hemolysis mechanism of dental adhesive ... 136 dental materials journal 9 (2): 136-146, 1990...

Download Hemolysis Mechanism of Dental Adhesive ... 136 Dental Materials Journal 9 (2): 136-146, 1990 Hemolysis

Post on 04-Apr-2020

0 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • 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.

    INTRODUCTION

    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

    thawing.

    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 (ĢH) 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.

    RESULTS

    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

Recommended

View more >