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High conversion self-curing sealer based on a novel injectable polyurethane system for root canal filling
Materials Science and Engineering C 33 (2013) 3138–3145
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
High conversion self-curing sealer based on a novel injectable polyurethane system for root canal filling Bin Sun a, Yi Zuo a,⁎, Jidong Li a, Li Wang a, Kuangyun Tang b, Di Huang a, Jingjing Du a, Peipei Luo a, Yubao Li a a b
Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China The State Key Laboratory of Oral Diseases and Orthognathic Surgery, Sichuan University West China College of Stomatology, Chengdu 610064, PR China
a r t i c l e
i n f o
Article history: Received 7 September 2012 Received in revised form 16 February 2013 Accepted 5 March 2013 Available online 16 March 2013 Keywords: Root canal filling Injectable and self-curing polyurethane High conversion degree Micro expansion Cytocompatibility
a b s t r a c t Low monomer–polymer conversion is the key factor leading to cytotoxicity for resin-containing restorative materials. This paper provides a new root canal filling system based on self-curing injectable polyurethane which can achieve high conversion in a short time. Traced FTIR spectra show more than 90% NCO group participated in the curing reaction after 4 h, and only about 5% remained after 24 h. The calculated data also testified the curing process supports a third-order reaction, and this efficient and sufficient reaction is postulated to weaken the toxic stimulation. By culturing with L929 murine fibroblasts, the PU sealer is shown to be favorable for cell attachment and proliferation. Then physicochemical properties of the injectable PU-based sealer were evaluated according to the Standard [ISO 6876:2001 (E)] for clinical application. A series of physicochemical properties of PU sealer have been tested comparing with AH Plus and Apexit Plus. And the results present that the self-curing PU sealer could not only match the clinic requirements, but even has better properties than the other two commercial sealers. We expect the high conversion PU sealer has a tremendous potential in the field of root canal filling after further biological evaluation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Hermetic 3-dimensional obturation of root canal system is of prime importance for long-term endodontic treatment success [1]. Sealers have been developed to eliminate oral cavity leakage. However, the main drawback of contemporary filling resins, such as polymethyl methacrylate, Resilon and Epiphany, is volumetric shrinkage during their polymerization [2,3]. Microscopic gap usually forms between material and tooth interface. The gap allows oral fluids leakage, leading to bacterial growth and penetration [4]. Even the most widely used root filling material, thermoplastisized Gutta-percha (GP), causes volume shrinkage after cooling [5]. Suitable material for root canal filling needs to be developed. Multiblock copolymer Polyurethane (PU) composed of soft segment and hard segment. In designing the injectable polymer sealer, tailored PU can offer many advantages [6], such as controlled fluid ability, strong bonding and fast curing. Specifically, polyurethane materials present volumetric dilatancy after polymerization, and this will meet with the requirement of hermetic obturation. To date, polyurethane based root canal filling materials have been developed by thermoplastic technique [6] and visible-light curable technique [7]. A set of experimental data proved that urethane acrylate-based sealers behaved better than Epiphany and EndoREZ for root canal filling. Unfortunately, the thermal initiators (2,2-azsobis-isobutyronitrile, AIBN or 2,2-azobis (2-methyl) ⁎ Corresponding author. Tel.: +86 28 85418178; fax: +86 28 85417273. E-mail address: [email protected] (Y. Zuo). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.03.014
butyronitrile, AMBN) and photoninitiators (camphorquinone and ethyl 4-dimethylaminobenzoate) is cytotoxic [8]. In addition, photoactivated PU-based sealer is not sufficient enough for root canal filling, its degree of conversion is no more than 70% after 2 weeks [9,10]. Other from previous thermo curable or visible-light curable polyurethane based sealers, a novel injectable and self-curing polyurethane sealer was developed. This study had three main purposes: (1) synthesis and analyze the curing mechanism of the novel PU sealer (2) evaluate the physical-chemical properties according to ISO standard and (3) evaluate the cytotoxicity of the novel sealer. 2. Materials and methods Polytetramethylene ether glycol (PTMEG 2000, Aladdin Reagent, Shanghai China), triethanolamine (Aladdin Reagent,Shanghai China), polyethylene glycol (600, Aladdin Reagent,Shanghai China) and Isophorone diisocyanate (IPDI, Aladdin Reagent,Shanghai China) were used as received. 1,4-butanediol (BDO, Aladdin Reagent, Shanghai China) was dehydrated under decompression under a vacuum of 1330 Pa at 120 °C. All reagents were of analytical reagent grade. 2.1. Polymers synthesis 2.1.1. Part A (prepolymer) Polytetramethylene etherglycol (PTMEG 2000) (90 g) was weighed and put into a dry three-neck flask equipped with a mechanical stirrer under nitrogen. Isophorone diisocyanate (IPDI) (22.41 g) was then
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added into the flask under nitrogen. The reaction mixture was heated to 70 °C and stirred for 5 h. Then 2 ml 1, 4-butanediol was added as a chain extender for crosslinking. The reaction was carried on for another 2 h and NCO-terminated PU prepolymer was yielded as Part A. The stoichiometric ratio (wt) of polyol/isocyanate is 1.5/1.
the column temperature was maintained at 40 °C. Molecular weight (Mw) calibrations were based on a linear calibration curve using polystyrene standards. The degree of polymerization ( X n ), which indicated the monomer numbers that the polymer molecules contains, is obtained as follows [12]:
2.1.2. Part B (curing agent) Part B was prepared by mixing stannous, triethanolamine, and polyethylene glycol (PEG 600) with a mass ratio of 2:3:8 at room temperature.
M0 ¼
2.1.3. Polymer preparation The PU sealer was achieved by mixing Part A and Part B at a mass ratio of 9:1 inside a two-pipe syringe and subjected to vigorous shaking for 10s (Amalgamator YY/T 0273–2009, Hangzhou Yinya new material Co. Ltd). Then the mixture was immediately dispensed into a teflon model (Φ6.0 mm × 10 mm). There would be a mild reaction between –NCO (in the prepolymer) and –OH (in the polyol). As the mixture cured in a few minutes at body temperature, samples were removed for future testing.
n X
ωn M n
ð3Þ
1
where M 0 is the molecular weight of the polymer unit, ωn is the molar ratio of different raw materials, Mn is the molecular weight of different raw materials.
Xn ¼
Mn
ð4Þ
M0
where X n is the degree of polymerization, M n is the number average molecular weight obtained through GPC, M 0 is the molecular weight of the polymer unit, and M 0 can be obtained from formula (3).
2.2. Analysis the conversion of polymerization
2.3. Physicochemical properties evaluation for root canal filling
Curing process of the self-curing polyurethane system based on PTMEG and IPDI was monitored by FTIR spectra. Gel permeation chromatography was used to analyze the molecular value of samples.
The flow, film thickness, setting time, solubility and dimensional change for the injectable PU sealer were determined according to the methods described in the International Organization for Standardization [ISO 6876:2001 (E)] [13], which was published for dental root canal sealing materials evaluation. The following tests (the setting time test was carried out at 37 °C and a relative humidity of 95%) were carried out at (23 ± 2)°C and at a relative humidity of (50 ± 5)%. Five samples per group were prepared for each of the following tests: flow, film thickness, setting time, solubility and dimensional change. And the materials were freshly mixed for each test. The main components of the sealers are shown in Table 1.
2.2.1. FTIR analysis To trace the chemical change during polymerization, PU sealer has been measured at different curing times. Five grams of Part A was dissolved in 250 ml dichloromethane, and Part B was dropped into the solution in proportion. Then a drop of the solution was immediately dropped onto the prepared potassium bromide disk. After dichloromethane volatilized, the functional groups of the sample was detected using a Nicolet 6700 FTIR spectrometer (Nicolet Perkin Elmer Co., America) from 500 cm −1–4000 cm −1. Baseline has been corrected for all absorption peaks. Subsequently, the polymerized procedure was investigated by magnifying the characteristic peaks of -NCO group of the PU sealer. And the degree of conversion was calculated by comparing the peak area of-NCO group at different curing time. The degree of conversion p can be expressed as following [11]: t
pðt Þ ¼ ∫0 dp
ð1Þ
Assuming no side reactions occur, the isocyanate conversion (p) can be used as the degree of curing: A −A∞ Isocyanate conversionðpÞ ¼ 1− t A0 −A∞
ð2Þ
Here, A0 is the integrated absorption area at the initial time, At is the integrated absorption area at time t during the curing process, and A∞ is the final integrated absorption area. As no isocyanate group will be available in a completely cured system, A∞ is zero. 2.2.2. Gel permeation chromatography (GPC) analysis The weight average molecular weights (Mw), number average molecular weight (Mn) and molecular weights dispersion (Mw/Mn) of the prepolymer and the cured PU sealer (the samples were placed in oven at 37 °C. for another 4 hours after setting) were respectively measured by gel permeation chromatography(GPC). Samples of 0.20 to 0.25 mg were dissolved in 3 ml tetrahydrofuran (THF) at ambient temperature. The GPC equipment consisted of a Waters 510 HPLC pump with a Super HM-H*2 (6.0 mm*15 cm) column, and a Waters 410 RI-detector. The flow rate was maintained at 0.6 ml/min, and
2.3.1. Flow Homogeneously mixed sealer of 0.5 ± 0.05 ml was taken with a syringe onto a piece of the glass plate. The glass plate was at least 40 × 40 mm 2 and the mass of one glass plate was approximately 20 g. Another glass plate (the same as the first one) was placed on top of the material centrally, followed by the weight to make a total mass on the plate of 120 ± 2 g. Ten minutes after the commencement of mixing, the weight was removed and the maximum and minimum diameters of the compressed disc of sealer were measured. If the diameters agree to within 1 mm, the result was recorded. The test was repeated if the two diameters differed by more than 1 mm. 2.3.2. Film thickness Film thickness was measured by two combined glass plates with a contact surface area of (200 ± 10) mm 2 to an accuracy of 1 μm. A portion of sealer was placed onto the center of one of the glass plates. As the area between the glass plates was completely filled with the
Table 1 Components of the root canal sealers. Sealer
Composition
AH Plus
Calcium tungstate, epoxy resins, zirconium oxide, iron oxide, silicone oil Calcium saltsa, hydrogenized colophony, disalicylate, bismuth saltsb, silicon dioxide, phosphoric acid PTMEG-IPDI based prepolymer as Part A, a mixture of polyglycol and catalystsc as Part B
Apexit Plus
PU sealer a b c
calcium hydroxide, calcium oxide, calcium phosphate. bismuth oxide, bismuth carbonate. stannous, triethanolamine.
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material, the other plate was placed on the sealer. After (180 ± 10) s, a load of 150 N was applied vertically on the top plate. The thickness of the plates was measured 10 min after the start of mixing. The results have been recorded by determining the thickness of the plates with and without sealer. 2.3.3. Setting time The setting time was tested with a Gillmore needle having a mass of (100 ± 0.5) g and a cylindrical end with a diameter of (2 ± 0.1) mm. The needle was vertically lowered on to the horizontal surface of the sealer. The needle tip was cleaned and the test was repeated with an interval of 10 min. Time was recorded when the indentation was invisible. 2.3.4. Solubility A shallow dish was weighted (W1) before the test. Two specimens with a diameter of (20 ± 1) mm and a height of (1.5 ± 0.1) mm were weighted (W2) and put into the shallow dish,taking care to avoid any contact between them and inner surface of the shallow. (50 ± 1) ml water was added, and then the dish was covered. The dish was placed in a cabinet for 24 h. And then the specimens were remove and washed with deionized water of 2 ml to 3 ml. The washings in the dish was examined as follows. The dish was put into an oven to evaporate the water without boiling. The dish was then weighted (W3) again. Then the results (W3 − W1) /W2 were recorded. 2.3.5. Dimensional change A toflon mould (Φ6.0 mm × 12 mm) was put on a polyethylene sheet back by a glass plate. Then 2 g fresh mixed sealer was injected into the mould. Another glass plate faced with a sheet of plastic was pressed on top of the sealer. Then the mould and the plates together were held firmly with a C-clamp. Five minutes after beginning the mix, the mould with the sealer and clamp was placed at an atmosphere of 95% to 100% relative humidity at (37 ± 1)°C for 24 h. After that, clamp and glasses were removed, the ends of the specimen was grinded flat by drawing the mould containing the specimen back and forth across fresh 600 grit wet sandpaper. Then the specimen was removed from the mould, the distance between the flat ends was measured to an accuracy of 10 μm. Then the specimen was remeasured to an accuracy of 10 μm after stored(a needle was used to hold the samples, the sample could neither touch the bottom nor exposed in the air) in distilled water at (37 ± 1)°C for 30 days. The change in length as a percentage of the original length was calculated and recorded.
according to the standard procedure for medical devices. They were used for cytotoxicity without any further manipulation. 2.4.2. SEM preparation L929 murine fibroblasts that cultured in DMEM media for three days were seeded on top of the specimens (1 × 10 4 cells per specimen). For allowing the cells to attach, the specimens were first placed in cell culture plate wells and left undisturbed in an incubator for 3 h, then an additional 1 ml culture medium was added into each well. The sealer/cell constructs were cultured in the incubator at 37 °C with 5% CO2/95% air atmosphere for 1 day and 5 day(s). The medium was changed every two days. Fixation, dehydrating and drying methods were followed from the previous work. Cells adhering on the PU sealer were chemically fixed using 2.5%(v/w) glutaraldehyde (Sigma-Aldrich, USA) for 2.5 h in 0.1 M potassium phosphate buffer with the pH 7.2. The sealer/cell constructs were then underwent a graded dehydration series of ethanol (20,40,50,60,70,80,90,100,100,100% for 10 min each) and then dried by the critical-point drying method. After drying, the samples were mounted on aluminum stubs, coated with gold and viewed by scanning electron microscopy (SEM, Jeol 6500LV, Japan) at 30Kv under high vacuum. 2.4.3. MTT Assay Cell metabolic activity was evaluated by MTT assay. Briefly, 10 g of AH Plus and PU were firstly sterilized by ethylene oxide gas exposure and then immersed in 100 ml DMEM media under 37 °C for 24 h respectively to form the leaching liquor. L929 murine fibroblasts that cultured in DMEM media for three days were seeded (5 × 103 cells) in 1 ml AH and PU leaching liquor. The experiment was run in triplicate, and the plastic well was set as control. The cells were cultured in the incubator at 37 °C with 5% CO2/95% air atmosphere for 1 day 3 day(s) and 5 day(s). The medium was changed every two days. The L929 cells cultured in leaching liquor were observed by an optical microscope (Nikon eclipse TE2000-u). And the proliferation of L929 cells was determined using MTT (3-{4,5-dimethylthiazol-2yl}-2,5-diphenyl-2H-tetrazoliumbromide) assay though a standard procedure [15]. 2.4.4. Statistical analysis Statistical significance was determined using SPSS (v10.0) software. Statistical comparison of two experimental groups was performed using the Student's t-test. Here, p b 0.01 was considered to be highly significant. 3. Results
2.3.6. Statistical analysis Statistical analysis of data for physicochemical properties were carried out by one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons using SPSS (v10.0) software to determine the values that were significantly different. Differences were considered statistically significant at p b 0.05. 2.4. Cytotoxicity 2.4.1. Cell culture Cytotoxicity of the PU sealer was evaluated by culturing with L929 murine fibroblasts(the L929 murine fibroblasts were obtained from the Medical Center of Sichuan University, China.). The cells grew routinely in DMEM medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin [14]. Cells were maintained at 37 °C with 5% CO2/95% air atmosphere, and subcultured for a week using trypsin/ PBS. The medium was changed every other day. Each specimen was made by injecting the homogeneous mixture of Part A and Part B into the teflon mold (Φ6.0 mm × 2.0 mm). After curing at 37 °C and relative humidity of 95% for 24 hours, the samples were packaged and sterilized by ethylene oxide gas exposure
3.1. Formation mechanism During the prepolymerization, the hydroxyl groups of PTMEG act as a proton donor. Firstly, PTMEG copolymerize with aliphatic IPDI. And then BDO was added as chain extender. IPDI and BDO form hard segment of the pre-PU molecular chain and PTMEG form the soft parts. When Part A mixed with Part B, there is a mild reaction between –NCO (in prepolymer) and –OH (in polyols and prepolymer). The homogeneous mixture is injectable at first, then it cure at body temperature. 3.2. FTIR spectra FTIR is widely used to study the curing process of polyurethane by monitoring the attenuation of the band at 2200–2300 cm−1. As shown in Fig. 1, isocyanate groups (at 2200–2300 cm−1) from the prepolymer and amino groups (at 3100–3600 cm−1) mainly from triethanolamine decrease with the cure time increasing, while bands at 1600–1760 cm−1 (attributed to C = O vibration) and bands at 1540 cm−1, 1376–1388 cm−1, 1226–1292 cm−1 (attributed to N-H,
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after curing is 13,658 and 37,161. And by calculation we can also get the dispersion index is 1.569 and 2.075, respectively. The degree of polymerization X n indicates the number of the constitutional repeating unit of the polymer. Thus, X n of the prepolymer and polymer after curing is 12.22 and 25.12, respectively. 3.3. Physicochemical properties of sealers
Fig. 1. The representative FTIR spectra of PTMEG/BD/IPDI in the zone 450–4000 cm−1 with cure time. NCO peak at 2350 cm−1 apparently decrease with the increasing of cure time.
C-N and C-C-N vibration) increase. Fig. 2 shows the decay of NCO with increasing cure time. The decrease of isocyanate absorption is attributed to its reaction with amino and hydroxyl groups. And the isocyanate absorption would not disappear if isocyanate monomer excess. The integrated peak areas were calculated and listed in Fig. 2 (inset in top right corner) to qualitatively evaluate the conversion changing against the time. It can be seen from Fig. 2 (inset in top right corner) that NCO group decay fast at the beginning, and then slower with the increasing cure time. It also suggests that more than 90% degree of conversion could be achieved within 280 min, and about 1470 min was needed to achieve 95% curing. 3.2.1. Gel permeation chromatography (GPC) analysis Average molecular weights (Mw) and molecular weight dispersion (Mw/Mn) for pre-PU and PU were obtained through GPC system. The average molecular weight (Mw) of the prepolymer and polymer
The physicochemical properties of AH Plus, Apexit Plus and PU sealer are presented in Table 2. Apexit Plus had the shortest setting time, followed by PU sealer and AH Plus. For PU sealer, no change was observed until after 120 min, after which some tackiness was noted. 200 min latter, a smooth indentation was observed, and no mark was left at about 240 min. Both flow and film thickness demonstrates the fluid ability of injectable materials. Here, novel injectable PU sealer demonstrated great fluid ability by flow and film thickness data. Statistical analysis demonstrated that the fluid ability of PU sealer were significantly higher than AH Plus and Apexit Plus. PU sealer also behaved good stability, for the solubility was only 0.13 ± 0.07% after 24 hours soaking in deionized water. Statistical analysis revealed that the results of PU sealer was significantly lower than AH Plus and Apexit Plus. All materials (AH Plus, Apexit Plus and PU sealer) were microdilatancy after immersed in deionized water for 4 weeks. Statistical analysis demonstrated similar results among AH Plus and PU sealer which were significantly higher than Apexit Plus. 3.4. Cytotoxicity Fig. 3 shows the SEM pictures of L929 murine fibroblasts cultured for 1 day, cells adhered on the surface of the material and appeared normal polygonal morphology. Also, it is interesting to note in Fig. 3(c) and (d) that, after culturing for 5 days, L929 cells greatly proliferated and adhered on the material. Evenly distribute and well spread cells on the surface of the material indicates that the PU sealer did not show significant cytotoxicity against L929 cells after incubation for 1 day and 5 days. The result of MTT assay is shown in Fig. 4. The proliferation of L929 cells in PU and Control groups steady increased with the culture time. However, there seemed to be no proliferation in AH group. In the prime of culture (day 1), the proliferation of cells in PU group is obviously higher than that of AH group (p b 0.01) and control group
Fig. 2. The representative FTIR spectra of sealer with cure time: 0, 2, 3, 5, 9, 12, 17, 20, 25, 30, 43, 59, 70, 90, 98, 144, 196, 224, 280, 1470 min. It shows the decay peaks of -NCO group. The inset in top right corner shows the integrated -NCO peak area vs. Time.
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Table 2 Physical–chemical properties of AH Plus, Apexit Plus and PU sealer.
Setting time(min) Flow (mm) Film thickness (μm) Solubility (%), 24 hours Dimensional change (%)
ISO standards
AH plus
Apexit Plus
PU sealer
When ≥30 min, ≤72 hr, within the range (min) ≥20 min ≥20 mm ≤50 μm ≤3% Expansion ≤0.1%
494 ± 25* 22.30 ± 0.68* 45.80 ± 1.30* 0.39 ± 0.21* 1.40 ± 0.71*
108 ± 21# 26.45 ± 0.84# 38.20 ± 2.59# 0.56 ± 0.27* 0.57 ± 0.33#
234 ± 19^ 32.79 ± 0.89^ 29.80 ± 3.27^ 0.13 ± 0.07^ 1.39 ± 0.38*
Values are mean ± standard deviation. Values followed by different superscript symbols in each row differ significantly.
(p b 0.01). At day 3, the proliferations of cells in PU group and control group is obviously higher than that of AH group (p b 0.01). At day 5, the proliferations of cells in control group is obviously higher than that of PU group (p b 0.01), and the proliferations in PU group is obviously higher than that of AH group (p b 0.01). Fig. 5 shows optical photomicrographs of L929 fibroblast cell interaction. The evaluation of cell morphology shows that cells could not attach and grew in the AH group, yet, the cells attached well in PU group. For 5 day(s), cells grew to confluence in PU groups, otherwise, cells in AH group could not even attach.
4. Discussion To avoid uncertain conversion rate and toxicity induced by polymerization [10], a novel injectable and self-curing system based polyurethane sealer has been developed in this study. The injectable system was designed into two parts for convenient manipulation and in situ self-curing. Part A was a prepolymer and was the reaction product of isophorone dissocyanate (IPDI) and polytetramethylene ether glycol (PTMEG). Isophorone dissocyanate (IPDI) was selected as the hard segment for it was considered nontoxic in degradation [16]. Whereas the soft segment was polytetramethylene ether glycol (PTMEG) for it’s excellent flexibility, hydrolysis resistance and resilience [17,18]. On the other hand, Part B was the curing agent which was the mixture of low molecular weight polyol and catalysts. Both
of the two parts were fluid slurries at ambient temperature and above. After mixing Part A and Part B at an appropriate proportion, the curable polymer would form. For root canal filling, cytotoxicity is related to short-term release of free monomers during the monomer–polymer conversion [8]. Many in vitro studies showed that incomplete polymerization should be directly responsible for the cytotoxicity on pulp and gingival cells. Efficient polymerization could lessen the cytotoxic by reducing unbound monomer. In recent years, LEE developed a series of PU-based root canal sealers to overcome the shrinkage of filling resins [7]. Neither thermoplastic nor visible-light curable polyurethane demonstrated good cytocompatibility for monomer remnants after reaction. Even after 2 weeks, photoactivated PU sealers obtained no more than 70% degree of conversion [9]. Thus urge us to develop a low cytotoxicity polyurethane with high conversion using for root canal filling. 4.1. Curing mechanism So, efficient polymerization in a short time is the key factor for resin based filling materials. In this study, a series of functional groups were detected using an FTIR spectrometer at different curing time. The peak of isocyanate group decayed with the increasing cure time. The degree of conversion was calculated by comparing the peak area of -NCO group at different curing time. Aforementioned
Fig. 3. Representative SEM micrographs of L929 co-cultured with the PU sealer for 1 day (a, b) and 5 days (c, d). After one day cell culture (a, b), fibrobalsts attached on the surface of PU and appeared a normal polygonal morphology; after 5 days (c, d), confluent cells greatly proliferated on PU.
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According to previous studies, a basic kinetic equation is dp ¼ kðT Þf ðpÞ dt
ð5Þ
and when presented in isothermal conditions dp −E ¼ A exp f ðpÞ dt RT
ð6Þ
Here, A is the pre-exponential factor and E is the activation energy. And the nth-order kinetics was chosen for modeling the cure kinetics. The nth-order kinetics can be expressed as
Fig. 4. MTT assay for proliferation of L929 murine fibroblasts cultured with leaching liquor of AH,PU, and control for 1,3, and 5 day(s) under same culture conditions. ** p b 0.01.
dp n ¼ k0 ð1−pÞ ð7Þ dt Here, k0 is constant which related to the rate constants, depends on the temperature. And p is the isocyanate conversion calculated by aforesaid formula (2). If n ¼ 1; lnð1−pÞ ¼ −k0 þ C
results qualitatively and quantitatively evaluate the conversion degree of polymerization. The data indicated that 90% NCO group participated in the curing reaction after 4 h, and only about 5% remained after 24 h. Almost all the -NCO group participated in the reaction in a short time, which ensure the minimal stimulation to the surroundings. According to GPC results, both weight average molecular weight and polymerization degree of PU sealer after curing were two times or more than that of the prepolymer. It is hypothesized that higher conversion degree of PU-based sealer may associate with lower cytotoxicity after curing in situ. As chemical reactions take place during the self-curing process, the understanding of the polymerization mechanism and kinetic are also crucial for the material changing from injectable gel to solid structure. Based on empirical rate laws, the rate of the curing degree for thermosetting systems can be expressed as a function of the cure degree and temperature [11,19].
If n ¼ 2;
1 ¼ k0 t þ C 1−p
If n ¼ 3;
1 ¼ k0 t þ C ð1−pÞ2
ð8Þ ð9Þ
ð10Þ
The curves in Fig. 6 are results calculated by the three equations applied for the curing process. The fitting results are summarized in Table 3. Comparing the results, it is evident to find that the curing process of PU sealer supports the third-order reaction. From above, this method attempts to adjust the equation to describe the polymeric reaction here, yet not suitable for the whole curing process. As the self-curing reaction proceeded, the liquid resin became solid slowly, and obviously its reaction mechanism changed. Kinetic equations could describe large part of the reaction but not the total
Fig. 5. Photographs of L929 murine fibroblasts cultured with different leaching liquor for 1 and 5 day(s). (a) AH 1 day, (b) AH 5 day(s), (c) PU 1 day, (d) PU 5 day(s).
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Fig. 6. Relationship between p and curing time of PU (a) n = 1; (b) n = 2; (c) n = 3.
process. For the reaction is controlled by different factors at different procedures. At the beginning, the rate is dominated by the reaction that between isocyanate groups and the amino groups from triethanolamine. After the entirely consume of the triethanolamine, the rate is controlled by the reaction between isocyanate groups and hydroxy groups. With the polymerization deepening on, the liquid gel gradually becomes solid. Ultimately, reaction rate is controlled by diffusion rather than chemical reaction. The efficient and sufficient third order reaction could assure minimal stimulation to the surrounding tissues. 4.2. Physicochemical properties For root canal filling, it is of great importance to design materials that are expected to meet some essential physicochemical requirements, Table 3 Rate constants and fitting results for the curing process of PU sealer (p, range of curing degree fitted to the reaction order; k, rate constants; SD, standard deviation of the fit). Model/parameters
p
k
SD
First-order Second-order Third-order
0.789–0.91 0.775–0.91 0.489–0.92
5.7 × 10−3 3.52 × 10−2 0.5085
0.6876 0.9140 0.9885
ranging from fluid ability and stability [20,21]. For further clinical application, a series technique tests have been systematic controlled by [ISO 6876:2001 (E)] to access physicochemical properties of the root filling materials. Although there is no definite standard setting time for sealers, it should be long enough for convenient clinic manipulation. In the present study, the setting time of PU sealer is between that of AH Plus and Apexit Plus. It is moderate for clinical application. The ability of the sealer to flow is an important feature that will allow the material to penetrate into irregularities and accessory canals [22]. From the results of flow and film thickness tests, we can see that PU sealers has better fluidity than AH Plus and Apexit Plus. It means that PU sealer can better full-fill the irregularities and accessory canals. However, excessive flow will increase the risk of extravasation, and also may lead to cytotoxicity and allergic-related reactions. Comprehensive evaluation, the fast setting and high conversion rate of PU sealer may reduce the risk of extravasation. The solubility and dimensional change were evaluated in order to measure the material stability. Simply, solubility means the loss of mass during a period of immersing into deionized water, and dimensional change indicates the shrinkage or expansion of the material after setting in percentage terms. The requirement for the International Standard of weight loss is no more than 3% after storage in distilled water for 24 h. All the three sealers met with the Standard. The solubility of PU sealer is much smaller than that of AH Plus and Apexit Plus. For most resin materials, the dimensional change came about during the first 4 weeks [23]. As material shrinkage will introduce gaps and channels, which over time may be large enough to permit micro-organisms to pass along, proper expansion is preferred. However, the development of the stress caused by expansion in deep, narrow root canal, may lead to a root fracture [24]. For PU sealer, an expansion of 1.4% was detected, which did not conform to the ISO standard (0.1%). Usually, setting and storage expansion of the sealer induces radial pressure on the pulpal aspect. However, the risk of fracture is governed by the associated tangential strain, which is affected by elastic modulus of dentine and filling material, and the percentage expansion of the material [25]. Taken in this sense, sealers with low elastic modulus have lower threat to dentine than those with high modulus. Therefore, the limitation introduced by material expansion should be highly depending on material characteristics. It is reported that the young's modulus for dentine is 13.2 ± 4.0 GPa [24]. And the modulus for the PU sealer is tested in advance to be 3.70(±0.42) MPa. The cured PU sealer has a low modulus which reduces the damage of root canal generated by volume expansion. And the low-modulus elastic material is also preferred to absorb some generated stress. As shown by the above evaluations, the new PU sealer system perfectly harmonizes with the requirements of root canal filling. 4.3. In vitro cytotoxicity For dental resin, unbound free monomers are considered to be directly responsible for cytotoxicity on pulp and gingival cells [8,25]. In other words, the release of the free monomers is correlated to the degree of polymerization conversion. To date, it has been reported that no full conversion could be achieved during the polymerization. Overwhelming, majority of dental resins have the degree of monomer–polymer conversion varied from 35% to 77% [26–29]. In this research, the PU sealer achieved high degree of conversion (95%) in a short time (24.5 h), thus effectively reduced the release of unbound free monomers. According to the cell experiment, L929 cells survived and grew on the bottom of all wells, they even could adhere and proliferate on the material. It means that the new PU sealer with high degree of conversion do not show significant cytotoxicity in the first 5 days. According to the preliminary cell culture results, cells routinely grow and proliferate within the culture time. It can be concluded that the new PU sealer shows good cytocompatibility during the culture process.
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5. Conclusion This paper introduces a novel injectable PU sealer. The two-part sealer is a self-curing system with high conversion degree. It is suitable for canal obturation that the material expands after polymerization in situ. The curing process was investigated by FTIR spectroscopy to monitor the decay of NCO group at different time. The chemical process contained three main periods, amino group controlled, hydroxy group controlled and last diffusionally controlled. Based on the research, the curing process of PU sealer supports the third-order reaction, and the efficient curing may minimal the cytotoxicity. Then physicochemical properties of the PU sealer were tested for further application, together with commercial AH plus and Apexit plus as control groups. The cell culture results estimated the new PU sealer has good biocompatibility. The new injectable sealer based on polyurethane may have good prospects in clinic. Acknowledgements This work is supported by 863 National Key Project (No. 2011AA030102) and National Natural Science Foundation of China (NSFC, 50972096, 51002099). References [1] Evanelos G. Kontakiotis, Giorgos N. Tzanetakis, Alexios L. Loizides, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 103 (2007) 854–859. [2] L. Fang, H. Jing-Wei, L. Zheng-Mei, L. Jun-qi, J. De-Min, Molecules 11 (2006) 953–958. [3] M. Francesca, O. Raquel, T. Manuel, F. Marco, David H. Pashley, R. Franklin, J. Dent. 38 (2010) 547–552.
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[4] M. Brannstrom, K.J. Nordenvall, J. Entomol. Res. 57 (1978) 3–10. [5] A. Gesi, O. Raffaelli, C. Goracci, D.H. Pashley, F.R. Tay, M. Ferrari, J. Endod. 31 (2005) 809–813. [6] K.H. Hsieh, K.H. Liao, E.H. Lai, J. Endod. 34 (2008) 303. [7] B.S. Lee, E.H. Lai, K.H. Liao, J. Endod. 34 (2008) 594–598. [8] M. Goldberg, Clin. Oral Investig. 12 (2008) 1–8. [9] L. Bor-sbiunn, W. Chenying, F. Yenyin, H. Kuohuang, L. Cbun-pin, J. Endod. 37 (2) (2011) 246–249. [10] N.C. Beriat, A. Ertan, Z.C. Cehreli, et al., J. Endod. 35 (2009) 110–112. [11] S. Li, R. Vatanparast, H. Lemmetyinen, Polymer 41 (2000) 5571–5576. [12] H.G. Elias, Macromolecules, Plenum Press, New York, 1977. [Chapter 8]. [13] International standard ISO 6876:2001(E). [14] A. Pizzoferrato, C.R. Arciola, E. Cenni, G. Ciapetti, S. Sassi, Biomaterials 16 (1995) 361–367. [15] H.N. Wang, Y.B. Li, Y. Zuo, J. Li, S. Ma, L. Cheng, Biomaterials 28 (2007) 3338–3348. [16] M.K. Lamba, K.A. Woodhouse, S.L. Cooper, Polyurethanes in Biomedical Applications, CRC Press, Washington, DC, 1998. [17] S. Gang, L. Yu, L. Hong, Z. Na, H. Keao, J. Appl. Polym. Sci. 105 (2007) 1611–1615. [18] In: C. Hepburn (Ed.), Polyurethane Elastomers, Allied Science Publishers, New York, 1997, p. 12. [19] S.Y. Li, R.V., E. Vuorimaa, H. Lemmetyinen, J. Polymer Sci., Part B: Polymer Phys. 38 (2000) 2213–2220. [20] L. Bor-shiunn, W. Chen-Ying, J. Endod. (2011) 37. [21] M.L. Zheng, Q.L. Jun, Y.F. Jun, L. Fang, W.H. Jing, J. Formos. Med. Assoc. 109 (11) (2010) 819–827. [22] M.-K. Wu, B. Fan, P.R. Wesselink, J. Endod. 26 (2000) 210–216. [23] O. Dag, N. Inger, John E. Tibballs, Dent. Mater. 17 (2001) 512–519. [24] H.H. Kuo, H.L. Ken, H.L. Eddie, L. Bor-Shiunn, Y.L. Chung, P.L. Chun, JOE (2008) 34. [25] J.L. Ferracane, J. Oral Rehabil. 21 (1994) 441–452. [26] J.L. Ferracane, Dent. Mater. Rev. Oral Biol. Med. 6 (1995) 302–318. [27] E. Asmussen, Scand. J. Dent. Res. 90 (1982) 490–496. [28] K. Chung, E.H. Greener, J. Oral Rehabil. 15 (1988) 555–562. [29] J.L. Ferracane, J.R. Condon, Dent. Mater. 6 (1990) 282–287.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
High conversion self-curing sealer based on a novel injectable polyurethane system for root canal filling Bin Sun a, Yi Zuo a,⁎, Jidong Li a, Li Wang a, Kuangyun Tang b, Di Huang a, Jingjing Du a, Peipei Luo a, Yubao Li a a b
Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China The State Key Laboratory of Oral Diseases and Orthognathic Surgery, Sichuan University West China College of Stomatology, Chengdu 610064, PR China
a r t i c l e
i n f o
Article history: Received 7 September 2012 Received in revised form 16 February 2013 Accepted 5 March 2013 Available online 16 March 2013 Keywords: Root canal filling Injectable and self-curing polyurethane High conversion degree Micro expansion Cytocompatibility
a b s t r a c t Low monomer–polymer conversion is the key factor leading to cytotoxicity for resin-containing restorative materials. This paper provides a new root canal filling system based on self-curing injectable polyurethane which can achieve high conversion in a short time. Traced FTIR spectra show more than 90% NCO group participated in the curing reaction after 4 h, and only about 5% remained after 24 h. The calculated data also testified the curing process supports a third-order reaction, and this efficient and sufficient reaction is postulated to weaken the toxic stimulation. By culturing with L929 murine fibroblasts, the PU sealer is shown to be favorable for cell attachment and proliferation. Then physicochemical properties of the injectable PU-based sealer were evaluated according to the Standard [ISO 6876:2001 (E)] for clinical application. A series of physicochemical properties of PU sealer have been tested comparing with AH Plus and Apexit Plus. And the results present that the self-curing PU sealer could not only match the clinic requirements, but even has better properties than the other two commercial sealers. We expect the high conversion PU sealer has a tremendous potential in the field of root canal filling after further biological evaluation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Hermetic 3-dimensional obturation of root canal system is of prime importance for long-term endodontic treatment success [1]. Sealers have been developed to eliminate oral cavity leakage. However, the main drawback of contemporary filling resins, such as polymethyl methacrylate, Resilon and Epiphany, is volumetric shrinkage during their polymerization [2,3]. Microscopic gap usually forms between material and tooth interface. The gap allows oral fluids leakage, leading to bacterial growth and penetration [4]. Even the most widely used root filling material, thermoplastisized Gutta-percha (GP), causes volume shrinkage after cooling [5]. Suitable material for root canal filling needs to be developed. Multiblock copolymer Polyurethane (PU) composed of soft segment and hard segment. In designing the injectable polymer sealer, tailored PU can offer many advantages [6], such as controlled fluid ability, strong bonding and fast curing. Specifically, polyurethane materials present volumetric dilatancy after polymerization, and this will meet with the requirement of hermetic obturation. To date, polyurethane based root canal filling materials have been developed by thermoplastic technique [6] and visible-light curable technique [7]. A set of experimental data proved that urethane acrylate-based sealers behaved better than Epiphany and EndoREZ for root canal filling. Unfortunately, the thermal initiators (2,2-azsobis-isobutyronitrile, AIBN or 2,2-azobis (2-methyl) ⁎ Corresponding author. Tel.: +86 28 85418178; fax: +86 28 85417273. E-mail address: [email protected] (Y. Zuo). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.03.014
butyronitrile, AMBN) and photoninitiators (camphorquinone and ethyl 4-dimethylaminobenzoate) is cytotoxic [8]. In addition, photoactivated PU-based sealer is not sufficient enough for root canal filling, its degree of conversion is no more than 70% after 2 weeks [9,10]. Other from previous thermo curable or visible-light curable polyurethane based sealers, a novel injectable and self-curing polyurethane sealer was developed. This study had three main purposes: (1) synthesis and analyze the curing mechanism of the novel PU sealer (2) evaluate the physical-chemical properties according to ISO standard and (3) evaluate the cytotoxicity of the novel sealer. 2. Materials and methods Polytetramethylene ether glycol (PTMEG 2000, Aladdin Reagent, Shanghai China), triethanolamine (Aladdin Reagent,Shanghai China), polyethylene glycol (600, Aladdin Reagent,Shanghai China) and Isophorone diisocyanate (IPDI, Aladdin Reagent,Shanghai China) were used as received. 1,4-butanediol (BDO, Aladdin Reagent, Shanghai China) was dehydrated under decompression under a vacuum of 1330 Pa at 120 °C. All reagents were of analytical reagent grade. 2.1. Polymers synthesis 2.1.1. Part A (prepolymer) Polytetramethylene etherglycol (PTMEG 2000) (90 g) was weighed and put into a dry three-neck flask equipped with a mechanical stirrer under nitrogen. Isophorone diisocyanate (IPDI) (22.41 g) was then
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added into the flask under nitrogen. The reaction mixture was heated to 70 °C and stirred for 5 h. Then 2 ml 1, 4-butanediol was added as a chain extender for crosslinking. The reaction was carried on for another 2 h and NCO-terminated PU prepolymer was yielded as Part A. The stoichiometric ratio (wt) of polyol/isocyanate is 1.5/1.
the column temperature was maintained at 40 °C. Molecular weight (Mw) calibrations were based on a linear calibration curve using polystyrene standards. The degree of polymerization ( X n ), which indicated the monomer numbers that the polymer molecules contains, is obtained as follows [12]:
2.1.2. Part B (curing agent) Part B was prepared by mixing stannous, triethanolamine, and polyethylene glycol (PEG 600) with a mass ratio of 2:3:8 at room temperature.
M0 ¼
2.1.3. Polymer preparation The PU sealer was achieved by mixing Part A and Part B at a mass ratio of 9:1 inside a two-pipe syringe and subjected to vigorous shaking for 10s (Amalgamator YY/T 0273–2009, Hangzhou Yinya new material Co. Ltd). Then the mixture was immediately dispensed into a teflon model (Φ6.0 mm × 10 mm). There would be a mild reaction between –NCO (in the prepolymer) and –OH (in the polyol). As the mixture cured in a few minutes at body temperature, samples were removed for future testing.
n X
ωn M n
ð3Þ
1
where M 0 is the molecular weight of the polymer unit, ωn is the molar ratio of different raw materials, Mn is the molecular weight of different raw materials.
Xn ¼
Mn
ð4Þ
M0
where X n is the degree of polymerization, M n is the number average molecular weight obtained through GPC, M 0 is the molecular weight of the polymer unit, and M 0 can be obtained from formula (3).
2.2. Analysis the conversion of polymerization
2.3. Physicochemical properties evaluation for root canal filling
Curing process of the self-curing polyurethane system based on PTMEG and IPDI was monitored by FTIR spectra. Gel permeation chromatography was used to analyze the molecular value of samples.
The flow, film thickness, setting time, solubility and dimensional change for the injectable PU sealer were determined according to the methods described in the International Organization for Standardization [ISO 6876:2001 (E)] [13], which was published for dental root canal sealing materials evaluation. The following tests (the setting time test was carried out at 37 °C and a relative humidity of 95%) were carried out at (23 ± 2)°C and at a relative humidity of (50 ± 5)%. Five samples per group were prepared for each of the following tests: flow, film thickness, setting time, solubility and dimensional change. And the materials were freshly mixed for each test. The main components of the sealers are shown in Table 1.
2.2.1. FTIR analysis To trace the chemical change during polymerization, PU sealer has been measured at different curing times. Five grams of Part A was dissolved in 250 ml dichloromethane, and Part B was dropped into the solution in proportion. Then a drop of the solution was immediately dropped onto the prepared potassium bromide disk. After dichloromethane volatilized, the functional groups of the sample was detected using a Nicolet 6700 FTIR spectrometer (Nicolet Perkin Elmer Co., America) from 500 cm −1–4000 cm −1. Baseline has been corrected for all absorption peaks. Subsequently, the polymerized procedure was investigated by magnifying the characteristic peaks of -NCO group of the PU sealer. And the degree of conversion was calculated by comparing the peak area of-NCO group at different curing time. The degree of conversion p can be expressed as following [11]: t
pðt Þ ¼ ∫0 dp
ð1Þ
Assuming no side reactions occur, the isocyanate conversion (p) can be used as the degree of curing: A −A∞ Isocyanate conversionðpÞ ¼ 1− t A0 −A∞
ð2Þ
Here, A0 is the integrated absorption area at the initial time, At is the integrated absorption area at time t during the curing process, and A∞ is the final integrated absorption area. As no isocyanate group will be available in a completely cured system, A∞ is zero. 2.2.2. Gel permeation chromatography (GPC) analysis The weight average molecular weights (Mw), number average molecular weight (Mn) and molecular weights dispersion (Mw/Mn) of the prepolymer and the cured PU sealer (the samples were placed in oven at 37 °C. for another 4 hours after setting) were respectively measured by gel permeation chromatography(GPC). Samples of 0.20 to 0.25 mg were dissolved in 3 ml tetrahydrofuran (THF) at ambient temperature. The GPC equipment consisted of a Waters 510 HPLC pump with a Super HM-H*2 (6.0 mm*15 cm) column, and a Waters 410 RI-detector. The flow rate was maintained at 0.6 ml/min, and
2.3.1. Flow Homogeneously mixed sealer of 0.5 ± 0.05 ml was taken with a syringe onto a piece of the glass plate. The glass plate was at least 40 × 40 mm 2 and the mass of one glass plate was approximately 20 g. Another glass plate (the same as the first one) was placed on top of the material centrally, followed by the weight to make a total mass on the plate of 120 ± 2 g. Ten minutes after the commencement of mixing, the weight was removed and the maximum and minimum diameters of the compressed disc of sealer were measured. If the diameters agree to within 1 mm, the result was recorded. The test was repeated if the two diameters differed by more than 1 mm. 2.3.2. Film thickness Film thickness was measured by two combined glass plates with a contact surface area of (200 ± 10) mm 2 to an accuracy of 1 μm. A portion of sealer was placed onto the center of one of the glass plates. As the area between the glass plates was completely filled with the
Table 1 Components of the root canal sealers. Sealer
Composition
AH Plus
Calcium tungstate, epoxy resins, zirconium oxide, iron oxide, silicone oil Calcium saltsa, hydrogenized colophony, disalicylate, bismuth saltsb, silicon dioxide, phosphoric acid PTMEG-IPDI based prepolymer as Part A, a mixture of polyglycol and catalystsc as Part B
Apexit Plus
PU sealer a b c
calcium hydroxide, calcium oxide, calcium phosphate. bismuth oxide, bismuth carbonate. stannous, triethanolamine.
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material, the other plate was placed on the sealer. After (180 ± 10) s, a load of 150 N was applied vertically on the top plate. The thickness of the plates was measured 10 min after the start of mixing. The results have been recorded by determining the thickness of the plates with and without sealer. 2.3.3. Setting time The setting time was tested with a Gillmore needle having a mass of (100 ± 0.5) g and a cylindrical end with a diameter of (2 ± 0.1) mm. The needle was vertically lowered on to the horizontal surface of the sealer. The needle tip was cleaned and the test was repeated with an interval of 10 min. Time was recorded when the indentation was invisible. 2.3.4. Solubility A shallow dish was weighted (W1) before the test. Two specimens with a diameter of (20 ± 1) mm and a height of (1.5 ± 0.1) mm were weighted (W2) and put into the shallow dish,taking care to avoid any contact between them and inner surface of the shallow. (50 ± 1) ml water was added, and then the dish was covered. The dish was placed in a cabinet for 24 h. And then the specimens were remove and washed with deionized water of 2 ml to 3 ml. The washings in the dish was examined as follows. The dish was put into an oven to evaporate the water without boiling. The dish was then weighted (W3) again. Then the results (W3 − W1) /W2 were recorded. 2.3.5. Dimensional change A toflon mould (Φ6.0 mm × 12 mm) was put on a polyethylene sheet back by a glass plate. Then 2 g fresh mixed sealer was injected into the mould. Another glass plate faced with a sheet of plastic was pressed on top of the sealer. Then the mould and the plates together were held firmly with a C-clamp. Five minutes after beginning the mix, the mould with the sealer and clamp was placed at an atmosphere of 95% to 100% relative humidity at (37 ± 1)°C for 24 h. After that, clamp and glasses were removed, the ends of the specimen was grinded flat by drawing the mould containing the specimen back and forth across fresh 600 grit wet sandpaper. Then the specimen was removed from the mould, the distance between the flat ends was measured to an accuracy of 10 μm. Then the specimen was remeasured to an accuracy of 10 μm after stored(a needle was used to hold the samples, the sample could neither touch the bottom nor exposed in the air) in distilled water at (37 ± 1)°C for 30 days. The change in length as a percentage of the original length was calculated and recorded.
according to the standard procedure for medical devices. They were used for cytotoxicity without any further manipulation. 2.4.2. SEM preparation L929 murine fibroblasts that cultured in DMEM media for three days were seeded on top of the specimens (1 × 10 4 cells per specimen). For allowing the cells to attach, the specimens were first placed in cell culture plate wells and left undisturbed in an incubator for 3 h, then an additional 1 ml culture medium was added into each well. The sealer/cell constructs were cultured in the incubator at 37 °C with 5% CO2/95% air atmosphere for 1 day and 5 day(s). The medium was changed every two days. Fixation, dehydrating and drying methods were followed from the previous work. Cells adhering on the PU sealer were chemically fixed using 2.5%(v/w) glutaraldehyde (Sigma-Aldrich, USA) for 2.5 h in 0.1 M potassium phosphate buffer with the pH 7.2. The sealer/cell constructs were then underwent a graded dehydration series of ethanol (20,40,50,60,70,80,90,100,100,100% for 10 min each) and then dried by the critical-point drying method. After drying, the samples were mounted on aluminum stubs, coated with gold and viewed by scanning electron microscopy (SEM, Jeol 6500LV, Japan) at 30Kv under high vacuum. 2.4.3. MTT Assay Cell metabolic activity was evaluated by MTT assay. Briefly, 10 g of AH Plus and PU were firstly sterilized by ethylene oxide gas exposure and then immersed in 100 ml DMEM media under 37 °C for 24 h respectively to form the leaching liquor. L929 murine fibroblasts that cultured in DMEM media for three days were seeded (5 × 103 cells) in 1 ml AH and PU leaching liquor. The experiment was run in triplicate, and the plastic well was set as control. The cells were cultured in the incubator at 37 °C with 5% CO2/95% air atmosphere for 1 day 3 day(s) and 5 day(s). The medium was changed every two days. The L929 cells cultured in leaching liquor were observed by an optical microscope (Nikon eclipse TE2000-u). And the proliferation of L929 cells was determined using MTT (3-{4,5-dimethylthiazol-2yl}-2,5-diphenyl-2H-tetrazoliumbromide) assay though a standard procedure [15]. 2.4.4. Statistical analysis Statistical significance was determined using SPSS (v10.0) software. Statistical comparison of two experimental groups was performed using the Student's t-test. Here, p b 0.01 was considered to be highly significant. 3. Results
2.3.6. Statistical analysis Statistical analysis of data for physicochemical properties were carried out by one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons using SPSS (v10.0) software to determine the values that were significantly different. Differences were considered statistically significant at p b 0.05. 2.4. Cytotoxicity 2.4.1. Cell culture Cytotoxicity of the PU sealer was evaluated by culturing with L929 murine fibroblasts(the L929 murine fibroblasts were obtained from the Medical Center of Sichuan University, China.). The cells grew routinely in DMEM medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin [14]. Cells were maintained at 37 °C with 5% CO2/95% air atmosphere, and subcultured for a week using trypsin/ PBS. The medium was changed every other day. Each specimen was made by injecting the homogeneous mixture of Part A and Part B into the teflon mold (Φ6.0 mm × 2.0 mm). After curing at 37 °C and relative humidity of 95% for 24 hours, the samples were packaged and sterilized by ethylene oxide gas exposure
3.1. Formation mechanism During the prepolymerization, the hydroxyl groups of PTMEG act as a proton donor. Firstly, PTMEG copolymerize with aliphatic IPDI. And then BDO was added as chain extender. IPDI and BDO form hard segment of the pre-PU molecular chain and PTMEG form the soft parts. When Part A mixed with Part B, there is a mild reaction between –NCO (in prepolymer) and –OH (in polyols and prepolymer). The homogeneous mixture is injectable at first, then it cure at body temperature. 3.2. FTIR spectra FTIR is widely used to study the curing process of polyurethane by monitoring the attenuation of the band at 2200–2300 cm−1. As shown in Fig. 1, isocyanate groups (at 2200–2300 cm−1) from the prepolymer and amino groups (at 3100–3600 cm−1) mainly from triethanolamine decrease with the cure time increasing, while bands at 1600–1760 cm−1 (attributed to C = O vibration) and bands at 1540 cm−1, 1376–1388 cm−1, 1226–1292 cm−1 (attributed to N-H,
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after curing is 13,658 and 37,161. And by calculation we can also get the dispersion index is 1.569 and 2.075, respectively. The degree of polymerization X n indicates the number of the constitutional repeating unit of the polymer. Thus, X n of the prepolymer and polymer after curing is 12.22 and 25.12, respectively. 3.3. Physicochemical properties of sealers
Fig. 1. The representative FTIR spectra of PTMEG/BD/IPDI in the zone 450–4000 cm−1 with cure time. NCO peak at 2350 cm−1 apparently decrease with the increasing of cure time.
C-N and C-C-N vibration) increase. Fig. 2 shows the decay of NCO with increasing cure time. The decrease of isocyanate absorption is attributed to its reaction with amino and hydroxyl groups. And the isocyanate absorption would not disappear if isocyanate monomer excess. The integrated peak areas were calculated and listed in Fig. 2 (inset in top right corner) to qualitatively evaluate the conversion changing against the time. It can be seen from Fig. 2 (inset in top right corner) that NCO group decay fast at the beginning, and then slower with the increasing cure time. It also suggests that more than 90% degree of conversion could be achieved within 280 min, and about 1470 min was needed to achieve 95% curing. 3.2.1. Gel permeation chromatography (GPC) analysis Average molecular weights (Mw) and molecular weight dispersion (Mw/Mn) for pre-PU and PU were obtained through GPC system. The average molecular weight (Mw) of the prepolymer and polymer
The physicochemical properties of AH Plus, Apexit Plus and PU sealer are presented in Table 2. Apexit Plus had the shortest setting time, followed by PU sealer and AH Plus. For PU sealer, no change was observed until after 120 min, after which some tackiness was noted. 200 min latter, a smooth indentation was observed, and no mark was left at about 240 min. Both flow and film thickness demonstrates the fluid ability of injectable materials. Here, novel injectable PU sealer demonstrated great fluid ability by flow and film thickness data. Statistical analysis demonstrated that the fluid ability of PU sealer were significantly higher than AH Plus and Apexit Plus. PU sealer also behaved good stability, for the solubility was only 0.13 ± 0.07% after 24 hours soaking in deionized water. Statistical analysis revealed that the results of PU sealer was significantly lower than AH Plus and Apexit Plus. All materials (AH Plus, Apexit Plus and PU sealer) were microdilatancy after immersed in deionized water for 4 weeks. Statistical analysis demonstrated similar results among AH Plus and PU sealer which were significantly higher than Apexit Plus. 3.4. Cytotoxicity Fig. 3 shows the SEM pictures of L929 murine fibroblasts cultured for 1 day, cells adhered on the surface of the material and appeared normal polygonal morphology. Also, it is interesting to note in Fig. 3(c) and (d) that, after culturing for 5 days, L929 cells greatly proliferated and adhered on the material. Evenly distribute and well spread cells on the surface of the material indicates that the PU sealer did not show significant cytotoxicity against L929 cells after incubation for 1 day and 5 days. The result of MTT assay is shown in Fig. 4. The proliferation of L929 cells in PU and Control groups steady increased with the culture time. However, there seemed to be no proliferation in AH group. In the prime of culture (day 1), the proliferation of cells in PU group is obviously higher than that of AH group (p b 0.01) and control group
Fig. 2. The representative FTIR spectra of sealer with cure time: 0, 2, 3, 5, 9, 12, 17, 20, 25, 30, 43, 59, 70, 90, 98, 144, 196, 224, 280, 1470 min. It shows the decay peaks of -NCO group. The inset in top right corner shows the integrated -NCO peak area vs. Time.
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Table 2 Physical–chemical properties of AH Plus, Apexit Plus and PU sealer.
Setting time(min) Flow (mm) Film thickness (μm) Solubility (%), 24 hours Dimensional change (%)
ISO standards
AH plus
Apexit Plus
PU sealer
When ≥30 min, ≤72 hr, within the range (min) ≥20 min ≥20 mm ≤50 μm ≤3% Expansion ≤0.1%
494 ± 25* 22.30 ± 0.68* 45.80 ± 1.30* 0.39 ± 0.21* 1.40 ± 0.71*
108 ± 21# 26.45 ± 0.84# 38.20 ± 2.59# 0.56 ± 0.27* 0.57 ± 0.33#
234 ± 19^ 32.79 ± 0.89^ 29.80 ± 3.27^ 0.13 ± 0.07^ 1.39 ± 0.38*
Values are mean ± standard deviation. Values followed by different superscript symbols in each row differ significantly.
(p b 0.01). At day 3, the proliferations of cells in PU group and control group is obviously higher than that of AH group (p b 0.01). At day 5, the proliferations of cells in control group is obviously higher than that of PU group (p b 0.01), and the proliferations in PU group is obviously higher than that of AH group (p b 0.01). Fig. 5 shows optical photomicrographs of L929 fibroblast cell interaction. The evaluation of cell morphology shows that cells could not attach and grew in the AH group, yet, the cells attached well in PU group. For 5 day(s), cells grew to confluence in PU groups, otherwise, cells in AH group could not even attach.
4. Discussion To avoid uncertain conversion rate and toxicity induced by polymerization [10], a novel injectable and self-curing system based polyurethane sealer has been developed in this study. The injectable system was designed into two parts for convenient manipulation and in situ self-curing. Part A was a prepolymer and was the reaction product of isophorone dissocyanate (IPDI) and polytetramethylene ether glycol (PTMEG). Isophorone dissocyanate (IPDI) was selected as the hard segment for it was considered nontoxic in degradation [16]. Whereas the soft segment was polytetramethylene ether glycol (PTMEG) for it’s excellent flexibility, hydrolysis resistance and resilience [17,18]. On the other hand, Part B was the curing agent which was the mixture of low molecular weight polyol and catalysts. Both
of the two parts were fluid slurries at ambient temperature and above. After mixing Part A and Part B at an appropriate proportion, the curable polymer would form. For root canal filling, cytotoxicity is related to short-term release of free monomers during the monomer–polymer conversion [8]. Many in vitro studies showed that incomplete polymerization should be directly responsible for the cytotoxicity on pulp and gingival cells. Efficient polymerization could lessen the cytotoxic by reducing unbound monomer. In recent years, LEE developed a series of PU-based root canal sealers to overcome the shrinkage of filling resins [7]. Neither thermoplastic nor visible-light curable polyurethane demonstrated good cytocompatibility for monomer remnants after reaction. Even after 2 weeks, photoactivated PU sealers obtained no more than 70% degree of conversion [9]. Thus urge us to develop a low cytotoxicity polyurethane with high conversion using for root canal filling. 4.1. Curing mechanism So, efficient polymerization in a short time is the key factor for resin based filling materials. In this study, a series of functional groups were detected using an FTIR spectrometer at different curing time. The peak of isocyanate group decayed with the increasing cure time. The degree of conversion was calculated by comparing the peak area of -NCO group at different curing time. Aforementioned
Fig. 3. Representative SEM micrographs of L929 co-cultured with the PU sealer for 1 day (a, b) and 5 days (c, d). After one day cell culture (a, b), fibrobalsts attached on the surface of PU and appeared a normal polygonal morphology; after 5 days (c, d), confluent cells greatly proliferated on PU.
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According to previous studies, a basic kinetic equation is dp ¼ kðT Þf ðpÞ dt
ð5Þ
and when presented in isothermal conditions dp −E ¼ A exp f ðpÞ dt RT
ð6Þ
Here, A is the pre-exponential factor and E is the activation energy. And the nth-order kinetics was chosen for modeling the cure kinetics. The nth-order kinetics can be expressed as
Fig. 4. MTT assay for proliferation of L929 murine fibroblasts cultured with leaching liquor of AH,PU, and control for 1,3, and 5 day(s) under same culture conditions. ** p b 0.01.
dp n ¼ k0 ð1−pÞ ð7Þ dt Here, k0 is constant which related to the rate constants, depends on the temperature. And p is the isocyanate conversion calculated by aforesaid formula (2). If n ¼ 1; lnð1−pÞ ¼ −k0 þ C
results qualitatively and quantitatively evaluate the conversion degree of polymerization. The data indicated that 90% NCO group participated in the curing reaction after 4 h, and only about 5% remained after 24 h. Almost all the -NCO group participated in the reaction in a short time, which ensure the minimal stimulation to the surroundings. According to GPC results, both weight average molecular weight and polymerization degree of PU sealer after curing were two times or more than that of the prepolymer. It is hypothesized that higher conversion degree of PU-based sealer may associate with lower cytotoxicity after curing in situ. As chemical reactions take place during the self-curing process, the understanding of the polymerization mechanism and kinetic are also crucial for the material changing from injectable gel to solid structure. Based on empirical rate laws, the rate of the curing degree for thermosetting systems can be expressed as a function of the cure degree and temperature [11,19].
If n ¼ 2;
1 ¼ k0 t þ C 1−p
If n ¼ 3;
1 ¼ k0 t þ C ð1−pÞ2
ð8Þ ð9Þ
ð10Þ
The curves in Fig. 6 are results calculated by the three equations applied for the curing process. The fitting results are summarized in Table 3. Comparing the results, it is evident to find that the curing process of PU sealer supports the third-order reaction. From above, this method attempts to adjust the equation to describe the polymeric reaction here, yet not suitable for the whole curing process. As the self-curing reaction proceeded, the liquid resin became solid slowly, and obviously its reaction mechanism changed. Kinetic equations could describe large part of the reaction but not the total
Fig. 5. Photographs of L929 murine fibroblasts cultured with different leaching liquor for 1 and 5 day(s). (a) AH 1 day, (b) AH 5 day(s), (c) PU 1 day, (d) PU 5 day(s).
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Fig. 6. Relationship between p and curing time of PU (a) n = 1; (b) n = 2; (c) n = 3.
process. For the reaction is controlled by different factors at different procedures. At the beginning, the rate is dominated by the reaction that between isocyanate groups and the amino groups from triethanolamine. After the entirely consume of the triethanolamine, the rate is controlled by the reaction between isocyanate groups and hydroxy groups. With the polymerization deepening on, the liquid gel gradually becomes solid. Ultimately, reaction rate is controlled by diffusion rather than chemical reaction. The efficient and sufficient third order reaction could assure minimal stimulation to the surrounding tissues. 4.2. Physicochemical properties For root canal filling, it is of great importance to design materials that are expected to meet some essential physicochemical requirements, Table 3 Rate constants and fitting results for the curing process of PU sealer (p, range of curing degree fitted to the reaction order; k, rate constants; SD, standard deviation of the fit). Model/parameters
p
k
SD
First-order Second-order Third-order
0.789–0.91 0.775–0.91 0.489–0.92
5.7 × 10−3 3.52 × 10−2 0.5085
0.6876 0.9140 0.9885
ranging from fluid ability and stability [20,21]. For further clinical application, a series technique tests have been systematic controlled by [ISO 6876:2001 (E)] to access physicochemical properties of the root filling materials. Although there is no definite standard setting time for sealers, it should be long enough for convenient clinic manipulation. In the present study, the setting time of PU sealer is between that of AH Plus and Apexit Plus. It is moderate for clinical application. The ability of the sealer to flow is an important feature that will allow the material to penetrate into irregularities and accessory canals [22]. From the results of flow and film thickness tests, we can see that PU sealers has better fluidity than AH Plus and Apexit Plus. It means that PU sealer can better full-fill the irregularities and accessory canals. However, excessive flow will increase the risk of extravasation, and also may lead to cytotoxicity and allergic-related reactions. Comprehensive evaluation, the fast setting and high conversion rate of PU sealer may reduce the risk of extravasation. The solubility and dimensional change were evaluated in order to measure the material stability. Simply, solubility means the loss of mass during a period of immersing into deionized water, and dimensional change indicates the shrinkage or expansion of the material after setting in percentage terms. The requirement for the International Standard of weight loss is no more than 3% after storage in distilled water for 24 h. All the three sealers met with the Standard. The solubility of PU sealer is much smaller than that of AH Plus and Apexit Plus. For most resin materials, the dimensional change came about during the first 4 weeks [23]. As material shrinkage will introduce gaps and channels, which over time may be large enough to permit micro-organisms to pass along, proper expansion is preferred. However, the development of the stress caused by expansion in deep, narrow root canal, may lead to a root fracture [24]. For PU sealer, an expansion of 1.4% was detected, which did not conform to the ISO standard (0.1%). Usually, setting and storage expansion of the sealer induces radial pressure on the pulpal aspect. However, the risk of fracture is governed by the associated tangential strain, which is affected by elastic modulus of dentine and filling material, and the percentage expansion of the material [25]. Taken in this sense, sealers with low elastic modulus have lower threat to dentine than those with high modulus. Therefore, the limitation introduced by material expansion should be highly depending on material characteristics. It is reported that the young's modulus for dentine is 13.2 ± 4.0 GPa [24]. And the modulus for the PU sealer is tested in advance to be 3.70(±0.42) MPa. The cured PU sealer has a low modulus which reduces the damage of root canal generated by volume expansion. And the low-modulus elastic material is also preferred to absorb some generated stress. As shown by the above evaluations, the new PU sealer system perfectly harmonizes with the requirements of root canal filling. 4.3. In vitro cytotoxicity For dental resin, unbound free monomers are considered to be directly responsible for cytotoxicity on pulp and gingival cells [8,25]. In other words, the release of the free monomers is correlated to the degree of polymerization conversion. To date, it has been reported that no full conversion could be achieved during the polymerization. Overwhelming, majority of dental resins have the degree of monomer–polymer conversion varied from 35% to 77% [26–29]. In this research, the PU sealer achieved high degree of conversion (95%) in a short time (24.5 h), thus effectively reduced the release of unbound free monomers. According to the cell experiment, L929 cells survived and grew on the bottom of all wells, they even could adhere and proliferate on the material. It means that the new PU sealer with high degree of conversion do not show significant cytotoxicity in the first 5 days. According to the preliminary cell culture results, cells routinely grow and proliferate within the culture time. It can be concluded that the new PU sealer shows good cytocompatibility during the culture process.
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5. Conclusion This paper introduces a novel injectable PU sealer. The two-part sealer is a self-curing system with high conversion degree. It is suitable for canal obturation that the material expands after polymerization in situ. The curing process was investigated by FTIR spectroscopy to monitor the decay of NCO group at different time. The chemical process contained three main periods, amino group controlled, hydroxy group controlled and last diffusionally controlled. Based on the research, the curing process of PU sealer supports the third-order reaction, and the efficient curing may minimal the cytotoxicity. Then physicochemical properties of the PU sealer were tested for further application, together with commercial AH plus and Apexit plus as control groups. The cell culture results estimated the new PU sealer has good biocompatibility. The new injectable sealer based on polyurethane may have good prospects in clinic. Acknowledgements This work is supported by 863 National Key Project (No. 2011AA030102) and National Natural Science Foundation of China (NSFC, 50972096, 51002099). References [1] Evanelos G. Kontakiotis, Giorgos N. Tzanetakis, Alexios L. Loizides, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 103 (2007) 854–859. [2] L. Fang, H. Jing-Wei, L. Zheng-Mei, L. Jun-qi, J. De-Min, Molecules 11 (2006) 953–958. [3] M. Francesca, O. Raquel, T. Manuel, F. Marco, David H. Pashley, R. Franklin, J. Dent. 38 (2010) 547–552.
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