It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification.

Timing and pattern of expression of alkaline phosphatase was examined during early differentiation of the 1st arch skeleton in inbred C57BL/6 mice. Embryos were recovered between 10 and 18 d of gestation and staged using a detailed staging table of craniofacial development prior to histochemical examination. Expression of alkaline phosphatase is initiated at stage 20.2 in the plasma membrane of mesenchymal cells in the distal region of the first arch. Expression is strongest in osteoid (unmineralised bone matrix) and presumptive periosteum at stage 21.32. Mineralisation begins at stage E23. Expression is present in the mineralised bone matrix. Secondary cartilages form in the condylar and angular processes by stage M24. The cartilaginous cells and surrounding cells in the processes are all alkaline phosphatase-positive and surrounded by the common periosteum, suggesting that progenitor cells of the processes, dentary ramus and secondary cartilages all originate from a common pool. Nonhypertrophied chondrocytes of Meckel's cartilage express alkaline phosphatase at stage M23. Expression in these chondrocytes is preceded by the expression in their adjacent perichondrium. This is true of chondrocytes in all other cranial cartilages examined. 3-D reconstruction of expression in Meckel's cartilage also revealed that the chondrocytes of Meckel's cartilage which express alkaline phosphatase and the matrix of which undergoes mineralisation are those surrounded by the alkaline phosphatase-positive dentary ramus. By stage 25, coincident with mineralisation in the distal section of Meckel's cartilage, most chondrocytes are strongly positive. The perichondria of malleus and incus cartilages express alkaline phosphatase at stage M24. Nonhypertrophied chondrocytes along these perichondria also express alkaline phosphatase. Superficial and deep cells in the dental laminae of incisor and 1st molar teeth become alkaline phosphatase-positive at the bud stage, stages 21.16 and 21.32, respectively. Dental papillae are negative until stage M24 when alkaline phosphatase expression begins in the dental papillae and follicles of the incisor teeth and the dental follicles of the 1st molar teeth. The dental papillae of the 1st molar teeth express alkaline phosphatase at stage 25. Expression in the dental papillae and follicles appears to coincide with cellular differentiation of follicle from papilla. The presumptive squamosal, ectotympanic and gonial membrane bones, lingual oral epithelial cells connected to the dental laminae of the incisor teeth, hair follicle papillae and sheath and surrounding dermis all express alkaline phosphatase in a stage-specific manner.