Ⅰ. INTRODUCTION
Osseointegration of dental implants, essential for long term-clinical success, involves the direct anchorage of the implant by the formation of bone without the growth of fibrous tissue at the bone-implant interface, and represents a dynamic process1-3) Although the higher success rate of titanium implants is reported, implant failure does occur as the result of multifactorial processes, and smoking is one of the factors often associated with higher implant failure rate4-11). The relationship between smoking and impaired wound healing in the oral cavity and the loss of endosseous implants was firstly reported12)in 1992. Tobacco smoking is associated with impaired alveolar bone healing after surgical treatment13), increased rate of bone loss and poor bone quality14-16), and higher incidence of peri- implantitis. Studies in animal models also showed a inhibitory influence of tobacco smoke on bone healing around implants17-19).
Nicotine as a major component of tobacco which is present in the plasma(4 to 73ng/ml), the saliva(96ng/ml to 1.6mg/ml) of tobacco smokers20-23), the crevicular fluid (5961ng/ml)24), can also be detected on the root surface of periodontally diseased teeth25). Nicotine administration appears to play a significant role in the negative effects of tobacco smoking in the bone regeneration26-28) because tissue ischemia due to peripheral vasoconstriction and decreased oxygen extension by nicotine reduce a potential osteoblast progenitor cell pool29), and contribute to impaired potentiality of bone tissue repair at implant interface.
Therefore, the purpose of this study was to evaluate the effect of nicotine on the proliferation and differentiation of normal human osteoblast from oral and maxillofacial area according to plasmatic and salivary levels of nicotine at the surface of screw-type plasma-sprayed titanium implants.
Ⅱ. MATERIALS and METHODS
A. Cell Culture
Commercially available screw-shaped titanium implants, 8mm in length, were used in this study with a titanium plasma spray(TPS) surface.
NHOst originated from oral and maxillofacial area were cultured in α-minimal essential medium(α-MEM) containing 10% FBS, 100IU/ml penicillin, 100mg/ml streptomycin, 2.5mg/ml fungizone and 50mg/ml ascorbic acid. Incubation was performed in a humidified atmosphere of 5% CO2 in air at 37°C, and the medium was changed twice a week. At 70-80% confluence, adherent cells were enzymatically detached(0.04% trypsin in 0.25% EDTA solution). 500ml of the cell suspension including a density of 104 cells/cm2 which was poured on the surface of each implant placed in 48-well culture plates pre-incubated with culture medium for 3hrs.
Upon seeding, cells were allowed to attach for 24hrs. At this stage, the culture medium was changed to a medium including β-glycerophosphate (5mM) and dexamethasone(100nM). Seeded implants were cultured during 21 days under the absence (control group) and under the presence of nicotine [(-)nicotine] [(-)-1-metyl-2-3-pyrrolidine] (Sigma N-3876, Sigma-Aldrich, Germany) 0.05, 0.1, 0.2, 0.3 and 0.5mg/ml (0.3-3mM). Cells were treated from 1 to 21 days with nicotine in the culture medium, which was renewed at each medium change three times a week. Control and nicotinetreated implants with attached cells were analyzed at 3, 7, 14 and 21 days for cell morphology, cell viability/ proliferation, alkaline phosphatase(ALP) activity, and ionized calcium(Cai) concentration for matrix mineralization.
B. Cell viability/proliferation, and ALP activity
For cell viability and proliferation of the seeded implants, the MTT assay for the cell viability was performed to oxidize MTT(3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-tetrazolium bromide) to an insoluble blue formazan product. Seeded implants were incubated with 0.5 mg/ml MTT for the last 4hrs under the culture period. After the implants were transferred to a new culture plate, the formed formazan salt was dissolved with dimethylsulphoxide, and the absorbance(A) was determined at 540nm, three times. Protein content was determined in cell lysates at 0.1M NaOH according to the method of Lowry using BSA as a standard.
ALP activity was assayed in cell lysates originated from the seeded implants treated with 0.1% triton by the hydrolyses of p-nitrophenol phosphate in alkaline buffer solution at pH 10.3 for 30min, and colorimetric determination of p-nitrophenol at 405nm, three times. Each ALP activity was expressed in nanomoles of p-nitrophenol produced per min/g of protein (nmol/min/μg protein).
C. Detection of Ionized Calcium(Cai) Concentration in the Culture Medium
The presence of calcium phosphate for matrix mineralization in the cell layer at the surface of implants could be inferred from the concentration of ionized calcium(Cai) in the culture medium during the experiment time.
Culture medium from control and nicotine-treated implants was collected, at each medium change, during the entire culture period, and analyzed Cai concentration(Sigma Diagnostic Kit(procedure N587), Germany), three times. Each Cai concentration was expressed in mmol/l of culture medium. Cai concentration in the culture medium originated from calcium phosphate compounds present in basal medium including osteogenic medium. Consumption of Cai from the medium reflected the formation of calcium phosphate deposits in the cell layer. Cai levels reflected cumulative changes at 3, 7, 14 and 21 days throughout the culture period, providing quantitative results regarding the rate of matrix mineral deposition in future.
Ⅲ. RESULTS
A. Cell morphology
Attached NHost spread on the implant surface and presented a polygonal shape with cytoplasmic processes attached to the surface irregular crevices. Proliferating NHost during the incubation time showed a compact cellular contact, and formed a flattened sheet attached to the surface of implant after 2-3 weeks. Continuous exposure to nicotine induced dose-dependent vacuolation of the cytoplasm. This effect was more pronounced under the culture condition at 0.05-0.1mg/ml nicotine, while it was transient exposed to 0.2-0.3mg/ml. The cells treated with 0.5mg/ml nicotine which became highly vacuolated, retracted, showed a tendency to detach from the implant surface and showed a dose- and time-dependent decrease in the number of attached cells. At higher concentrations, very few cells remained attached to the implant surface at 21 days.
B. Cell viability/proliferation and ALP activity
NHosts grown in control conditions proliferated gradually throughout the experiment time, with a higher proliferation rate during the first 3 weeks(Fig. 1). Exposure to 50ng/ml nicotine(plasma concentration) increased the MTT reduction values, with statistical significance at 21 days(Fig. 1). Exposure up to 0.2mg/ml(salivary concentration) caused a dose-dependent increase of MTT reduction, indicating stimulation on cell proliferation(Fig. 1). MTT values at 21 days were about 15%, 15% and 38% higher than those of control cultures in the seeded implants treated with 0.05, 0.1 and 0.2mg/ml nicotine, respectively(Fig. 1). A small inhibitory effect showed about 70% with 0.3mg/ml nicotine at 21 days, but in the last week MTT reduction was similar to those in control(Fig. 1). Higher nicotine levels(0.5mg/ml) caused a dose-dependent decreased cell growth and presented reductions of about 90% in the MTT assay as compared with control at 21 days(Fig. 1).
ALP activity measured in control was low during the first 2 weeks but increased significantly during the 3rd week, and maximum values were observed around 21 days(Fig. 2). The presence of 50ng/ml nicotine resulted in a small stimulation from the 2nd week onwards, although not statistically significant(Fig. 2). Dose-dependent induction in ALP activity during the 2nd and 3rd weeks in the cultures treated with 0.2mg/ml showed peak levels at 21 days, increased about 3 folds(Fig. 2). Exposure to 0.3mg/ml nicotine caused an inhibitory effect in ALP activity, reflected by slightly lower levels during the first 3 weeks, and were similar to those of control. Higher nicotine concentrations(0.5mg/ml) resulted in decreased ability to synthesize ALP(Fig. 2).
C. Detection of Ionized Calcium(Cai) Concentration in the Culture Medium
In control group, Cai levels in the medium began to decrease around 14 days(Fig. 3). Cultures exposed to 0.1 and 0.2mg/ml nicotine showed prominent calcium concentration at 21 days, while the presence of 0.3 mg/ml resulted in a slower rate of calcium concentration(Fig. 3). Cai levels treated at 0.5 mg/ml did not change during the culture period(Fig. 3).
Ⅳ. DISCUSSION
The success of dental implants depends on the efficient and stable interaction between bone cells and the biomaterial of implant surface. Although the relationship of smoking and increased rates of implant failures has been controversial, nicotine of tobacco compound itself is known to affect the intense vasoconstrictor action observed with low concentrations26), with evident direct and indirect inhibitory effects on bone metabolism27). The present work described the effect of nicotine on the behavior of NHost from oral and maxillofacial area28,29) cultured at the surface of plasma-sprayed titanium implants. The concentration range of nicotine used(50ng/ml-1mg/ml) in this study was representative of that reported in the plasma(4-73ng/ml) and saliva(96ng/ml-1.6mg/ml) of tobacco users20-23). This concentration range in the tobacco users took in account that both systemic and local(oral cavity) effects appeared to play a role in the osseointegration at the surface of dental implants6). NHosts cultured under the presence of ascorbic acid, β-glycerophosphate, and dexamethasone presented a gradual proliferation for 3 weeks followed by a senescence process, a significant increased ALP activity during 3 weeks, and the formation of calcium phosphate deposits intimately associated with NHost layer at late incubation times. This culture condition suggested that a progressive differentiation could represent for the establishment of normal osteoblastic phenotype30-33).
Nicotine appeared to affect NHost in a dose-dependent manner, that is, stimulatory effects in the cell proliferation and ALP activity associated with an earlier onset of matrix mineralization with levels up to 0.2mg/ml, and inhibitory effects at higher concentrations. Nicotine caused a dose-dependent vacuolation of the cytoplasm at concentrations higher than 0.2mg/ml in this stusy. The numerous vacuole of osteoblast from chick embryo calvariae34) was previously observed because nicotine were known to accumulate in lysosomes by membrane permeation and trapping by protonation35) followed by osmotic water enterance, inducing large vacuoles.
In this study, it suggested that the pattern of in vitro osteogenesis might be related with the dose-dependent effect of nicotine in cell proliferation and ALP activity. It is known that proliferation of osteoblastic cells is functionally related to the synthesis of an appropriate collagenous matrix which is essential to osteoblastic differentiation30), because an increased amount of the collagenous matrix is associated with a higher ALP production30,36). This enzyme plays an important role in the mineralization of the collagenous bone matrix by providing the increased local phosphate concentration with hydrolyzing β-glycerophosphate required for the initiation of hydroxyapatite nucleation and crystallization37-39).
A significant increased cell growth rate, an induced ALP activity and an increased matrix mineralization were seen at levels up to 0.2mg/ml. Exposure to 0.3mg/ml caused a slight initial inhibitory effect but NHosts were still able to recover, although with later ALP maximal levels and delayed mineralization. It suggested that ALP activity and matrix mineralization might appear to be closely related. SEM observation showed matrix mineralization as the fibrillar cell layer on implants in close association with the cell layer at 21 days, and X-ray microanalyses of the mineral deposits showed the presence of calcium and phosphorus peaks40).
A trend for an increase in the extent of mineralization was apparent in the presence of 0.05-0.2mg/ml. NHosts exposed to 0.3mg/ml showed delayed mineralization at 21 days, and cells treated with 0.4mg/ml presented no signs of mineralized structures40).
The onset of mineral deposition followed maximal ALP activity at 0.3mg/ml and higher ALP activity also showed an increased extent of matrix mineralization at 0.05-0.2mg/ml nicotine, as compared with control. 0.5mg/ml nicotine caused dose-dependent cytotoxic effects including decreased cell growth and cell contact, low ALP activity and the absence of mineral deposition, and detachment from the implant surface and cell death. In the present work, it suggested that the anticipation of mineral deposition by NHost resulting from exposure to 0.05-0.2mg/ml nicotine might be associated with the positive effect on cell proliferation with expected increase in the amount of the extracellular matrix produced. Cultured osteoblastic cells from embryonic chick calvariae exposed to 0.05-0.6mg/ml nicotine previously showed an increase in tritiated thymidine incorporation and a decrease in collagen synthesis and ALP activity34). ROB-C26 and MC3T3-E1 cells treated with 0.06-1.5mM nicotine responded with an increase in ALP activity and calcium deposition or a decrease in both parameters, respectively41). In present study, the behaviour of NHost seeded at the surface of TPS titanium implants was affected by the continuous exposure to nicotine, with inhibitory effects at levels 0.3mg/ml. Low levels(50ng/ml) as the nicotine plasma concentrations reported in smokers, did not cause significant effects in the cell behaviour, although with a trend for a small positive effect. Salivary levels up to 0.2mg/ml could promote the growth and differentiation of NHosts.
In an attempt to explain the mechanism involved in the cell response to nicotine, at 0.1-1.0M levels demonstrated the presence of the nicotinic receptor subunit a4, and Dtubocurarine(a nicotinic receptor antagonist) inhibited nicotine-induced cell proliferation42). It was speculated that changes in the intracellular concentration of Ca+2 ions, following binding of nicotine to its receptor may modulate the effects of nicotine on bone metabolism42), and increased ALP activity of mouse osteoblastic cells43). Also it is known that osteoblastic cells respond to calcium in a time and concentration manner, regarding proliferation and expression of several markers of osteoblastic cells43,44). These observations suggested that NHost response to nicotine might be mediated by changes in cellular levels of calcium ion. Although the effect of nicotine on the osseointegration of titanium implants in animal models did not find significant effects in bone healing around the implants in the conditions used17,20,21), some clinical studies strongly suggested that local exposure of the peri-implant tissues to tobacco smoking be the main factor leading to an overall greater failure rate in smokers17,45). Considering the high tissue diffusion potential of nicotine with a small and lipophilic molecule29) and the permeability features of the gingival implant epithelium7,46,47), higher levels in saliva of tobacco users could play a role in a direct modulation of osteoblast activity as a contributing factor to the effect of nicotine on the bone formation at the surface of dental implants. In conclusion, nicotine caused a dose-dependent effect on Cai concentration for matrix mineralization by NHost cultured at the surface of plasma-sprayed titanium implants, reflected by an earlier onset of cell mediated calcium phosphate deposition at 0.1-0.2mg/ml, and delayed and absence of mineral deposition at higher concentrations.
