|Year : 2014 | Volume
| Issue : 4 | Page : 279-285
Association of physical properties and maintenance of sterility of primary teeth in human tooth bank
Nitika Bajaj1, Navneet Grewal2, Prashant Monga3, Sukreet Grewal2
1 Department of Pedodontics and Preventive Dentistry, Genesis Institute of Dental Sciences and Research, Ferozepur, Punjab, India
2 Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College and Hospital, Amritsar, Punjab, India
3 Department of Conservative Dentistry and Endodontics, Genesis Institute of Dental Sciences and Research, Ferozepur, Punjab, India
|Date of Web Publication||17-Sep-2014|
Departments of Pedodontics and Preventive Dentistry, Genesis Institute of Dental Sciences and Research, Ferozepur, Punjab
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: This study was carried out to evaluate the sterility and structural integrity of stored primary teeth in artificial saliva over a storage period of 12 weeks (3 months) in human tooth bank (HTB). Materials and Methods: A total of 80 freshly extracted teeth were taken and were categorized into four groups of 20 each. The samples were stored at 4°C for 12 weeks (3 months) in the refrigerator. During their storage time of 3 months, they were repetitively evaluated for their sterility, enamel hardness, calcium and phosphate solubility and color stability at regular intervals of 3 weeks. Observations and Results: That teeth stored in artificial saliva maintained their sterility throughout the storage time of 12 weeks, but changes in their physical and chemical properties occurred with an increase in storage time. Conclusion: Storage time not more than 9 weeks is recommended for deciduous teeth to be stored in artificial saliva in a HTB.
Keywords: Artificial saliva, biological restorations, human tooth bank
|How to cite this article:|
Bajaj N, Grewal N, Monga P, Grewal S. Association of physical properties and maintenance of sterility of primary teeth in human tooth bank
. J Indian Soc Pedod Prev Dent 2014;32:279-85
|How to cite this URL:|
Bajaj N, Grewal N, Monga P, Grewal S. Association of physical properties and maintenance of sterility of primary teeth in human tooth bank
. J Indian Soc Pedod Prev Dent [serial online] 2014 [cited 2020 Jul 13];32:279-85. Available from: http://www.jisppd.com/text.asp?2014/32/4/279/140939
| Introduction|| |
Complete oral rehabilitation of children affected by early childhood caries has always been a daunting task for a pediatric dentist. For centuries, scientists have been searching for an ideal restorative material and in this pursuit dental material science has introduced a large diversity of materials into the market. However, no material has yet been found to match the properties of natural tooth tissue in all its biomechanical aspects.  Use of human teeth for therapeutic purposes is a practice that has been employed since ancient times.  To enable a large-scale use of this precious tissue, there is an increasing interest in the preservation of teeth and the concept of "tooth bank."
An important aspect of a human tooth bank (HTB) is, storing the teeth in appropriate medium and conditions so as to maintain the sterilization and structural integrity. Tooth Bank was started in 2004 and established in 2006 in the Department of Pedodontia and Preventive Dentistry, Punjab Government Dental College and Hospital, Amritsar where research was carried out on permanent teeth stored in Hanks Balanced Salt Solution (HBSS) for up to 1 year to see the effect of storage on the structural integrity and sterilization.  As was observed in that study, there was a quantitative loss of calcium from the enamel of stored teeth in HBSS after 3 months, thereby making it a prerogative to try out an alternative storage media that could better simulate the oral environment.
The present study was carried out to evaluate the following parameters of stored primary teeth in artificial saliva in a HTB till they show significant changes:
- Sterility status.
- Enamel hardness.
- Calcium and phosphorous solubility of enamel.
- Color stability.
- All the above parameters after autoclaving.
| Materials and Methods|| |
About 80 freshly extracted retained or indicated for extraction therapy primary teeth were collected. The teeth selected were:
- Free from caries.
- Free from any morphological defects.
- Free from any discolorations.
- Without any restorations.
Each tooth donor was asked to provide confidential histories by filling a proforma kept in the tooth bank record along with the duly signed consent form [Annexure 1 and [Figure 1]. Teeth were kept in storage following the protocol of the tooth bank vis-a-vis. scrupulous cleaning, scaling and pulp extirpation. The prepared samples were then placed in artificial saliva until subjected to the initial cycle of cleaning and sterilization in an ultrasonic cleaner.
Samples were then categorized into the following groups for their analysis at successive 3 weeks interval. Twenty teeth for each group were selected to be analyzed for the effect of storage on:
- Group I: Preservation of sterility of teeth
- Group II: Enamel hardness
- Group III: Enamel solubility by determining the amount of soluble calcium and phosphorous
- Group IV: Color stability of teeth using Vita 3D (Vita zahnfabrik, H. Router GmbH & co. KG, D79713 Bad Sackingen, Germany) master shade guide.
The samples were put in autoclaved culture tubes containing 5 ml of 6% (w/v) H 2 O 2 . Tubes were then placed in an ultrasonic cleaner filled with water so that the tubes were fully immersed. Each of the sample was sonicated for 30 min (5 working cycles of 6 min) at 42 GHz and 100 W output [Figure 2]. Artificial saliva used had 0.4% carboxymethylcellulose, 6% sorbitol, 0.062% KCl, 0.085% NaCl, 0.005% MgCl 2 , 0.016% CaCl 2 , 0.08% K 2 HPO 4 , 0.2% nipagin and distilled deionized water. ,
|Figure 2: Tooth subjected to the high-powered digital ultrasonic sterilization at frequency 42 GHz at 5-working cycles|
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Group I: Twenty samples (microbial examination)
The samples were analyzed for the presence of any aerobic, anaerobic and fungal growth. The cultures for aerobic and facultative anaerobes were MacConkey's agar and blood agar medium [Figure 3]. For the anaerobic examination, a loopful from each sample was incubated on brain heart infusion agar in an anaerobic jar for 48 h. The culture for fungal growth was made on Sabourad's dextrose medium. A loopful of each sample was taken and was inoculated on the Sabourad's dextrose with antibiotics and was incubated at 25°C and 37°C. The growth was observed daily. It was incubated for 1 week before declaring it negative. Above mentioned procedure was followed each time successively after 3 weeks interval up to throughout its storage of 12 weeks.
|Figure 3: Cultures for aerobic and facultative anaerobes made on MacConkey's agar and blood agar medium|
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Group II: Twenty samples (enamel hardness)
Each sample was prepared, in a way, so as to expose only a small flat enamel surface [Figure 4]. The hardness of enamel was tested with a Knoop indenter with rhomboid base pyramidal diamond point to a constant load of 100 g. The above mentioned procedure was followed each time successively after 3 weeks interval up to 12 weeks of its storage.
Group III: Twenty samples (calcium and phosphorous solubility)
Each sample was prepared to expose only a small window of enamel (3 mm diameter) [Figure 5]. It was then subjected to 30 min. decalcification in 10 ml of 0.2 N acetic acid adjusted at pH 4 by 1N-NaOH. Later 1-2 ml of the solution was pipetted out in small tubes to be placed in autoanalyzer. The amount of soluble calcium and phosphate in acid solution indicated enamel solubility and was determined quantitatively by Roche automated clinical chemistry analyzer. After measuring the enamel solubility, the area of decalcification was painted, so that new area could be exposed at the next evaluation. The above mentioned procedure was followed each time successively after 3 weeks interval up to 12 weeks of its storage.
|Figure 5: Sample prepared for testing calcium and phosphorous solubility of enamel|
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Group IV: Twenty samples (color stability)
Visual inspection of each sample was made, and its corresponding shade from the shade guide was selected. Color matching was done in a natural daylight keeping the shade guide and the sample parallel to each other at eye level. A double-blind evaluation by two additional operators was carried out to rule out the interexaminer variation in color match. Evaluation of color stability by visual inspection using modified Cvar/Ryge criteria were also done for the clinical applicability of color of the teeth stored in HTB.
Rating used was:
(A): No shade mismatch in room light in 3-4 s
Perceptible mismatch but clinically acceptable
Esthetically unacceptable (clinically unacceptable).
Samples were stored at 4°C and during their storage time they were repetitively evaluated for the various parameters at 3 weeks successive intervals.
At the end of the study, all the group samples were subjected to autoclaving to determine its effect on the stored samples. Data were obtained was compiled and put to statistical evaluation.
| Results|| |
Group I: Twenty teeth microbial growth
No microbial growth (aerobic, anaerobic, and fungal) was noticed until 6 weeks of storage. Only 5% (1 out of 20) samples showed aerobic growth after 6 weeks which was statistically insignificant. However, 95% of the samples maintained their sterility till the end of 12 weeks storage. About 100% sterility was observed after autoclaving at the end of 12 weeks.
Groups II and III: Twenty teeth each enamel hardness and enamel solubility (calcium and phosphorous solubility)
Mean of all the readings at consecutive 3 weeks intervals were calculated till significant results obtained and at the end of 12 weeks of storage samples were subjected to autoclaving to determine its effect on enamel hardness. Various statistical measures such as standard deviation and coefficient of variance in percentage were computed. Besides this confidence intervals at 99% were also computed and tabulated [Table 1] and [Table 2].
|Table 1: Basic statistic values for enamel hardness (KHN), calcium and phosphorous solubility at successive 3 weeks intervals and after autoclaving|
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|Table 2: Level of signifi cance of enamel hardness, calcium and phosphorous solubility using paired t-test|
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Group IV: Twenty teeth color stability
It was observed that all the teeth selected for storage in tooth bank after undergoing sonication procedure showed a marked change of color to lighter bleaching shades as compared with Vita 3D master bleaching shade guide. However, after placement of sonicated teeth in artificial saliva the shade remained same for 65% of samples over 12 weeks storage with only slight discoloration observed in 35% of samples which was of not much significance. After autoclaving, teeth did show minor color changes but again were of not much significance.
According to the modified Cvar/Ryge criteria 40% of the stored teeth in HTB were rated as B, that is, perceptible mismatch, but clinically acceptable and 60% as C, that is, clinically unacceptable. Further after autoclaving when the color was examined according to modified Cvar and Ryge criteria, 55% category was seen under C = Charlie and 45% under B = Bravo when compared to 60% and 40% before autoclaving.
| Discussion|| |
Primary teeth with extensive carious lesions are routinely observed in clinical practice and should be properly restored to re-establish their anatomy and hence their masticatory, phonetic, esthetic and space-maintainer functions in the dental arches. Over the years, clinicians have tried various procedures to restore mutilated primary teeth. Dentists and material scientists have long been searching for the "holy grail" of restorative dentistry, a material that is biocompatible, esthetically matches tooth structure and exhibits properties similar to those of tooth enamel and dentin. 
However, until date no material has been found to match the properties in all its biomechanical aspects.  Natural enamel and dentin are used as the "gold standard" in search for an ideal restorative material. Hence, why not use the "gold standard" itself that is, a natural enamel and dentin? To enable a large-scale use of this precious tissue, there is an increasing interest in the preservation of teeth and the concept of "tooth bank."
A HTB was set up in the Department of Pedodontia and Preventive Dentistry in 2006 as a nonprofit institution for research purposes. However, the storage medium used was HBSS as suggested by Habelitz et al. which does not simulate the oral environment. 
Hence, the present study was undertaken to evaluate the sterility status, calcium and phosphate solubility of enamel, hardness and color stability of stored deciduous teeth in artificial saliva. Artificial saliva was selected as a storage medium to simulate the oral environment. Application of artificial saliva for various in-vitro studies of dental materials was reported as early as in 1931, but little research has been done on determining the stability of sound tooth tissue stored in it.  For storing teeth in tooth bank, it is mandatory to clean and sterilize them, as freshly extracted teeth are rapidly contaminated by several bacteria species. In the present study, sonication in 6% H 2 O 2 w/v was carried out and teeth were subjected to sterility check after storage in artificial saliva over successive 3 weeks intervals. Nineteen samples out of 20 remained sterile, whereas one sample showed microbial growth after 9 weeks interval. This could be attributed to mishandling of the samples during transfer to the media. Enamel hardness of human teeth has been determined by variety of methods, including abrasion, scratch and indentation techniques.  Methods using a micro-scratch or micro-indentation have been preferred, and Knoop diamond indenter is commonly used and recommended. ,, In the present study Knoop Hardness Number was used as criteria for testing enamel hardness at various intervals of time, and a significant change in hardness was observed between 9 and 12 weeks of storage. Mechanical properties of calcified tissues are related to their mineral content.  A wide range of techniques have been used to measure the change in mineral status of dental enamel. Among these are the chemical analysis of calcium and phosphorous appearing in a buffer solution during dissolution, surface profilometry, scanning electron microscopy, polarized light microscopy, microradiography and atomic force microscopy.  Application of most of these methods requires specific preparation of tooth samples such that they cannot be used in successive experiments.  The present study made use of chemical analysis of calcium and phosphorous in acetic acid buffer solution (pH 4) as this was best suited for successive evaluation of the same sample.
Attempts have been made to show the correlation, between physical and chemical properties of enamel. An inverse relationship between hardness and calcium and phosphate solubility of enamel was found. An indication of parallelism between mineral loss and decrease in hardness had also been previously reported by Koulourides and Reed.,
Shades of all the samples subjected to initial sterilization cycle became whiter than the original shades and matched to bleaching shades of the Vita 3D Master shade guide. It was attributed to 6% w/v H 2 O 2 in which the teeth were immersed for 30 min during sonication cycle. This observation suggested that the original shade of a tooth selected for storage in HTB does not remain the same after undergoing sonication. Storage and autoclaving did show minimal effect on the shade of teeth but were of not much significance.
Grewal and Seth  observed that when teeth taken from HTB were clinically used as biological restorations there was an initial color mismatch with the adjacent teeth. This mismatch however no longer persisted when exposed to exogenous stains in the oral cavity, and 87% of restorations matched well after re-evaluating at 3 months.
American Dental Association and Centers of Disease Control and prevention suggest autoclaving as the best sterilization method for materials exposed to body fluids.  Stored teeth though maintained their sterility during their storage time but still before using them as biological restoration it is recommended they be autoclaved. However, evidence-based literature exists to show that changes in enamel calcium and phosphate content affect its hardness. Hence, it was important to evaluate the effect of autoclaving on the tooth structure after having stored it in artificial saliva.
Stored samples were subjected to autoclaving for 40 min at 240°F and 20 psi pressure as recommended by Dominici et al.  A significant reduction in micro hardness and an increase in the calcium, phosphorous solubility of enamel occurred after autoclaving of the stored teeth in a tooth bank.
| Conclusion|| |
Due to changes in the physical and chemical properties of primary teeth stored in HTB, storage time of not more than 9 weeks is recommended for primary teeth to be stored in artificial saliva in an HTB.
A highly significant decrease in enamel hardness is observed when these teeth are subjected to autoclaving thus increasing their brittleness which does not allow them to be used for their clinical use. Hence further research could throw light on the effects of autoclaving teeth stored for a lesser time in HTB.
Moreover, shade matching is an important criterion which was highly variable before and after sonication. Hence, a longitudinal study on the color stability of HTB stored teeth when used clinically in-vivo is suggested.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]