osteoblast cell to osseointegration in dental implant thrugh the New Zealand rabbits that had already been exposed to nicotine. Nicotine chosen because of the main ingredient of cigarettes is contains tobaccos, whereas 95% nicotine. It shows that nicotine significantly prevent or slow down the bone regeneration6.
MATERIALS AND METHODS
This research is an experimental study with a post-test control group design.
Observations were made at the firts week (inflammatory period) after the second implant
96 planted in the jaw bone and the second obervation were made at the eighth week (a period of consolidation and osseointegration) after the first implant planted. The samples were the left and right lower jawbone of the New Zealand rabbits, which met the inclusion criteria, which is healthy, male, aged 3.5 to 5 months and weight 2.5 to 3.5 kg.
The study was conducted in several different laboratories. The making and preparation of block tissue and HE staining performed in Anatomical Pathology laboratory RSU Haji Surabaya. Animal care and the placement of the implants were performed in the Institute of Tropical Disease, Airlangga University, Surabaya.
Insertion of dental implants. Prior to the implant placement, all the rabbits were weighed to determine the dose of anesthesia. All the rabbits anesthetized with total dose of 0,15 ml xylazine per kg body weight and 0,2ml ketamil per kg body weight by intramuscular injection. The jaw bone was marked before the implants implanted, then the bone was drilled with a speed of 1,200 rpm and with a depth of 7 mm. Implants with a diameter of 3.3 mm and a length of 7 mm were implanted the same as bone level with the speed of 30 rpm. Then do the torque was done with the power of 20 Newton and then the implant was closed with screw cover. The flap sewn and treated with antiseptic. Lastly, the rabbits were injected with long acting antibiotics (Medoxy LA) 0,1 ml per kg body weight.
Method of nicotine exposure. Nicotine powder ((-) Nicotine hydrogen tartrate salt) from Sigma-Aldrich, United Kingdom product, weighing 2.5 mg / kg then dissolved with 0.2 ml of sterile distilled water. After that, the liquid nicotine is taken with syringe and injected in the subcutan rabbits neck. Nicotine was injected every morning for 9 weeks, until the study ended.
Method and material processing work. The rabbits were killed by overdosed anesthetic agent (Ketamine). Then the mounted implant area made flap for measurements the value of osseointegration with Osstell tool. Lower jaw bone removed and cleaned from the soft tissue, and then implants with their jaws were fixed with formalin 10% for 1 day and formiat acid 5% for 14 days, followed by a series of bone processes of histological preparation. Number of osteoclasts and osteoblasts counted in preparation stained with Hematoxylen eosin (HE).
Finally, the osteoclast and osteoblast cells calculated by looking at 10 different fields of electron microscopic view with magnification 400x.
97 RESULTS
Data on the number of osteoclasts and osteoblasts derived from observations of the number of cells in the bone tissue around the implant with histological methods. The results of observations made in the control group and the treatment group who had been given nicotine exposure can be seen in Figure 1.
Description of HPA : Cells are marked with arrows osteoclasts and osteoblasts drop marked with arrows. A) The control group at week 1 (k1). B) treatment group were exposed to nicotine at week 1 (KN1). C) The control group 8 weeks (k8). D) were exposed to nicotine treatment group at week 8 (kn8). Nikon E 100 400x magnification.
Data were analyzed using SPSS version 13.0 for Windows. Statistical methods examination with 95% confidence intervals, used Kolmogorov-Smirnov normality test, Lavene test, MANOVA different test and Pearson correlation test. To determine differences between groups, statistical analysis was done by Tukey HSD test.
A B
C D
Fig 1. Osteoblast and osteoclast cells around the implant
98 Table 1. The value of osseointegration at week 1 and week 8.
Variable Time Group (n=8) Mean SD P Osseointegration Week 1 Control 51.38 0.744 0,997
Nicotine 51.13 0.991
Week 8 Control 65.63 2,134 0,000 Nicotine 52.13 2,031
Significance : p<0,05
Table 2. The number of osteoclasts and osteoblasts cells at week 1 and week 8
Variable
Time Group (n=8) Mean Number of
SD P
Osteoclast Week 1 Control 13.00 2,390 0,000 Nicotine 18.63 4.104
Week 8 Control 10.50 1,773 0000 Nicotine 29.63 2,875
Osteoblast Week 1 Control 21.13 2,360 0,000 Nicotine 10.50 1,770
Week 8 Control 27.88 2,950 0000 Nicotine 6.88 2,360
Significance: p<0,05
The statistical test showed no significant difference for osseointegration variables between study groups at week 1 (p = 0.997), whereas at week 8 significant difference (p = 0.000). For osteoclast variable, results of data analysis at week 1 shows, there is an increase in the average 13.00 to 18.63. Similarly, at week 8, where the mean of the control group and the treatment group 10.50 to 29.63. The statistical tests for variables significantly different between the two study arms osteoclasts (p = 0.000). Results of data analysis for osteoblast variable at week 1 shows, there is a mean decrease 21.13 to 10.50. Similarly, at week 8, where the mean of the control group and 27.88 in the treatment group decreased to 6.88. The statistical tests for variables significantly different between the two study arms osteoblasts (p
= 0.000).
DISCUSSION
Results of counting the number of osteoblasts in this study indicate that the number of osteoblasts in nicotine exposed group was lower than in the control group, both at the first
99 and eighth week. Thus proved that nicotine exposure inhibits osteoblast cell development and osteoblastogenesis.
In this study also proved, nicotine exposure accelerates osteoclastogenesis. Number of osteoclasts in the treatment group in this study, more than the control group, both in observations at the first week and at the eighth week (p = 0.000). The results support Henemyre7 which states, exposure to nicotine will increase the number of osteoclasts.
Nicotine may affect osteoclastogenesis through different ways and result in bone metabolism changing8.
Nicotine suppress osteoblast proliferation and inhibit the expression of osteogenic and angiogenic mediators. Nicotine causes iskhemia and directly affect osteoblast cell growth9. Nicotine affects the expression of osteogenic and angiogenic genes in rabbit bone regeneration. Nicotine also affects the further development of osteoclasts through indirect mechanisms, namely through osteoblasts6.
According to Pereira 10, exposure to nicotine negatively affects osteoblast activity.
Nicotine has the effect of stimulators and inhibitors of bone metabolism. The low concentrations of nicotine can stimulate osteoblast cell proliferation, increased expression of osteocalcin, type 1 collagen and alkaline phosphatase, but it will be the opposite effect in higher concentrations11.
Osteoblastic activity is influenced by the microenvironment of the cells, systemic factors and local factors. In this study, exposure to nicotine in the treatment group resulted in the microenvironment of the cells changed. With decreased osteocalcin, it can be said osteoblast cell activity also decreased due to the decreased number of osteoblasts. The results support previous research that states that high nicotine concentrations in smokers or low osteocalcin expression obtained.
In this study, the results of measurements of osseointegration at the first week in the control group and the treatment there was no significant difference (Table 1). This is due the first week of going soft callus formation and osseointegration has not happened yet. The average value of osseointegration in the control group was 51.38, while the treatment group was 51.13. This value has not shown the existence of osseointegration and not ready to be a burden.
At the eighth week measurements, lower value osseointegration was found in the treatment group was 52.13 while in the control group reached an average value of osseointegration 65.63 (Table 1). Cellular mechanisms are very sensitive to systemic and
100 local factors including nicotine. In this study proved that exposure to nicotine at a dose of 2.5 mg / day / kg body weight, inhibit osseointegration of dental implants in New Zealand rabbits.
Osseointegration can be found by laboratory examination and clinical examination. By laboratories, osseointegration can be seen with a microscope which saw new bone into the area of thread 5. Therefore, to see whether osseointegration has occurred in people, can only be done by clinical and radiological examination. How to measure clinically osseointegrated can use the torque strength implants look when played. When the torque of implant is 20N, mean there has been a move osseointegration1. However, this method can only estimate it based on perception. In addition done the torque will damage the bone healing process.
The right tool for measuring osseointegration is the ISQ Osstell 12. ISQ is a measurement scale that is used together with the method of RFA (resonance frequency analysis) to determine the stability of the implant. Osseointegration measurements needed to determine the readiness and the right time to receive an implant load. This tool uses the resonance frequency and is expressed on a scale of 1-100. Implant stabilization progress can also be monitored with ISQ. When from time to time ISQ values increased, meaning the process of osseointegration is underway. Conversely, if the value of ISQ fixed or declining, then the osseointegration process does not run as expected and will likely implant treatment failure. An implant is stable if the ISQ values above 55, and if the value reaches 70, then the implant well in osseointegration and ready to receive the load 12, 13, 14.
CLINICAL RECOMMENDATION
In this study it can be concluded that the implantation of dental implants in the study subjects were exposed to nicotine will cause delays the implant osseointegration because there are an increase the number of osteoclast, and decreased the number of osteoblast.
Research needs to be done to look for levels of concentrations of nicotine in humans that inhibition affect on osseointegration dental implant. Further research also needs to be done to explore the biomoleculer mechanisms of nicotine effects to osteoclast and osteoblast cells.
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