|Year : 2022 | Volume
| Issue : 1 | Page : 7-15
In vitro evaluation of cyclic loading on retention strength of Retention.sil and O/ring attachments for tooth supported overdenture
Raadhikka Karthhik, Bharath Raj, GP Surendra Kumar
Department of Prosthodontics, V.S. Dental College and Hospital, Bengaluru, Karnataka, India
|Date of Submission||28-May-2021|
|Date of Acceptance||27-Oct-2021|
|Date of Web Publication||04-Jan-2022|
#13, 6th C Cross, Sairam Layout, Attur, Yelahanka, Bengaluru - 560 064, Karnataka
Source of Support: None, Conflict of Interest: None
Statement of Problem: Numerous different attachments are used to retain overdentures. Hence, there is a need to assess new chairside attachment system based on polyvinylsiloxane (Retention.sil [R.S]) to know its in vitro behavior to be used clinically.
Purpose: To assess and compare the retentive force of two female matrix retention system (R.S with a conventional O-ring [OR] attachment system) in tooth-supported overdenture.
Materials and Methods: Completely edentulous mandibular model with two canine root-anchored ball attachments for overdentures were fabricated. A total of 30 mandibular dentures were made and divided to form two groups, each with a different attachment design. One group (test) received 5 pairs of R.S attachments while the other group (control) received 15 pairs of ORs. Retention force was measured by subjecting them to 540 cycles of insertion and de-insertion cycles (representing 6 months functional life) in axial direction before and after thermocycling.
Results: Statistical analysis comprised Kolmogorov–Smirnov test, Friedman test, and Wilcoxon signed-rank test (α = 0.05). The initial retention of R.S attachments was 5.90 ± 0.77 N and of ball/OR 17.36 ± 3.81 N. The final retention of R.S attachments was 5.41 ± 0.72 N and of ball/OR 12.91 ± 3.18 N. The OR developed higher retentive force as compared to the R.S attachments (P < 0.05). However, no significant changes in retention force were observed for R.S groups after repeated dislodging and thermal undulation.
Conclusion: Both systems presented acceptable retention capacities after 540 cycles. The newly developed R.S attachments can provide an alternative to OR attachments for short-term retention. Clinical studies are however required to elucidate the long-term performance of these materials.
Keywords: O-ring, retention and stability, retention silicon, root-anchored ball attachment
|How to cite this article:|
Karthhik R, Raj B, Surendra Kumar G P. In vitro evaluation of cyclic loading on retention strength of Retention.sil and O/ring attachments for tooth supported overdenture. J Oral Res Rev 2022;14:7-15
|How to cite this URL:|
Karthhik R, Raj B, Surendra Kumar G P. In vitro evaluation of cyclic loading on retention strength of Retention.sil and O/ring attachments for tooth supported overdenture. J Oral Res Rev [serial online] 2022 [cited 2023 Mar 27];14:7-15. Available from: https://www.jorr.org/text.asp?2022/14/1/7/334830
| Introduction|| |
Retentive characteristics of overdenture attachment systems represent a critical factor in clinical outcomes of attachments on root-supported overdentures. Overdentures are retained using different conventional attachments such as splinting attachments (bars and clips) or nonsplinting attachments (ball and sockets, magnets, telescopic copings, and stress breaker attachments)., Attachment with ball and O-rings (ORs) is the most common approach that has been used. However, they have major drawbacks such as cost factor, complex fabrication, rapid wear, limited rotational freedom, and require routine maintenance and periodic repair.,,,
Recently, a new female matrix material attachment called Retention.sil (R.S, Bredent Medical, Germany) has been introduced based on its chemical composition (polyvinylsiloxane [PVS]) as silicon matrices attachment for implant overdentures, in place of the attachment system component in the denture's base. It is resilient and has a high tenacious strength that secures the prosthesis in place through mechanical interlocking with frictional contact. In addition, it has good shock absorbing ability, easy to repair, reduces cost, and decreases chances of aspiration of conventional ORs attachment. It comes with three options according to the detachment force desired (200, 400, and 600 g/f).,
Previous studies have reported that retention silicon-based overdenture attachment with TiSi. snap abutments (based on the design of bollards) as a suitable matrix product for resilent retention of implant overdenture due to its positive biological, physical, and retention properties. Based on the consideration above, it can be contemplated that retention silicones in combination with root-anchored ball attachments would provide as the easiest and reliable method to guarantee the retention of overdentures. However, to our knowledge, till date, the effectiveness of R.S as a female matrix attachment system on root-anchored mandibular overdenture has never been investigated before, and thus, it is unclear on the retentive force of R.S in root-supported overdenture attachment. Therefore, the present in vitro study is undertaken to investigate the retentive force of two female matrix retention systems (R.S and widely employed ORs) in tooth-supported overdenture, relative to each other.
| Materials And Methods|| |
Mandibular-overdenture model fabrication - Experimental model
An acrylic resin model of an edentulous mandible without alveolar undercuts was fabricated with heat polymerized polymethyl methacrylate resin (DPI Heat Cure, DPI, Mumbai, Maharashtra, India). For standardization purposes, this in vitro study was conducted on the same edentulous mandibular acrylic resin model.
Two extracted natural noncarious canine teeth were inserted (placed 22 mm apart) in 2 recesses prepared in the canine areas of the model. The teeth were aligned parallel to each other and perpendicular to the horizontal plane. To increase the retention of the root in the acrylic resin model during the pull-out test, each root will be notched on buccal and lingual surfaces with tungsten carbide bur and secured with auto-polymerizing acrylic resin inside the model. The custom post coping and patrix (ball attachment) were fabricated and cemented with self-cure bonding resin cement (Rely X, 3M USA) after verifying the ball attachment parallelism in model with the aid of surveyor (B2; Bio-ArtDental Equipment Ltd). The thickness of the copings was not more than 1 mm. Master cast was duplicated using agar and agar for fabrication of samples.
Preparation and grouping of overdenture-simulating blocks
Thirty heat cure acrylic edentulous mandibular models were fabricated and divided into as follows:
- Test group (n = 15, R.S): Impression of denture made with light body to mark the position of ball attachments. A hole was trimmed with a large round carbide bur. The hole was then conditioned using a primer (Multisil, Bredent, GmbH&Co, Senden, Germany). According to the manufacturer's instructions, the R.S was allowed to cure for 3 min and excess silicone was trimmed with a special cutter (GSQ-Cutters, H 251 GSQ, Komet Dental, Lemgo, Germany) [Figure 1]
- Control Group (n = 15, OR):The OR (Paltop Implants, Burlington, MA, USA) was placed on the ball attachment which is then picked up by the intaglio of the mandibular overdenture with metal housing secured with auto-polymerizing resin (direct pickup technique). The excess material is trimmed and polished with acrylic trimming and polishing burs [Figure 2].
|Figure 1: Master cast of mandibular dental arch with two natural teeth retained ball superstructure. Fitting surface of Retention.sil-retained mandibular overdentures with Retention.sil 600 in place|
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|Figure 2: Master cast of mandibular dental arch with two natural teeth retained ball superstructure. Fitting surface of O-ring-retained mandibular overdentures with Metal housing and O-ring in place O-ring-retained maxillary overdentures|
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Retention force testing before thermocycling (vertical dislodging)
We adopted the methodology utilized by many other studies as Gamborena et al., Fromentin et al., Botega et al., da and Evtimovska et al. for evaluation of retention force of the selected attachments. Metal chains were attached to the hook-on overdenture in the faciolingual surfaces at two points in the right and left premolar region. To determine the retention against vertically directed dislodging forces that are parallel to path of insertion, two-point vertical pull was used. The edentulous acrylic model (analog base block) was attached to the lower compartment of the universal testing machine position parallel to the horizontal plane and securely tightened to the machine, while overdenture-simulating blocks were attached to the upper moving arm of the universal testing machine. Dislodgement of the denture from the attachments was performed using the universal testing machine parallel to the long axis of the ball abutments. The universal testing machine (MTS E 43, Guangdong, China) controlled by a computer to interface the (MTS test suite software) was used to apply maximum seating and dislodging forces [Figure 3]. The crosshead speed was adjusted at 50 mm/min as this has been reported to approximate the speed of the movement of the denture away from the ridge during mastication. To simulate repeated insertions and removals of the overdenture over a 6-month period (assuming three daily removals and insertions of the overdenture for the purpose of hygiene), each overdenture was pulled out 540 times and insertion of 4 mm magnitude of movement at a frequency of 12 cycles/min. Retention forces as indicated on the digital indicator were calculated three times (initially, after each 90 cycles). Three records of maximum load needed to dislodge the experimental overdenture from the mandibular test model (retentive force) were recorded in Newton (N) and averaged for each specimen as the retention value after the simulation of 6 months of use. A specimen was considered failed if showed separation between attachment components and overdenture-simulating blocks, or separation of attachment components itself. This model permitted to perform and evaluate dislodgement in axial direction only.
|Figure 3: Experimental overdenture assembly connected to universal testing machine for applying the repetitive dislodging cycles (vertical dislodging) and the simultaneous measurement of the retentive forces|
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Thermocycling of all the overdentures with the attachments placed on the edentulous models were subjected to manual thermocycling using SUPolytubs (Schuler Dental GmbH & Co. KG, Germany); one maintained at 5° ± 1° and other at 55° ± 1°. The test samples were subjected to a total of 540 cycles with each cycle equivalent to 30 s of dwell time in each temperature-controlled tub with a transfer time of 10 s, with 540 thermal cycles being equivalent to 6 months of service in the oral cavity [Figure 4]. None of the samples failed.
|Figure 4: All the samples tested were subjected to manual thermocycling using S–U–Polytubs Manual thermocycling unit (Two SUPolytub, Schuler Dental)|
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Retention force testing after thermocycling
Each of the models were again subjected to 100 pulls each to dislodge the overdenture from the acrylic model and values as indicated were tabulated.
| Results|| |
The present in vitro study was carried out to investigate the retentive force of two female matrix retention systems (R.S and widely employed ORs) in tooth-supported overdenture, relative to each other.
Thirty mandibular canine root-anchored ball attachments for overdentures were fabricated to form two groups, each with a different attachment design. Group 1 (test) received 15 pairs of ball/R.S attachments, while the other Group 2 (control) received 15 pairs of ball/OR. Retention force was measured by subjecting them to 540 cycles of insertion and de-insertion cycles (representing 6 months functional life) in the axial direction.
All the tested samples showed positive retention values. None of the tested samples failed. The data are calculated as a mean of the three readings collected as three retention values for each sample measured at initial and final removal and insertion cycles. 40 retention values were measured for each group in this study.
Statistical analysis was performed using the Statistical Package for Social Science (SPSS ver 12) software. The normality of the data was accessed using Kolmogrov–Smirnov and Shapiro–Wilk test. The null hypothesis for this test is that there is no difference between test and control group in retention values. Data analyzed show that samples do not follow normal distribution. Hence, normal distribution is not assumed and test for assessment of nonparametric data is applied for further calculations.
To test the hypothesis of equality of means, among the two groups, Group I and Group II with respect to retention values, nonparametric Friedman test and Chi-Square test was carried out, and P < 0.05 was considered to be statistically significant. Wilcoxon signed-rank test was used for intragroup comparison. The Mann–Whitney U test is used to compare differences between two independent groups (inter-group comparison).
In Group 1 (R.S system), χ2 (1) = 2.996, P > 0.001. The mean ranks obtained for retention values of (±standard deviation [SD]) were 1.59 before thermocycling (BT) and 1.33 at after the completion of 540 thermal cycle thermocycling (AT). The mean retention fell from 5.90 ± 0.77 at baseline to 5.41 ± 0.72 N after 540 cycles and the percentage of retention loss was 8.28% ± 2.98%. No significant decrease was seen between AT and baseline (Z = −1.91 P > 0.001). Hence, the hypothesis of equality of means is accepted at 5% level of significance (P > 0.05), which signifies that means are not statistically significant. The Mann–Whitney test applied to compare initial and final retention identified no significant differences (P = 0.11) between mean values. The post hoc analysis with Wilcoxon signed-rank test was conducted with a Bonferroni correction applied, resulting in no significance level set at P < 0.001. This implying less retention loss with R.S system throughout the cyclic load testing cycles. Results are tabulated in [Table 1], [Table 2], [Table 3], [Table 4], [Table 5] and [Figure 5] and [Figure 6].
|Table 4: Intragroup comparison of retention values within the Retention.sil group (before thermocycling and after thermocycling) using Wilcoxon signed-rank test ranks|
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|Figure 5: Comparison of change in mean retention level of Retention.sil group and O-ring group before thermocycling at 540 cycles|
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|Figure 6: Comparison of change in mean retention level of Retention.sil group and O-ring group after thermocycling at 540 cycles|
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In Group II (OR system), χ2 (1) = 11.45, P < 0.001. The mean ranks obtained for retention values of (±SD) were 1.64 BT and 1.21 AT. The mean retention fell from 17.36 ± 3.81N BT to 12.91 ± 3.18 N AT and the percentage of retention loss was 25.79% ± 5.61%. A significant decrease was seen between AT and BT (Z = −3.111, P < 0.001). Hence, the hypothesis of equality of means is rejected even at 5% level of significance (P < 0.05), which signifies means are statistically significant. The post hoc analysis with Wilcoxon signed-rank test was conducted with a Bonferroni correction applied, resulting in significance level set at P < 0.001.
Further, for multiple comparison, i.e., Group I versus Group II, Mann–Whitney U-test was carried out to find out which pair or pairs differ significantly at 5% level of significance. When Groups I and II were compared, at the end of the study (540 cycles), the differences in retention were statistically significant. P< 0.001 is of statistical significant with retentive values of AT 5.41 ± 0.72 N and 12.91 ± 3.18 N for R.S attachments and OR, respectively. The R.S group showed a mean percentage loss of 8.28% ± 2.98% of the initial retention and the OR group showed total loss of 25.79% ± 5.61% of the initial retention. The results suggest that there is significant difference in retention values in both the tested group.
The OR attachment showed the highest mean retentive force of 17.36 ± 3.81 N and 12.91 ± 3.18 N BT and AT, respectively. The maximum retentive force was exhibited by the OR, 12.91 ± 3.18 N (cycle no. 546) followed by R.S group attachment, 5.41 ± 0.72 N (cycle no. 546). A decrease in the retention force was observed in both attachment systems after subjecting them to thermal cycles. However, this decrease was found to be statistically significant (P < 0.05) only in OR attachment. On the other hand, there are no significant differences in the mean retention force of R.S group as a female matrix system for overdenture retention. The results obtained are summarized in [Table 6].
| Discussion|| |
Over the past decades, retaining a natural tooth to and utilizing it as an abutment for overdentures have gained popularity. To add, with the advent of attachments, it has become one of the most sought-after treatment modalities. Questions regarding the most effective mode of attachment between the overdentures and supporting implants remain unanswered. Attachment systems that permit ease of prosthesis placement and removal and those that are readily hygienic may be preferable for elderly patients. Furthermore, they should permit the atraumatic and even distribution of stress to both the mechanical and biological supporting structures. PVS material called R.S has been introduced as silicone matrix for implant overdenture, in place of the attachment system component in the denture's base. It is proposed to be both constantly retentive as well as easy to remove. In addition to an economical chairside technique to attach the matrix to the prosthesis, it offers higher chewing comfort because of the material's flexibility and accompanied by low adherence of microbial plaque as well as decreased stress to the supporting tissues. Further, it provides greater latitude of movement and comfort to the patients who are Bruxer.
However, scientific data are scarce for large-scale use. The retentive PVS-RS attachments used in this study have never been evaluated before on root-supported overdenture. Therefore, we presume that, if the R.S attachment system being investigated in this study can achieve a minimal retention force of overdenture retention, it is likely to be suitable for clinical applications. Hence, the current in vitro study was carried for the first time to evaluate the retentive property of retentions OR in root-supported overdenture.
- As there are many factors that contribute to overdenture retention and stability intraorally, in this in vitro study design, we have tried to closely simulate real conditions of intraoral mode. (1) The analogs were placed 22 mm apart as it had been reported that natural canines are separated by the same distance. However, Doukas et al. stated that there are no standardized rules for the ideal distance between attachments to achieve optimum retention. (2) Traditional Instron testing machines are the most common instrument used to replicate the vertical separation of the denture from the mouth. It had been accepted as reliable and valid instrument to test peak load forces in vitro.,, (3) Retentive and stabilizing properties also depend on dislodgement speed. 50 mm/min dislodgement speed was selected in our study to compare results with other studies, as majority of them have used similar testing conditions. (4) Since overdentures generally do not have a distinct path of insertion or removal, retentive properties were tested under standardized conditions during linear dislodgement in order to get more constant results. (5) The acrylic resin was used in the construction of the mandibular model because of the similarity in modulus of elasticity between the compact bone and acrylic resin as suggested by Ichikawa et al. (6) Previous studies have measured retentive force of overdenture attachments with a variety of dislodgement speeds ranging from 0.5 mm/min to 150 mm/s.,,,, The 50 mm/min dislodgement speed was selected for easier comparison of results with those from the majority of previous studies. (7) The presence of saliva and constant occlusal load may affect the rate of attachment wear and thus the retentive values. The presence of soft tissue resiliency may increase the load on the abutments and therefore affect the retentive values. However, artificial substitutes are not considered to be as efficacious as human saliva or soft tissue. Furthermore, simulation of such factors is difficult in an in vitro study, and those factors are better evaluated in clinical trials. Although in vitro studies differ from clinical studies, they allow standardization of the tests by excluding oral conditions, and therefore, they provide important information.
In our study, a total of 20 specimens each with R.S 600 and OR female attachment were tested for loss of retention after being subjected to 540 cycles (6 months) cyclic dislodgement with thermocycling to investigate its effect on the retention force. After cyclic dislodgement, the results showed in the OR group a strong negative linear relationship between the cyclic times and retention force (P < 0.0001). On the other hand, in R.S group, there is no significant change in relationship between the cyclic times and retention force (P > 0.0001).
The present study obtained final retention values, after 540 cycles, as 5.90 ± 0.77 for the R.S attachments compared to before treatment values of 5.41 ± 0.72, indicating only a slight loss of 0.49 N. Furthermore, the most interesting finding of this study is the significantly lower percentage (8.28% ± 2.98%) of retention loss for R.S. In our study, the reasons for this slight decrease in retention force of R.S 600 attachment is not clear, it can be attributed to the fact that repeated dislodgement might have reduced the hardness and stiffness of PVS. This would have lead to low wear of the surface ball attachment and decrease in retention force loss.
Our results showed good retention forces of RS overdenture attachment for the tested 540 cycles (6 months), which is in accordance with previous investigations of overdenture attachments.,,,, Since there is no in vitro conducted comparing RS with OR attachments in root-supported overdenture, no clinical data exist at this time. Hence, we have attempted indirect comparison with implant-supported ball/OR with PVS attachments. Klamper, 2013 used R.S for implant prosthesis and reported that patients were particularly satisfied with comfort and fit and suggested that it could be a potential alternative to hard snaps in hybrid prosthesis. Li et al., 2020 proposed a new clinical concept of applying a polyetheretherketone (PEEK) post core restoration combined with a PVS attachment overdenture system. It was concluded that the PEEK post-core restoration with PVS attachments showed the favorable retention force, which corresponds to an appropriate retention force for 10 years of clinical use.
However, the most interesting finding of this study is the significantly lower percentage of retention loss (8.28%) for R.S. Nonetheless, the retention forces of PVS attachments (R.S 600) after 540 cycles are constantly higher than 5 N, probably sufficient to the minimum requirements (>5 N) for overdenture stability. Based on this information, the retention forces of R.S attachment systems tested in the present study would be acceptable after 6 months of usage.
Another interesting property of R.S noted in the literature is that, shorter time needed for disconnection of PVS attachments compared to other attachments. Considering these facts, Petropoulos et al. recommended, in cases of patients suffering from bruxism where excessive lateral forces are already present, selecting a less retentive attachment system. The PVS system is less retentive and thus delivers less force to its abutments. Being more elastic as mechanic attachments, but more stable than magnetic attachments, it will release easily compared to more retentive attachments when subjected to excessive lateral forces. Since the time needed for disconnection is less, this might be an advantage to repeat itself in the final position before separating completely when viscous food is chewed. Another clinical situation where an attachment of lower retention might be desirable is a patient with dexterity problems, who might have difficulty inserting and removing the overdenture. Besides this, they can also find use in a geriatric setting.
The ball abutment with OR attachment system has long been used for overdentures and has been clinically proven providing favorable results. It is commonly used due to their simplicity in design, ease in cleaning, and minimum leverage. The technical work required is minimal and can be carried out at chairside, thus making it cost-effective.,, However, clinical studies have reported that overdentures supported by ball and OR attachments need maintenance more often due to wear of the polymeric ring component of the OR, which needs to be exchanged to maintain the retention of the prosthesis.,
The present study obtained final retention values, after 540 cycles, as 17.36 ± 3.81 N and significantly higher percentage of retention loss, 12.91% ± 3.18% for the OR attachments. Further, the OR attachments exhibited the highest peak as well as the highest mean retention force at the end of the study. Retention slowly decreased at the end of cycle but still significantly higher than RS attachment. This could be attributed to high retentive power of OR and faster wear and plastic deformation of the nylon inserts.,
Our results were in agreement other studies. Rodrigues et al. evaluated the retention force characteristics of two-implant embedded in polyester resin with OR attachments in different implant angulations. At 0° implant angulation, the retention loss was 16.6% over a simulation of 6 months. After simulation of 12 months, the retention loss was 36.9%. While after simulation of 24 months, there was increasing in retention loss, which was 57.1%, indicating that the elastomeric OR is worn out when used more. Reda et al. compared the retention force of three different types of overdenture attachment systems used in implant-retained mandibular complete overdentures. Each specimen was subjected to 5500 cycles of insertion and removal in the presence of artificial saliva, representing 5 years of usage. They found that regardless of the initial retention level of overdenture attachment, gradual loss of retention values is inevitable.
The thermocycle aging treatment on the retention forces revealed that the retention forces before aging were significantly higher than those after aging for OR attachment system. Retention forces affected by the aging treatment, probably caused by the repeated wear and artificial saliva. In a study by Rutkunas et al. reported similar findings: OR attachment groups showed a significant decrease of retentive force in the range of 21%–62% after artificial aging. In an another study, a decrease of 73.9% of the initial retentive force was recorded after 5400 insertion–removal cycles of locator attachments, whereas no significant changes in retentive force were observed for magnet attachments.
Our results have to be interrupted with caution as it has been performed under a controlled experimental simulation and there are few inherent limitations. (1) The sample size of the specimen used was relatively small but was in accordance with previous similar experiments. (2) This study was performed under a controlled experimental simulation to evaluate the insertion and removal cycles only in centric loading. This might lead to lower aging effects compared to eccentric loading. Further, the factors such as oral environment and saliva could have also contributed an influence on the results when simulated in the oral conditions. (3) SEM analyses of pattern of wear were not investigated, as it was beyond the scope of this study. (4) The manual aging thermocycling treatment was performed at room temperature, which cannot mimic the thermal cyclic in oral conditions. (5) Different angulation of ball attachment and the influence of various fluids on retentive power were not investigated. (6) Only 540 cycles stimulating 6 months short-term usage was investigated.
Hence, future researches have to be directed to clarify: (1) how long retention silicones can ensure the retention of the dentures in clinical conditions. (2) In the case of PVS attachments, the experimental setup might be extended. According to Depprich et al., PVS used for maxillary obturator prostheses was highly affected bacterial colonization, supporting gingival and mucosal inflammation. Therefore, further in vitro and in vivo investigations should take this aspect into account to verify the longevity of PVS attachment systems. (3) Retention force has to be checked with variable fluid environments, multidirectional force application, load–unload conditions, the effects of fatigue on material properties, and influence of fungus growth, hygiene procedures, and chemical attack. (4) Development of retention silicones with the possibility of generating higher retention power. (5) Understand the cause for loss in retention force of R.S attachment systems. (6) Evaluate the retentive properties of these components for longer periods of cyclic fatigue. (7) To test the retention properties of the R.S attachment systems with different ball angulations.
Within the limitations of this in vitro study design, the following conclusions can be drawn:
- The OR attachments showed higher retention than RS attachments
- The RS attachments provide constant favorable retention forces >5 N for simulated 540 cycle. This corresponds to an appropriate retention force for 6 months of stimulated clinical usage and the minimum required for stabilizing the prosthesis. Hence, it can be considered as a cost-effective new attachment option with ease of doing and thus might be considered as a promising concept for overdenture retention in short- and medium-term management.
HEMPEGOWDA Institute of Medicak Sciences, Institutional ethicas Committe (registerd under CDSCO vide file no. ECR/307/KRIS/Inst/Kar/2013).
The authors thank the Laboratory of Jyothi Institute of Technology for the facilities.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]