|Year : 2013 | Volume
| Issue : 2 | Page : 74-81
Reaction of rat subcutaneous tissue to mineral trioxide aggregate and Portland cement: A secondary level biocompatibility test
P Karanth1, MK Manjunath2, Roshni3, ES Kuriakose4
1 DDS, Private practice, Charlotte, USA
2 Department of Conservative Dentistry and Endodontics, J.S.S Dental College and Hospital, Mysore, India
3 Department of Conservative Dentistry and Endodontics, Vydehi Institute of Dental Sciences and Research Centre, Bangalore, India
4 Department of Conservative Dentistry and Endodontics, Sri Sankara Dental College, Varkala, Kerala, India
|Date of Web Publication||26-Jul-2013|
No 12, F Block, J.P. Nagar, Mysore
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: This secondary-level animal study was conducted to assess and compare the subcutaneous tissue reaction to implantation of white mineral trioxide aggregate (MTA) and white Portland cement. Study Design: Polyethylene tubes filled with either freshly mixed white MTA (Group I) or white Portland cement (Group II) were implanted subcutaneously into 12 Wistar Albino rats. Each animal also received an empty polyethylene tube as the control (Group III). After 7, 14, 21 and 30 days, the implants, together with surrounding tissues were excised. Two pathologists blinded to the experimental procedure, evaluated sections taken from the biopsy specimens for the severity of the inflammatory response, calcification and the presence and thickness of fibrous capsule surrounding the implant. Statistical analysis was performed using the Cross-tabs procedure, Univariate analysis of the variance two-way and the Pearson product moment correlation to assess inter-rater variability between the two evaluators. Results: At 7 days, there was no significant difference in the severity of inflammation between the control group, white MTA, and white Portland cement groups. In the 14 day, 21 day and 30 day test periods, control group had significantly less inflammation than white MTA and white Portland cement. There was no significant difference in the grading of inflammation between white MTA and white Portland cement. All materials exhibited thick capsule at 7 days and thin capsule by 30 days. Conclusion: Both white MTA and white Portland cement were not completely non-irritating at the end of 30 days as evidenced by the presence of mild inflammation. However, the presence of a thin capsule around the materials, similar to the control group, indicates good tissue tolerance. White MTA and white Portland cement seem to be materials of comparable biocompatibility.
Keywords: Biocompatibility, white mineral trioxide aggregate, white Portland cement
|How to cite this article:|
Karanth P, Manjunath M K, Roshni, Kuriakose E S. Reaction of rat subcutaneous tissue to mineral trioxide aggregate and Portland cement: A secondary level biocompatibility test. J Indian Soc Pedod Prev Dent 2013;31:74-81
|How to cite this URL:|
Karanth P, Manjunath M K, Roshni, Kuriakose E S. Reaction of rat subcutaneous tissue to mineral trioxide aggregate and Portland cement: A secondary level biocompatibility test. J Indian Soc Pedod Prev Dent [serial online] 2013 [cited 2021 Feb 27];31:74-81. Available from: https://www.jisppd.com/text.asp?2013/31/2/74/115698
| Introduction|| |
The original mineral trioxide aggregate (MTA) material, which was grey in color, has been used world-wide, with several clinical applications such as pulp capping, apical barrier, perforation repair, root-end filling material, and orthograde filling material.  This material has been tested thoroughly in vivo, through implantation tests and usage tests. It has been shown to consistently allow for regeneration of the periodontal ligament, apposition of a cementum-like material and formation of bone. 
The grey MTA has been patented as being composed of American Standards for Testing Materials Type I Portland cement (ordinary grey Portland cement) with a 4:1 addition of bismuth oxide added for radiopacity.  This generated interest in the evaluation of grey Portland cement as an alternative to MTA as grey Portland cement costs less and is widely available. Comparative analysis revealed no significant difference between the different elements in grey Portland cement and grey MTA except there was no detectable quantity of bismuth in grey Portland cement. 
The biocompatibility of grey MTA and grey Portland cement has also been compared and both materials have been found to be biocompatible. 
Grey MTA has been associated with the occasional staining of teeth when used in pulpotomy and pulp capping procedures.  Therefore, a white MTA material has been developed recently to overcome this concern. As with other endodontic materials, the white MTA should undergo the same extensive testing as grey MTA. 
Review of the literature reveals only limited research carried out on this new material. It was reported in one study that the behavior of osteoblasts is very different in contact with the surface of white MTA compared to grey MTA.  Furthermore, it is unclear if white MTA was formulated using the white Portland cement as base material.  Therefore, the objectives of the present study were to evaluate and compare the biocompatibility of white MTA and white Portland cement by implanting them in the subcutaneous tissue of rats as a secondary level biocompatibility test [Table 1].
| Materials and Methods|| |
A total of 12 healthy, male, 6-month-old Wistar Albino rats (Rattus Norvegicus albinus) weighing 220-230 g were selected and kept in well-ventilated cages. The animals were maintained on a normal diet during the period of the study. All recommended points by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Chennai, in care and use of laboratory animals were observed at all stages of the project.
Polyethylene infant feeding tube size 06, GS-4008 (Romsons, Agra, India) with an outer diameter of 2 mm and inner diameter of 1.1 mm was cut into 36 pieces of 10 mm length each. 12 of these were selected, for use as negative control (Group III). In the remaining 24 pieces, a window of 6 mm length and 0.5 mm width was cut on the body of the pellet with a Bard Parker (BP) blade no. 11. These tubes were to be used for the experimental cements and the window was to ensure controlled area of contact of the test materials with the surrounding tissues. All the tubes were sterilized by formaldehyde gas [Figure 1].
|Figure 1: Diagrammatic representation of the prepared polyethylene tube for implantation of experimental cements|
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ProRoot MTA powder from the pouch was dispensed onto a mixing pad. Contents of the ProRoot liquid micro-dose ampoule were squeezed onto the mixing pad next to the powder. The liquid was gradually incorporated into the cement using a plastic spatula and mixed for 1 min, according to the manufacturer's instructions.
1 g of white Portland cement powder, previously sterilized with formaldehyde gas, was dispensed onto a glass slab. A total of 0.4 ml of distilled water was placed next to the powder. Mixing was performed with a cement spatula for 30 s as described in previous studies. ,
The pastes were then filled into the polyethylene pellets using the plastic filling instrument and endodontic plugger. Moist gauze was placed on top of the mixed cements to extend their working time so as to ensure that cement in the freshly mixed state was implanted in the animals.
Total 3 implantation sites were selected on the dorsum of each rat, about 2 cm laterals to the vertebral column; 2 sites directly posterior to the forelimb and 1 site approximately 3 cm posterior to the first site.
The site directly posterior to the left forelimb was used for implantation of white MTA pellet (Group I) and the site directly posterior to the right forelimb was used for implantation of white Portland cement pellet (Group II). The site posterior to the second site was used for implantation of empty polyethylene tube (Group III). The 12 animals were randomly divided into 4 subgroups A, B, C, and D of 3 animals each, for biopsies to be taken at 7, 14, 21, and 30 days after the implantation respectively.
The rats were anesthetized with diethyl ether. The surgical sites were shaved and scrubbed with 70% isopropyl alcohol. Three stab incisions 2 mm wide were made in relation to the limbs as shown in [Figure 2]. The trochar cannula was inserted through each incision up to about 1 inch away from the incision. The trochar was removed, leaving cannula inside the subcutaneous tissue. The pellet was inserted into the cannula such that the window faced away from the muscle layer. Using a smaller sized cannula as plunger, the pellet was pushed to be deposited in the tissue. A gradual withdrawal of the cannula when the pellet was being pushed inside helped in the gentle positioning of the pellet in the tissue.
|Figure 2: The three incisions and sites of implantation on the dorsum of the rat|
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The animals were sacrificed with an overdose of ether anesthesia. Following shaving and scrubbing with isopropyl alcohol, the pellets, along with surrounding tissue were dissected out with the help of scissors and gently soaked over a sterile gauze piece to remove any blood. The specimens were placed in 10% formalin for fixation. After 6 h, the specimens were taken out of the solution and the polyethylene tubes were carefully removed through a small incision cut for the purpose. Removing the tube at this stage helped to assure that the partly fixed tissue retained the space created by the tube; thus, facilitating interpretation of the microscopic picture. Removal of the tube permits easier cutting of paraffin sections.  Fixation was completed by another 42 h of immersion in the 10% formalin solution.
After 48 h of fixation and histologic processing, serial 6 μm sections were made at right angles to the long axis of the tube space, using a rotary microtome. 3 sections, representative of the center and ends of the tube were stained with hematoxylin and eosin and mounted for evaluation. A total of 108 slides were examined.
All the slides were examined and rated by two pathologists who had no knowledge of the materials used in the experiment. The slides were scanned at low power (×4) for representative areas with the highest density of inflammatory cells. Then, inflammatory responses were graded by counting the number of inflammatory cells at high power (×40) in the field showing maximum inflammatory cell infiltrate as described in previous studies , [Table 2].
In addition, the slides were evaluated for the presence of edema, necrosis, vascular congestion, calcification, and fibrous capsule. Fibrous capsule was categorized as thin when thickness was < 150 μm and thick at > 150 μm. 
Statistical methods applied
- Cross-tabs procedure (Contingency coefficient test)
- Univariate analysis of variance two-way
- Pearson product moment correlation.
| Results|| |
The animals tolerated the surgical procedures well. No apparent adverse events occurred during the study period of 30 days. Since, there was a significant correlation (P < 0.001) between the histopathologic scores given by the two evaluators, only results assessed by one evaluator are presented. [Table 3], Graph 1 [Additional file 1] and [Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7] and [Figure 8].
|Figure 3: Photomicrograph showing severe inflammation, necrosis (n) and calcification (c) (×40)|
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|Figure 4: Photomicrograph showing moderate inflammation, calcification (c) (×40)|
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|Figure 6: Photomicrograph showing no inflammation and thin capsule (t) (×40)|
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|Figure 7: Photomicrograph showing thick capsule (t) (c) calcification, ×10|
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Statistical analysis (Cross tabs procedure) revealed that there was no significant difference in the grading of inflammation between the materials in the 1 st week. There was a significant reduction in inflammation for all the materials (groups) at week 2, 3, 4 (P < 0.001). However, within the subgroups B, C and D (14, 21, and 30 days), control group had significantly lower grading of inflammation than white MTA and white Portland cement groups (P < 0.001). Two-way ANOVA revealed that there was no significant difference in the effect of white MTA and white Portland cement over inflammation.
| Discussion|| |
Biocompatibility can be assessed by in vitro and in vivo methods. The in vivo subcutaneous implantation method possesses several of the qualities desired of a test for biologic evaluation of endodontic materials. Relatively uncomplicated, it permits a comparative interpretation of data within one animal, with the least number of variables. The inflammatory response is a characteristic phenomenon common to all fibrous connective tissue and varies little from tissue to tissue or from animal to animal among the higher species. 
Whilst materials to be tested were directly applied subcutaneously in some studies, the implantation of materials in polyethylene, dentin, silicon or Teflon tubes is advocated in others. In the 1960's Torneck showed that subsequent to the implantation of polyethylene tubes, fibrous tissue repair occurred with no lasting inflammation.  Placement of test materials in polyethylene tubes controls the quantity and form and prevents the material from major disintegration, eliminating these variables.  It stabilizes the material in place and achieves standardization of material-tissue interface. A model that resembles the root canal system is also obtained.  Therefore, polyethylene tubes 10 mm length were used in this study as 10 mm length tubes were found to be least irritating when compared to other lengths.  A window on the body of the polyethylene tube was for a fixed amount of material and its controlled release.
Techniques for subcutaneous implantation include injection of a known quantity of a drug or the implantation of materials through incisions in the skin. In this study, the modified implantation technique on the dorsal of rats was performed as pioneered by Bhat and Walvekar.  This technique permits implantation to be performed with minimum trauma. Surgery and anesthesia are short and minimal and mortality of experimental animals is reduced. 
Inflammatory reaction is less intense in adipose or muscle tissue than in the highly vascular connective tissue. Therefore, care was taken to see that the window on the polyethylene tube always faced the dermis and not the muscle when the tubes were being implanted.
It was decided to make observations for both the grade of inflammation and fibroblastic response and capsule formation as the inflammatory process is closely intertwined with the process of repair. Inflammation serves to destroy, dilute or wall off the injurious agent, but it, in turn, sets into motion a series of events that as far as possible, heal and reconstitute damaged tissue. 
In the control group, there was severe inflammation at the 7 day test period. Trauma from the surgical procedure as well as the reaction to a foreign substance could be the reason for this observation. A thick capsule was noted in all specimens attributable to enhanced collagen synthesis cytokines (Interleukin-1 [IL-1], IL-4) and growth factors (Platelet-Derived Growth Factor PDGF and Tumor Growth Factor TGF-β) secreted by leucocytes.  By the 3 rd week, most of the specimens exhibited no inflammation and thin capsule and by 30 days, all specimens were devoid of inflammation and had thin capsule. This is in agreement with previous work.  The thinning of capsule seen in the latter test periods is a sign of repair by fibrosis. Growth factors that stimulate collagen synthesis also modulate the remodeling of the connective tissue frame-work ultimately converting the tissue into a scar composed of dense collagen. 
In the white MTA group, the severe response noted at 7 days was most likely multi-factorial with high pH,  heat generated during setting and generation of inflammatory cytokines (IL1 and 6),  contributing to the process. The presence of inflammatory cells in the tissue adjacent to the material compared to control at the 3 rd and 4 th week can be taken as evidence of tissue irritability through the cumulative effect of operative trauma, toxicity of the material and continued seepage of particles of the material into surrounding tissues. Capsule surrounding the tube was thick for the 1 st 2 weeks. It was thin by the 4 th week, similar to the control group indicating that the material was well-tolerated by the tissues.
The observation of focal areas of calcification in the capsule surrounding the implant in all four test periods is in agreement with previous studies.  According to Holland et al.  the granulations seen in tissues surrounding MTA were calcite crystals originating from a reaction of calcium from the calcium hydroxide with carbon dioxide from tissue. Sarkar et al.  found that MTA underwent dissolution in synthetic tissue fluid releasing calcium, which led to the precipitation of hydroxyapatite. They argued that since tissue fluid is rich in phosphate ions and its carbonate content is relatively low such a milieu chemically favored the formation of hydroxyapatite and not calcite.
Reactions to white Portland cement were similar to that of white MTA. Although the average inflammatory grading for white MTA were generally lower than for the white Portland cement group, the difference was not statistically significant. The higher alkalinity,  and more heavy metals in the composition of white Portland cement,  could have played a role in the observed difference. The focal areas of calcification noted at all test periods can be attributed to the bioactivity of white Portland cement in the presence of phosphate containing fluids, leading to the formation of carbonated apatite via an initial amorphous calcium phosphate phase.  The inflammation was mild and the capsule surrounding the white Portland cement implants were thin at the 30-day evaluation period suggesting that the material was well-tolerated by the tissues.
The mechanism of action of MTA and Portland cement favoring hard tissue deposition is similar to that of calcium hydroxide.  On hydration, MTA and Portland cement produce silicate hydrate gel and calcium hydroxide.  Thus, this similarity in their mode of biologic action stems from their propensity to release calcium and ability to form hydroxyapatite. 
None of the tested materials was "bland" or completely non-irritating by the end of the study period of 30 days. However, for a root-end filling material, initially some type of cytotoxic reaction may be partially beneficial. A mild irritant could stimulate both a fibrous and a calcific deposition, which could wall off the root canal filling from the periapical region as seen in apexification procedures using the calcium hydroxide.  Further studies, of longer duration are necessary to determine if the white MTA and the white Portland cement eventually become bland.
The results from this study suggest that white Portland cement has the potential to be developed as a root-end filling material. However, while white MTA is manufactured under strict regulations conforming to international standards and is approved for use by the FDA, industrially manufactured white Portland cement is not. Additional studies of longer duration and on bone reactions of animals are recommended to ensure that white Portland cement meets the medical device requirements set out by the FDA.
| Conclusion|| |
Within the limits of this study:
- White MTA and white Portland cement were surrounded by fibrous connective tissue indicating that they were well-tolerated by the tissues
- There were no significant difference in inflammatory responses between white MTA and white Portland cement at 7, 14, 21, and 30 days, following implantation.
| Acknowledgment|| |
We are grateful to Dr. Shivabasavaiah, Professor, PG Department of studies and research in Zoology, University of Mysore, for his help in procuring the ethical clearance and in helping to perform the experimental procedures. We wish to express gratitude to Dr. Lakshman, Consultant Pathologist, Kannan Diagnostic Center, Sayyaji Rao Road, Mysore, Dr. Paranjyothi, Head, Department of Oral Pathology, J.S.S. Dental College, and her team including Dr. Shylaja, Dr. Anju and Dr. Vidya for their insightful guidance and help in the histopathological evaluation. We gratefully acknowledge Mr. Paul Ovsepian and Dentsply-Maillefer for donating the ProRoot MTA material for this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]
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