Alterations in morphology and composition of ossifying turkey leg tendons were followed using biophysical and histochemical procedures in combination with improved methods of tissue preparation. The principal biophysical changes accompanying calcification were loss of water, increase in shrinkage temperature, shifts in form birefringence curves and increased resistance to the disrupting effects of potassium iodide. Fibroblasts of the tendon were modified to cuboidal cells; at the same time, intracellular lipid increased and presumptive secretion of glycoproteins and lipids into the contiguous matrix was observed. As the bony tendon matured, osteocytes appeared and the stainability of the matrix with the periodic acid-Schiff reagent and dinitrofluorobenzene was markedly diminished. The matrix changes were interpreted as representing increased cross-linking of a negatively charged biological polyelectrolyte. Concurrently, diminished binding of calcium, the formation of hydrophobic lipid-containing phases and the lowering of dielectric constant favor the precipitation of bone salts. During calcification, barriers to diffusion, including low vascularity, tend to create a closed, or partially closed, system in the tissue. Then, the mineral phases form with minimal transfer of electrolytes to the circulation. According to the phase rule, the formation of insoluble bone salts stabilizes the heterogeneous biological structure.The morphological changes in cells and matrix occurring during the direct conversion of a soft connective tissue to bone can be observed at a simple level in the ossification of tendon. This process, as it occurs in the limb tendons of many birds, has been described in detail at least since the middle of the last century. The papers of Lieberkiihn (1860Lieberkiihn ( , 1863 and of Ranvier (1889a, b ) Between the twelfth and sixteenth week, tendons of the turkey thigh and leg undergo a distinct metaplasia. With the exception of those parts which pass through joints such as the tibio-tarsal, the collagenous tissues are transformed to a type of bone. This change is accompanied by an increased infiltration or appearance of lipid in cells and in portions of the extracellular matrix.In earlier electrochemical studies (Engel et al., '62), it was shown that the tendon behaves as an amphoteric colloid which is negatively charged at physiologic pH. This biological polyelectrolyte has the capacity to bind calcium. The degree of calcium binding appeared to diminish with the onset of ossification. These results stimulated further studies employing histochemical and biophysical methods. The following account is based on a study of changes in the tendons in a se-
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MILTON B. ENGEL AND EUGENIO ZERLOTTIries of animals ranging in age from 4 to 40 weeks. The methods of tissue preparation are substantially different from those followed by previous workers. Most of the material which is described here was cut in a cryostat at very low temperature and then dried in the frozen state. In this way, denaturat...