Influence of Hyperbranched Multi-methacrylates for Dental Neat

Qichun Wan, Deborah Rumpf, Scott R. Schricker, Angelo Mariotti, and Bill M. Culbertson*. College of Dentistry, The Ohio State University, 305 West 12t...
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Biomacromolecules 2001, 2, 217-222

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Influence of Hyperbranched Multi-methacrylates for Dental Neat Resins on Proliferation of Human Gingival Fibroblasts Qichun Wan, Deborah Rumpf, Scott R. Schricker, Angelo Mariotti, and Bill M. Culbertson* College of Dentistry, The Ohio State University, 305 West 12th Street, P.O. Box 182357, Columbus, Ohio 43218-2357 Received October 2, 2000; Revised Manuscript Received November 13, 2000

We have previously shown that hyperbranched multi-methacrylate (H-MMA)-modified dental resins have VLC activities, lower polymerization shrinkage, and improved mechanical properties, compared to the 2,2bis[4-(2-hydroxy-3-methacryloyolxypropoxy)phenyl]propane/triethyleneglycol dimethacrylate (BisGMA/ TEGDMA) neat resin. The results are due to the unique molecular structure and high molecular weight of H-MMA intermediates. The purpose of this study was to evaluate the biocompatibility of H-MMA-modified dental neat resins. The cell proliferation of three human gingival fibroblast strains on either H-MMA, BisGMA/ TEGDMA, or a polystyrene disk was examined. Following 10 days of cell proliferation, there was no statistical difference in cell number between H-MMA-modified and unmodified resin disks. H-MMA-modified resins had less free monomer leaching than the unmodified resin but showed similar properties in water sorption and contact angle values. All these results suggest that the biocompatibility of H-MMA-modified dental neat resins is as good as that of commercially used BisGMA/TEGDMA resin and H-MMA has potential applications in dental composites. Introduction The current widely used 2,2-bis[4-(2-hydroxy-3-methacryloyolxypropoxy)phenyl]propane (BisGMA)- and triethyleneglycol dimethacrylate (TEGDMA)-based dental composite resins have exhibited satisfactory clinical performance, especially for anterior restoratives. However, there are some deficiencies associated with using this resin matrix, such as susceptibility to water sorption, incomplete conversion of double bonds, and high polymerization shrinkage. Over the past several decades, extensive efforts have been made to develop new resin systems, including BisGMA derivatives,1-3 fluorinated dimethacrylates,4-6 dimethacrylates with high molecular weight and rigid structure,7-9 urethane dimethacrylate,10-12 and spiro-ortho-ester based monomers.13-16 Among them, high molecular weight multi-methacrylates have been considered as one of the most promising resin system to reduce the polymerization shrinkage and improve the mechanical properties. Significantly, recently developed hyperbranched multi-methacrylates have been proved to be useful as comonomer candidates in formulating dental resin systems.17-19 In recently published papers, we reported the syntheses, characterization, and copolymerization of hyperbranched multi-methacrylates and evaluation of these hyperbranched polyesters as modifiers for formulating dental composites.20,21 It is generally considered that modification of dental composites with high molecular weight polymers will reduce their polymerization shrinkage and improve their mechanical properties, but at the expense of increasing formulation viscosities and decreasing the polymerization activities. * To whom correspondence should be addressed. Tel. (614) 292-0777. Fax: ext. 9422. E-mail: [email protected].

Hyperbranched polymers could improve the mechanical properties and decrease the polymerization shrinkage while maintaining lower viscosity due to their unique molecular structure. However, little is known about their influences on the biocompatibility with the vital tissues, especially gingival fibroblasts, since such dental composites often come into intimate contact with periodontal tissue. There are three stages for the biocompatibility evaluation: initial screening test; small animal testing; long-term mammals or human subject testing.22 Among them, the in vitro cell culture technique is the easiest and cheapest method and has been used to evaluate the cellular behavior on dental composites.23-25 Generally, the biocompatibility of dental composites is good, especially when they are highly filled.26,27 However, the release of unreacted monomers and components or degradation products of dental materials is considered a major factor causing tissue inflammation such as pulp sensitivity and death or cell lipid metabolism activity.28-32 Hanks et al.29 studied the inhibitory effects of resin components on DNA and protein synthesis of Balb/c 3T3 mouse fibroblasts. They found that initiators such as camphorquinone and ethoxylated Bisphenol A were the most cytotoxic components, while BisGMA, UDMA, TEGDMA, and Bisphenol A showed moderate cytotoxicity. Ratanasathien et al.33 found that the cytotoxicity ranking of the unpolymerized monomers were BisGMA > UDMA > TEGDMA >>> HEMA. It was also found that the monomethyl ether of hydroquinone (MEHQ) and DMAEMA could inhibit the hamster oral epithelial growth and BPO and DMAEMA could affect cell lipid synthesis.32 The cytotoxicity of acrylates was correlated to their lipophilicity, indicating that monomer might incorporate into

10.1021/bm000101p CCC: $20.00 © 2001 American Chemical Society Published on Web 12/21/2000

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Figure 1. Structures of BisGMA, TEGDMA, and H-MMA monomers used in this study

the lipid bilayer of the cell membrane. It was also found that acrylates were more toxic than the methacrylates with similar structure and the presence of a hydroxyl group also increased the cytotoxicity.34 The impurity in commercial BisGMA may have an estrogenesis effect on the development, growth, and normal function of estrogen-sensitive tissues.35 It was found that BisGMA exerts a small but detectable effect on estrogensensitive tissues. This may be due to existence of a known xenoestrogene Bisphenol A, a starting compound for making BisGMA. For the visible-light-cured dental resins, Tiba compared the biocompatibility of a multi-methacrylate-containing resin with the conventional BisGMA/TEGDMA, using cell culture techniques of human gingival fibroblasts. The results revealed that the multi-methacrylate-based resins favored the human gingival fibroblast cell growth.36 Young37 evaluated the effect of the amount of TEGDMA in dental resin on the cell proliferation by using a fluorometric cell proliferation assay and a radiolabeled protein assay. It was found that materials without TEGDMA resulted in the greater cell proliferation.37 The objectives of this initial study were to examine the influence of dental neat resins with H-MMA modifiers on proliferation of human gingival fibroblasts. The acetone extraction, water sorption, and contact angle have also been studied. It is hypothesized that hyperbranched multi-methacrylates, with less free monomer leaching, could be used as modifiers in dental applications. BisGMA/TEGDMA resins modified with hyperbranched, multi-methacrylates

should have less free monomer(s) in the polymerized formulations because the H-MMA-based multi-methacrylates have significantly higher molecular weight than BisGMA or TEGDMA dimethacrylates and the large number of methacrylate double bonds on the hyperbranched polymers should tether more of the BisGMA and TEGDMA molecules into the cross-linked matrix. Materials and Methods Materials. 2,2-Bis[4-(2-hydroxy-3-methacryloyolxypropoxy)phenyl]propane (BisGMA) and triethyleneglycol dimethacrylate (TEGDMA) were received from Cook Composites and Polymers (Kansas, MO) and Aldrich Chemical Co. (Milwaukee, WI), respectively. Camphorquinone (CQ) and 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA) were also used as received from Aldrich Chemical Co. Hyperbranched multi-methacrylates (H20-MMA and H30-MMA) were synthesized from commercial (Boltorn) hyperbranched polyesters, supplied by Perstorp Corp. In the published procedures,20,21 the Boltorn polyesters were esterified with methacryloyl chloride or methacrylic anhydride, with the polymers purified and characterized by FT-IR, NMR (1H and 13C), and gel permeation chromatography (GPC).20,21 Molecular weights (MW) of the multi-methacrylates, estimated by GPC, were 3075 and 5340, compared to BisGMA having 512. The general structure of the monomers used or prepared in this study are shown in Figure 1. Preparation of VLC Disk Samples. All of the resin formulation contained 0.5 wt % of initiator CQ and 1.0 wt

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Biocompatibility of Modified Dental Composites Table 1. Cell Strain Information strain

donor sex

donor age

passages used

A B C

M M F

39 75 36

T3 T3 T3

% of co-initiator DMAEMA. Two experimental resins were formulated by mixing 10% of hyperbranched multi-methacrylates H20-MMA or H30-MMA with BisGMA/TEGDMA (50:50 wt/wt). BisGMA/TEGDMA (50:50 wt/wt) served as control. In addition, tissue culture polystyrene disks were used as a negative control. To make the polymer disks, a stainless steel mold with an inner diameter of 15 mm and a thickness of 2 mm was used. The mold was put against a clean glass slide. After the above mixture was added to the mold, another glass slide was placed on top of the mold. It was then cured for 1 min by using a light curing unit (Belle Glass HP TEGLITE, 400 mW/cm2) on each side and further cured for another 4 min using a Triad 2000 light oven. In the disk preparation, care was given to avoid any surface defects since they would influence the cell attachment and behavior. Isolation and Culture of Human Gingival Fibroblasts. Fibroblasts used in these experiments were obtained from three different maxillary gingival papilla biopsies (see Table 1). Cells were cultured and propagated as previously described.38 Cells used for these experiments were thawed from The College of Dentistry Cryogenic Storage Facility and grown in minimal essential media (MEM) supplemented with 10% fetal bovine serum, L-glutamine, penicillin (100 units/mL), and streptomycin (100 mg/mL). Only fibroblasts between the third and fifth passages were used in the experiments described. Cell Proliferation Assay. Cell proliferation was measured over a 10-day period on dental neat resins. By use of a Coulter counter to determine cell number, cells were plated at 20 000 cells per disk in 1 mL of MEM supplemented with 10% fetal bovine serum, L-glutamine, penicillin (100 units/ mL), and streptomycin (100 mg/mL) on BisGMA/TEGDMA, H20-MMA, H30-MMA, or polystyrene control disks. Each disk was used multiple times, and disks were cleaned between uses as follows. Sixteen disks were put into a 250mL beaker with 100 mL of 1% Alconox detergent solution and ultrasonically cleaned (Jelenko Ultrasonic Cleaner, Armonk, NY) at 44-48 kHz for 20 min. The Alconox was then removed and 100 mL of distilled water was added, followed by ultrasonic treatment for another 20 min. Disks were rinsed with 50 mL of distilled water approximately 15 times to make sure all the Alconox detergent was removed. The clean disks were pop-dried by using Kleenex tissue paper and stored in a clean container. The silicon O-rings were cleaned using the same procedure. Prior to use, all disks and silicon O-rings were sterilized overnight in ultraviolet light. The disks were then placed into the wells of 24-well cell culture plates, and silicone O-rings were used to hold disks in place. At days 1, 3, 5, 7 and 10, the cell number was determined using the Alamar Blue assay. Alamar Blue (AB) is a nondestructive method measuring cell metabolism activities.39 After incubation with

cells, the Alamar Blue is removed, a new fresh medium is added. Cells are still alive for the next period testing. This method is shown very effective in testing the cell metabolism over a period of time intervals. The AB dye was diluted from the stock solution with growth medium in a 1:10 dilution. After the cell plating incubated for the desired time period, it was taken out from the incubation. The growth medium was removed from each well, and 1 mL of AB-media solution was added. Then the cell culture plates were incubated for 3 h. During the same time, the dilution plates were prepared. That is, 1.9-mL of double distilled water was pipetted into a clean 24-well cell culture plate. After incubation, 100 µL of liquid was drawn from each well and added to the above dilution plates, containing 1.9 mL of distilled water. This gave a dilution of 1:20. Next, 100 µL from each dilution well was placed into a corresponding well of an opaque 96-well plate. The fluorescence intensities were measured using a Perkin-Elmer luminescence spectrometer LS 50 B (Perkin-Elmer, USA). The excitation wavelength was 560 nm with a slit width of 2.5 nm, and the emission wavelength was 590 nm with a slit width of 5 nm. Relative fluorescence intensity is the intensity of samples with cells minus that of the Alamar Blue blank with no cells. Contact Angle Measurement. The contact angle was measured by placing a 2-µL drop of double distilled water onto the surface of the material using a NRL contact angle goniometer (Rame-Hart, Inc., Mountain View, NJ). The preparation and cleaning procedure used for the disk samples were the same as those for cell proliferation testing stated above. Three disk specimens were tested with each on four different locations. Thus, 12 readings were made for each material. Water Sorption. For each resin, three visible-light-cured disks (15 mm in diameter and 0.8 mm in thickness) were conditioned to a constant weight in a desiccator and then immersed in distilled water at 37 °C. At different time intervals, the specimens were removed from the water, lightly blotted with a paper tissue, and weighed. After immersion for about 1 year, the specimens were removed and reconditioned to a constant weight in a desiccator. The water sorption for each specimen was determined from the difference in weight between the specimen immersed for the predetermined time intervals and the reconditioned specimen. The BisGMA/TEGDMA neat resin served as control. Acetone Extraction. Disk samples (15 mm in diameter and 0.8 mm in thickness) were photopolymerized for a total of 5 min, weighed (W0), and then extracted by acetone for 4 days. Since the matrix is organic, it has some affinity with organic solvents such as acetone. After the extraction, the samples were immediately weighed (Wa). The weight increase from the starting weight due to absorption of acetone was calculated. Then the samples were put into vacuum oven at room temperature for 3 days and weighed again. After one more day in a vacuum oven, samples were weighed again. Repeated dry and weight processes were done until the difference of the last two weights was less than 0.0005 g. The weight was recorded as Wf. The percentage of

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Figure 2. Cell growth curves for four materials over 10 days (average of three cell strains).

absorption is calculated by (Wa - Wf) × 100%/Wf and the percentage weight loss is calculated by (W0 - Wf) × 100%/ W0. Results and Discussion Since AB is relatively new for testing the effects of dental materials on gingival fibroblast cells, the repeatability of the Alamar Blue fluorescence intensity procedure used here was first examined. It was found that the AB fluorescent reading had very good repeatability. In addition, a very good association exists between the cell number and fluorescent reading for each cell strain. That is, for each cell strain, the fluorescent reading could be explained to a great extent by the appearance of cell numbers. The results of the average relative fluorescence intensity of three cell strains over the period of 10 days are presented graphically in Figure 2. It can be seen from Figure 2 that cell growth occurred on all materials over the 10-day period. Generally, for the first 5 days, only a slight increase was found. Then, dramatic changes occurred for days 7 and 10. This trend held for every material. However, different materials showed a different extent of change. Polystyrene (Pst) control always had highest fluorescent intensity of the four materials. Hyperbranched H20-MMA- or H30-MMA-modified resins had fluorescent intensity similar to that of the unmodified BisGMA/TEGDMA. Figure 3 provides a comparison of relative fluorescent intensity of four materials contacted with three strains at day 10. For all strains, the Pst showed significantly higher fluorescent intensity than the other three experimental materials, indicating that Pst had the best interaction for the cell growth. The fluorescent intensities of modified and unmodified experimental resins were not statistically different. The results suggested that hyperbranched multi-meth-

Figure 3. Relative fluorescent intensity of four materials contacted with three cell strains at day 10.

acrylate-modified resins have cell interactions or biocompatibility at least as good as the BisGMA/TEGDMA control. While it has become commonly accepted that hybridized dentin acts as a barrier to protect dentin and pulp from residual monomers,40 there remains a worthy goal of having no leached monomers in the oral cavity and restoratives that allow for normal cell growth. Thus, biocompatibility, as used above, says only that the hyperbranched-based materials prepared in this work allows cell growth as good or better than the commonly used BisGMA/TEGDMA control, an important consideration in the possible use of such materals. Many factors will influence the interactions of materials with the cells, including the material surface physical or chemical characteristics, amount and properties of leachable or degradable materials, cell properties, etc. The surface properties include the surface roughness, wettability, and electronic charge and van der Waals forces.41 It is known that surface charges could promote cell attachment. For example, commercial polystyrene tissue culture plates have been plasma or corona discharge treated to give a surface

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Biocompatibility of Modified Dental Composites

Figure 4. Water sorption of VLC neat resins in distilled water at 37 °C for about 1 year. Table 2. Contact Angle Test for Neat Resins resins

contact angle (deg)

BisGMA/TEGDMA 10% H20-MMA 10% H30-MMA polystyrene

63.2 (2.5) 65.1 (2.2) 65.3 (3.0) 78.9 (3.6)

with a negative charge, which is suitable for most cell attachment and growth.42,43 In the present study, disk samples were prepared against the smooth glass surface and washed ultrasonically by distilled water. Presumably all the sample disks had similar surface topography and features such as hills or pits. Another important factor for cell interaction is the surface chemical characteristics including functional groups and hydrophilicity. Some functional groups such as -OH, -COOH, -COOR, -CHO, -CONH2, and -NH2 will influence the cell attachment or proliferation. For our materials, BisGMA contains some hydroxyl groups, while others (TEGDMA and H-MMAs) have only ether or ester groups. Modified and unmodified resins have similar chemical features. This could be seen from the contact angle test (Table 2). Modified and unmodified resins have similar surface chemical features. Another major consideration may be the amount or properties of materials leaching to the media, especially for the VLC neat resins, which are polymerized at room temperature with some free monomers trapped in the polymer matrix. When contacted with water or other media, the neat resins will absorb water or other liquids. This process facilitates free monomers leaching out of the resins, especially for small molecules such as TEGDMA and other additives.44 In general, the amounts of leachable free monomers and water sorption play important roles in the biocompatibility.

Table 3. Extraction of Neat Resins of BisGMA/TEGDMA (50/50, wt/wt) Modified by 10% H-MMAa

BisGMA/TEGDMA control 10% H20-MMA !0% H30-MMA 10% H40-MMA

weight loss (%)

absorption (%)

0.92 (0.37) 0.82 (0.38) 0.79 (0.45) 0.73 (0.51)

9.52 (0.10) 9.28 (0.28) 7.12 (0.34) 1.00 (0.47)

a BisGMA/TEGDMA 50/50 (w/w) with 0.5 wt % initiator (CQ) and 1.0 wt % co-initiator (DMAEM). Resin specimens were 15.0 mm in diameter × 0.7 mm in thickness; each entry is the mean value (standard deviation) for a group of five specimens (N ) 5).

The results of percentage weight loss and absorption are given in Table 3. It can be seen from Table 3 that all the samples absorbed acetone with the amount of absorption about 10%. Materials with addition of 10% of H-MMAs showed less absorption than the BisGMA/TEGDMA. However, compared to the high acetone absorption, all materials had a smaller amount of weight loss. H-MMA-modified resins had about 20% less weight loss than the BisGMA/ TEGDMA. This may be due to the high functionality of H-MMAs, which could tether more monomers into the polymer network. Low free monomers usually produces less toxicity, locally or systemically. When dental composites are in contact with water or oral fluids, two processes, absorption of water and leaching of small molecules, occur simultaneously. This is shown through the water sorption curves (Figure 4). First, neat resins quickly absorbed water during the first few days with a weight increase. Small molecules then began to leach out. When the leaching rate became higher than the absorption rate, the weight began to decrease. Finally these two processes reached a steady state and the percentage weight change remained constant. In our study, H-MMA-modified or unmodified resins showed similar water sorption trends

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over the period of 1 year and had a similar amount of water sorption. This could partly explain why modified and unmodified resins had similar cell interactions. Conclusions Hyperbranched multi-methacrylate-modified resins had a smaller amount of free monomer leaching out than the unmodified resin but showed similar properties in water sorption and contact angle. In addition, in contact with three human gingival fibroblasts over the 10 days, the H-MMAmodified resins had similar cellular responses or proliferation to the commercially used BisGMA/TEGDMA resin systems. On the basis of this preliminary testing, it could be concluded that hyperbranched polymers have acceptable biocompatibility and have the potential to be used in dental applications. All these results are in favor of our initial hypothesis. Future studies will include developing a better way to synthesize and purify the hyperbranched multi-methacrylates. In addition, the modified mixtures will be combined with suitable fillers to form composites and their properties will be tested. Moreover, a longer-term biocompatibility test should be conducted. Acknowledgment. The authors express appreciation to NIH (DE 13433) and OSU (an Interdisciplinary Seed Grant) for partial support of this work. References and Notes (1) Kalachandra, S.; Taylor, D. F.; DePorter, C. D.; Grubbs, H. J.; McGrath, J. E. Polymer 1993, 34, 778. (2) Kawaguchi, M.; Fukushima, T.; Horibe, T. Dent. Mater. J. 1988, 7, 174. (3) Holter, D.; Frey, H.; Mulhaupt, R. Polym. Prepr. 1997, 38 (2), 84. (4) Tanaka, J.; Inoue, K.; Masamura, H.; Matsumura, K.; Nakai, K.; Inoue, K. Dent. Mater. J., 1993, 12 (1), 1. (5) Stansbury, J. W.; Choi, K. M.; Antonucci, J. M. Polym. Prepr. 1997, 38 (2), 96. (6) Wang, G.; Culbertson, B. M.; Xie, D.; Seghi, R. R. J. Macromol. Sci., Pure Appl. Chem. 1999, A36 (2), 225. (7) Culbertson, B. M.; Tong, Y.; Wan, Q. Polym. Prepr. 1997, 38 (1), 217. (8) Culbertson, B. M.; Xu, J.; Tiba, A. Polym. AdV. Technol. 1999, 10 (4), 206. (9) Sankarapandian, M.; Shobha, H. K.; Kalachandra, S.; Taylor, D. F.; Shultz, A. F.; McGrath, E. Polym. Prepr. 1997, 38 (2), 92. (10) Antonucci, J. M.; Brauer, G. M.; Termini, D. J. J. Dent. Res. 1980, 59 (1), 35. (11) Matsukawa, S.; Hayakawa, T.; Nemoto, K. Dent. Mater. 1994, 10, 343.

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