To evaluate the biocompatibility of the Indian Portland cement with potential for use in dentistry: An animal study.

AIMS: This study evaluated the biocompatibility of the Indian Portland cement with potential for use in dentistry.
MATERIALS AND METHODS: This study was performed in Swiss albino mice, by implanting the Indian Portland cement pellets subcutaneously. After 1, 3, and 6 weeks the tissue specimens were prepared for histological examination.
RESULTS: The histological analysis showed moderate to severe inflammation at 1 week. The inflammation gradually decreased by 6 weeks, with most of the specimens showing the absence of inflammatory reaction.
CONCLUSIONS: According to these experimental conditions, the tested Indian Portland cement was biocompatible.

INTRODUCTION

Calcium hydroxide, suggested by Herman in 1920, has been used as an intra-canal medicament in different clinical situations such as to promote apexification, to repair perforation, to enhance healing of periapical lesions, to control root resorption, and to control exudation in teeth with persistent periapical inflammation. The ability to stimulate mineralization associated to the antimicrobial effectiveness, histocompatibility and a highly alkaline pH confers on it, the current success as an endodontic medication.[1]

When Ca(OH)2 is used in various applications, therapy may extend from months to years before the desired effects are achieved. Furthermore, it has been observed that Ca(OH)2 treated immature teeth show a high failure rate because of an unusual preponderance of root fracture and it has been suggested that changes in the physical properties of dentin by the Ca(OH)2 medicament may be responsible for this.[2] This chemical has several disadvantages, such as variability of treatment time (average 12.9 months),[34] difficulty of the patient's recall management, delay in the treatment, and increase in the risk of tooth fracture after dressing with calcium hydroxide for extended periods.[4567]

Alternatives to calcium hydroxide have been proposed to overcome these problems; the most promising being mineral trioxide aggregate (MTA). The advantages of this material are multiple:

Possibility to restore the tooth with a minimal delay in treatment time, and

It also avoids changes in the mechanical properties of dentine.[8] Camilleri et al. showed that both white and grey MTA have a similar chemical constitution to Portland cement except for the addition of bismuth oxide to make it radiopaque.[910] MTA is a type of hydraulic cement that can set in the presence of water. It is a mineral powder that consists of hydrophilic particles, whose principal components are tricalcium silicate, tricalcium aluminate, tricalcium oxide, and other mineral oxides. It has a pH of 12.5 and sets in the presence of moisture in approximately 4 h. It presents acceptable sealing ability, biocompatibility, and low cytotoxicity and induces odontoblasts to form hard tissue barriers.[11] MTA was developed at the University of Loma Linda (USA) to seal communications between the root canal system and the external tooth surface at all levels. The elevated cost of this product has not allowed its use in all levels of health attention.[12]

Therefore, the possibility of using Portland cement as a less expensive alternative to MTA in dental practice should be considered. Portland cement is made from materials mined from the earth and is processed using energy provided by fuels; and, therefore, may contain trace amounts of naturally occurring minerals which might be detected during chemical analysis.

Biocompatibility is the ability of a material to perform with an appropriate host response in a specific application. This means that the tissue of the patient that comes into contact with the materials does not suffer from any toxic, irritating, inflammatory, allergic, genotoxic, or carcinogenic action.[13] MTA is a derivative of Portland cement containing certain other additives. Although Portland cement shows similarities with MTA in its main components (tricalcium silicate, dicalcium silicate, tricalcium aluminate, and tetracalcium aluminium ferrite), few reports have dealt with biocompatibility or antimicrobial aspects of the Portland cement. However, there are no studies till date to confer the biocompatibility of the Indian Portland cement.

Thus, the aim of this study is to assess the biocompatibility of the Indian Portland cement for dental clinical use.

MATERIALS AND METHODS

Thirty mice (Swiss Albino), aged 6 weeks to 8 weeks old and weighing 25-40 g, maintained at the small animals section of Institute of Animal Health and Veternary Biologicals, Bengaluru were used for this study. Ethical clearance was obtained for the undertaken study. There was one group of 10 animals each for an experimental period of 1, 3, and 6 weeks.

Portland cement (Birla White) was sterilized in an autoclave. Holes measuring 5 mm diameter and 2 mm thickness were drilled in thermoforming sheets to prepare Portland cement pellets. The cement was mixed with distilled water and pellets were made, which were again autoclaved. The animals were anesthetized by the administration of Ketamine HCL (Aneket) (44 mg/kg body weight) and Xylazine (Indian Immunologicals Ltd.) (4-6 mg/kg body weight) intramuscularly using insulin syringes. The back of the animals were shaved and disinfected with a povidone-iodine solution (Betadine, Win Medicare Pvt., Ltd.,). Incisions (5 mm) were made on either side of the dorsum, and pockets were prepared by a blunt dissection.

Portland cement pellet was placed in one pocket, and the incision was closed with surgical nylon sutures [Figure 1a and b]. Suturing was also performed on the opposite side without a pellet, which was used as a control. After the experimental stage, the animals were kept in cages according to the group and placed in isolation. The animals after implantation were maintained on a balanced diet. At the end of each period (1, 3, and 6 weeks), the animals were anesthetized and the pellets were removed along with the surrounding tissue and immersed in 10% buffered formalin [Figure 1c and d]. Normal tissue on the opposite side was also sectioned and stored in 10% formalin. The animals were then euthanized with an anesthetic overdose. After fixing for 48 h, the tissue was processed for paraffin embedding and serial sections were cut and stained with hematoxylin and eosin.

Figure 1

(a) Indian Portland cement pellets being implanted. (b) Implantation of the pellets and suturing. (c) Overlying skin excised to reveal the pellet embedded on the subcutameous tissue. (d) The specimen after excision

The tissue responses were graded as being mild, moderate, and severe according to the criteria already published by previous authors and correlating the changes to the normal tissue specimens.

The criteria for scoring the inflammatory tissue response are as follows:

Grade 0 - no inflammation.

Grade 1 - slight inflammation.

The thickness of the reaction zone is similar to or only slightly wider than the normal tissue, no or a few inflammatory cells.

Grade 2 - moderate inflammation.

An increased reaction zone in which macrophages, plasma cells, or both are present.

Grade 3 - severe inflammation.

An increased reaction zone in which macrophages, plasma cells, occasional foci of neutrophil granulocytes, lymphocytes, or both are present.

The results were analyzed statistically by Chi-square/Fisher Exact test. The interpretations of the results were based on statistical analysis of the data to determine whether the material could be accepted for clinical use in dentistry.

RESULTS

The following findings were observed in Group I (sacrificed 1 week after surgery): Mild to the moderate inflammatory reaction in most of the specimens and severe inflammation in one specimen. The fibrous tissue, congested vessels, engorged capillaries, and lymphocytes were observed. No signs of degeneration and necrosis were seen in any specimen. There was an evidence of intact adipose tissue. The specimens in Group I suggested an acute inflammatory response to Portland cement [Figure 2a].

Figure 2

(a) Large dilated capillaries with engorged RBC's, mild inflammatory infiltrate and normal adipose tissue. (b) Mild inflammatory cell infiltrates with few capillaries seen admixed with collagen fibres. Areas of myxoid and adipose tissue seen. (c) Mild inflammatory cell infiltrates with few capillaries, loose connective tissue stroma with lymphoid aggregate. (d) Superficial loose connective tissue showing collagen bundles with fibroblasts and capillaries and deeper connective tissue shows muscle bands

Group II (sacrificed 3 weeks after surgery): Granulomatous reaction, few capillaries, and chronic inflammatory reaction with a predominance of lymphocytes, fibroblasts, and few plasma cells. Neutrophils, eosinophils, and macrophages were not seen. There was no evidence of degeneration and necrosis, except for a few areas with dystrophic calcification and granulation. Reparative reactions were evident with wall formation and intact adipose tissue depicting the absence of infection [Figure 2b].

Group III (sacrificed 6 weeks after surgery): There was decreased inflammation with well-organized granulation tissue. Few specimens showed few lymphocytes with plasma cells. Enlarged and decreased number of capillaries and tertiary lymphoid follicles with no signs of necrosis and degeneration was evident [Figure 2c].

The normal tissue on the opposite side was also examined, which showed superficial loose connective tissue showing collagen bundles with fibroblasts and capillaries and deeper connective tissue with muscle bands. Loose myxoid tissue with adipocytes and capillaries was also evident [Figure 2d].

Based on the scores [Table 1], 80% of the specimens showed signs of slight inflammation, 10% showed signs of moderate and 10% showed signs of severe inflammation in Group I.

Table 1

Scores of the inflammatory response at 1, 3 and 6 weeks

In Group II, 50% specimens showed slight inflammation, 20% demonstrated moderate, 10% showed severe inflammation, and 20% specimens did not show any signs of inflammatory reactions.

In Group III, 20% of the specimens displayed slight inflammation, moderate inflammation was observed in 20% specimens, and there was complete resolution of the inflammatory process in 60% specimens (Graph).

Graph

Results of the percentage of bio-compatibility for all experimental groups

Statistical analysis of the degree of the inflammatory reaction was evaluated using the Chi-square and Fisher Exact test. Individual comparison between groups revealed that there was no statistical significance (P ≥ 0.05) in the percentage of inflammatory reactions in all the groups, except for the “no inflammation” category in Group III which showed a statistical significance with P = 0.029.

DISCUSSION

Before any dental material is employed on human beings, it must be submitted to several studies in order to demonstrate that it may be applied in a safe and efficient manner. Investigations on the Portland cement have recently been initiated in dentistry since it was observed that this cement basically had the same chemical composition as the MTA, besides the addition of bismuth to provide radiopacity.[14]

The biocompatibility of Portland cement has previously been documented.[15] Holland et al. have shown similar mechanisms of action of Portland cement, MTA and calcium hydroxide.[16] Saidon et al. claimed that both MTA and Portland cement have the same physical, chemical, and biological properties.[17] Camilleri et al. have demonstrated that both Portland cement and MTA are composed of tricalcium and dicalcium silicate which on hydration produce calcium silicate hydrate gel and calcium hydroxide.[9] In another study, it was shown that it is the cement elution rather than the cement itself that was biocompatible. Both Portland cement and MTA release calcium hydroxide as a reaction by-product, and this is leached out of the material at an early stage.[10] Elutions collected from cement after 1 and 7 days demonstrated a high degree of biocompatibility.

The findings of previous investigations demonstrated the biocompatibility of this cement after implantation of this material in polyethylene tubes in the subcutaneous connective tissue of rats during 7, 12, and 60 days. In this study, an attempt has been made to evaluate the biocompatibility of the Indian Portland cement, to make it suitable for clinical dental applications.

There are three basic types of tests used to measure the biocompatibility of dental materials: The in vitro test, the animal test, and the usage test performed either in animals or in humans. The greatest disadvantage of in vitro tests is their potential lack of relevance to the in vivo use of the material. The in vitro environment also lacks the complex coordination of systems that are present in an organism such as an immune system, an inflammatory system, and a circulatory system. Regardless of the type of test used, the advantage of an animal test is its ability to allow an intact biological system to respond to a material. The material may interact with the many complex biological systems within the animal, and a complete biological response is, therefore measured.[18]

This study was conducted in rats because this experimental model met the basic criteria required for such study and also a 1-week life span of a rat correlated to one and a half years to that in humans. Hence, the results could be obtained at the shorter duration of intervals. furthermore, the implantation of endodontic filling materials into the subcutaneous connective tissue of rats is a valid screening method for testing biocompatibility according to some studies done earlier.[19] It is proposed that in vivo implantation of materials in laboratory animals provides more information about the inflammatory and immune responses developed by the test material.[20]

The test material may be directly injected or implanted (either directly or within Teflon, silicone or polyethylene tubes) into various tissues, such as the subcutaneous connective tissue, muscle or bone of rats, rabbits, guinea pigs, hamsters, and ferrets.[21] In this study, Portland cement pellets were implanted directly over the tissue rather than placing polyethylene tubes impregnated with the cement mix, to aid in better contact of the material with the tissue, so that tissue reactions were more pronounced.

Based on histopathological examination of the specimens, congested vessels were observed in Group I, revealing the inflammatory status of the specimens. It should be noticed that the Portland cement displayed a tendency toward chronic inflammation in Group II, therefore indicating biocompatibility. These findings are in agreement with Holland et al. (2002).[16] Group III showed few inflammatory cells and evident reparative reaction. It is significant to note that the initial severe reaction decreased over time and had been resolved at the end of the experiment although a few persistent inflammatory cells still remained.

The results of this work have disclosed that the evaluated material presented biocompatibility in rat subcutaneous tissue.

CONCLUSION

At the given intervals of the study, Indian Portland Cement is a biocompatible material to be used for various clinical applications in dentistry and medicine.

Financial support and sponsorship

Institute of Animal Health and Veternary Biologicals, Hebbal, Bengaluru.

Conflicts of interest

There are no conflicts of interest.