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 Table of Contents  
ORIGINAL RESEARCH
Year : 2020  |  Volume : 12  |  Issue : 7  |  Page : 13-18

Comparative evaluation of the effect of artificial aging on the marginal leakage of cast crowns luted with three cements: An in vitro study


1 Zayed Military Hospital, Abu Dhabi, United Arab Emirates
2 Department of Prosthodontics and Crown and Bridge, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India

Date of Web Publication17-Jan-2020

Correspondence Address:
Dr. Mahesh Mundathaje
Associate Professor Department of Prosthodontics and Crown and Bridge, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Light House Hill Road, Mangaluru 575001, Karnataka.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_96_19

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  Abstract 

Aims and Objectives: This study was carried out to evaluate the effect of artificial aging on the marginal leakage of crowns and the credibility of three different luting cements in preventing marginal percolation. Materials and Methods: Thirty-six extracted intact premolars were selected for the study. Tooth preparation was carried out to receive complete cast crown. Castings were microblasted with 50 μm alumina powder. Group I was cemented with zinc polycarboxylate (Poly-F® Dentsply De Trey GmbH, Konstanz, Germany), Group II with glass ionomer (Fuji I, Fuji I GC Corporation, Tokyo, Japan), and Group III with resin-modified glass ionomer (Fuji II Plus, Fuji II Plus GC Corporation, Tokyo, Japan). The specimens in the subgroup I served as the control group and those in the subgroup II were subjected to thermocycling to simulate the intraoral conditions. They were artificially aged using artificial saliva as a medium of thermocycling, with a temperature variation of 5°C–55°C. All specimens were treated with 0.5% basic fuchsin dye for 24h. The crowns were then embedded in clear self-cure acrylic resin and sliced buccolingually. The sectioned halves were then placed under the optical vision microscope, and the depth of dye penetration in the proximal region was qualitatively evaluated. Results: The results showed the mean marginal leakage score is highly significant in thermocycled group as compared to that in non-thermocycled group. The results were subjected to one-way analysis of variance and post hoc Tukey test. It was found that artificial aging did have an effect on the marginal leakage of zinc polycarboxylate group and glass ionomer group. The resin-modified glass ionomer group, however, did not show much significant difference. Conclusion: The artificial aging did have an effect on the marginal leakage of polycarboxylate group and glass ionomer group, whereas resin-modified glass ionomer group did not show much statistical significance. Zinc polycarboxylate cement had maximum micro-leakage followed by glass ionomer cement. Resin-modified glass ionomer showed minimum leakage and can be regarded as the best cement for clinical use.

Keywords: Cast Crowns, Luting Cement, Microleakage, Thermocycling


How to cite this article:
Mathew VP, Mundathaje M, Rodrigues SJ, Shetty TB, Pai UY, Saldanha S. Comparative evaluation of the effect of artificial aging on the marginal leakage of cast crowns luted with three cements: An in vitro study. J Int Oral Health 2020;12, Suppl S1:13-8

How to cite this URL:
Mathew VP, Mundathaje M, Rodrigues SJ, Shetty TB, Pai UY, Saldanha S. Comparative evaluation of the effect of artificial aging on the marginal leakage of cast crowns luted with three cements: An in vitro study. J Int Oral Health [serial online] 2020 [cited 2020 Apr 1];12, Suppl S1:13-8. Available from: http://www.jioh.org/text.asp?2020/12/7/13/276089


  Introduction Top


Restoring a diseased or fractured tooth is the most common procedure carried out in dental science for the oral health of the individual. The integrity and durability of the marginal seal has always been of prime concern in the investigation of the performance of dental restorative materials. Despite improvements in materials and techniques, crowns do not adapt perfectly to the prepared teeth, resulting in the marginal opening at the junction of crown and the tooth. So in regular practice, dental luting cements are used to compensate for this marginal discrepancy and to fix the restoration in a proper position.[1]

The clinically undetectable passage of bacteria, fluids, molecules, or ions between the tooth surface and the restorative material has been defined as microleakage.[2] Penetration of bacteria and their products through the potential gaps along the tooth restoration interface accounts for the pathologic component of microleakage that results in recurrent caries and subsequent pulpal pathosis. The toxic products released by such microorganisms may irritate the pulp and cause inflammatory pulpal lesions.

Time-honored cements, such as zinc phosphate, are rapidly diminishing in popularity as luting cements because of problems associated with their high degree of solubility in oral fluids and microleakage.[3] This has led to the emergence of adhesive luting materials such as polycarboxylate and glass ionomer. Since the introduction of glass ionomer material in 1972, this widely used material has been the subject of numerous studies to improve its properties. Advances in the mechanical properties of resin coupled with adhesive qualities and biocompatibility of glass ionomer resulted in the introduction of resin-modified glass ionomer cements (GICs). Their credibility as a luting cement is yet to be proved.

Previous studies have suggested that the marginal percolation was caused by a difference between the coefficient of thermal expansion of the restorative material and the dental tissues and by the thermal expansion of the fluids occupying the crevice between the tooth and restoration.[4],[5],[6] Thermocycling in vitro is a common way of testing dental materials. It aids in establishing their suitability for clinical use. The temperature range used for thermocycling should reflect the temperatures that exist intraorally while taking hot and cold food substances. This is referred to as artificial aging.[7] Similar studies were conducted to evaluate the marginal leakage of provisional crowns with different luting cements.[3] One of the study evaluated the effect of finish lines and luting agents on marginal fit and microleakage in direct metal laser-sintered copings.[1]

The aim of this study was to evaluate the effect of artificial aging on the marginal leakage of crowns. The purpose of this study was also to compare and evaluate the credibility of three luting cements, namely zinc polycarboxylate, glass ionomer, and resin-modified glass ionomer in preventing marginal percolation.


  Materials and Methods Top


The study was approved by the Institutional Ethics’ Committee with Reference Number: 16070. The purpose of this study was to evaluate and compare the effect of thermocycling on the marginal leakage of nickel–chromium (Ni-Cr) full crowns luted with three cements. This in vitro study was undertaken at the Department of Prosthodontics and Crown and Bridge, Manipal College of Dental Sciences, Mangaluru, India. This study was carried out for one year (2018–2019).

Armamentarium used for the study included mouth mirror, explorer, tweezers, metal scale, divider, scissors, B. P. blade, high-speed handpiece (J. Morita, Tokyo, Japan), low-speed handpiece (J. Morita), diamond abrasives, Carborundum discs, diamond discs, burs–diamond (Shofu Dental Shofu Inc., Kyoto, Japan), tungsten carbide, plastic bowl, metal spatula, brush, carving and waxing instruments, rubber cups, and polishing instruments.

The materials used for the study include autopolymerizing resin (Stellon, DPI - India), polyvinyl siloxane impression material (Reprosil, Dentsply), tray adhesive (Caulk, Dentsply), die stone (Kalrock, Kalabhai Karson Pvt., Ltd, India), die spacer (Pico-fit, Renfert, Hilzingen, Germany), separating media (Stellon, DPI), inlay wax type II (Harvard Dental International GmbH, Hoppegarten, Germany), investment material (Powercast, Whipmix, Lousiville, KY, USA), alloy capsules (Remanium® G-soft, Dentaurum GmbH & Co. Isringen, Deutschland), nail varnish, cements, and basic fuchsin dye.

The sample size of 36 extracted premolars were selected for the study. All these premolars were intact. The teeth were extracted for the purpose of orthodontic treatment and were collected from the Department of Oral Surgery. The teeth were kept in normal saline from the time of extraction. Each tooth was mounted with their long axis vertically positioned in self-cure resin. Tooth preparation was carried out in all the selected teeth to receive complete cast crown. The teeth were prepared using a high-speed handpiece (with water spray) and a medium round-ended tapered diamond bur. The occlusal surfaces were reduced following the anatomical form of the tooth. Then the axial walls were made near parallel (contact angle 4°–6°) with a custom-made paralleling device, which aided in attaining a consistent taper for each crown preparation. A uniform preparation height of 3 mm was measured at the midbuccal surface. A cervical chamfer finish line of 0.5 mm was established at the cementoenamel junction, and all the preparations were terminated in dentin. A uniform cervical finish line contour was maintained for all the surfaces.

After completing the tooth preparations, impressions were made with polyvinyl siloxane material. Light body impression material was injected around the tooth and a special tray loaded with heavy body material was placed over the teeth. Using die pins, separable dies were prepared in improved stone. The dies were then painted (leaving 1 mm at the margins) with three coats of die spacer. Wax patterns using inlay wax were fabricated with axial contour simulating natural teeth and an occlusal surface that was made flat. Good marginal adaptation was confirmed using ×10 microscope. The patterns were immediately sprued and invested in a phosphate-bonded investment and cast in non-precious alloy (Ni-Cr), using a standard lost wax technique and an induction casting machine. Well-finished castings were microblasted with 50 μm alumina powder for 15s at 80 lb/n2 pressure.

Before crown cementation, the tooth surface and the crowns prepared were thoroughly cleaned with water and dried with filtered compressed air. The teeth with the castings were randomly grouped into three groups of 12 each. First group was cemented with zinc polycarboxylate (Dentsply), second with glass ionomer (Fuji I), and third with resin-modified glass ionomer (Fuji II Plus). Luting agents were manipulated according to manufacturer’s recommendations. Each casting was then filled with sufficient cement to evenly cover the inner surface, seated on the tooth with digital pressure, and sustained under 90N static load for 10min with a loading device[8] [Figure 1]. Care was taken to see that excess cement was flown out all around. Using an explorer, the excess cement was carefully removed.
Figure 1: Standardized cementation using a loading device (UFIB 200; Ibertest)

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Teeth cemented with crowns were stored in normal saline at 37°C for seven days. Each group of 12 each was then subgrouped into two of six teeth each. The specimens in the subgroup I served as the control group and specimens in subgroup II were subjected to thermocycling. This is carried out to simulate the intraoral conditions. They were artificially aged using artificial saliva [Table 1] as a medium of thermocycling, with a temperature variation of 5°C–55°C.[9],[10] Each specimen was thermocycled for 500 cycles with a dwell time of 10s.[11]
Table 1: Composition of artificial saliva

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After artificial aging, the surfaces of the samples were painted with two coats of nail varnish leaving 1 mm at the margins to prevent staining of the root. Then all specimens (control and thermocycled) were treated with 0.5% basic fuchsin dye for 24h. After exposure to the dye, the specimens were thoroughly washed. The crowns were then embedded in clear self-cure acrylic resin and sliced buccolingually using diamond and Carborundum discs. The sectioned halves were then placed under the optical vision microscope [Figure 2] and the depth of dye penetration in the proximal region was qualitatively evaluated at ×10 magnification using reflected light.
Figure 2: Leitzaristoplan optical vision microscope (Leitz; Martin Microscope Company)

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Evaluation of microleakage: Marginal microleakage is defined as the linear penetration of basic fuchsin dye from the external margin, where margins of the restoration interfaced with the tooth. The extent of dye penetration from the external margin was evaluated and scored according to the following scale.[12]

  • 0: No microleakage


  • 1: Microleakage up to one-third of the axial wall


  • 2: Microleakage up to two-third of the axial wall


  • 3: Microleakage up to full length of the axial wall


  • 4: Microleakage extending on to the occlusal surface


The marginal microleakage value of each group was determined by averaging the scores recorded.

  1. Control group: (12 specimens for each cement)


  2. Thermocycled group: (12 specimens for each cement)


Statistical analysis: The obtained scores were then subjected to one-way analysis of variance (ANOVA) with the Statistical Package for the Social Sciences software (SPSS, version 22, IBM, Chicago, Illinois). The scores from both groups were combined and ranked in order of increasing size. For intergroup comparison, post hoc Tukey test was used.


  Results Top


The mean marginal leakage score for polycarboxylate cement in non-thermocycled group was 0.667 with a standard deviation of ±0.492 and for the thermocycled group, it was 1.9 and ±0.996, respectively. The mean marginal leakage score for GIC in the non-thermocycled group was 0.417 with a standard deviation of ±0.515 and for thermocycled group, it was 1.167 and ±0.389, respectively. For the resin-modified glass ionomer, it was 0.33 and ±0.492 for non-thermocycled and 0.667 and ±0.492 for thermocycled group as shown in [Graph 1].
Graph 1: Mean values of marginal leakage in non-thermocycled and thermocycled groups

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The three luting cements were compared with each other under the categories of thermocycling present and absent using one-way ANOVA and post hoc Tukey test. The results show that the thermocycling group shows significant difference between the zinc Poly-f and GIC as well as zinc Poly-f and resin-modified GIC

Comparison of the effect of thermocycling within the three cement groups was carried out using independent t-test. Zinc Poly-f and GIC showed significantly higher values of microleakage in thermocycling groups, whereas the resin-modified GIC was more stable showing nonsignificant association as shown in [Table 2].
Table 2: Comparison of marginal leakage with three luting cements with non-thermocycled and thermocycled conditions

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  Discussion Top


Preventing the development of secondary caries and maintaining the health, form, and function of the restored teeth are the goals of the fixed prosthetic treatment. Microleakage is the main cause for the development of secondary caries. Lots of researches are carried out to try and achieve perfect marginal fit of the crowns. In spite of having sound clinical knowledge and best materials available, we are not able to prevent the development of secondary caries completely. Despite these advancements, castings cannot be made to adapt perfectly to the margins of the prepared teeth. For this reason, we must still depend on the integrity of the cement to maintain the marginal seal.

Luting cements vary considerably in solubility, strength, and the ability to adhere to tooth structure. Several previous studies have found significant differences between luting agents in their ability to prevent interfacial leakage between the restoration and the teeth surface.[3],[13],[14],[15]

The work carried out by Myers et al.[16] found that the glass ionomer is superior to polycarboxylate and zinc phosphate with respect to solubility and microleakage. Some of the recent studies by Crim[17] and Sidhu[18] showed that resin-modified GICs appear to display substantially better adaptation to dentin than conventional materials and they are fairly resistant to microleakage in in vitro studies. Similar studies were conducted to evaluate the marginal leakage of provisional crowns with different luting cements.[3] One of the study evaluated the effect of finish lines and luting agents on marginal fit and microleakage in direct metal laser sintered copings.[1]

The differences in linear coefficient of thermal expansion of the cast alloy, the luting cement, and the adjoining tooth structure are presumed to cause marginal microleakage in cast restorations.[4],[5],[6] It would seem reasonable that ingesting hot or cold substances is the cause of the most extreme temperature variations in the oral cavity. To simulate the intraoral conditions, exposing the dental materials to the range of temperature is the common part of dental material testing.[15],[19],[20],[21]

Crim and Mattingly[20] compared the effectiveness of four thermocycling techniques with basic fuchsin and radioactive calcium as tracers and found that no significant differences existed among those four thermocycling techniques. Crim[17] stated that the use of dye as a tracer will be more convenient for the researchers because the depth of the dye penetration is readily visualized.

White et al.[22] who performed an in vitro study of the marginal leakage of luting cements concluded that the results they obtained were in agreement to prior in vitro studies. They suggested that in vitro model systems using artificial aging may accurately predict clinical performance. This in vitro study was undertaken to evaluate the effect of artificial aging on the marginal leakage of cast crowns looted with three cements. It also evaluated and compared the extent of marginal leakage, which had occurred after thermal cycling on the three cements used, namely polycarboxylate, glass ionomer, and resin-modified glass ionomer. The tooth preparation was carried out using a custom-made paralleling device. This was fabricated by attaching a high-speed handpiece to the vertical arm of the surveyor. This gave constant taper and standardization for all the preparations. A chamfer finish line was given for all the preparations with uniform width.

To avoid misreading caused by possible differences in the diffusion of dye along the enamel and dentin, all preparations were terminated in dentin. Similar diamond points were used in an attempt to obtain uniform depth of the margins of the preparations and consequently about equal thickness of the metal. Three coats of die spacer were used on the dies with each coat measuring 7–8 µm in thickness (from the manufacturer’s literature), totaling an average cement space between 20 and 35 µm.[23]

All castings were fabricated using common standardized procedures. Inside portion of all finished castings was microblasted using alumina powder to enhance the micromechanical retention of luting cements to the metal. Finished castings were randomly grouped into three of 12 each and cemented using polycarboxylate, glass ionomer, and resin-modified glass ionomer, respectively. Castings were then cemented using a standardized force as proposed by Grieve.[8] The castings were first seated using digital pressure and sustained under a static load of 90N for 10min using a loading machine.

Cemented specimens were again subgrouped into two, one group was subjected to thermocycling, whereas the other group remained as control. Evaluation of microleakage in cast crowns must include thermocycling to simulate intraoral conditions. The value of thermocycling in investigation of microleakage is found in its ability to show the relative effectiveness of different restorative materials and techniques to prevent microleakage of the tooth restoration interface. In this study, the thermocycling technique used by Eakle[11] in determining the microleakage was followed.

An aqueous solution of basic fuchsin is the most widely used dye in laboratory investigation of microleakage. Bearing this in mind, 0.5% basic fuchsin dye was used in this study. The criteria for evaluation of the extent of marginal leakage were scored according to the scale followed by Tjan et al.[12] The results of the study showed that artificial aging did have an effect on marginal leakage of polycarboxylate and glass ionomer, whereas in resin-modified glass ionomer, it did not show much statistical significance. This indicated that after thermocycling, the specimen cemented with polycarboxylate has undergone more microleakage. This may be attributed to the high solubility of polycarboxylate, disparity in the linear coefficients of thermal expansion of the different materials involved, and the shrinkage of cements on setting. In case of glass ionomer, microleakage after thermocycling was relatively less compared to that of polycarboxylate. Even though it has got resistance to solubility and coefficient of thermal expansion similar to tooth structure, the microleakage shown may be due to its early solubility in water and dilute acids caused by the slow formation of a strong gel salt. This is in confirmation to the previous studies by White et al. in 1995.[15],[24]

Resin-modified glass ionomer showed maximum resistance to microleakage after thermocycling. This may be attributed to the high solubility-resistant resin matrix present in resin-modified glass ionomer. This is in confirmation to the previous studies by Crim[17] and Sidhu.[18] Among cements, polycarboxylate showed more microleakage than glass ionomer and resin-modified glass ionomer. This may be due to the weak matrix formed with zinc ions, which are less resistant to erosion compared to glass ionomer and resin-modified glass ionomer. This is in confirmation to the previous studies by White et al.[14]

As marginal leakage can lead to secondary problems and ultimately pulpal pathosis, it is very much essential to prevent this phenomenon. From the result of this study, we observed the ability of resin-modified glass ionomer luting cements in preventing marginal leakage. So in this regard, further research into resin-modified GICs in longitudinal and clinical studies are proposed to find out its clinical acceptability.

The results of this study conclude that artificial aging did have an effect on the marginal leakage of polycarboxylate group and glass ionomer group, whereas resin-modified glass ionomer group did not show much statistical significance. Among the cements used, zinc polycarboxylate had the maximum microleakage followed by glass ionomer and resin-modified glass ionomer. Resin-modified glass ionomer showed minimum leakage and can be regarded as the best cement for clinical use.

Ethical consent

As the presented study is an in vitro study, so ethical consent is not applicable.

Data availability

The data set used in this study is available on request from corresponding author Dr. Mahesh M at mahesh.m@manipal.edu.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Chavan P, Shetty T, Mundathaje M, Rodrigues SJ. Evaluation of effect of different finish lines and luting agents on marginal fit and microleakage in direct metal laser sintered copings—An in-vitro study. Indian J Forensic Med Toxicol 2018;12:323-7.  Back to cited text no. 1
    
2.
Hafezeqoran A, Koodariyan R, Esmaili A, Noori H, Shahbaz A. Marginal adaptation of metal ceramic crowns cast from four different base metal alloys before and after porcelain application. Adv Biosci Clinical Med 2015;3:30-6.  Back to cited text no. 2
    
3.
Nemane V, Akulwar RS, Meshram S. The effect of various finish line configurations on the marginal seal and occlusal discrepancy of cast full crowns after cementation: An in vitro study. J Clin Diagn Res 2015;9:ZC18-21.  Back to cited text no. 3
    
4.
Arora SJ, Arora A, Upadhyaya V, Jain S. Comparative evaluation of marginal leakage of provisional crowns cemented with different temporary luting cements: In vitro study. J Indian Prosthodont Soc 2016;16:42-8.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Sener I, Turker B, Valandro LF, Ozcan M. Marginal gap cement thickness and microleakage of 2 zirconia crown systems luted with glass ionomer and MDP based cements. Gen Dent 2014;62:67-70.  Back to cited text no. 5
    
6.
Asmussen E. The effect of temperature changes on adaptation of resin fillings. Acta Odont Svcand 1974;32:329.  Back to cited text no. 6
    
7.
Palmer DS, Barco MT, Billy EJ. Temperature extremes produced orally by hot and cold liquids. J Prosthet Dent 1992;67:325-7.  Back to cited text no. 7
    
8.
Grieve AR. Study of dental cements. Br Dent J 1969;4:405-9.  Back to cited text no. 8
    
9.
Tai Y, De Long R, Goodkind RJ, Douglas WH. Leaching of nickel, chromium, and beryllium ions from base metal alloy in an artificial oral environment. J Prosthet Dent 1992;68:692-7.  Back to cited text no. 9
    
10.
Johansson BI, Lemons JE, Hao SQ. Corrosion of dental copper, nickel, and gold alloys in artificial saliva and saline solutions. Dent Mater 1989;5:324-8.  Back to cited text no. 10
    
11.
Eakle WS. Effect of thermal cycling on fracture strength and microleakage in teeth restored with a bonded composite resin. Dent Mater 1986;2:114-7.  Back to cited text no. 11
    
12.
Tjan AHL, Dent DR, Dunn JR, Grant BE. Marginal seal of injection molded ceramic crowns cemented with three adhesive systems. J Prosthet Dent 1989;61:24-7.  Back to cited text no. 12
    
13.
Mc Comb D. Retention of casting with glass ionomer cement. J Prosthet Dent 1982;48:285.  Back to cited text no. 13
    
14.
White SN, Sorensen JA, Kang SK, Caputo AA. Microleakage of new crown and fixed partial denture luting agents. J Prosthet Dent 1992;67:156-61.  Back to cited text no. 14
    
15.
White SN, Furuichi R, Kyomen SM. Microleakage through dentin after crown cementation. J Endodont 1995;21:9-12  Back to cited text no. 15
    
16.
Myers ML, Staffanau RS, Hembree JH, Wiseman WB. Marginal leakage of contemporary cementing agents. J Prosthet Dent 1983;50:513-5  Back to cited text no. 16
    
17.
Crim GA. Effect of aging on microleakage of restorative systems. Am J Dent 1993;6:192-4  Back to cited text no. 17
    
18.
Sidhu SK. Marginal contraction gap formation of light-cured glass ionomers. Am J Dent 1994;7:115-8.  Back to cited text no. 18
    
19.
Tjan AH, Miller GD, Whang SB, Sarkissian R. The effect of thermal stress on the marginal seal of cast gold full crowns. J Am Dent Assoc 1980;100:48-51.  Back to cited text no. 19
    
20.
Crim GA, Mattingly SL. Evaluation of two methods for assessing marginal leakage. J Prosthet Dent 1981;45:160-3.  Back to cited text no. 20
    
21.
Blum J, Weiner S, Berendsen P. Effects of thermocycling on the margins of transitional acrylic resin crowns. J Prosthet Dent 1991;65:642-6.  Back to cited text no. 21
    
22.
White SN, Yu Z, Tom JF, Sangsurasok S. In vitro microleakage of luting cements for cast crowns. J Prosthet Dent 1994;74:333-8.  Back to cited text no. 22
    
23.
Shillingberg HT, Hobo S, Whitsett LD. Fundamentals of Fixed Prosthodontics 2nd ed. Chicago: Quintessence Publishing; 1981. p. 318.  Back to cited text no. 23
    
24.
Park JK, Lee WS, Kim HY, Kim WC, Kim J. Accuracy evaluation of metal copings fabricated by computer aided milling and direct metal laser sintering systems. J Adv Prosthodont 2015;7:122-8.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Graph 1]
 
 
    Tables

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