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. 2025 May 28;25:839. doi: 10.1186/s12903-025-05972-w

Effect of glazing and hydrothermal aging on the fracture strength of metal-ceramic single restorations after additional firing: an in vitro study

Zahra Parvizi 1,2, Elham Ansarifard 3,✉, Masumeh Taghva 4,✉, Seyed Ali Mosaddad 5,6
PMCID: PMC12121120  PMID: 40437420

Abstract

Background

Additional firing to repair a lost proximal contact or porcelain chipping might affect the fracture strength of a metal-ceramic crown. This study aimed to evaluate the effect of glazing and hydrothermal aging on the fracture strength of metal-ceramic crowns after additional firing.

Methods

A total of 40 metal-ceramic crowns were fabricated and divided evenly into five groups: not aged/glazed (G0), 3-month-old/glazed (G3), 24-month-old/glazed (G24), 3-month-old/unglazed (nG3), and 24-month-old/unglazed (nG24). Autoclaving was used as the aging protocol. All groups underwent an additional firing cycle, and fracture strength was measured. Data were analyzed using ANOVA and independent t-tests.

Results

G0 demonstrated the highest fracture strength (4405.00 ± 1528.20 N), followed by G3, nG3, G24, and nG24, respectively. There was a significant difference between G0 and G24 (P =.01), G0 and nG3 (P =.00), and G0 and nG24 (P =.00).

Conclusion

Glazing does not significantly affect the fracture strength of a metal-ceramic crown undergoing additional firing; however, aging has a significant adverse effect on fracture strength. This negative effect is not significant until after 3 months.

Keywords: Fracture strength, Metal ceramic restorations, Ceramics, Dental prosthesis, Dental prosthesis repair

Introduction

Fixed dental prostheses, particularly metal-ceramic crowns, are widely used to restore missing or prepared teeth due to their durability and aesthetic appeal [1]. These crowns are favored for benefiting from the combination of high tensile and compressive strength, wear resistance, and corrosion resistance from the metal alloy, along with the aesthetic appeal of ceramics. They meet both functional and aesthetic demands, offering durability, good corrosion behavior in the oral environment, and biocompatibility with oral tissues [2].

However, technical complications such as proximal contact loss and porcelain chipping are common, potentially leading to patient discomfort, periodontal issues, and compromised longevity of restorations [3, 4]. Proximal contact loss [4, 5], which affects around 29% [6] of metal-ceramic crowns may lead to a cascade of secondary problems, including food impaction, periodontal disease [7], and increased susceptibility to caries, emphasizing the need for effective repair strategies that extend the life of the restoration and minimize the need for replacement [8, 9]. Addressing these issues is crucial, as they directly affect clinical outcomes and the long-term success of restorations in dental practice, highlighting the importance of regular monitoring and maintenance of contact quality [10, 11]. Applying occlusal guards and orthodontic retainers has also been suggested to prevent open proximal contacts [12].

When an open proximal contact is detected in a metal-ceramic crown, several options are available for restoring it. While fabricating a new crown is one solution, it is typically not the first choice due to the associated cost and time [6]. Another suggested repair method involves using composite resin materials to restore chipped ceramic areas, but this approach has shown high failure rates due to the insufficient bond between ceramic and composite, which can lead to further chipping or discoloration over time [13, 14]. As an alternative, re-firing to add porcelain can achieve a better bond, but it may impact the restorations’ properties. The addition of porcelain to an existing crown requires additional firing cycles to achieve the desired quality [15]. However, repeated firing cycles can lead to the formation of leucite crystals in the porcelain, increase the thermal coefficient of expansion of the ceramic, elevate surface stress, and potentially create micro-cracks thereafter [16]. Furthermore, the mechanical and structural properties of the crown may be adversely affected [17]. Additionally, the indirect repair procedure may reduce the accuracy of the contact restoration [10, 13]. In a metal-ceramic crown, firing cycles can also impact the oxide layer and reduce the bond strength as a consequence [18, 19].

In this context, glazing which is often applied after ceramic firing to enhance the surface characteristics of the restorations, needs additional firing. The individual effects of all these additional firing cycles on crown bond strength [15–18, 20–22], and glazing and aging on crown mechanical strength [23–26] have been studied separately in the literature. It has been demonstrated that in addition to enhancing aesthetics, glazing creates a smooth and hygienic surface for the crown [27]. A glazed surface typically exhibits lower thermal expansion and helps compress the surface, reducing porosity and creating a denser, more durable finish [23]. While the overall impact of glazing on the mechanical properties of the crown remains debated, it is generally believed that glazing helps reduce surface micro-cracks, thereby strengthening the crown [23]. Additionally, these restorations are also subjected to aging in the oral environment that can accelerate degradation, potentially offsetting the benefits of glazing. Aging, resulting from continuous exposure of the crown to oral conditions including humidity, temperature variations, and mechanical loading, is believed to decrease the fracture strength of a metal-ceramic crown. This reduction is attributed to the formation of micro-cracks, likely caused by a mismatch in the coefficient of thermal expansion between the metal and porcelain components [28].

Despite these findings, limited research exists on fracture strength post-firing, specifically examining how glazing and aging interact when crowns undergo additional firing cycles for porcelain repair. Given this gap in the literature, this study aimed to assess how glazing and hydrothermal aging affect the fracture strength of metal-ceramic crowns after additional firing cycles when repairs are necessary post-delivery. The first null hypothesis posited that glazing does not affect the fracture strength of metal-ceramic crowns following multiple firing cycles, while the second null hypothesis suggested that aging has no effect on the fracture strength of these crowns under the same conditions.

Methods

The study received approval from the Ethics Committee of Shiraz University of Medical Sciences under ethical code IR. SUMS.DENTAL.REC.1401.016.

A total of 40 metal-ceramic molar crowns were fabricated. Based on the effect size (f = 1.54), the calculated post hoc power for the one-way ANOVA F-test was 99.9%. The samples were then randomly divided into the following groups based on the time since insertion and whether or not they were glazed: not aged and glazed (G0 / control), three months old and glazed (G3), twenty-four months old and glazed (G24), three months old and unglazed (nG3), twenty-four months old and unglazed (nG24). Figure 1 illustrates the flow diagram of the samples. Based on findings by Wei et al. [5] that open contact in metal-ceramic crowns often occurs around three months after insertion, with peak contact loss by the 24th month, three and 24 months were chosen as key time points to assess the effects of aging on fracture strength.

Fig. 1.

Fig. 1

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Flow diagram of the study design and grouping

Preparation of the metal-ceramic samples

A lower right mandibular typodont tooth was prepared on a lathe (CNC350; Arix Co., Tainan Hsin, Taiwan) (Fig. 2A) to serve as the master model. The preparation included a 1 mm wide, smooth, continuous margin without irregularities, with an occlusal convergence angle of 6 degrees and a height of 7 mm to support each crown [29]. Additionally, a ledge was created at the occlusal-axial line angle of the master die to function as an anti-rotational feature.

Fig. 2.

Fig. 2

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(A) The brass metal die; (B) The stone model

To simulate proximal contacts, a lower right mandibular second premolar and second molar were mounted on a stone cast, with the metal die positioned as the first molar. A metal-ceramic restoration was fabricated on this die (Fig. 2B). The restoration process involved taking an impression with a one-stage putty-light body silicone impression material (Elite HD+, Zhermack, Italy) and pouring it with type IV stone (Fujirock; GC Europe N.V.) to create a master die for designing and casting the metal framework.

The metal framework was then fabricated using nickel-chromium (Ni-Cr) alloy by first preparing a wax pattern on the metal die model. This pattern was invested in a high-heat-resistant mold (Bellavest® SH, Bego, Germany) and placed in a burnout oven to eliminate the wax. After burnout, a Ni-Cr alloy (Wiron 99®, Bego, Germany) was melted and cast into the mold using an induction casting machine (Fornax® T, Bego, Germany) to create the metal framework. The cast copings were then divested, cleaned, and finished to ensure proper fit and smoothness. Each coping underwent sandblasting with 110 μm aluminum oxide particles (Korox® 110, Bego, Germany) to create a rough surface, optimizing the bonding area for the porcelain application [30, 31]. One opaque layer (Vintage PRO Basic Paste Opaque Set; Shofu Inc., Kyoto, Japan) was applied to Ni-Cr copings and fired in a calibrated porcelain furnace, a process repeated twice. A dentin ceramic (Vintage PRO Dentin Paste, Shofu Inc.) was applied according to manufacturer recommendations for porcelain layering. Subsequently, two dentin firings were carried out. The porcelain thickness was standardized to 2 mm in all specimens using a putty index.

In the final step, the crowns in groups G0 (control), G3, and G24 underwent glazing (Vintage Art Glazing Paste; Shofu Inc.). Glaze-firing was performed at 1 atm pressure, with a controlled temperature ramp rate of 56 °C per minute, starting from 593 °C and reaching a final temperature of 850 °C. Specimens were removed from the furnace at this final temperature. All firing cycles adhered strictly to the manufacturer’s recommended protocols to ensure consistency and minimize potential variability in atmospheric conditions within the furnace. Figure 2B illustrates the prepared samples. Table 1 outlines the specific firing cycles for the opaque, dentin, and glaze ceramics, including the recommended parameters from the manufacturer.

Table 1.

Ceramic firing cycles and manufacturer recommendations

Firing Stage Material Temperature Ramp Rate Starting Temperature Final Temperature Holding Time Atmosphere
Opaque Firing Opaque Ceramic (Vintage PRO Basic Paste) 60 °C per minute 500 °C 960 °C 1 min 1 atm
Dentin Firing Dentin Ceramic (Vintage PRO Basic Paste) 50 °C per minute 650 °C 910 °C 1 min 1 atm
Glaze Firing Glaze Ceramic (Vintage Art Glazing Paste) 50 °C per minute 600 °C 850 °C Immediate Removal 1 atm
Repair Firing Low-Fusing Ceramic for Repair (Vintage LD) 55 °C per minute 450 °C 760 °C 1 min 1 atm
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Resin dies were fabricated by casting inlay pattern resin (DuraLay; Reliance Dental MFG Co., Worth, IL) into silicone impressions. All prepared specimens were cemented onto the resin dies for the fracture strength test.

Simulation of the aging process

The hydrothermal aging method was employed to simulate aging in the samples, using an autoclave (Melag Vacuklav® 24-B, Melag Medizintechnik, Germany) as a reactor controller to regulate temperature and pressure. Aging conditions included a temperature of 122 °C (± 1 °C) and a pressure of two bars, following previous protocols [32]. Specimens in groups G3 and nG3 were subjected to these conditions for 15 min, simulating approximately 3 months of aging, while specimens in groups G24 and nG24 were aged for two hours to simulate approximately two years.

Repairing the crowns with the addition of porcelain

The procedure involved reducing the distal surface of the adjacent second premolar tooth on the model by one millimeter to open the mesial contact of the first molar crown, simulating proximal contact loss on the mesial side. This reduction was measured using a Boley gauge. Subsequently, an equivalent amount of porcelain (Vintage LD; Shofu Inc.) was added to the mesial contact area of the first molar crown across all study groups, followed by refiring the samples in the furnace.

The unglazed aged specimens (nG3 and nG24) underwent a total of five firing cycles: two for the opaque layer, two for the dentin layer, and one for the repaired porcelain. For the control group (G0) and the aged-glazed groups (G3 and G24), an additional glaze firing was applied after the aging protocol, resulting in a total of six firing cycles.

Cementation

Before cementing the crowns onto the resin dies, the dies were sandblasted with 110 μm aluminum oxide powder at a pressure of 2 bars and subsequently steam cleaned to improve retention [33]. All crowns were then cemented onto Duralay dies using a self-curing luting resin cement (Multilink Automix, Ivoclar Vivadent AG). To minimize variability in the cementation process, each crown was initially seated using finger pressure, followed by careful removal of excess cement under magnification. A standardized seating load was then applied: a static load of 15 N was maintained for 5 min [34] on each crown in the direction of insertion during cementation to ensure consistent pressure and positioning. Finally, all crowns were stored in a moist environment for 24 h to allow the cement to fully set before mechanical testing [35].

Fracture strength test

All specimens were secured in a test jig and subjected to fracture testing using a universal testing machine. The load was applied to the occlusal surface of the crowns using a 2.5 mm stainless steel ball, with a 1 mm thick plastic foil placed between the ball and the crown. The crosshead speed was set at 0.255 mm/min, and the fracture was defined as either a visible crack or an acoustic event, whichever occurred first. The fracture test was conducted by a researcher (Z.P) who was blinded to the study design and group assignments.

Statistical analysis

Data were analyzed using the SPSS (Statistical Package for the Social Sciences) program (version 26, IBM Corporation, Armonk, NY, USA). Statistical tests, including Levene’s test for homogeneity of variances, normality test, independent t-test, ANOVA, and post hoc Tukey HSD test, were performed. A significance level of P <.05 was used to determine statistical significance.

Results

The normality test indicated no deviation from the normal distribution for the parameters, allowing for parametric data analysis. ANOVA test results revealed a significant difference between the groups (P =.02).

Table 2 presents the mean ± SD fracture strength, along with the minimum and maximum values for each group, where all specimens were loaded to fracture. Figure 3 illustrates the mean fracture strength of each group via a bar chart. G0 (control) exhibited the highest load-bearing capacity before fracture, followed by G3, while the lowest fracture strength was observed in group nG24. Levene’s test confirmed no significant variance differences among the groups (P =.299).

Table 2.

Mean fracture strength (N) of specimens in each group

Groups Number Mean ± SD Minimum Maximum
G0 (control) 8 4405.00 ± 1528.20 3127.39 5682.61
G3 8 3660.00 ± 858.354 2942.40 4377.60
G24 8 2891.25 ± 592.801 2395.66 3386.84
nG3 8 2635.00 ± 428.886 2276.44 2993.56
nG24 8 2470.00 ± 761.127 1833.68 3106.32
Total 40 3212.25 ± 1134.432 2849.44 3575.06
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Fig. 3.

Fig. 3

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Bar chart of the mean fracture load of the specimens in each group

The Tukey HSD test results presented in Table 3 indicated significant differences between specific groups, as highlighted in Fig. 3 with asterisks. According to both Fig. 3; Table 3, statistically significant differences were observed between G0 and G24 (P =.01), G0 and nG3 (P =.00), and G0 and nG24 (P =.00).

Table 3.

Pairwise comparison of the groups according to the Tukey HSD test

Group (I) Group (J) Mean Difference (I-J) Sig.
G0 (control) G3 745.00 0.49
G24 1513.75* 0.01
nG3 1768.75* 0.00
nG24 1935.00* 0.00
G3 G0 (control) -745.00 0.49
G24 768.75 0.45
nG3 1023.75 0.19
nG24 1190.00 0.09
G24 G0 (control) -1513.75* 0.01
G3 -768.75 0.45
nG3 255.00 0.98
nG24 421.25 0.88
nG3 G0 (control) -1768.75* 0.00
G3 -1023.75 0.19
G24 -255.00 0.98
nG24 166.25 0.99
nG24 G0 (control) -1935.00* 0.00
G3 -1190.00 0.09
G24 -421.25 0.88
nG3 -166.25 0.99
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* The mean difference is significant at the 0.05 level

Discussion

Given the frequent occurrence of open contacts and fractures in tooth- or implant-supported metal-ceramic crowns and the need for additional firing during repair processes [36], this study aimed to assess how additional firing affects the fracture strength of metal-ceramic crowns of varying ages. Beyond the initial three firing cycles involved in sample preparation using the layering technique, an additional firing was conducted to add porcelain to the proximal area. The findings showed that G0 exhibited the highest fracture strength, followed by G3, nG3, G24, and nG24, respectively, with a significant difference in fracture strength observed between G0 and all other groups except G3.

Based on the study findings, the first null hypothesis regarding the effect of glazing on the fracture strength of metal-ceramic crowns after additional firing was accepted. Previous research has indicated that glazing might strengthen crowns by reducing surface thermal expansion and crack depth [37]. However, the results show no significant difference in fracture strength between glazed and unglazed crowns, whether aged for three months or 24 months, after additional firing. Thus, glazing does not appear to increase the fracture strength of metal-ceramic crowns in this context.

In a prior study, it was observed that glazed high-translucent zirconia full ceramic crowns exhibited greater flexural strength compared to unglazed ones [23]. The discrepancy in the results compared to theirs might stem from differences in the type of restoration (metal-ceramic vs. full ceramic) and the potential impact of sample polishing, which could induce phase transformation and affect the coefficient of thermal expansion [23]. Another study highlighted that regardless of mechanical aging, glazing, whether on one or both sides, enhanced the flexural strength of Y-TZP zirconia full ceramic crowns [24]. Differences in restoration type (metal-ceramic vs. full ceramic) and glazing protocols might explain the discrepancy between our results and theirs; they utilized an over-glazing approach, whereas we adjusted the firing temperature without additional glazing layers.

Based on the results obtained, the second null hypothesis was partially accepted. It was observed that aged metal-ceramic crowns exhibited lower fracture strength compared to new ones following an additional firing cycle. Aging emerged as a significant determinant affecting fracture strength, with a progressive impact over time. Specifically, after two years, whether a crown was glazed or not before additional firing did not influence its fracture strength. However, the hypothesis was not completely rejected, as no significant difference in fracture strength was found between G0 and G3. This finding suggests that aging does not significantly reduce fracture strength in metal-ceramic crowns after an additional firing cycle of up to three months. Previous studies have indicated that hydrothermal aging can diminish fracture strength by creating interface cracks attributed to differences in mechanical properties between metal and porcelain, such as elastic modulus and coefficient of thermal expansion [28]. In this study, G0 demonstrated significantly higher fracture strength compared to G24, nG24, and nG3, while no significant differences were observed among the intervention groups (G3, G24, nG3, nG24). The reduction in fracture strength observed after hydrothermal aging may be attributed to several microstructural changes occurring within the veneering porcelain. Aging has been reported to promote the propagation of microcracks and interfacial degradation due to thermal mismatch between the metal substructure and ceramic veneer [38, 39]. Furthermore, water-induced degradation may penetrate the glaze layer and accumulate in flaws or voids at the metal-ceramic interface, exacerbating crack initiation and growth under load [40]. These phenomena are likely to contribute to the decreased mechanical performance in aged crowns. Although this study did not include microscopic evaluation techniques, future investigations employing scanning electron microscopy (SEM) or X-ray diffraction (XRD) could further elucidate the material-level failure mechanisms involved.

In a previous investigation, it was noted that monolayer zirconia crowns without aging exhibited significantly greater fracture resistance compared to both aged and non-aged bilayer zirconia crowns. Our findings did not indicate a significant effect of aging after three months; however, after two years, our results aligned with theirs, showing a significant adverse effect of aging [41]. Another study employed a chewing simulator to simulate five clinical years and reported that unaged monolithic zirconia and monolithic lithium disilicate crowns had higher fracture strength than their aged counterparts [37]. Additionally, a study using a thermal cycling protocol to simulate one year of oral conditions found that unaged screw-cement-retained implant-supported zirconia-based restorations demonstrated significantly higher fracture resistance regardless of crown type (full ceramic or metal-ceramic) [25].

This study focused on metal-ceramic crowns, which differ significantly from monolithic zirconia and lithium disilicate crowns in composition and mechanical behavior. Metal-ceramic crowns consist of a strong metal core (typically nickel-chromium or cobalt-chromium) veneered with feldspathic porcelain, offering high fracture resistance but introducing a bonding interface prone to microcracking and debonding over time, especially with multiple firing cycles [40]. This interface, where the metal and ceramic layers meet, is a known weakness, particularly during repairs​. Monolithic zirconia crowns, by contrast, are single-layer restorations made entirely of polycrystalline zirconium dioxide, known for its high flexural strength (~ 1000 MPa) and resistance to catastrophic fracture due to its transformation toughening property [42]. However, zirconia is inherently brittle and cannot be easily repaired once fractured [43]. Lithium disilicate, another monolithic material, is a glass-ceramic with lower strength (around 400 MPa) but superior esthetics and translucency [44]. Unlike metal-ceramics, both zirconia and lithium disilicate lack a metal-ceramic interface, offering more uniform stress distribution but lower repair flexibility and potentially higher susceptibility to fractures under concentrated loads [45, 46]​. These differences highlight that, while findings from monolithic ceramics offer insights, the layered structure and bonding interface in metal-ceramics present unique challenges in durability and repairability, making direct comparisons limited [47].

The findings of this study have important clinical implications for the repair and longevity of metal-ceramic crowns. Our results indicate that while glazing has a minimal impact on fracture strength, hydrothermal aging significantly reduces it, especially after prolonged periods. Clinicians should consider that repairs requiring additional firing cycles could weaken the structure of metal-ceramic restorations over time, potentially compromising their durability in the oral environment. For patients requiring repairs to proximal contacts or minor chipping, the decision to re-fire or add porcelain should be weighed against the potential long-term reduction in fracture strength, particularly in cases where high occlusal forces are expected. Clinicians might consider alternatives, such as polishing repaired areas instead of additional glazing, to minimize thermal stress on the ceramic structure. Understanding these limitations can aid in better case selection, helping clinicians decide when to proceed with repairs versus replacement, ultimately improving patient outcomes by selecting interventions that best balance aesthetic and functional longevity.

Various protocols for simulating low-temperature degradation to mimic the oral environment have been developed, including storage in distilled water, exposure to acidic solutions, autoclaving, and mechanical cycling [48]. In a comparative study of five different aging protocols (mechanical cycling, thermal cycling, thermomechanical cycling, autoclaving, and storage in distilled water), it was found that mechanical cycling and thermomechanical cycling resulted in lower flexural strength values compared to the other methods [49]. This research utilized autoclaving under 2 bars of pressure for 5 h, as per the ISO standard protocol for zirconia aging (ISO standard 13356:2008) [48]. Resin cement was chosen for this study due to its high bonding strength and fracture resistance, which make it suitable for simulating the clinical performance of metal-ceramic crowns. Its strong bond with the resin die provides stability under mechanical loading, allowing for an accurate assessment of the crowns’ fracture strength in conditions that closely mimic those encountered in clinical practice [50, 51].

While this in vitro study was carefully designed and statistically powered to evaluate the fracture strength of metal-ceramic crowns following additional firing, certain methodological limitations should be acknowledged. First, the sample size, though sufficient based on power analysis (99.9% power), may benefit from expansion in future research to reduce variability and improve generalizability. Second, although hydrothermal aging using an autoclave is an established method for simulating low-temperature degradation, it does not fully replicate the complex intraoral environment, which includes cyclic loading, temperature fluctuations, and biochemical challenges. Additionally, the fracture strength was assessed using static loading, which may not fully represent dynamic masticatory forces. While these parameters allowed controlled and repeatable testing, future studies incorporating thermomechanical cycling and fatigue testing would be valuable. Microstructural analyses (e.g., SEM and XRD) were beyond the scope of this study but could reveal underlying mechanisms of failure and material transformation. Surface roughness analysis and adhesion testing were also not performed, but their inclusion in future studies would enrich our understanding of the effects of glazing and repeated firing on both surface characteristics and interfacial bonding.

Conclusions

Despite the limitations of the current study, it is concluded that during an additional firing cycle of a metal-ceramic crown, glazing does not significantly increase its fracture strength. Conversely, aging significantly reduces the fracture strength of metal-ceramic crowns after additional firing. Notably, this detrimental effect becomes significant only after 3 months post-insertion.

Acknowledgements

The authors gratefully acknowledge the Vice Chancellor for Research of Shiraz University of Medical Sciences and Dr. Asmarian for his valuable assistance with the statistical analysis of this project.

Author contributions

Conceptualization: E.A., M.T., and Z.P.; Methodology: E.A., M.T., and Z.P.; Software: S.A.M; Validation: S.A.M and M.T.; Formal analysis: Z.P.; Investigation: E.A., M.T., and Z.P.; Resources: E.A., M.T., and Z.P.; Data Curation: S.A.M.; Writing - Original Draft: E.A., M.T., and Z.P.; Writing - Review & Editing: S.A.M.; Visualization: S.A.M; Supervision: M.T.; Project administration: E.A. and M.T. All authors have read and approved the published version of the manuscript.

Funding

This study was funded by Shiraz University of Medical Sciences, Grant number 24860.

Data availability

The data presented in this study are available on request from the corresponding author.

Declarations

Ethics approval and consent to participate

The study received approval from the Ethics Committee of Shiraz University of Medical Sciences under ethical code IR. SUMS.DENTAL.REC.1401.016.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Elham Ansarifard, Email: ansarifard.elham@gmail.com.

Masumeh Taghva, Email: taghvam@yahoo.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

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