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Basic ResearchTechnology

Effects of Bone Graft Materials on the Microhardness of Mineral Trioxide Aggregate


Erick Y. Sato, DDS, Timothy Svec, DDS, MS, Brian Whitten, DDS, and Christine M. Sedgley, MDS, MDSc, PhD
Abstract
Introduction: Large through-and-through lesions have been reported to heal faster and better when lled with bone graft material at the time of an apicoectomy. It is unknown what effect these have on retrograde lling materials such as white mineral trioxide aggregate (WMTA). In this study, the null hypothesis was tested that the presence of bone graft materials does not affect the microhardness of WMTA. Methods: Freshly mixed WMTA was condensed into acrylic cylinders and preincubated aerobically at 37 C for 1 hour. Cylinders were immersed in simulated body uid in close proximity to graft materials: xenograft (Bio-Oss, n = 60), freezedried bone allograft (MinerOss, n = 60), demineralized freeze-dried bone allograft (OraGraft, n = 40), and allograft (Puros, n = 60). Knoop microhardness of half the samples in each group was evaluated after 2 weeks of incubation and the remainder at 4 weeks. The values for each group were then compared with 2-way analysis of variance and Bonferroni post hoc tests. Results: WMTA microhardness values for Bio-Oss, MinerOss, and Puros groups were lower than those for OraGraft and control groups regardless of incubation period (P < .001); values for the OraGraft group were higher than those for the control group at 2 weeks (P < .001), with no difference at 4 weeks. Microhardness values were higher at 4 weeks compared with 2 weeks for MinerOss (P < .05), OraGraft (P < .01), and control (P < .001), with no differences for Bio-Oss and Puros groups. The null hypothesis was rejected. Conclusions: Demineralized and mineralized graft materials appear to have a differential effect on the microhardness of WMTA. (J Endod 2012;38:700703)

recent survey of the members of the American Association of Endodontists revealed that bone replacement grafts are used by 85.6% of respondents who use guided tissue regeneration techniques (1). Graft materials used include xenografts (eg, bovine bone) (2), freeze-dried bone allograft (FDBA) (3), and demineralized freeze-dried bone allograft (DFDBA) (4). The placement of resorbable graft materials might facilitate hard tissue healing after apicoectomies, especially for large lesions (5), lesions that violate both buccal and lingual cortical plates (6), and in apicomarginal defects (7). Other studies show no reliable benets of bone graft material when used after apicoectomies (2). There is no information on the possible effect graft materials have on the physical properties of retrograde lling materials. White mineral trioxide aggregate (WMTA) has been used as a retrograde lling material (8, 9). Surface microhardness of WMTA is related to its hydration process (10) and can be affected by the pH and the presence of ions (11, 12). It is feasible that the presence of ions such as calcium, phosphate, and carbonate, inorganic components of bone graft material, might likewise affect the hydration process of WMTA. Proprietary methods of graft processing have the potential to alter the mineral composition of the graft materials; indeed, DFDBA is intentionally demineralized. These differences in mineral composition might alter the hydration of WMTA to varying extents. Although no benets of bone graft placement have been denitively demonstrated in large clinical studies, it is worthwhile to explore the possibility that WMTA retroll might be inuenced to varying degrees depending on the type of graft material used. If the hydration of WMTA is affected by the presence and type of graft material, this might be a consideration when deciding on the application of hard tissue grafts in situations that involve their placement against freshly condensed WMTA retrolls. In this study the microhardness of WMTA exposed to different types of bone graft materials was evaluated. The null hypothesis was tested that the presence of bone graft materials does not affect the microhardness of WMTA.

Materials and Methods


Commercially available white ProRoot MTA (WMTA) (Dentsply/Tulsa Dental, Tulsa, OK) from the same lot was used. WMTA was mixed according to the manufacturers recommendations with a powder to liquid ratio of 3:1 and then condensed with an amalgam carrier and hand condensers into 280 custom-made, hollow, acrylic cylinders (external diameter of 4.8 mm, internal diameter of 1.6 mm, and height of 3 mm). Condensation of WMTA into the cylinders was performed against a clean glass slab to prevent extrusion of material out the opposite end of the cylinder. Markings were made on the cylinder to indicate which side was facing the glass slab and which side was facing the condenser during condensation. Attempts were made to use consistent

Key Words
Bio-Oss, bone graft materials, Knoop microhardness, MinerOss, MTA, OraGraft, Puros, white mineral trioxide aggregate

From the Department of Endodontology, School of Dentistry, Oregon Health and Science University, Portland, Oregon. Supported by the AAE Foundation and the OHSU Department of Endodontology Les Morgan Endowment Fund. Address requests for reprints to Dr Christine M. Sedgley, Department of Endodontology, School of Dentistry, Oregon Health and Science University, 611 SW Campus Drive, Portland, OR 97239. E-mail address: sedgley@ohsu.edu 0099-2399/$ - see front matter Copyright 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.02.019

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condensation forces by 1 operator (E.Y.S.), because condensation forces have been shown to affect microhardness of WMTA (13, 14). Care was taken to ensure that the nished surface of WMTA was ush with the end of the acrylic cylinder and that the surface of WMTA was not physically disturbed during the incubation period. Cylinders lled with WMTA were preincubated in 100% humidity at 37 C for 1 hour to allow initial hydration of WMTA. Four different bone graft materials were used, each from 1 lot of material: Bio-Oss (Osteohealth/Luitpold Pharmaceuticals, Shirley, NY), Puros (Zimmer Dental, Inc, Carlsbad, CA), MinerOss (BioHorizons/Osteotech, Inc, Eatontown, NJ), and OraGraft (LifeNet Health, Inc, Virginia Beach, VA) (Table 1). An equal amount (60 mg) of graft material mixed with 20 mL of 0.9% sterile saline solution was placed into the base of individual wells of 96-well plates (Bio-Oss, n = 60; MinerOss, n = 60; OraGraft, n = 40; and Puros, n = 60). Control wells (n = 60) contained no graft material. Simulated body uid (SBF) (165 mL) was added to each well 10 minutes after graft material placement; SBF has a pH of 7.40 and contains concentrations of the following ions similar to that found in human blood: Na+, K+, Mg2+, Ca2+, Cl, HCO3, HPO42, SO42, and was prepared as previously described (15). indentation were (1) an elongated diamond indentation with equilateral sides and (2) well-delineated opposing corners. Analysis was performed 1 group at a time. The microhardness of 1 of the 2 surfaces of exposed WMTA was analyzed for each cylinder; 1 surface only was included per cylinder to ensure independent samples. The surface to be analyzed was randomly selected by a coin toss. Five separate indentations were made at distances no closer than twice the width or length of neighboring indentations. The mean of the readings from the 5 indentations was taken as the microhardness of that specimen. After 4 weeks of incubation, the remaining cylinders from control (n = 30), Bio-Oss (n = 30), MinerOss (n = 30), OraGraft (n = 20), and Puros (n = 30) groups were analyzed as described above.

Incubation of WMTA with Graft Materials Cylinders were condensed with WMTA and preincubated for 1 hour in batches of 9 cylinders. After preincubation, cylinders were placed randomly among the various experimental and control groups to reduce the effects of batch preparation on the nal results. Cylinders with WMTA were completely submerged in SBF in each of the prepared wells as shown in Figure 1. Cylinders were placed with the condensed WMTA facing the sides so that each of the 2 sides of the condensed WMTA were exposed to nominally similar ionic environments in the well and not in direct contact with the graft material. An adhesive lm was placed over the 96-well plates to prevent evaporation of the solutions. Plates were incubated at 37 C in a humidied incubator. To facilitate the even distribution of ions in solution during the duration of the experiment, the 96-well plate was gently rocked every other day. Microhardness Tests After 2 weeks of incubation, cylinders from control (n = 30), BioOss (n = 30), MinerOss (n = 30), OraGraft (n = 20), and Puros (n = 30) groups were recovered. Both ends of each cylinder were wetground at room temperature with 2400-grit silicon carbide paper mounted on a rotating polishing table. The cylinders were polished so that the opposing surfaces of the cylinder remained parallel, because this is critical for microhardness testing. The polished surfaces were then rinsed with tap water and air-dried. A Duramin-5 Knoop microhardness tester (Struers, Inc, Cleveland, OH) was used with a load of 100 g and dwell time of 10 seconds; this load and dwell time were optimized for use with 40 magnication. The shape of the indentation made on any material by a Knoop microhardness tester can be described as an equilateral parallelogram or elongated diamond with a 172 internal angle. The distance between the furthest corners of the indentation is measured, and the Knoop microhardness is calculated by software. The criteria for a quality
TABLE 1. Bone Graft Materials Used in This Study Graft
Bio-Oss MinerOss OraGraft Puros

Statistical Analyses Pilot studies with Portland cement as a substitute for WMTA showed that 30 samples per group were required to obtain a statistical power of 80% with 95% condence interval. Subsequent preliminary experiments with WMTA showed a sample size of 20 gave a statistical power of 95% with 95% condence interval. The Kolmogorov-Smirnov test veried normality of the data sets. The t test conrmed that microhardness values of the 2 surfaces of the cylinder did not differ. The microhardness values between groups at each incubation time period (2-week and 4-week) were compared with 2-way analysis of variance (ANOVA) and Bonferroni post hoc tests. Statistical signicance was set at P <.05. A post hoc power analysis was performed.

Results
The mean Knoop microhardness values of WMTA after 2 and 4 weeks of incubation with different bone graft materials are listed in Table 2. Two-way ANOVA showed that both graft material and incubation period had highly signicant effects on WMTA microhardness (P < .0001).

Comparison of Graft Groups At both 2 and 4 weeks, WMTA microhardness values for the Puros, Bio-Oss, and MinerOss groups were signicantly lower than for the OraGraft and control groups (P < .001). Two-week values for WMTA incubated with OraGraft were signicantly higher than those for the control group (P < .001), and at 4 weeks there was no difference between OraGraft and control groups. Incubation Period WMTA microhardness values were signicantly higher at 4 weeks of incubation compared with 2 weeks of incubation with MinerOss (P < .05), OroGraft (P < .01), and control (P < .001). No differences in WMTA microhardness were noted between the 2-week and 4-week time frames for Puros and Bio-Oss groups. Post hoc power analyses indicated a statistical power of at least 97% with 95% condence interval between the 2-week experimental and control groups and of 100% with 95% condence interval between

Graft type
Xenograft (bovine) Allograft (FDBA) Allograft (DFDBA) Allograft (Tutoplast)

Mineral content
Mineralized Mineralized Demineralized Mineralized

Particle type
Cancellous Cortical and cancellous Cortical Cancellous

Particle size
0.251 mm 0.61.25 mm 0.250.71 mm 0.251 mm

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lous bone. The preparation of OraGraft (DFDBA) used in this study was demineralized to contain 1.0%4.0% residual calcium by using a patented Pulsatile Acid Demineralization technology. The surface area of the graft materials potentially affects the ionic interactions with SBF. For this reason, an attempt was made to keep the particle size as similar as possible to reduce variations in surface area. Microhardness of MTA was assessed, because it has been associated with the hydration process of MTA (10). The hydration products occupy more than twice the volume of the anhydrous cement during the setting process (16). As a result, anything in the environment that changes the hydration process of WMTA has the potential to signicantly change WMTAs physical properties (11, 12). Products of the hydration process of MTA are calcium silicate hydrate and calcium hydroxide (16). In this study, it is possible that calcium ions released from the mineralized graft material saturated the solution and inhibited the release of calcium silicate and calcium hydroxide. Inhibiting the production of these hydration products might have interfered with the hydration process of WMTA. Likewise, the absence of calcium in the demineralized graft material might have allowed the formation of hydration by-products in solution. Another plausible explanation for the experimental results involves the formation of calcium phosphate precipitates similar to hydroxyapatite. It is possible that the precipitate formed on WMTA could inuence its surface microhardness. Previous studies have demonstrated the formation of precipitate on the surface of setting WMTA when exposed to phosphate-containing solutions (17). The pH of WMTA has been reported to rise to 12.5 about 3 hours after mixing (18). As the pH rises, the solubility of calcium and phosphate falls, forcing the calcium and phosphate to precipitate out of solution. It is possible that the existing apatite crystals on the mineralized graft material served as a template onto which the calcium and phosphate readily precipitated. Because demineralized graft material has relatively few existing apatite crystals to serve as a template, deposition of calcium and phosphate into the surface layers of WMTA might be forced to occur to a greater degree. The cause of the alteration in hydration behavior of WMTA in the presence of various graft materials in this study remains speculative. Future studies might investigate the changing concentrations of free hydrogen, calcium, and phosphate ions in SBF over time. Previous studies have demonstrated that pH plays an instrumental role in the hydration of MTA (19). Scanning electron microscopic analysis and energy dispersive x-ray spectroscopy might be used to characterize the surface of WMTA when set under these experimental conditions. These analyses might help to elucidate the roles of graft material and SBF in the hydration process of WMTA in this experimental setup. The 2-week and 4-week time frames were chosen because other studies have observed changes in WMTAs properties during periods of weeks after mixing (2023). The results of this study are

Figure 1. Experimental setup for incubation of WMTA with graft materials in assay well of 96-well plate.

the 4-week experimental and control groups. The null hypothesis was rejected.

Discussion
Within the limitations of this study, the results demonstrate that close proximity of bone graft materials inuences the microhardness of WMTA. Although the microhardness of WMTA was higher after incubation with demineralized bone graft material compared with controls, the opposite was found with the other materials tested. After 4 weeks of incubation, the microhardness of the control and demineralized graft groups equilibrated, but the microhardness of WMTA incubated with mineralized graft materials remained lower than control. Respondents to a recent American Association of Endodontists survey reported usage of DFDBA (67.3%), FDBA (29.9%), alloplasts (22.2%), xenografts (15.7%), and autogenous bone replacement grafts (11.1%) (1). In this study, the graft materials investigated similarly varied with respect to source, treatment, mineral content, and particle type (Table 1). Bio-Oss is a xenograft composed of proteinized bovine bone. Puros (allograft) is treated by a patented Tutoplast process. MinerOss (FDBA) is supplied as a mixture of crushed cortical and cancel-

TABLE 2. Knoop Microhardness Values (mean standard deviation) of WMTA Incubated with Bone Graft Materials Bone graft material Incubation period
2 weeks 4 weeks

Control*
61.6 9.4 77.4 14.8

Bio-Oss
51.2 8.8 46.7 6.9

MinerOss
40.8 6.0 49.78.0

OraGraft
72.4 9.1 80.1 10.8

Puros
49.1 7.1 53.3 7.6

*Higher at 4 weeks compared with 2 weeks (P < .001). Higher at 4 weeks compared with 2 weeks (P < .01). Higher at 4 weeks compared with 2 weeks (P < .05). Lower than control at same incubation period (P < .001). Higher than control at same incubation period (P < .05).

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consistent with those of Nekoofar et al (23), who found an increase in WMTA microhardness during an extended period (ie, between 4 and 28 days). In this study, the amount of WMTA used in each sample was relatively small in comparison with other studies that have evaluated the physical properties of this material (11, 24, 25), but it might be a closer representation of the volume and dimensions used clinically. Another aspect of the protocol that differs signicantly from other studies is the incubation of WMTA with SBF instead of deionized water, saline, or phosphate-buffered saline. This was done to more closely mimic plasma ion concentrations, because ionic concentrations can have an effect on the setting of WMTA (11, 26). Clinically, the coagulum in the center of the osteotomy remains devoid of inammatory and reparative cells for several days (27). As such, no active circulation exists around the retroll material during this time period; however, there is likely to be at least some passive diffusion of ions away from the setting WMTA. In the present study, no attempt was made to compensate for ionic diffusion to or from the surgical site. The circulatory exchange of uids that develops during the later stages of healing was also not simulated. It is possible that these ion exchanges in a clinical environment allow the hydration process of WMTA to proceed unhindered. The indication for bone graft use in apical crypts after apicoectomies is controversial (1, 28). There are reported benets of grafting in the treatment of apicomarginal defects (7), large lesions (5), and lesions that violate both buccal and lingual cortical plates (6). However, a study by Bernab e et al (29) showed that placement of graft material in the crypt has no effect on the histologic appearance of osseous tissue approximating retrolls consisting of gray MTA in 6 dogs. It has also been reported that placement of graft material in routine apicoectomies associated with smaller lesions carries no benet (2). There are potential risks associated with graft use; recent reports of the presence of prions in bovine xenograft preparations have raised concerns about possible transmission to patients (30). Certainly, graft materials raise the cost of the procedure for the patient. Potential benets of graft material use should be weighed carefully against these risks and costs. Microhardness is only 1 of many interdependent, measurable properties of WMTA. Although the data from the current study show that demineralized and mineralized graft materials appear to have a differential effect on the microhardness of WMTA, the clinical significance of this is not known. As such, future clinical studies could take the potential for these effects into consideration.
5. Pecora G, Kim S, Celletti R, Davarpanah M. The guided tissue regeneration principle in endodontic surgery: one-year postoperative results of large periapical lesions. Int Endod J 1995;28:416. 6. Taschieri S, Testori T, Azzola F, Del Fabbro M, Valentini P. [Guided-tissue regeneration in endodontic surgery]. Rev Stomatol Chir Maxillofac 2008;109:2137. 7. Dietrich T, Zunker P, Dietrich D, Bernimoulin JP. Periapical and periodontal healing after osseous grafting and guided tissue regeneration treatment of apicomarginal defects in periradicular surgery: results after 12 months. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:47482. 8. Saunders WP. A prospective clinical study of periradicular surgery using mineral trioxide aggregate as a root-end lling. J Endod 2008;34:6605. 9. Christiansen R, Kirkevang LL, Horsted-Bindslev P, Wenzel A. Randomized clinical trial of root-end resection followed by root-end lling with mineral trioxide aggregate or smoothing of the orthograde gutta-percha root lling: 1-year follow-up. Int Endod J 2009;42:10514. 10. Lee YL, Lin FH, Wang WH, Ritchie HH, Lan WH, Lin CP. Effects of EDTA on the hydration mechanism of mineral trioxide aggregate. J Dent Res 2007;86:5348. 11. Giuliani V, Nieri M, Pace R, Pagavino G. Effects of pH on surface hardness and microstructure of mineral trioxide aggregate and Aureoseal: an in vitro study. J Endod 2010;36:18836. 12. Shie MY, Huang TH, Kao CT, Huang CH, Ding SJ. The effect of a physiologic solution pH on properties of white mineral trioxide aggregate. J Endod 2009;35:98101. 13. Nekoofar MH, Adusei G, Sheykhrezae MS, Hayes SJ, Bryant ST, Dummer PM. The effect of condensation pressure on selected physical properties of mineral trioxide aggregate. Int Endod J 2007;40:45361. 14. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature reviewpart I: chemical, physical, and antibacterial properties. J Endod 2010;36: 1627. 15. Oyane A, Kim HM, Furuya T, Kokubo T, Miyazaki T, Nakamura T. Preparation and assessment of revised simulated body uids. J Biomed Mater Res A 2003;65: 18895. 16. Camilleri J. Hydration mechanisms of mineral trioxide aggregate. Int Endod J 2007; 40:46270. 17. Bozeman TB, Lemon RR, Eleazer PD. Elemental analysis of crystal precipitate from gray and white MTA. J Endod 2006;32:4258. 18. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end lling material. J Endod 1995;21:34953. 19. Lee YL, Lee BS, Lin FH, Yun Lin A, Lan WH, Lin CP. Effects of physiological environments on the hydration behavior of mineral trioxide aggregate. Biomaterials 2004; 25:78793. 20. Camilleri J. Characterization of hydration products of mineral trioxide aggregate. Int Endod J 2008;41:40817. 21. Yildirim T, Er K, Tasdemir T, Tahan E, Buruk K, Serper A. Effect of smear layer and root-end cavity thickness on apical sealing ability of MTA as a root-end lling material: a bacterial leakage study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e6772. 22. Danesh G, Dammaschke T, Gerth HU, Zandbiglari T, Schafer E. A comparative study of selected properties of ProRoot mineral trioxide aggregate and two Portland cements. Int Endod J 2006;39:2139. 23. Nekoofar MH, Aseeley Z, Dummer PM. The effect of various mixing techniques on the surface microhardness of mineral trioxide aggregate. Int Endod J 2010;43: 31220. 24. Saghiri MA, Lot M, Joupari MD, Aeinehchi M, Saghiri AM. Effects of storage temperature on surface hardness, microstructure, and phase formation of white mineral trioxide aggregate. J Endod 2010;36:14148. 25. Saghiri MA, Lot M, Saghiri AM, Vosoughhosseini S, Aeinehchi M, Ranjkesh B. Scanning electron micrograph and surface hardness of mineral trioxide aggregate in the presence of alkaline pH. J Endod 2009;35:70610. 26. Aggarwal V, Jain A, Kabi D. In vitro evaluation of effect of various endodontic solutions on selected physical properties of white mineral trioxide aggregate. Aust Endod J 2011;37:614. 27. Harrison JW, Jurosky KA. Wound healing in the tissues of the periodontium following periradicular surgery: IIIthe osseous excisional wound. J Endod 1992;18:7681. 28. Lin L, Chen MY, Ricucci D, Rosenberg PA. Guided tissue regeneration in periapical surgery. J Endod 2010;36:61825. 29. Bernabe PF, Gomes-Filho JE, Cintra LT, et al. Histologic evaluation of the use of membrane, bone graft, and MTA in apical surgery. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:30914. 30. Kim Y, Nowzari H, Rich SK. Risk of prion disease transmission through bovinederived bone substitutes: a systematic review. Clin Implant Dent Relat Res 2011. Dec 15. doi: 10.1111/j.1708-8208.2011.00407.x.

Acknowledgments
The authors thank Dentsply/Tulsa Dental, Osteohealth/Luitpold Pharmaceuticals, Inc, and BioHorizons/Osteotech, Inc for supplying materials. The authors deny any conicts of interest related to this study.

References
1. Naylor J, Mines P, Anderson A, Kwon D. The use of guided tissue regeneration techniques among endodontists: a web-based survey. J Endod 2011;37:14958. 2. Taschieri S, Del Fabbro M, Testori T, Weinstein R. Efcacy of xenogeneic bone grafting with guided tissue regeneration in the management of bone defects after surgical endodontics. J Oral Maxillofac Surg 2007;65:11217. 3. Saad AY, Abdellatief EM. Healing assessment of osseous defects of periapical lesions associated with failed endodontically treated teeth with use of freeze-dried bone allograft. Oral Surg Oral Med Oral Pathol 1991;71:6127. 4. Chen G, Fang CT, Tong C. The management of mucosal fenestration: a report of two cases. Int Endod J 2009;42:15664.

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