Practical Investigations of Radical Polymerization of ...

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Practical Investigations of Radical Polymerization of Glycolmethacrylate as an Embedding Medium Daniel chappard*', Zenagui h en as car"^, Christian ~lexandre'and Jean-Perre ~ o n t h e a r d '~aboratoire ~, de Biologie de Tissu Osseux, Faculte de Mkdecine, 15 rue Ambroise Pare, 42023 Saint Entienne, Cedex 2 France, and '~aboratoirede Chimie MacromolCculaire, Faculte des Sciences, 42023 Saint Entienne, CCdex France. Abstract Glycolmethacrylate(GMA) is now a widely accepted plastic embedding medium for histopathological uses although technical conditions of polymerization are not fully understood. We found that benzoyl peroxide initiated GMA medium accelerated with dimethylaniline (NN-DMA) polymerizes at different rates according to the volume left to polymerize and the size of the embedding molds. This phenomenom appears to be sustained by an unexpected and complex thermodynamical process that we were unable to resolve. However, we did find that NN-DMA concentration is a polynomial function of the initial GMA volume, and the proposed equation may help in adjusting the techniques when varying the amount of methacrylate embedding medium or when changing the size of the embedding molds. (The J Histotechnol 1289, 1989.) Key words: embedment, glycol methacrylate, polymerization. Introduction The application of glycolmethacrylate (GMA) as an embedding medium for histopathologicaldiagnosis has raised a great amount of enthusiasm and consequently a lot of technical problems (1). A rapid survey of the histopathological literature shows that numerous formulations and polymerization systems have recently been reported. Clearly, this indicates that all parameters occurring during GMA polymerization are not fully understood nor controlled. Non-reliable results are often obtained when investigators try to adapt a method available in literature to their own problems or specimens. This paper points out the differencesobserved in the polymerization rate of GMA with respect to the volume of monomer to be polymerized and other experimental conditions. Material and Methods Solutions The present study was composed of two experiments. The embedding medium used in all the experiments was prepared by mixing *Author to whom reprint requests should be addressed. This work was supported by grants from the Ministkre de la Recherche et de I'Enseignement Sup6rieur (Project Industrial-UniversitaryResearch No 87-08/86-55)

The Journal of Histotechnolo~ylVol.12, No. 2IJune 1989

GMA (2-hydroxyethly-methacrylate) . . . . . . . . . . . 250 ml Polyethylene glycol 400 D . . . . . . . . . . . . . . . . . . . . 50 ml BPO (benzoyl peroxide; moistened with 25% H20) . . 1.2 g All components were dissolved with a magnetic stirrer and stored at 4OC until use. The accelerating solution was composed of NN-dimethylaniline (NN-DMA), 1 ml, in propanol-519 ml. All reagents, of laboratory grade, were purchased from MerckB. In previous experiments, an amount of 4 0 0 ~ 1of the NN-DMA solution was reported to polymerize 25 ml of this GMA embedding medium at 4OC, giving a final concentration of 1.6% of the NN-DMA solution (2). Automatic pipettes (Gilson Pipetman P5000 and P200) were used for reagent handling. Experiment 1 750 ml of the initiated GMA medium was accelerated with 1.6% of the amine solution and immediately distributed in 2 series of glass flasks of the same model. Series A was composed of 5 flasks receiving 5,10,15,20, and 25 ml of the acceleratedintitiated GMA medium. The 5 flasks of the B series were similarly distributed with the medium, and a layer of paraffin oil was poured onto the top of the medium to exclude oxygen (a polymerization inhibitor). The flasks were then screwcapped and left polymerized at 4OC in a precision oven (Kotterman 2770). A waterbath was used to limit the temperature peak during polymerization. The reaction was controlled at 24 h and the amount of polymerized GMA was measured and expressed as a percentage of the intitial volume of the medium. Each experiment was repeated 5 times. Experiment 2 In the second study, the initiated GMA medium was distributed in 5 series of glass flasks (same model as that used in experiment 1). Flasks of the A series were filled with 5 ml of the GMA medium; series B: 10 ml; series C: 15 ml, series D: 20 ml; and series E: 25 ml. Increasing volumes of the accelerating solution (i.e., with a 0.2%step) were distributed in each series to give final concentrations ranging from 1.4 to 4%in series A; 0.4 and E. Flasks were left polymerized at 4OC, and the amount of polymer was measured. This experiment was also repeated 5 times. 89

1

-r I

PIETHACRYLIC ACID CH2 = C(CH3) COOH

-

aiNZOIC ACID

I

0

5

10

15

20

25


ml ot G M A medium

Figure 1. Experiment 1. Diagram showing how the initial volume of accelerated-initiated GMA medium can affect the amount of final polymerization.

Figure 2. Experiment 1. Gas chromatograph mass spectometric analysis of the unpolymerized brown supernatant showing a peak of benzoic acid derived from benzoyl peroxide fragmentation.

Statistical analyses of the data were performed on a Macintosh+ microcomputer by regression analyses.

appears to be a complex function of monomer volume (x) according to the following equation:

Results Experiment I The 5 trials in each series gave similar results (Figure 1).The polymerization was fully achieved within 24 hours at 4OC in the glass vials containing 20 and 25 ml of medium whether or not paraffin oil was added. Small volumes of accelerated initiated GMA expressed only a slight tendency to polymerize (0-10%in volume) at the bottom of the flask, and the remaining liquid supernatant turned brown. Intermediate volumes presented incomplete polymerization with 3 superimposed phases: from the bottom to the top of the flask, a hard polymer (75-80% of initial volume); a gel phase (10-15%);and a brown supernatant. Except for the 5 ml volumes, no differences could be observed between series A and B. The brown unpolymerized supernatants were collected and pooled, and a gas chromatograph mass spectrometric analysis (GC-MS) was performed with a chromatograph Perkin - Elmer Sigma 3B. A capillary column BP20 (50 m) was used with helium as carrier gas. Column temperature was programmed from 60°C to 125OC (2°C per mn) and to 240°C (5OC per mn). The mass spectrometer VG 30 F had a 70 eV electron impact, source 200°C, and emission current 100mA. Shimadzu integrator coupled to the flame ionization detector was used to calculate the peak area percentage. The mean peaks detected (Figure 2) was methacrylate acid (impurity of starting GMA), benzoic acid (product of BPO decomposition) and unreacted GMA.

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Experiment 2 The five trials in each series gave similar results. The amount of polymer formed in each series during 24 hours was found to be non-linearly related with the amine concentration (Figure 3). The minimal NN-DMA concentration resulting in a complete polymerization was checked as a function of the initial volume of monomer. The results show clearly that a non-linear relationship exists (Figure 4). A polynomial regression was found (r = .977) and the slope of amine concentration (y)

Discussion The usual process used for the formation of methacrylate polymers is a chain polymerization formed by the stepwise addition of monomericmolecules to a specially reactive species. Free radicals are most frequently used (3). The entire polymerization process consists of four steps: Fragmentation of the initiator in a redox system Resin polymerization involves the consumption of the initiators; the term "catalyst" often used in the literatureshould be avoided. Peroxides (BPO in this study) are polymerization initiators (I) which can be degraded by heat, metallic ions, UV light or substituted anilines (e.g, NN-DMA) and this fragmentation produces free radicals (R'). The speed of this reaction (kd) is temperature dependent according to the ArrhCnius' law and this was found useful to infiltrate large specimens (4). I >2 R* kd Decomposition of peroxides by amines is extremely rapid and can be explosive. The mechanism of fragmentation was first described by Homer and Schlenk (5). Chain initiation The primary free radicals (R') then react with the C = C bond of a monomer molecule (M) and produce a propagating chain; some of them may react either with one another or with an entire peroxide molecule to form inactive products (7). Studies of decomposition of BPO in various solvents have shown that carbon dioxide is almost always produced with benzoic acid and various phenolates (3,7). Chain propagation Once a propagating chain R - M' is generated, it quickly reacts with another monomer molecule to form long polymer chains with C - C links: Radicular Polymerization of GMAlChappard et al

$5 1

% of activating solullon (NN- DMA)


Initial volume

Figure 3. Experiment 2. Relationshipbetween the amount of polymer obtained after 24 hand the initial concentration of NNdimethylaniline. (2 series, 15 and 25 ml are shown. Other volumes also gave a sigmoidal response).

R-M'+M

>R-M-M'

kp The propagation reaction rate (kp) remains almost constant except at the end of polymerization when the viscosity suddenly rises and impairs the recruitment of new monomer molecules, by limiting their diffusivity. This phenomenon is known as the Trommsdorff's effect (3,6). Chain termination Chain termination occurs when two propagating chains react together. Two types of chain termination are observable: *combination: implies that the two chains react and combine in a simple chain: R-M'n+R-M'm >R-Mn+m-R ktc *disproportionation: leads to the formation of two unreactive chains but leaves an unreacted terminal C = C bond at the end of each chain. R-M'n+R-M'm >R-Mn+-R-Mm ktd It was reported that combination is the most important mechanism at low temperature (70°C), a condition that is not really compatible with good cellular preservation. The type of chain termination may modify the hardness and the mechanical properties of the polymers by producing long chains of high molecular weight. For histotechnological purposes, GMA and other acrylic resins are bulk polymerized, a condition rarely used by chemists who usually dilute monomers in solvents for studying polymerization processes. In the present study, we found that polymerization of a given accelerated-initiated medium varies according to the initial volume (Experiment 1). This phenomenon had to be taken into account when embedding specimens of various size in different molds. Because polymerization is an exothermic reaction, heat may accumulate in the center of the blocks and induce various artifacts. On the other hand, smaller molds may lead to unpolymerized or tacky blocks unsuitable for sectioning The Journal of HistotechnologyIVol. 12, No. 2lJune 1989

;

; 10

OO

20

of

30 rnl

initiated monomer

Figure 4: Experiment 2 Relationship between the initial volume of initiated GMA medium and the amount of NN-dimethylanilinenecessary for inducing a 100%polymerization.

if the concentration of the initiator system has not been raised. To our knowledge, these differences in the ability of a given accelerated initiated-methacrylate medium to polymerize have not been reported in the histotechnical literature. Futhermore, we have been unable to find a rational explanation in recent and classical books on polymerization thermodynamics (7,8). As shown in Experiment 1, the effect of atmospheric oxygen could be eliminated because only variations in the volume of monomer can influence polymerization. The brown unreacted supernatant analyzed by GC-MS was shown to contain large amounts of benzoic acid and unreacted GMA. It seems that small volumes of medium are associated with an excess BPO fragmentation providing unreactive species or oligomers (i.e., minute chains that cannot be detected with GC-MS) and leave numerous GMA monomer molecules unreacted. Attempts have been made to determine simple equations that could correct this phenomenon. We found that NN-DMA concentration (producing a 100%polymerization of a given monomer volume) was not a linear function of this initial volume. Experiment 2 and the above function estimating the amount of NN-DMA have been repeatedly reproduced in this laboratory and can help others in adjusting their own methods. Surface contact is known to be an important parameter in catalytic chemistry and the interface area between the medium and the flask could interfere with BPO fragmentation either by increasing kd or by inducing side reactions leading to inactive species. Futhermore, glass is a reactive material, which can itself interact with peroxide or amines. Finally, this paper reporting current and real problems of bulk polymerization in a histotechnological point of view may be underlined by a complex thermodynamical problem we have beem unable to solve by ourselves. Acknowledgements The authors thank Professor JC Healy for helpful discussions and Mr. Monin (SPECIA Laboratories) for his interest in this study. References 1. Bennett HS, Wyrick AD, Lee SW, McNeil JH: Science and art in

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preparing tissue embedded in plastic for light microscopy with special reference to glycol methacrylate, glass knives and simple stains. Stain Technol 20:71-97, 1976. 2. Chappard D: Uniform. polymerization of large and very large blocks in glycol methacrylate at low temperature with special reference to enzyme histochemistry. Mikroskopie 42: 148-150, 1985. 3. Louie BM, Carratt GM, Soong DS: Polymerization of methylmethacrylate. J Appl Polym Sci 30: 3985-4012, 1985. 4. Chappard D, Alexandre C, Camps M, et al: Embedding iliac bone biopsies at low temperature using glycol and methylmethacrylates.

Stain Technol 58: 299-308, 1983. 5. Horner L. Schlenk E: The acceleration of peroxide decomposition by amines. Angew Chem 61:411, 1949. 6. Sigwalt P: RCactions de polymCrisation. In Chimie Macromol~culaire, Champetier G (ed), Hermann, Paris, 1970, pp 80-135. 7. Tobolsky AV, Mesrobian RB: Organic Peroxides. Interscience Publ, New York, 1954. 8. Allen PE, Patrick CR: Kinetics and Mechanism of Polymerization Reactions; Application of Physico Chemical Principles. Halsted Press, 1974.

Second Annual Leitz-NSH Medical Photography Contest Winner: Roy Bloebaum, PhD, Veteran's Administration Medical Center, University of Utah School of Medicine, Salt Lake City, Utah.

"Hard Heart"

Microphotograph of Cancellous Bone With Arteriole Central Polarized Light, X20.

Radicular Polymerization of GMAlChappard et al