| Abstract|| |
Aim: To evaluate the biodegradability of preformed stainless steel crowns at varying salivary pH and the cytotoxic effect of leached out elements on fibroblasts. Methodology: A total of 243 stainless steel crowns were selected and were divided into 3 groups (I, II, III) based on Ph of immersion media. The pH of samples in group I, II, III were 4.3, 5.5 and 6.3 with 81 crowns in each group. Each group has 9 samples with 8 crowns in each sample. All samples were immersed in polyethylene bottles containing 10ml of artificial saliva and incubated at 37°C for 4 weeks. All the samples were analyzed on 1,7,14 and 21 days by atomic absorption spectrophotometer for the quantitative assement of Ni, Cr and Fe. Fibroblast tissue culture was used to assess the cytotoxicity of the samples. Statistical Analysis: Analysis of variance. Results: Maximum release of Ni, Cr, Fe ions were observed at pH 4.3 followed by pH 5.5 and least release of ions were observed at pH 6.3 from SS crowns. The cytotoxic results showed that the least cell viability of cells was seen at pH 4.3. Conclusion: With decrease in pH, there is an increase in ion release from stainless steel crowns and the mean release of nickel, chromium and iron were very much below the average dietary intake. But the allergic manifestations of ions like nickel can't be ruled out.
Keywords: Atomic absorption spectrophotometer, biodegradability, chromium, iron, nickel, stainless steel crowns
|How to cite this article:|
Anusha K, Sridevi E, Sai Sankar A J, Sridhar M, Sankar K S, Chowdary K H. An In Vitro evaluation of biodegradability of stainless steel crowns at various salivary pH. Indian J Dent Res 2020;31:569-73
|How to cite this URL:|
Anusha K, Sridevi E, Sai Sankar A J, Sridhar M, Sankar K S, Chowdary K H. An In Vitro evaluation of biodegradability of stainless steel crowns at various salivary pH. Indian J Dent Res [serial online] 2020 [cited 2022 Jan 22];31:569-73. Available from: https://www.ijdr.in/text.asp?2020/31/4/569/298417
| Introduction|| |
Prefabricated stainless steel crowns (SSCs) are one of the most common dental devices used to restore primary molars which are severely damaged or endodontically treated and as an abutment tooth for a space maintainer. The use of preformed crowns for primary tooth restoration reduces treatment time and number of visits to clinics. The chemical composition is 65–73% iron, 17–20% chromium, 8–13% nickel and less than 2% manganese, silicon and carbon. The frequent use of preformed crowns has led to concerns that heavy metals in the crowns could be released into the oral cavity and accumulate in the body.
Oral release of dental metals is caused by mechanical stimulation due to abrasion and by chemical and thermal stimulation from eating and drinking. Dental metals were found to be cytotoxic to DNA and cultured cells, although the amounts of such metals released into the oral cavity were not harmful to human health. However, systemic accumulation of heavy metals could trigger allergies.
The major corrosion products of stainless steel are iron (Fe), chromium (Cr), and nickel (Ni). Although all three elements potentially have adverse effects, Ni and Cr have received the most attention because of their reported potential for producing allergic, toxic, or carcinogenic reactions. Approximately 10% of the general population exhibit hypersensitivity to Ni, with females being reported to be 10 times more sensitive than males presumably because of sensitization from nickel-containing jewelry. In pediatric patients, the prevalence of sensitization is around 15–16%.
It has been well established that these metals possess the propensity to produce hypersensitivity, dermatitis, asthma, and ulcers of the oral mucous membrane. In addition, a significant carcinogenic and mutagenic potential have been demonstrated for the compounds of these metals. Metals leaching out of crowns can cause toxicity reactions if they exceed the maximum recommended daily intake levels.
Hence, the purpose of the present study was to assess the leach out of Ni, Cr, and Fe at varying salivary pH on 1, 7, 14, and 21 days and to assess the cytotoxicity of these leached out elements on cultured fibroblasts (L929 cells).
| Methodology|| |
A total of 216 stainless steel crowns were selected and divided into three groups (I, II, III) based on pH of immersion media with 72 crowns in each group. The pH of samples in group I, II, and III were 4.3, 5.5, and 6.3, respectively. Each group has 9 samples with 8 crowns in each sample. All the samples were immersed in polyethylene bottles containing 10 ml of artificial saliva and incubated at 37°C for 4 weeks.
Artificial saliva was prepared by dissolving 0.8 g of NaCl (Sodium chloride), 2.4 g of KCl (Potassium chloride), 1.5 g NaH2 PO4 2H20 (Sodium dihydrogen phosphate dihydrate), 0.1 g Na2S, 9H20 (Sodium sulphide), 2 g CO (NH2)2(Urea) in 2000 ml of distilled deionized water. The pH of the artificial saliva was measured by pH meter, with the addition of an acid/and base. The pH was adjusted to obtain the different pH ranges designated for the study.
The samples were placed in the solution on day 0. After day 1 and every 7 days, they were taken out from the solution and placed in another container with fresh artificial saliva in order to avoid saturation of solution with released ions. Following immersion the samples were shaken gently to ensure bathing of all crowns in saliva and to obtain a uniform solution.
The amounts of released elements were measured on days 1, 7, 14, and 21 by Atomic absorption spectrophotomer (AA- 6300, Shimadzu, Kyoto, Japan).
Cell culture and MTT assay
The fibroblast (L929) were plated separately using 96-well plates with the concentration of 1 × 104 cells/well in Dulbecco's Modified Eagle's medium (DMEM) with 1 × Antibiotic-Antimycotic Solution and 10% fetal bovine serum (Himedia, India) in a CO2 incubator at 37°C with 5% CO2. The cells were washed with 200 μL of 1 × Phosphate-buffered saline (PBS), then the cells were treated with various test concentration of IB, IIB, IIIB samples on the 1st and 7th day in serum-free media and incubated for 24 hrs. The medium was aspirated from cells at the end of the treatment period. 0.5 mg/mL MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) prepared in 1 × PBS was added and incubated at 37°C for 4 hrs using CO2 incubator. After the incubation period, the medium containing MTT was discarded from the cells and washed using 200 μL of PBS. The formed crystals were dissolved with 100 μL of Dimethyl sulfoxide (DMSO) and thoroughly mixed. The development of color intensity was evaluated at 570 nm. The formazan dye turns to purple-blue color. The absorbance was measured at 570 nm using a microplate reader.
| Results|| |
The results depicted that maximum release of Ni was observed from group I samples on all the experimental day's. The values were 0.272 ppm, 0.330 ppm, 0.218 ppm, 0.185 ppm on 1, 7, 14 and 21 days, respectively. By comparing the release of Ni on all days at three designated pH, the maximum leach out was seen on the 7th day with 0.330 ppm, 0.264 ppm, 0.243 ppm and there was a gradual declination to 21st day with values being 0.185 ppm, 0.114 ppm, 0.100 ppm at 4.3, 5.5, and 6.3 pH, respectively [Graph 1].
The maximum release of Fe was observed at pH 4.3 with 0.673 ppm, 0.650 ppm, 0.563 ppm, 0.516 ppm on 1, 7, 14, and 21 days, respectively. By comparing the release of Fe on all days at 3 selected pH, the maximum leach out was seen on 1st day with values 0.673 ppm, 0.509 ppm, 0.425 ppm and there was a gradual declination to 21st day with 0.516 ppm, 0.414 ppm, 0.375 ppm at 4.3, 5.5, and 6.3 pH, respectively [Graph 2].
In case of Cr, maximum release was observed at pH 4.3 with 0.476 ppm and 0.173 ppm on 1st and 7th day, respectively. There was a maximum leach out of Cr on 1st day with 0.476 ppm, 0.43 ppm and 0.003 ppm at 4.3, 5.5, and 6.3 pH, respectively. By the 7th day the leach out had declined to 0.173 ppm at 4.3 pH -0.13 ppm at 5.5 and -0.0259 ppm at 6.3 pH, respectively [Graph 3].
Evaluation of mean cell viability of cultured fibroblasts on day 1 at 4.3, 5.5 and 6.3 pH is 80.8, 84.5, 88.7, respectively and on day 7 is 66.6, 94.2, 109.9, respectively. The values do not represent any significant alteration in the viability of cells; however, least cell viability was observed at pH 4.3 on both 1st and 7th days [Table 1].
|Table 1: Mean cell viability of L929 cells at 4.3,5.5,6.3 pH on 1st and 7th day|
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Few numbers of fibroblasts were seen on day 7 at pH 4.3 using Phase contrast microscopy magnification 100x with reflected light fluorescence system at different pH on 1st and 7th day [Figure 1].
|Figure 1: Phase contrast microscopy magnification 100 × with reflected light fluorescence system of fibroblasts at different pH on 1st and 7th day|
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| Discussion|| |
In pediatric dentistry, the stainless steel crowns are widely used which are made of a base metal alloy mainly containing nickel, chromium and iron as main constituents. The oral environment is particularly an ideal climate for the biodegradation because of its microbiologic and enzymatic phenomena. The general mechanism for the corrosion and subsequent release of metal ions from stainless steel involves loss of the passivating layer of chromium oxide and chromium hydroxide that forms on the surface upon contact with oxygen.
Nickel is a strong sensitizer and one of the most common cause for contact allergies and claimed to have carcinogenic and mutagenic effects. The ability of nickel to induce allergic reaction has been contributed to the high haptenic capacity of released metal. Allergic response to nickel-containing alloys is mainly type 4 hypersensitivity reaction. It has been suggested that long-term exposure to nickel-containing dental materials may adversely affect both human monocytes and oral mucosal cells. In addition to nickel, chromium and cobalt ions also cause hypersensitivity and dermatitis. These metals can also induce cytotoxicity and genotoxicity. WHO (1988) and WHO (1991) stated that 0.2 mg/kg body weight of nickel and 50 mg/kg body weight of chromium can cause systemic manifestations.
Hence, the purpose of the present study was to assess the leach out of Ni, Cr, and Fe which are the main components of preformed stainless steel crowns used in restoring the primary teeth at varying salivary pH and to assess whether the corrosion products are within the acceptable limits.
In the oral cavity, the temperature and pH variations caused by diet, decomposition of foods, cell debris, oral microflora and their by-products are the important factors to be considered while evaluating the clinical behavior of orthodontic components that remain in the oral cavity for months or years. So in the present study three pH variants - 4.3 was considered to simulate a condition that can occur when people feed on acidic foods or drinks; 5.5 is the critical pH and 6.3 is the normal pH of human saliva. In each sample eight crowns were taken, considering the fact that a maximum of eight crowns can only be placed excluding anterior teeth.
Corrosion is measured in a number of ways such as electro-chemical tests that measure elemental release indirectly through the flow of the released electrons current or by tests that measure of the elements directly by spectroscopic methods. Perhaps the most relevant measure of corrosion from the standpoint of biocompatibility is identifying and quantifying the elements that are released. Thus, atomic absorption spectrophotometer was used in the study to measure the leach out of the elements.
In the present study, maximum release of Ni, Cr, Fe ions was observed at pH 4.3 followed by pH 5.5 and the least release of ions were observed at pH 6.3. This observation illustrate that as the pH of saliva increases, the leach out of ions from the stainless steel crowns decreases. Similar findings were observed in the studies conducted by Menek et al. (2012) and Tiwari et al. (2016). Huang et al. (2004) observed elevated levels of nickel released from stainless steel brackets at lower pH. The reason attributed to this could be that acidic conditions provide a reducing environment in which the stainless steel oxide film required for corrosion resistance is less stable.
When the concentrations of nickel were measured at the various time intervals, peak level was noticed on day 7 and the concentration levels showed a progressive decline on day 14 and 21. Park and Shearer (1983) and Menne et al. (1987) also found that the corrosion of the appliances reached a plateau after 6 days and did not increase appreciably thereafter. Two explanations for this behavior can be contemplated. First, the nickel present on the surface of the stainless steel may quickly corrode during the first 7 days of the experiment. Subsequently, the rate of release falls as the surface nickel is depleted. Second, the corrosive products formed on the surface after 7 days slows down the corrosion of nickel. When the overall findings including those of chromium levels are taken into consideration, the first hypothesis is the more appropriate.
The release of Cr and Fe ions in the present study are same as the study conducted by Matos de Souza (2008) where they assessed the invivo release of Ni, Cr, Fe ions into saliva by three commercial metallic brackets and the results showed that both Cr and Fe levels were maximum on the 1st day and declined gradually. In contrast to these results Barret et al. (1993) demonstrated increased release of Cr during the first two weeks and levelled off in the subsequent weeks inIn Vitro conditions. This difference in the leach out of ions could be due to methodological differences such as storage medium, sample size and the study variables.
The amount of Ni, Cr, and Fe released in all the test solutions were below the critical level so its systemic toxic effects are so improbable. However, even this low amount of Ni has the ability to induce allergic reactions. For the occurrence of allergic reaction in the mucosa the antigen should be 5-12 times stronger than what is required to create an allergic reaction on the skin. This amount of Ni can be enough to induce an allergic reaction due to high haptenic capacity of the released metal.
When a patient is suspected of allergy, a thorough history taking and clinical examination should be done. Prick test and scratch test are used to confirm immediate hypersensitivity while patch test confirms delayed hypersensitivity and MELISA (memory lymphocyte immuno-stimulation assay test) is used to measure the sensitization induced by metals.
To evaluate the cytoxicity of the leached out elements, cultured fibroblasts (L929 cells) were used. MTT, an enzyme-based method which relies on a reductive coloring reagent and dehydrogenase in a viable cell was used to determine the cell viability with a colorimetric method. The MTT assay determines the functional state of mitochondria that indicates cell viability. A mitochondrial dehydrogenase enzyme in living cells reduces the yellow tetrazolium salt MTT to blue MTT formazan, which is precipitated in uninjured cells. The MTT assay is frequently used to evaluate the biocompatibility of dental materials.
The mean cell viability of L929 cells on day 1 at 4.3, 5.5, and 6.3 pH were 80.8, 84.5, 88.7, and on day 7 it was 66.6, 94.2, 109.9, respectively. This represents that there is no significant alteration in the cell viability however the least cell viability was seen at pH 4.3 on both 1st and 7th days which could be due to the maximum release of ions.
The results of thisIn Vitro study are limited and extrapolation to the clinical situation is difficult because the methodologies used are unable to reproduce precisely the highly complex and dynamic oral environment. The interaction between the characteristics of the human saliva, alterations of pH due to food variety, bacterial colonization and its by-products make the oral cavity an extremely favorable environment to corrosive process and is difficult to reproduce in laboratory conditions. Moreover, an important factor in metal corrosion is the flow rate of saliva. Most of the studies in the literature used static conditions, but more metal release could be observed in real life because of the composition and fluidity of the saliva and also because oxide layers are removed by tooth brushing.
| Conclusion|| |
- Initial leach out of the tested elements was noticed at different salivary pH.
- The mean release of Ni, Cr and Fe were very much below the average dietary intake (200–300 μg/day; 50–200 μg/day and 18 mg, respectively) and are not capable of causing any toxic effects.
- pH of saliva and exposure period is indirectly proportionate to the leachability of the ions.
Even though the ion leach out is negligible, there is possibility of allergic reactions in few cases so it is upto the clinician to explore alternative viable options.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. A J Sai Sankar
Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh
Source of Support: None, Conflict of Interest: None