A histochemical method for the demonstration of calcifying cartilage [PMIDJ3145130]

Methods
Enchondral calcification and ossification is of particular interest in developmental biology, growth, and fracture healing. The histochemical demonstration of calcifying cartilage in enchondral bone formation is not commonly done due to methodological difficulties. These technical problems include providing sufficient embedding medium support for sectioning of relatively hard tissue and appropriate staining to demonstrate histochemical characteristics of both calcified and uncalcified cartilage while preserving cell detail. The availability of polymethylmethacrylate embedding for sectioning of calcified tissue has largely obviated the problem
Key words: Enchondral bone formation - - Histochemical - - Staining - - Mineralized, unmineralized - - Embedding.
of providing sufficient support. In this communication we describe our method of processing calcifying cartilage and our technique of histochemical staining which permits the examination of calcification in histochemically distinct cartilage while at the same time preserving chondrocyte histologic detail. The induction of enchondral ossification by the implantation of decalcified bone matrix was initially described by Reddi and Huggins [1]. We, and others, have applied this model as a test system for physical and pharmacological agents that affect the enchondral ossification process [2, 3]. The developmental sequence of the model is biochemically well characterized. The application of our histological techniques has permitted a detailed morphological description of the system (R. K. Aaron et al., unpublished data).
Calcif Tissue Int (1988) 43:244-249
Calcified Tissue International
9 1988Springer-VerlagNew YorkInc.
A Histochemical Method for the Demonstration of Calcifying Cartilage
Embedding and Fixation
The specimens were fixed in 10% formaldehyde in 0.1 M sodium cacodylate at pH 7.2 for 24 hours at 4~ They were then dehydrated in an upgraded series of alcohols of 30, 70, and 85% for 1 hour each with two more changes in 100% alcohol for 1 hour each. The specimens were placed into equal volumes of absolute alcohol and cellosolve for 1 hour and then in cellosolve overnight. They were then transferred to freshly prepared methylmethacrylate containing 15% dibutyl phthalate and 2.5% benzol peroxide and were placed in a vacuum of 20" Hg at room temperature for 30 min, this process was repeated three times. After the last change, the specimens were stored at 4~ overnight. Freshly prepared embedding media was partially polymerized by heating to 85-90~ with constant stirring. When small bubbles appeared that were not caused by stirring, it was immersed in ice water to lower the temperature to 40-45 ~. The process was repeated until there was a slight increase in the viscosity of the methylmethacrylate. The specimens were then placed in this partially polymerized medium at 4~ for 24 hours after which time they were allowed to polymerize at room temperature for 2 days. The specimens were cut on a Polycut microtome (Reichert-Jung) with a tungsten carbide knife moistened with 40% alcohol, and 5 ixm sections were prepared. The sections were placed with 95% alcohol on slides that had been pretreated with 0.4% gelatin, and pressed fiat. A total of 6 days was required for processing and full polymerization
Send reprint requests to Roy K. Aaron, M.D., 154 Waterman Street, Providence, RI 02906, USA.
Decalcified Bone Matrbc-Induced Enchondral Ossification
Decalcified bone matrix was prepared from the diaphyses of femurs and tibias of adult CD rats [1]. The bones were defatted with ethanol and ether, milled frozen in liquid nitrogen to a particle size of 75-400~m, decalcified with 0.5 N hydrochloric acid, and extracted with ethanol and ether and air dried. Twenty-five milligrams of the decalcified bone matrix powder was implanted subcutaneously along the thoracic musculature in immature CD rats. Animals were sacrificed periodically and ossicles were taken for biochemical and histological study. Prior to sacrifice, animals were injected intraperitoneally with 1 i~Ci/g body weight of 35sulfate for proteoglycan analysis. On day 2 following implantation, mesenchymal cells migrated into the implant. On days 4 through 8, differentiation to chondrocytes occurred primarily on the surfaces of the bone chips. Chondrogenesis was maximal on days 8-10. Calcification of the cartilage began on about day l0 with the formation of bony trabeculae from days 16 through 20.