hemolysis mechanism of dental adhesive ... 136 dental materials journal 9 (2): 136-146, 1990...
Post on 04-Apr-2020
Embed Size (px)
136 Dental Materials Journal 9 (2): 136-146, 1990
Hemolysis Mechanism of Dental Adhesive Monomer (Methacryloyl- oxydecyl Dihydrogen Phosphate) Using a Phosphatidylcholine Liposome System as a Model for Biomembranes
Seiichiro FUJISAWA*, Yoshinori KADOMA** and Yasuo KOMODA** * School of Dentistry , Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyou-Ku, Tokyo, 113 Japan
** Institute for Medical and Dental Engineering , Tokyo Medical and Dental University, 2-3-10. Kanda- Surugadai, Chiyoda-Ku, Tokyo 101, Japan
Received on July 2, 1990
Accepted on September 3, 1990
To clarify the mechanism of interaction of dental adhesive monomers with biological membranes at the molecular level, we studied the interaction of methacryloyloxydecyl dihydrogen phosphate (MDP) and methacrylic acid (MAA) with the dipalmitoylphosphatidylcholine (DPPC) liposome system using NMR and DSC. MDP-DPPC interaction became apparent through broadening of the DPPC phase transition as pH decreased, finally the enthalpy of MDP-DPPC (1:1mol ratio) reduced to zero at pH 2.5. Proton chemical shifts of MDP enhanced shielding and proton signals due to the phosphatidylcholine polar group (O-CH2-CH2 -N bond) of DPPC were observed . MAA-DPPC interaction was smaller than that of MDP-DPPC, even at low pH. It was concluded that the strong hemolytic activity of MDP may be due to its interation with the
phospholipid bilayers of erythrocyte membranes.
Key words: Phosphate Monomer, Hemolytic Activity, Phospholipid Liposomes.
Bonding agents are widely used in dentistry for pit and fissure sealants, restorative
materials, orthodontic and prosthetic devices1,2). To bond composite resins to dentin, bonding
agents must be directly applied to dentin and then the agents must penetrate or react with
the dentin structure. In particular, acid etching of dentin is liable to cause injury to dental
pulp due to the increase in dentin permeability3). Hence, dentin bonding agents in the
restorative system may directly affect the dental pulp. Thus, residual monomers, initiators
and other small molecules in bonding agents may be involved in pulpal responses. Recently,
phosphate monomers (phenyl-P) were used as one of components of the enamel and dentin
bonding agents4). This adhesive resin-system was found to be biocompatible with the dental
pulp when the infected outer layer of carious dentin was removed and then the phosphate
monomer system was placed upon the etched dentin5)6). However, this adhesive system
induced severe cytotoxicity7). The in-vivo usage test for restorative resin-system only
provides a rough measure of pulp irritation and the clinical histopathologic experimentation
does not allow quantification8). Therefore, it is important to evaluate the biological prop-
erties of materials before clinical application7,8). On the other hand, it appears that the
cause-and-effect relationship between restorative resin materials and pulpal responses is not
clearly correlated with biological properties of materials, microleakage, bacterial contamina-
tion, or any other single factor or multiple factors and that the absence of the •gtoxicity•h in
HEMOLYSIS MECHANISM OF MDP 137
a material does not make it biocompatible9).
The mechanism or components of the dentin-pulp complex which afford protection to the
dental pulp from resin systems have not been clarified. Dental pulp is a complex tissue
consisting of cells and abundant extracellular matrix. Components in dentinal tubules may
create a barrier against the diffusion of toxic substances of dental materials10-12). Dentin
permeability may be an important factor of pulpal responses caused by dental materials. Dentin and pulp tissues which are derived from mesoderm are known to be very sensitive to
acids13). Hydrophilic acid monomer, MAA used in self-curing methyl methacrylate (MMA)
resin-system was known to be toxic in the dental pulp3). Also, this showed the high degree
of acute toxicity in mice and of tissue toxicity14,15) MDP which has been used in the
commercial resin-system as a new bonding agent, is also an acid monomer16). The bonding
agents with MDP showed a larger cytotoxicity compared with those without MDP17) and had
an adverse effect on the dental pulp18). Therefore, it is of particular interest to determine how
the molecular structure of phospholipids in biological membranes is modified by interaction
with MDP compared to MAA.
In the present study, we examined the hemolytic activity caused by MDP, MAA and
MMA. NMR and DSC were used to investigate the degree and nature of interaction of MDP
or MAA with the DPPC liposome system as a model for biomembranes. The mechanism of
the interaction of MDP with erythrocyte membranes was discussed.
MATERIALS AND METHODS
L-ƒ¿-dipalmitoylphosphatidylcholine (DPPC)#, 3-(trimethylsilyl) propionic acid sodium
salts-D4 (TMSPA)## and deuterium oxide (D2O)## were used without purification. Metha-
crylic acid (MAA)* and methyl methacrylate (MMA)* were used after purification by means
of high performance liquid chromatography (HPLC)**.
Synthesis of MDP19). 10-Hydroxydecyl methacrylate was prepared from 1, 10-decanediol
and methacryloyl chloride. The product was isolated by column chromatography on silica
gel. The purified alcohol and triethylamine were slowly added to phosphorous oxychlor-
ide. After the termination of the reaction, the mixture was extracted with ether and the
solvent was evaporated. The resulting viscous MDP was washed with n-hexane several times
and dried (yield about 74%). The purity of MDP was examined by HPLC, indicating a single
peak. The structure of MDP was identified by NMR and infrared spectra.
Hemolysis studies. Human erythrocytes from normal male donors were isolated from
blood anticoagulated with EDTA by centrifugation (x1000) for 10min. The plasma and buffy
coat were then removed and erythrocytes were washed three times with PBS (phosphate
buffer solution 100mM, pH 7.4) before being dispersed in PBS. An appropriate concentration
of MAA, MDP and MMA were prepared in PBS. One ml of the solution obtained was placed
in each assay tube and 1ml of the cell suspension was added to it and incubated at 37•Ž for
# Sigma Chemical Co., St. Louis, MO., USA. ## Merck Chemical Co. Darmstadt, Germany.
* Tokyo Kasei Chemical Co . Tokyo, Japan. ** Nippon Millipore Ltd . Waters Chromatography Division, Tokyo, Japan.
138 S. FUJISAWA, Y. KADOMA and Y. KOMODA
an appropriate time. After incubation, the tubes were centrifuged at 2000rpm 10min and the
absorbance of supernatant at a wave length of 545nm was measured by using a UV-210A
spectrometer.+ The hemolysis percentage was calculated on the basis of the measured
absorbance for the original cell suspension after hemolyzing had taken place by freezing and
Preparation of the liposomes for NMR. An appropriate amount of DPPC was dissolved
in chloroform and dried under vacuum. The test monomer (MAA or MDP) was added to the
dried lipid film and was dispersed in D2O by vortexing on a Vortex shaker at 45•Ž for 2-3min
and then was sonicated under a nitrogen atmosphere for 30min at 45•Ž. The molar ratio of
DPPC to test compound was 1:1. Suspensions containing approximately 10% DPPC
liposomes were prepared.
NMR spectroscopy. Proton NMR spectra were measured at 30•Ž and 52•Ž under JEOL
JNM-GX 270 spectrometer***, at 270MHz. The chemical shifts (ƒÂH) of MDP, and MAA are
reported in ppm downfield from the external standard, TMSPA, as previously described12).
DSC studies. The DPPC liposome system was prepared in a manner similar to that
described above. All samples for DSC studies were 75mM DPPC, 140mM sodium phosphate
and the appropriate concentration of MDP and MAA. Each 10ƒÊl sample of both DPPC and
test compound was sealed in a DSC container. Also, some samples of MAA and MDP were
sealed in a container with D2O. The sample was then allowed to equilibrate for 14h at 5•Ž
and finally shaken again for 1min by hand at 25•Ž. The sample (20ƒÊl) was scanned in a sealed
calorimetric container on a DSC-Rigaku calorimeter### operating at a heating rate of 5•Ž/
min with a range setting of 0.5mcal/s20). The transition enthalpy (ƒ¢H) was calculated from
the area under the curve which was determined by cutting out the DSC-curves and weighing
the chart paper. Enthalpy measurements of the DPPC liposome system were run at least in
triplicate. Error limits on enthalpy are given as •}1 standard deviation.
Since in general, the enthalpy of pretransition is small and has a behavior similar to that
of the main transition, we discuss only the main transition21).
p H measurement of solution. pH was determined with a COM-8 pH meter++ at 23•Ž.
Hemolysis studies. The percentage of hemolysis caused by MDP, MAA and MMA is
shown in Table 1. In case of MDP, the time and concentration required to produce about 50
% hemolysis were 10min with 19mM and 60min with 2.4mM, respectively. MAA and MMA also showed a small degree of hemolysis (1-2%) at the same time and concentration.
From this, i