CALCIFICATION AND OSSIFICATION

II. Control of Calcification in the Fracture Callus in Rachitic Rats

¹ Johns Hopkins Medical School, Baltimore, Maryland; Department of Physiology, University of Chicago, Chicago, Illinois

1. The early stages of healing of fractures in rachitic rats resemble the same process in the previously normal animal in every respect except that of calcification. Formation of the fibrocartilaginous callus, and the production of subperiosteal and subendosteal intramembranous osseous tissue are not influenced by the complete absence of bone salt, in the first four to five days following a fracture.

2. The influence of the absence of calcification on the healing of fractures in rachitic rats begins to manifest itself by about the fourth to the fifth day following a fracture. Invasion of the fibrocartilaginous callus by the new intramembranous osseous tissue is delayed, often until the ninth to the twelfth day. This delay is comparable to the delay in initiation of the rachitic type of cartilage removal in the epiphyseal cartilage following the onset of rickets. When invasion of the callus cartilage occurs, it closely resembles the similar process in the epiphyseal cartilage in rickets.

3. Healing of fractures in rachitic rats, in the absence of calcification may be further complicated by the embedding or encapsulation of the callus cartilage in a dense mass of fibrous tissue or fibrocartilage. This forms a barrier to the advance of the uncalcified osseous tissue.

4. Spontaneous calcification in the intramembranous osseous tissue of the callus without calcification in the epiphyseal cartilage or in the rachitic metaphysis begins in the rat at about the tenth to the fifteenth day following the injury, and leads eventually to union of the fracture by partially calcified osteoid.

5. Calcification in the osteoid of the callus, as well as in the epiphyseal cartilage and in the osteoid of the rachitic metaphysis, may be initiated by a single dose of phosphate, in a dose of 2.5 cubic centimeters of one-tenth molar phosphate mixture per 100 grams of body weight. Following such treatment the initial deposit of bone salt in time subperiosteal osteoid occurs some four to eight hours later than in the epiphyseal cartilage .

6. Time distribution of bone salt in the epiphyseal cartilage, metaphysis, and callus following administration of phosphate is the same whether fixation is with neutral formalin or by the Altmann-Gersh freezing-drying method. Its localization is also identical, whether visualized by staining with silver nitrate, by intra vitam staining with sodium alizarin sulfonate, by observation of unstained sections with the aid of polarized light, or by the fluorescence seen in calcified areas by ultraviolet light.

7. As in previously normal animals, calcification of the matrix of time callus cartilage, following the administration of phosphate, occurs only when hypertrophic cartilage is in contact with the periosteum, or with advancing subperiosteal or subendosteal bone. When the callus has become encapsulated in the dense fibrous tissue or fibrocartilage, which forms a barrier to the advance of the new osseous tissue, there is a delay of some days in the initiation of calcification of the callus cartilage following the administration of repeated doses of phosphate. When phosphate is given at the time of the fracture, or earlier, so that no barrier is formed, newly formed but poorly calcified osseous tissue gains contact with the callus cartilage without delay, and cartilage removal proceeds in an essentially normal fashion, with normal time relationships.

8. If phosphate is administered at longer intervals than twenty-four hours there is interruption of calcification, and resumption of the rachitic type of cartilage removal in the epiphyseal cartilage. A twenty-four-hour period between doses is barely sufficient, in the majority of instances, to maintain a continuous process of calcification. When phosphate administration is forced at the maximum possible rate, calcification in the callus is greatly accelerated, but an increased rate of phosphate administration or an increased initial dose, has no demonstrable effect on the time of appearance of the initial calcification in the epiphyseal cartilage or in the callus.

9. A diet inadequate in phosphate, but with a supplement of twoper-cent. cod-liver oil, does not produce the gross lesions of rickets, or the manifestations of absence of calcification in the fracture callus. The distribution of the bone salt deposited in the bone and in the callus are the same whether large doses of viosterol, or phosphates are administered. Still larger doses of viosterol initiate pathological resorption of bone and of osteoid, and seriously interfere with the healing of fractures.

10. When an adult rat with a previously normal skeleton is transferred to a rachitogenic phosphate-deficient diet, calcification in the callus is adequate to produce bony union at the normal time, and there is only a very slight lag in calcification in the osseous tissue of the callus.

11. It is suggested that the observations upon the retardation of the healing process in the absence of sufficient mineral to produce calcification in the callus, and upon the delay in the initiation of a normal type of response when treatment is initiated, which results from encapsulation of the callus cartilage in a dense mass of connective tissue and fibrocartilage, may have a bearing upon certain problems of delayed union or non-union.

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