|Year : 2012 | Volume
| Issue : 1 | Page : 6-13
Biocompatibility assessment of modified Portland cement in comparison with MTA® : In vivo and in vitro studies
I Khalil1, J Isaac2, C Chaccar3, JM Sautier2, A Berdal2, N Naaman3, A Naaman1
1 Department of Endodontics, Saint Joseph University, Beirut, Lebanon
2 Laboratory of Molecular Oral Physiopathology, Team 5, UMRS 872, Research Center of Cordeliers, Paris, France
3 Department of Periodontology, Saint Joseph University, Beirut, Lebanon
|Date of Web Publication||10-Dec-2012|
Department of Endodontics, Faculty of Dental Medicine, Saint-Joseph University, P.O. Box 166255, Beirut
Aim: The aim of our study is to elaborate a new cement based on Portland cement (PC), Modified Portland Cement (MPC) with modified chemical and physical properties that allow easier clinical manipulation and faster setting time than MTA® and then to evaluate its cytotoxicity in vitro and its biocompatibility in vivo in comparison with MTA® . Materials and Methods: Elaboration of MPC: Portland cement powder slenderly grinded to homogenize the particles, mixed with a radiopaque element and a setting time accelerator. A comparative in vitro study (MTS test) of the toxic effect of MTA® and MPC with culture isolated from the calvaria of 18-day-old fetal Swiss OF1 mice are done. A comparative in vivo study of the biocompatibility of MTA® and MPC: Under general anaesthesia, three holes (2.5 mm) were made in both the left and right femurs of six White New Zealand rabbits. In the first hole MPC is placed, in the second MTA® and the third one is left empty (negative control group). Three weeks after implantation, two rabbits are sacrificed, then two other rabbits over six weeks and the last two after twelve weeks. The neck of the femur is trimmed and prepared for undecalcified histological studies. Mann-Whitney test was used to analyze the results. Results: The cell viability test according to the morphological observations suggested the biocompatibility of the two biomaterials tested. The in vivo test showed similar biocompatibility between MTA® and MPC. Bone healing and minimal inflammatory response adjacent to MTA® and MPC implants were observed at all experimental periods (3, 6 and 12 weeks), suggesting that both materials are well tolerated. Conclusion: This pilot comparative study of MTA® and MPC showed no or very limited toxic effects of both cements in vitro and similar biocompatibility in vivo. However, additional in vivo and clinical studies should be done on MPC before it can be introduced in our clinical practice.
Keywords: Biocompatibility, cytotoxicity, MTA® , Portland cement
|How to cite this article:|
Khalil I, Isaac J, Chaccar C, Sautier J M, Berdal A, Naaman N, Naaman A. Biocompatibility assessment of modified Portland cement in comparison with MTA® : In vivo and in vitro studies. Saudi Endod J 2012;2:6-13
|How to cite this URL:|
Khalil I, Isaac J, Chaccar C, Sautier J M, Berdal A, Naaman N, Naaman A. Biocompatibility assessment of modified Portland cement in comparison with MTA® : In vivo and in vitro studies. Saudi Endod J [serial online] 2012 [cited 2015 Mar 27];2:6-13. Available from: http://www.saudiendodj.com//text.asp?2012/2/1/6/104415
| Introduction|| |
Mineral trioxide aggregate (MTA® ) was developed as a root-end filling material for periapical surgery and for sealing communications between the root canal system and the surrounding tissues.  MTA® was shown to be superior to other commonly used root-end filling materials such as amalgam, IRM® , and Super-EBA® in studies of marginal adaptation and leakage.  However, despite MTA® qualities like high biocompatibility and sealing ability, it is often difficult to manipulate (sandy consistency) and often requires a second appointment for the placement of a restoration in order to allow for setting. 
Any ability to accelerate the setting of the material to within a single appointment time frame, and the ability to manipulate and rinse around the MTA® without the possibility of displacement would be greatly beneficial. In fact, the principal component of MTA® is Portland cement (PC) that sets in the presence of moisture and blood.  Given the similarity between MTA® and PC, the question is raised whether PC additives are interchangeable with MTA® . Several studies have proven that MTA® and PC have many similar physical, chemical, and biologic properties, that PC shows promising potential as an Endodontic material and that data on it may be used for the further development or modification in order to improve its physical characteristics and expand its scope of clinical applications but none suggested that it could simply replace MTA® mainly because PC is not radiopaque and has a longer setting time. ,,
Biological compatibility of all root canal cements is of importance as these materials frequently come into contact with periapical tissues and the tissue response to these materials may influence the final outcome of the root canal treatment.  MTA was reported to be biocompatible in many in vitro and in vivo studies.
In vitro cytotoxicity experiments involve the use of cell and tissue culture methods. Cell expression and growth have been used by several investigators to evaluate the biocompatibility of root-end filling materials including MTA. ,, Materials tested should have at least one flat surface and should be sterilized prior to subjecting it for biocompatibility testing.
In vivo biocompatibility method involves the use of animal models. ,,, MTA activity of animal tissues can be studied by histological evaluation of the tissues involved.
The purpose of this study was to evaluate, in vitro and in vivo, the cytotoxicity as well as biocompatibility of the MPC in comparison with MTA.
| Materials and Methods|| |
Elaboration of modified portland cement
By far the most common and most important hydraulic cement in modern construction is PC, the basic ingredient of concrete which is made primarily of calcium, silicon, aluminum, iron and small amounts of other ingredients to which gypsum is added in the final grinding process to regulate the setting time of the concrete, in a high temperature process that drives off carbon dioxide and chemically combines the primary ingredients into new compounds. The basic raw materials for PC are calcium oxide or lime (CaO), silica (SiO 2 ), alumina (Al 2 O 3 ) and iron oxide (Fe 2 O 3 ). These components are appropriately proportioned to produce various types of PC. In manufacturing PC, the selected raw materials are crushed, ground, and proportioned for the desired composition then blended. 
The PC is composed of particles with a wide range of size, whereas for the new cement we obtained a uniform and smaller particle size because chemical and physical properties of cements depend mainly on the manner and degree to which they are ground and the resulting particle size. The fineness of a cement is indicated by the cement's Blaine number, which represents the ratio of the cement's particle surface area to its weight (square centimeters of surface per gram). Faster setting cements, like that preferably utilized in the present study, have a Blaine number in the range of 4,000-5,500 cm 2 /g.
The mixture is then fed into a rotary kiln where it is heated to temperatures of up to 1400°-1650°C. In this process the mixture is chemically converted to cement clinker which is subsequently cooled and pulverized. A small amount of gypsum (CaSO 4.2H.sub.2 O) is added to the cement to control the setting time of the concrete. The resulting cement consists principally of tricalcium silicate (3CaOSiO 2 ), dicalcium silicate (2CaOSiO 2 ), tricalcium aluminate (3CaOAl 2 O 3 ) and tetracalcium aluminoferrite (4CaOAl 2 O 3.Fe.sub.2 O 3 ).
The characteristics of the cement composition depend upon the size of the particles, the powder-water ratio, temperature, presence of water, and entrained air. PC hardens by reactions with water, which are called hydration reactions. These reactions are complex, but principally involve the reaction of tricalcium silicate (3CaOSiO 2 ) and dicalcium silicate (2CaOSiO 2 ) with water. When these compounds react with water during the hardening of the cement, the principal hydration product is tricalcium silicate hydrate. This material is a colloidal gel of extremely small particles (less than 1 μm). The tricalcium silicate hardens rapidly and is most responsible for the early strength of Portland cement. The dicalcium silicate has a slower hydration reaction and is mainly responsible for strength increases beyond one week. Tricalcium aluminate, which plays a lesser role in the hardening process, hydrates rapidly also and contributes to early strength of development. 
PC is not naturally radiopaque, so a radiopaque component should be added to render it radiopaque for purposes of dental diagnostics. Barium chloride (BaCl 2 ) has been found to be a suitable compound. In addition, calcium carbonate (CaCO 3 ) was also combined with the present cement composition to help facilitate its manipulation. The powders are sterilized (under 180°C for 2 hours).
In vitro cytotoxicity evaluation of MPC in comparison with MTA
Osteoblasts were isolated enzymatically from the calvaria of 18-day-old fetal Swiss OF1 mice. Briefly, calvaria were aseptically dissected and fragments were incubated in phosphate-buffered saline solution (Invitrogen, San Diego, CA, USA) with 0.25% collagenase (Sigma, St Louis, MO, USA) for 2 hours at 37°C. Then, cells were dissociated from the bone fragments by mechanical pipetting, washed in PBS and plated at 4 × 10 4 cells/cm 2 . The cells were grown in Dulbecco's Modified Eagle Medium (Invitrogen, San Diego, CA, USA) supplemented with ascorbic acid (50 g/ml) (Sigma, St Louis, MO, USA), 10 mM β- glycerophosphate (Sigma, St Louis, MO, USA), 50 UI/ml Penicillin-Streptomycin (Invitrogen, San Diego, CA, USA), and 10% fetal calf serum (Invitrogen, San Diego, CA, USA). The cells were maintained at 37°C in a fully humidified atmosphere at 5% CO2 in air. Cells were grown in 6-well plates and 24 later MTA and MPC powders were added (10 mg/well). Cells were photographed using phase contrast microscopy to visualize their morphology.
Cell viability analysis
To evaluate cell viability, we used the Cell Titer 96 Aqueous One solution cell Proliferation Assay from Promega (Promega, Madison, USA), where the formation of formazan from methyl tetrazolium salt (MTS) in living adherent cells was quantified by colorimetric measurement at 490 nm. Absorbance was measured at 490 nm using a 96-well plate reader and corrected by subtraction of background absorbance (same amount of medium without cells). Preliminary measurements using MTA and MPC powders without cells were performed and no significant variations of absorbance were observed (data not shown). Measurements were performed 24 hours and 72 hours after the addition of MTA® and MPC powders to the cell cultures. All experiments were performed in triplicates and repeated three times.
Results of one representative experiment are shown, expressed as mean ± the standard deviation. For statistical analysis, a Mann-Whitney test was used. A difference between experimental groups was considered to be significant when P < 0.05.
In vivo biocompatibility evaluation of MPC in comparison with MTA®
A total of six male New Zealand white rabbits were used (weight: 4 to 5 Kg). General anaesthesia was obtained by intramuscular injection of 45 mg/kg of Ketamine (Kétalar® ) and 8 mg/kg of Xylasine. Left and right proximal femur region of each animal was shaved and completely disinfected with Betadine TM . This surgical site was chosen because of its appropriate bone thickness and accessibility. Skin incision was made with a blade 15 from the lateral side, the muscles and the soft tissues were elevated. Using a twist drill and torque control unit and under saline irrigation, three holes of 2 mm depth were drilled with a round burr in order to provide enough material thickness.
MTA® and MPC were mixed separately with sterile saline water. An excess of water was eliminated with a gauze compress. Cements were then inserted with an "MTA gun®". One hole received MPC, the second one MTA® and the third was kept empty (negative control group) [Figure 1].
|Figure 1: Preoperative view of the 3 drilled cavities: The first one received modified Portland cement, the second MTA® and the third were kept empty (×400)|
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After replacement of the elevated soft tissues, the flap with the muscle layer were repositioned and sutured. Each rabbit received 10 ml of Amoxycillin 250 mg/5 ml/24 h for 7 days.
On the day 21 (3 weeks), two rabbits were sacrificed, two others on day 42 (6 weeks), and the last two on day 84 (12 weeks). Rabbits were euthanized to obtain bone-implant specimens in both the left and right femurs. Once trimmed, the bone samples including the three holes were stored in 10% formalin solution for one week and prepared for histologic examination.
Eight weeks after surgery, the animals were euthanized with a lethal injection of anesthetics. The skin was dissected, the femur removed and immediately immersed in a 10% tempered solution of formaldehyde. Undecalcified histological sections were obtained according to the method of Donath for mineralized tissues. After fixation, the specimens were dehydrated, embedded in glycol methacrylate (Technovit 7200VLC, Kulzer and Co GmbH, Wehrheim, Germany) then polymerized for eight hours (Light Polymerisation Unit, Exakt-Apparatebau GmbH and Co. KG, Norderstedt, Germany).
Blocks were then glued to plexiglas and sections of ∼100 μm were obtained using a diamond saw with coolant (Cutting machine Exakt-Apparatebau GmbH and Co. KG, Norderstedt, Germany). Sections were then reduced to a final thickness of 15 to 20 μm using the abrasion machine (Exakt-Apparatebau GmbH and Co. KG, Norderstedt, Germany) and subsequently stained with Giemsa-Paragon. Sections were examined under a light microscope (Olympus BX 60, Olympus Corporation, Tokyo, Japan) connected to a digital camera (Nikon Coolpix 4500, Nikon, Tokyo, Japan). After calibration, new bone formation was quantified using Image Tool 3.0 software (UTHSCSA, San Antonio, TX, USA). Histology slides of the tissue adjacent to the implanted materials were evaluated under a light microscope (Olympus BX 60 related to a digital camera jhu Coolpix 4500) by 2 examiners who were blinded to the type of material used. During histological analysis, details were considered regarding the type of tissue (bone or connective), the presence/absence of inflammation and bone apposition.
| Results|| |
In vitro experiment
The mouse calvarias cells were seeded at a density of 4 × 10 4 cells/cm 2 alone and in the presence of MTA and MPC granules [Figure 2]. Phase contrast microscopy allowed for in vitro analysis of the morphological changes associated with the days of culture. At 24 h in control cultures, cells attached and spread on the culture dishes, exhibiting spindle-shape morphology. After 48 hours of control cultures the cells have proliferated and exhibited polygonal morphology, and finally reached confluence 72 after platting. Similarly, in cultures in the presence of MTA and MPC, the cells did not show any evidence of cytotoxicity and formed a subconfluent layer around the granules of both materials. However, we noticed differences in the geometry and size of the biomaterial granules, though no morphological differences were observed between the three types of cultures.
|Figure 2: Examination of cell cultures using phase contrast microscopy 24 hours, 48 hours and 72 hours after the addition of MTA or MPC powders. (Bar = 50 ìm). No cytotoxic effect of powder was observed|
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Quantification of MTS conversion indicated a decreased MTS conversion by cells cultured in the presence of materials (MTA and MPC) in comparison with control cultures at 24 hours and 72 hours. In addition, after three days of culture, MTS conversion was significantly stimulated by cells cultured in the presence of MPC in comparison with MTA. These differences may be explained by a pH modification in the culture medium due to the ion release by biomaterials. Indeed, it was shown that calcium and silica were released by MTA during the first days of culture but with no inhibition of osteoblasts differentiation during the following days. 
Finally, the cell viability test according to the morphological observations suggested the biocompatibility of the two biomaterials tested [Figure 3].
|Figure 3: MTS conversion test: Effect of MTA and modified Portland cement powder on cell viability. Cells were plated at a density of 4 × 104 cells per cm2 24 hours (a) and 72 hours (b) after the addition of MTA and MPC powders P<0.05 indicates a significant difference between cells grown on the culture dish (control) as compared with cells cultured in the presence of MTA or MPC powder P<0.05 indicates a significant difference between cells grown in the presence of MTA as compared with MPC powder NS: Not significant, OD: Optical density|
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In vivo experiments
The total number of investigated bony cavities was 36, considering that three cavities in each femur of each rabbit were prepared.
Similar results are shown between MPC and MTA® . We noticed the presence of an unmineralized bone matrix that separates both cements from the surrounding bony walls, inflammatory cells and multinucleated giant cells are numerous [Figure 4]. In the negative control group, we noticed no bone apposition with the presence of inflammatory cells filling the empty cavity [Figure 5].
|Figure 4: At three weeks, similar results are shown between MPC and MTA®: Presence of unmineralized bone matrix (formed between each cement and bone) that is resorbed and incubated by inflammatory cells and giant cells (×400)|
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|Figure 5: At three weeks, there is no bone apposition while inflammatory cells fill the empty cavity (×400)|
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MPC and MTA® are now in direct contact with bone and inflammatory and multinucleated giant cells are in direct contact with each cement. Limited bone formation can be observed at this stage [Figure 6] and [Figure 7]. In the negative control group, bone apposition is also present [Figure 8].
|Figure 6: At six weeks, MPC is in direct contact with bone and inflammatory and giant cells are in direct contact with the cement. Limited bone formation is observed (×400)|
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|Figure 7: With MTA, at six weeks there is presence of inflammatory cells, with giant cells and new bone formation (×400)|
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|Figure 8: At six weeks, in the control group, bone apposition is present (×400)|
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Histologic sections show similar results for both cements. Bone remodelling is the obvious and little amount of inflammatory cells is noticed. [Figure 9], [Figure 10]. In the negative control group, we notice bone apposition in the empty cavity (not shown).
|Figure 9: At twelve weeks bone separates the MPC from the newly formed bone with some inflammatory cells. And in other sites we can observe unmineralized bone matrix (×400)|
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|Figure 10: At twelve weeks, we can observe unmineralized bone matrix surrounding the MTA with the presence of inflammatory cells (×400)|
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| Discussion|| |
Materials used in root treatments are frequently in direct contact with soft and hard tissues of the periodontium. Therefore it is necessary to be highly compatible and not toxic. The new cement elaborated in this study (MPC), based on PC, showed faster setting time and easier manipulation. Additional components were known to be no cytotoxic if introduced in the security margin. The addition of Calcium carbonate (reduces the setting time) and of Barium chloride (radiopaque agent) did not interfere with biocompatibility while the PC and MTA® biocompatibility was already proven.
In vitro test
It is generally accepted that in vitro tests are essential prior to conducting in vivo trials. While recognizing that their main limitation is their relative simplicity compared to the complex interactions that occur in vivo, their sensitivity in discriminating between biocompatible and non-biocompatible materials (with proper control materials) can be used to reject unsatisfactory materials before performing in vivo implantation studies. In vitro biocompatibility of a material is usually assessed against two controls: A negative control material, which does not produce a cytotoxic response and a positive control material, which provides a reproducible cytotoxic response. The purpose of the negative control is to demonstrate background response while that of the positive control is to demonstrate appropriate test system response. Tests are performed either on the material itself or on material extracts. Extraction vehicles include culture medium with and without serum, or physiological saline. 
In this experimental in vitro test, none of the cells examined showed a round shape, evidence of premature senescence or cellular suffering. Osteoblast-like cells cultured in the presence of the materials tested showed the typical osteoblast morphology, well spread, and quite close to the confluence. Examination of cells seeded in presence of MTA® and MPC cements by phase contrast microscopy provided a visual confirmation of the positive interaction between the cells and the experimental materials. Furthermore, the morphological observations suggesting no toxic effect of the two materials were confirmed by the MTS test, widely used as a rapid and sensitive method for the assessment of cell viability cultured in the presence of materials. In addition, all experiments were performed three times using independent cell cultures, with the measurements performed in triplicate. Furthermore, results of one representative experiment are expressed statically.
Our results are in line with others studies using different osteoblastic cell lines. Mitchell et al. reported in a study of osteoblasts biocompatibility of mineral trioxide aggregate that MTA® is biocompatible and suitable for clinical trials. Saidon et al.  compared the cytotoxic effect in vitro of mineral trioxide aggregate and Portland cement. Millipore culture plate inserts with freshly mixed or set material were placed in the culture plates with already attached L929 cells. After an incubation period of 3 days, the cell morphology and cell counts were studied. The results showed no difference in cell reactions in vitro. Scott in 2004 showed that the addition of an APC in the amount of 5.0% can accelerate the setting reaction of MTA® significantly faster than MTA® alone.  Camilleri et al. in 2005 studied the chemical constitution and biocompatibility of accelerated PC for Endodontic use.  Biocompatibility testing of the cement eluants showed the presence of no toxic leachable from the grey or white MTA and that the addition of bismuth oxide to the accelerated PC did not interfere with biocompatibility. Finally, both experimental cements tested to support the growth of osteoblast-like cells, showing suitable properties to be used as canal sealers and root-end filling materials. Gandolfi et al. studied the cellular response of new PC cement-based materials for Endodontics mixed with articaine solution and found that all experimental cements supported the growth of bone-like cells, showing suitable properties to be used as canal sealers and root-end filling materials.
In vivo test
Generally, biocompatibility tests on bone biomaterials such as those applied in Endodontics should be performed in the special bones identically to their clinical applications for bone healing process and its quality depend on the location of the implanted material. The International Standard Organization (ISO) recommends bones as the tibia, femur and the mandible of laboratory animals for material implantation investigation and among small animals, rabbits, rats, guinea pigs, and cats are more popular.  In this experimental test, the type of animals used (rabbits for in vivo) is in conformity with ISO recommendations.
In spite of a plethora of possible test methods, the protocols currently required by North American and European regulatory agencies in the evaluation of biocompatibility do not incorporate quantitative test procedures aimed at determining the nature and intensity of cellular reactions. Only qualitative assessments are recommended, and these involve the estimation of acute toxicity through the gross microscopic examination of tissue, which relies on the subjective expertise of investigators in identifying cell morphology and the severity of the reaction.  Bone healing and minimal inflammatory responses adjacent to MTA® and MPC implants were observed at all experimental periods (3, 6 and 12 weeks), suggesting that both materials are well tolerated and express similar biocompatibility.
Generally, the presence of inflammatory reaction, after 3 weeks around the implanted materials was an indicative of their initial stimulating effects due to alkaline caustic properties and foreign body reaction. 
Our results are in line with other in vivo experimentations on comparing biocompatibility and histologic responses between MTA and PC when implanted in bones of laboratory animals.
Torabinejad et al. studied in 1998 the tissue reaction to MTA implantation in both tibia and mandible of guinea pigs.  As in every specimen, it was free of inflammation. Saidon in 2003 studied the tissue reaction of ProRoot MTA and Portland cement in bone implantation in the mandibles of guinea pigs.  Bone healing and minimal inflammatory response adjacent to ProRoot and PC implants were observed in both experimental periods (2 and 12 weeks), suggesting that both materials are well tolerated.
Razmi and Zarabian in a histologic evaluation of tissue reaction to three implanted materials (MTA® , Root MTA® and PC) in the mandible of cats found no statistically significant differences in the degree of inflammation, presence of fibrotic capsule, severity of fibrosis, inflammation thickness and in bone formation between the three materials (P > 0.05) and that all of these three materials are biocompatible. 
According to the methodology proposed and based on the results of this study, it may be concluded that MPC has the potential to be used in clinical situations similar to those in which MTA® is being used. Although the results are very encouraging, extrapolations to the clinical situation must be made with caution. More studies in vivo and in human beings need to be conducted before unlimited clinical use can be recommended.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]