Repair of Fractured Lower Jaws in the Spotted Salamander: Do Amphibians Form Secondary Cartilage?

Secondary cartilage forms on avian and mammalian dermal bones, both during normal ontogeny and during repair of fractures, but it has not been observed in any other vertebrate class. We fractured the left lower jaws of adult spotted salamanders, Ambystoma maculatum, to see whether secondary cartilage would form during fracture repair. It did not. Instead, periosteal hyperplasia produced a callus from which new dermal bone formed to bridge the fracture. Meckel's cartilage underwent superficial dissolution but showed a minimal chondrogenic response. A large callus cartilage did form, but it appeared to arise by metaplasia from connective tissue adjacent to the bone. Thus, the environment within the fracture is conducive to chondrogenesis but the periostea of the dermal bones either are not able to respond to that environment or are unable to synthesize cartilage-specific products. Among recent vertebrates, the ability to form secondary cartilage is limited to birds and mammals and is not a primitive property of the periostea of dermal bones shared by "lower" vertebrate classes. The impetus for undertaking this study on fractured urodele lower jaws was to determine whether secondary cartilage would differentiate during the reparative process. Secondary cartilage is a class of cartilage which forms from periosteal cells of dermal bones after the process of intramembranous ossification has been initiated. Developmentally it is therefore distinct from the primary cartilages which precede osteogenesis during endochondral ossification, and has been so recognized for a long time (Schaffer, '30). Histologically, secondary cartilage consists of hypertrophic chondrocytes in a sparse extracellular matrix, the latter often only comprising 5-10% of the volume of the cartilage. Advantages which accrue from the ability to form secondary cartilage include formation of shock-absorbing articulations between dermal bones and reduction of damage to periostea at points of attachment of muscles or ligaments; quick immobilization of fracture with secondary callus cartilage; and the developmental plasticity which comes from being able to shift the site of an articulation and still form a normal joint. The best known examples are the cartilages on the condylar and coronoid processes of the mammalian dentary (Durkin, '72; Vinkka, '82; Silbermann and Frommer, '72) and those on the dermal bones of the avian craniofacial skeleton (Murray, '63; Hall, '70, '78). Beresford ('81) has devoted a book to a very extensive treatment of secondary cartilage and two major developmental processes in which secondary cartilage is involved, viz., the formation of chondroid bone and metaplasia. No unequivocal evidence has been presented for the existence of secondary cartilage in vertebrates other than birds and mammals. There is one unconfirmed report of secondary cartilage on the pterygoid of the lizard Lacerta uiuipara, but one of us (B.K.H.) could find none in the skull of the Australian tiger snake, Notechis scutatus (see Hall, '84, for a discussion of the absence of secondary cartilage in reptiles). Ismail et al. ('82) have described a type of secondary cartilage on the parasphenoid and upper pharyngeal jaws of a cichlid fish, Astatotilapia elegans, but histochemical and ultrastructural analysis failed to show similarities between this tisJames Hanken's present address is Environmental, Population, and Organismic Biology, University of Colorado, Boulder, CO 80309. Address reprint requests to Brian Hall, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H4J1. ($1 1985 ALAN R. LISS, INC 360 B.K. HALL AND J. HANKEN sue and the secondary cartilage of birds and mammals (Huysseune et al., '81) and no similar cartilage has been observed during repair of fractured dermal bones in fish (Moss, '62; Goss, '69). Beresford ('81) regards such tissue as chondroid bone. Cartilages are sometimes associated with the dermal opercular bones of fish, but they arise independently from the bone and only subsequently fuse to it. They are clearly not secondary cartilages, and likely are also chondroid bone (Moss, '61; Murray, '63; Patterson, '77; Beresford, '81.) We know of no reference to secondary cartilage in any amphibian. These results corroborate Patterson's ('77) contention that secondary cartilage is confined to endothermic tetrapods (birds and mammals). Patterson suggested that a search for secondary cartilage during repair of fractured dermal bones in amphibians and reptiles would provide an excellent test of whether the ability to form secondary cartilage was restricted to birds and mammals. The rationale for his proposal is that secondary cartilage is mechanically induced-periosteal cells, which would have become osteoblasts, become secondary chondrocytes when exposed to intermittent pressure and tension (Hall, '67, '68, '79; Meikle, '73; Petrovic, '72). A fractured dermal bone provides a mechanically active environment in which secondary cartilage can form both in birds (Hall and Jacobson, '75) and in mammals (Jolly, '61). Therefore, we fractured the lower jaw of the spotted salamander, Ambystoma maculatum, to determine whether secondary cartilage would form during repair of the dentary, a dermal bone. It did not. MATERIALS AND METHODS Experimental procedures Adult Ambystoma maculatum (Amphibia: Ambystomatidae) were collected near Halifax International Airport, Halifax, Co., Nova Scotia, during the spring breeding migration in late April 1983. Upon return to the laboratory they were housed in shallow plastic trays (40 x 27 x 10 cm) with 1-2 cm of dechlorinated tap water and maintained at 14"C, a temperature similar to that in the wild. Prior to the experiments the water was changed and the animals fed live earthworms three times a week; following jaw fracture the water was changed regularly but the animals were not fed. Ten individuals were anaesthetized by immersion in a 0.02% aqueous solution of MS222 (Ethyl m-Aminobenzoate; Sigma No. E1626) and then placed ventral-side-up on a moistened paper towel. The left lower jaw was fractured with a single snip of a dissecting scissors a t a point approximately halfway between the posterior angle of the jaw and the anterior symphysis. In all cases the jaw was severed completely; in some specimens, the anterior portion of the fractured jaw immediately bowed outward and away from the posterior portion, leaving a distinct gap separating the cut ends of the fracture. Typically there was little or no bleeding following fracture, and all animals recovered from anaesthesia.