The rapidly evolving field of materials science is providing dentistry with new treatments and alternatives. Calcium phosphate materials have been increasingly employed in orthopedic and dental applications. Recently, much attention has been paid to calcium phosphate cements (CPCs) because of their advantages in comparison with calcium phosphate bioceramics, regarding
Moreover, in the present state, CPCs do not compare favorably with currently available dental cements in terms of setting time. Therefore, modification of powder composition and properties, introduction of reinforcing materials into liquids, and/or changing the mixing methods are necessary for improvements of CPCs to meet the requirements for dental applications, and thus replace calcium hydroxide pastes currently used in dentistry with more durable materials bearing setting time (2–8) minutes and a high compressive strength (50–80 MPa) while retaining their biological advantages.
In this study, we tested the hypothesis that incorporation of several polymeric acids into traditional CPCs would produce formulations with promising physical, mechanical and biological properties to permit wide dental applications. The present study tests this hypothesis by measuring: (1) the physical chemical characteristics of three novel CPC formulations derived from a mixture of CPC powder with three aqueous solutions of polymeric acids (modified polyacrylic acid, light-activated modified polyalkenoic acid, and 35% w/w polymethyl vinyl ether maleic acid as compared to a clinically available zinc polycarboxylate cement, (2) the compressive and diametral tensile strength of these polymeric cements, and (3) using the direct contact cell culture format, we compare the cytotoxic properties of these CPC formulations and one clinically available zinc polycarboxylate cement. (The biocompatibility examined on human gingival fibroblast cells and the cell viability quantified using MTT assay.)
Three polymeric calcium phosphate cements and zinc polycarboxylate cement were evaluated (Table
Materials used in this study and their manufacturers.
Material | Composition | Trade Name | Manufacturers |
---|---|---|---|
(1) Zinc polycarboxylate cement | Powder: Zinc oxide with traces of Mg oxide and Sn oxide. | G.C.R. | Advanced Research IncDental division England |
(2) Glass Ionomer liquid light cured modified polyalkenoic acid | A light sensitive aqueous solution of polyalkenoic acid modified with methacrylic group. | Vitremer | 3M Dental products St. Louis, USA |
(3) Monocalcium phosphate monobasic (MCPM) | Calcium Phosphate Monobasic | Sigma Chemical Co., Aldrich GmbhGermany | |
(4) Calcium oxide (CaO) | Adwic Laboratory Chemical | ||
(5) Polymethyl vinyl ether maleic anhydrate copolymer (white powder) PMVE-Ma | Sigma-chemical Laboratories St. Louis, USA | ||
(6) Synthetic hydroxylapatite (SHAP6) | Prepared at the Department of Dental Materials of the Medical College of Georgia, Augusta, Ga, USA |
Both the monocalcium phosphate monohydrate (MCPM) and the calcium oxide (CaO) powder were crushed separately in an agate mortar and then sieved to obtain an average particle size of 80
Three types of polymeric liquids (Table
Several pilot studies were performed to select the best powder to liquid ratio (4 : 1) that produced good handling characteristics and working time. Zinc polycarboxylate cement was mixed according to the manufacturer’s instructions and considered as an additional control group. These newly formulated calcium phosphate cements composed of one form of powder and three different types of liquid were evaluated and compared to the control group (zinc polycarboxylate) cement in regards to the following.
The initial setting time of each of the cement mixtures under investigation was determined according to the method described in the ANSI/ASTM-C-191-1977 [
A total of 120 cylindrical specimens of 6mm diameter and 12 mm height were prepared according to the ISO specification no. 4104 [
The split molds were covered with glass plates, and specimens were kept undisturbed for 60 minutes at 37°C under 100% relative humidity before separation from the mould [
For the diametral tensile strength test, 120 disc specimens of 6 mm diameter and 3 mm height were prepared for each type of cement [
The compressive and diametral tensile strength test of each type of cement were determined after 1 hour, 24 hours, 1 week, 4 weeks, and 8 weeks storage in distilled water using a Universal Testing Machine (Com-Ten Industries, Inc., Fla, USA) at a cross-head speed of 0.5 mm/min. The compressive strength was measured by dividing the maximum load in compression on the ends of the cylindrical specimens by the original cross-sectional area of the test specimen [
All data recorded were subjected to one-way analysis of variance (ANOVA) followed by (where appropriate) the Tukey-
Fragments of the set cements used for the compressive strength test (24 hours after mixing) were ground and weighed to obtain 1 gram of each set material and were mixed with a measured amount of potassium bromide in an agate mortar. After pressing the mixtures into rigid pellets, IR analysis was performed using a (Perkin Elmer IR spectrometer, Model 1403, USA) at a wavelength range between 4000 and 600 cm−1 [
The CPC powder components before and after mixing with the liquid and setting were manually ground to a fine powder in an agate mortar for X-ray diffraction analysis (XRD) analysis. The XRD patterns were collected on a theta-theta PANalytical X’Pert Pro X-ray diffractometer. The instrument was scanned over a 5–70° 2
Compound search-match identification was performed with jade soft ware (Version 9) from Materials Data Inc. using the latest inorganic PDF4 powder diffraction data base from the International Centre for Diffraction Data (ICDD).
The evolution in morphology of the crystalline structures formed during the process of cement setting (24 hours after mixing) was observed by examining the longitudinal and the fractured surface of the samples using scanning electron microscopy (SEM) (LEO 1450VP, Carl Zeiss SMT, Oberkochen, Germany).
Sample preparation was performed aseptically to prevent the risk of biological contamination during the cytotoxicity testing [
Six discs for eachcement (Types I, II, and III CPCs and zinc polycarboxylate cement) were fabricated in sterile Teflon molds 5.5 mm in diameter and 3 mm thick. The materials were packed into the mold and allowed to set at room temperature (25°C) before testing. Teflon discs were used as a negative control.
Specimens (
Cellular activity was assessed by measuring mitochondrial succinic dehydrogenase (SDH) activity via the MTT colorimetric assay [
For mitochondrial activity, the means and standard deviations of the MTT-formazan optical densities to Teflon controls were calculated. Statistical difference between the calcium phosphates and the controls was determined using analysis of variance (ANOVA) with Tukey
The results of the initial setting time are presented in (Table
The initial setting time (in minutes) of zinc polycarboxylate cement and the three polymeric calcium phosphate cements (CPCs).
Zinc polycarboxylate (control group) | Polymeric calcium phosphate | L.S.D.5% | |||
Type I | Type II | Type II | |||
Mean setting time (in minutes) ± SD | 5 ± 1 | 5 ± 1 | — | 9 ± 1 | 1.30* |
NB: Type II polymeric CPC (no setting reaction) VLC type.
*Significant at 5% level.
The mean values of compressive strength and diametral tensile strength of zinc polycarboxylate cement (control group) and the three polymeric calcium phosphate cements (Types I, II, and III) are listed in (Tables
Mean compressive strength and standard deviation of zinc polycarboxylate cement and the three polymeric calcium phosphate cements (CPCs) in MPa.
Cement types | 1 hour | 24 hours | 1 week | 4 weeks | 8 weeks | ||
---|---|---|---|---|---|---|---|
Control zinc polycarboxylate | 46.85 ± 3.51 | 50.83 ± 4.50 | 50.87 ± 2.65 | 51.88 ± 2.80 | 52.60 ± 2.95 | 4.33* | |
Calcium phosphate cements (CPCs) | Type I | 40.42 ± 3.33 | 44.87 ± 3.25 | 49.80 ± 2.75 | 48.74 ± 2.80 | 46.91 ± 3.65 | 3.90* |
Type II | 66.86 ± 1.38 | 67.13 ± 1.30 | 67.15 ± 1.38 | 67.20 ± 1.30 | 67.12 ± 1.22 | 1.74* | |
Type III | 71.68 ± 3.15 | 75.12 ± 3.55 | 75.56 ± 2.75 | 73.62 ± 2.95 | 73.59 ± 3.10 | 4.09* | |
LSD 5% | 3.81* | 4.22* | 3.19* | 3.30* | 3.32* |
*Significant at 5% level.
Mean diametral tensile strength and standard deviation of zinc polycarboxylate cement and the three polymeric calcium phosphate cements (CPCs) in MPa.
Cement types | 1 hour | 24 hours | 1 week | 4 weeks | 8 weeks | ||
---|---|---|---|---|---|---|---|
Control zinc polycarboxylate | 5.65 ± 1.91 | 5.92 ± 2.35 | 4.49 ± 1.91 | 5.50 ± 1.99 | 4.80 ± 1.51 | 2.55* | |
Calcium phosphate cements (CPCs) | Type I | 4.70 ± 1.84 | 5.85 ± 2.41 | 6.41 ± 2.04 | 5.55 ± 2.36 | 4.58 ± 1.84 | 2.77* |
Type II | 7.39 ± 1.99 | 8.74 ± 1.74 | 8.80 ± 2.02 | 8.63 ± 2.51 | 8.57 ± 2.96 | 2.96* | |
Type III | 11.43 ± 2.37 | 13.81 ± 2.22 | 14.03 ± 1.95 | 12.77 ± 1.89 | 12.59 ± 1.57 | 3.66* |
*Significant at 5 % level.
Histogram showing the mean compressive strength of zinc polycarboxylate cement and the three polymeric CPCs in MPa.
Histogram showing the mean diametral strength of zinc polycarboxylate cement and the three polymeric CPCs in MPa.
At all the storage periods, from 1 hr to 8 weeks, the compressive strength values of polymeric calcium phosphate cement (Type III) derived from 35% w/w aqueous solution of PMVE-Ma were higher than that of zinc polycarboxylate cement (control) and the other two polymeric calcium phosphate cements (
The mean value of compressive strength of (Type II) derived from visible light cured (VLC) polyalkenoic acid slightly increased from 1 hour (66.86 MPa) to one week (67.15 MPa) and remained nearly constant to the end of the eighth week (67.12 MPa).
The mean value of compressive strength of (Type III) polymeric, CPC derived from (PMVE-Ma), significantly increased from 1 hour (71.68 MPa) up to one week (75.56 MPa) then slightly decreased at the end of the eighth week (73.59 MPa).
At all the storage periods from 1 hour to 8 weeks, the mean diametral strength values of polymeric calcium phosphate cement (Type III) and Type (II) were higher than that of zinc polycarboxylate cement and (Type I) polymeric calcium phosphate cement (
The mean value of diametral tensile strength of (Type I) polymeric CPC significantly increased from 1 hour (4.70 MPa) up to one week (6.41 MPa) then significantly decreased at the end of the eighth week (4.58 MPa). The mean diametral strength value of (Type II) polymeric calcium phosphate cement significantly increased from 1 hour (7.39 MPa) up to one week (8.80 MPa) and slightly decreased at the end of the eighth week (8.57 MPa). The mean diametral tensile strength value of (Type III) polymeric CPC cement significantly increased from 1 hour (11.43 MPa) up to one week (14.03 MPa) then decreased at the end of the eight week (12.59 MPa).
The infrared spectrum of modified polyacrylic acid, visible light-cure modified polyalkenoic acid, 35% (w/w) aqueous solution of polymethylvinylether-maleic acid (PMVE-Ma) and the set cements derived from their mix with CPC powder are presented in (Figures
(A) Zinc polycarboxylate cement; (B) Type I; (C) Type II; (D) Type III. For all cements tested, IR spectra of polymeric acids (modified polyacrylic acid, modified polyalkenoic acid, 35% w/w aqueous solution of PMVE-Ma (a) showed the absorption bands of carboxylic group (C=O) between 1635 to 1640 cm−1 cm (arrows). IR spectra of set cements (b) showed the absorption bands between 1558 and 1401 cm−1 indicating the formation of carboxylic salts (arrows).
The infrared spectrum of the modified PA acid and the set zinc polycarboxylate cement (control group) (Figure
The infrared spectrum of the modified PA acid and the set cement (Type I) (Figure
The infrared spectra of the 35% w/w aqueous solution of PMVE-Ma acid and the set cement (Type III) are shown in (Figure
Two phases (zincite-ZnO and cassiterite-SnO2) which exhibited the characteristic peaks around 2
(a) X-ray diffraction pattern of unreacted zinc polycarboxylate powder. (b) X-ray diffraction pattern of set zinc polycarboxylate cement.
X-ray diffraction pattern of the three types of polymeric cements (Type I, Type II, and Type III CPCs). (A) Unreacted powder component. (B) Set product of Type I cement. (C) Set product of Type II cement. (D) Set product of Type III cement. Different arrows indicating the characteristic peaks of the different crystalline phases of the X-ray diffraction pattern; cross-hatched arrows indicate the crystal phase of calcium hydrogen phosphate hydrate, black arrows portlandite, grey arrows hydroxyapatite, and gradient arrows calcite.
The SEM photomicrographs in (Figures
SEM microphotograph of the longitudinal top surface of the four types of cements. (a) Zinc polycarboxylate cement; (b) Type I; (c) Type II; (d) Type III after setting.
A mixture of thin needle- or rod-shaped microcrystals characteristic of hydroxyapatite were identified on the top surface of Type II cement. These hydroxyapatite crystals were precipitated on the cement surface together with plate-like crystals as shown in (Figure
SEM microphotograph of the fractured surface of the four types of cements. (a) Zinc polycarboxylate cement; (b) Type I; (c) Type II; (d) Type III after setting.
Cellular mitochondrial suppression induced by the CPCs and zinc polycarboxylate cement (control group) is illustrated in (Figure
Mitochondrial suppression induced by zinc polycarboxylate, Type I, Type II, and Type III calcium phosphate cements as function of aging time. Cytotoxicity was measured by succinic dehydrogenase activity and expressed as a percentage of Teflon controls (defined as 100%). There were six replicates per condition. Different letters indicate a statistically significant difference between the materials (ANOVA, Tukey intervals
The most difficult challenge in designing and manipulating dental materials is being able to mimic the complex physical and functional characteristics of natural tissues. Development of a replacement material that either mimics natural tissue properties and performance and/or one that is eventually resorbed and replaced by equivalent new tissue is the final goal of restorative dentistry.
Thus, hydroxyapatite (HA) materials combined with organic compounds are promising dentin replacing materials. Various calcium phosphate derivatives, for example, hydroxyapatite (HA), tricalcium phosphate (TCP), octacalcium phosphate (OCP), dicalcium phosphate (DCP), and monocalcium phosphate (MCPM) have been studied in the last decade because of their biocompatibility, osteoconductivity, and self-hardening properties which are desirable in a broad range of dental and biomedical application [
In the present study, experiments have been undertaken in order to develop some nontraditional dental cementing materials. The principal compounds [synthetic hydroxyapatite (SHAp6) and calcium oxide (CaO)] that have been used, proved to have (as far as dentistry is concerned) encouraging properties which will certainly open an avenue for the material scientists to overcome some of the drawbacks encountered with well known dental cements. In the present study, all materials selected for the preparation of polymeric calcium phosphate cements: calcium oxide (CaO) monocalcium phosphate monohydrate (MCPM), and synthetic hydroxyapatite (SHAp6) powders as well as modified polymeric liquids (Polyacrylic acid “PA”), visible light cured polyalkenoic acid (VLC), and polymethyl vinyl ether maleic acid (PMVE-Ma), are all of medical grade, commercially available, and have a well-established compatibility.
Calcium oxide (CaO) is known to react rapidly with water and plays and important role in the hydration reaction of the set cement (a linear relationship was found to exist between the strength and the degree of hydration of dental cements) [
As for monocalcium phosphate monohydrate (MCPM) it is often used as the acid calcium phosphate in hydraulic calcium phosphate formulations, but commercial MCPM is not pure, contains a small amount of orthophosphoric acid and moisture, is consequently difficult to mill, and the powder is sticky and presents aggregates.
Because granularity influences the mechanical properties of the hardened cement, it was, therefore, necessary to premix MCPM with CaO before grinding it though a rapid decrease in the amount of (MCPM) was observed during mechanical grinding by a solid-solid reaction with CaO [
Ginebra et al. (2004) stated that the cement cannot be univocally related to the degree of reaction without considering the microstructural features [
Therefore, in the present work, in order to overcome the disadvantages of mixing with water, the aqueous solutions of modified (polyacrylic acid, polyalkenoic (VLC) and polymethylvinyl ether maleic acid) were used. Their setting reaction was found to be biphasic, the first step during the mixing time, (MCPM) reacted with CaO immediately to give dicalcium phosphate dihydrate (DCPD) which in the second step, reacted more slowly with the remaining CaO to give hydroxyapatite.
An essential criterion was also considered in relation to the molecular weight and concentration of the aqueous solutions used for mixing the powder, as higher molecular weights tend to result in cements with shorter setting times and a higher compressive, diametral, and biaxial flexural strengths than lower molecular weight counter parts [
The polymethyl vinyl ether maleic anhydride (PVME-Ma) is a commercial copolymer offered in several molecular weights and can be dissolved by hydrolysis of the anhydride group in water to form the corresponding maleic acid copolymer (polymethyl vinyl ether maleic acid). This copolymer has already a number of nondental applications in hair sprays and surgical adhesives, which suggests potential favorable biocompatibility for dental and other biomedical uses [
In order to optimize the powder liquid ratio of the mixing powder, extensive preliminary testing of various powder mixtures ratios was performed resulting in the optimal ratio of 4 : 1.
The setting time of zinc polycarboxylate cement (control group) used in this study agreed with those values previously reported in the literature (Table
The setting time results of the three formulated polymeric cements (Table
As for Type II polymeric CPC cement mixed with polyalkenoic acid (VLC), the setting time (Table
In Type III CPC mixed with (PMVE-Ma acid), a setting time of 9 min was observed. This may be attributed to the multifunctional nature of the PMVE-Ma acid which appears to react forming insoluble products which coat the cement particle. This encapsulation slightly retards their dissolution which may be attributed to residual maleic anhydride units as observed in the IR spectral data (Figure
Results of the compressive and diametral tensile strength values of the newly formulated polymeric calcium phosphate cements (CPCs) as shown in (Tables
In this study, the reaction between CPC powder and 35% (w/w) (PMVE-Ma) aqueous solution (Type III) resulted in a polymeric CPC with a compressive strength of 71.68 MPa and diametral strength of 11.43 MPa one hour after mixing. These results are in accordance with Matsuya et al. [
The cement prepared from CPC powder and visible light cure polyalkenoic acid (VLC) Type II had a compressive strength value of 66.86 MPa and diametral tensile strength of 7.39 MPa, 1 hr after mixing and reached its maximum value at the end of 24 hours. These results coincide with Miyazaki et al. [
Storage in distilled water at 37°C slightly affected the mechanical properties, therefore suggesting a stable formula that can resist disintegration in the oral environment.
Multifunctional acids such as polymethylvinyl ether maleic acid (PMVE-Ma) and polyacrylic acid (PA) are characterized by the presence of carboxylic groups [
The use of total reflectance of infrared spectroscopy makes it possible to monitor the setting reaction and the transformation of the COOH group to COO groups.
The infrared spectra of the set polymeric CPC Types (I, II, and III) 24 hours after mixing showed the absence of the stretching peak of carboxylic group (–COOH) and the appearance of two new carboxylate stretching peaks indicating the formation of the polyacrylic salts (Figures
The X-ray diffraction analysis of zinc polycarboxylate unreacted powder identified two crystalline phases ZnO and SnO2. The presence of tin oxide depends on the commercial cement initially used: it is present in small quantities in some cements and not in others. No zinc polycarboxylate reflections were seen, since this compound is amorphous. Therefore, the cement must be a composite of unreacted oxides ZnO (mainly) and SnO2 and the amorphous zinc polycarboxylate matrix resulting from the setting reaction of these oxides with polyacrylic acid and water. These results are in accordance with the core link structure proposed for such cements [
During the hydration of cements consisting of nominally 60wt % monocalcium phosphate monobasic and calcium oxide and 40 wt% hydroxyapatite, two dibasic phosphate compounds, (monetite and its dihydrate, brushite) were identified in the sample. Monocalcium phosphate monobasic (MCPM) reacted with excess water forming monetite and/or brushite by
Although mechanical and physical properties are of great concern for dentin regenerating pulp capping, lining, or base material, biocompatibility is another critical issue. The current study established that 4 weeks aging of the developed formulations of calcium phosphate cements may significantly change their ability to alter cellular function. However, the effect was not uniform for all formulations. Type I calcium phosphate cement (CPC mixed with polyacrylic acid) showed less mitochondrial suppression with time. It is possible as cytotoxic elements leached from the material, they either complexed with other molecules in the medium or broke down into smaller components, in each case rendering it less cytotoxic.
For Type II calcium phosphate cements mixed with resin modified glass ionomer (Vitremer), suppressed cellular activity was ongoing, suggesting that leaching of components with biological liabilities remained even after 4 weeks of aging. The Vitremer liquid is a light sensitive, aqueous solution of a modified polyalkenoic acid and contains 2 hydroxy ethyl methacrylate (HEMA). In most dental resin modified glass ionomer cements, HEMA is often used as a comonomer to render the resin modified polyacid compatible with water [
We prepared the CPCs by mixing calcium oxide, calcium phosphate monohydrate (MCPM), and synthetic hydroxyapatite. Calcium oxide (CaO) is known to react rapidly with water and plays an important role in the strength and degree of hydration of dental cements. As for monocalcium phosphate monohydrate (MCPM), it is often used as the acid-calcium in hydraulic calcium phosphate formulations. Calcium phosphate biomaterials are thought to generally be biologically well tolerated, because the main inorganic constituents of bone, hydroxyapatite is comprised of calcium and phosphate [
Interestingly, the cytotoxicity of the three different formulations is dependent on the composition of the polymeric acid used for mixing. In our experimental design, cytotoxicity was estimated by mitochondrial succinate dehydrogenase activity (SDH) activity in the MTT assay and expressed as a percentage of the Teflon negative control value (100%) being equivalent to Teflon with no evident cytotoxicity.
On the other hand, the biocompatibility of zinc polycarboxylate cement (control group) has also been investigated. Our data show that zinc polycarboxylate cement was the most cytotoxic of the tested materials in accordance with previous data [
Type III CPC presented reasonable setting time, significantly higher compressive, and diametral tensile strengths when compared to zinc polycarboxylate cement (control group).
By virtue of these characteristics coupled with its biocompatibility, Type III CPC cement shows promise for dental applications.