Photopolymerized 2-hydroxyethylmethacrylate as a mounting medium preserving immunocytochemical reaction and nuclear counterstain.

Immunocytochemical or cytoenzymological techniques often make use of coupling reactions between a substituted naphtol and a diazonium salt. A positive reaction results in an azo dye precipitate. Unfortunately, this precipitate is easily soluble in alcohols and organic solvents. Thus, usual mounting media are not usable and permanent preparations cannot be obtained. A stable mounting medium such as Apathy's syrup can be used, but nuclear counterstaining disappears in a few days. We tested the hydrophilic monomer 2-hydroxyethylmethacrylate, containing various photopolymerizing agents, as a permanent mounting medium. 2-2 Dimethoxy 2-phenyl acetophenone proved to be the most useful photopolymerizer. The cytocentrifugation slides must be dried before mounting to avoid recrystallization artifacts. The azo dye precipitate and nuclear counterstaining can be preserved perfectly long-term. The cost of these media is very low.

Free radicals and side products released during methylmethacrylate polymerization are cytotoxic for osteoblastic cells.

Polymerization of orthopedic cements makes use of a peroxide initiator which is decomposed by an accelerator to provide free radicals. Free radicals which act on the monomer molecules are also known to induce cell lesions and cell death. We used an in vitro model of cement polymerization to study the effects of free radicals release on osteoblast-like cells. Initiation of methylmethacrylate was done with benzoyl peroxide and acceleration by N,N-dimethylaniline. Bulk polymerization was done in calibrated test tubes which were left aging until use. Polymers (aged from J1 to J31 days after completion of the polymerization process) were sawed to produce slices. Slices were rinsed in distilled water and free radical release was measured by spectrophotometric titration with p-iodonitrotetrazolium. Saos-2 osteoblast-like cells were cultured in parallel on the slices. Cells appeared to be round and were altered when grown on slices prepared freshly after polymerization. Cytomorphometric analysis of the cell shape (surface area and form-factor polyethylene confirmed that they spread and flatten on slices prepared a long time after polymerization. Free radical release from polymethylmethacrylate cements is a long-lasting event that can induce bone cells alterations in their neighborhood. Two cytotoxic mechanisms were evidenced: (a) polymer slices released a stable toxic component which could be removed by extensive washing; (b) they released free radicals which were still detectable several days after the end of polymerization. The titration curve was a negative exponential.

Embedding iliac bone biopsies at low temperature using glycol and methyl methacrylates.

A mixture of pure and anhydrous glycol methacrylate and methyl methacrylate is used as an embedding medium for iliac bone biopsies. Infiltration is carried out at -20 C with the embedding medium and a cold inactivated catalyst-initiator system. Raising the temperature to 4 C initiates polymerization and limits the peak temperature of polymerization to 25 C. In this way, such thermolabile enzymes as osteoclastic acid phosphatase are preserved. After staining, sections are dehydrated in polyethylene glycol 400 30% in 2-propanol. This gives flat sections and improves staining properties.

A simple and reliable method for purifying glycol methacrylate for histopathological studies.

Pure glycol methacrylate (GMA) is an excellent embedding medium which allows High Performance Optical Microscopic studies. However presence of methacrylic acid (MA) small amounts (usually 1 to 3%) leads to intense background staining of the section. A simple and reliable chemical purification procedure is exposed which leads to an extra-pure GMA free of MA and hydroquinone.