in layers on the trabeculæ of calcified cartilage, which they envelop with osseous tissue.

5. Osteoclasts cause the absorption of many of the smaller osseous trabecula; others become thickened by a deposition of new layers of osseous tissue. Osteoblasts are enclosed in bonetissue and become bone-cells. In this way there is formed embryonic cancellous bone, bounding Haversian spaces inclosing embryonic marrow.

6. In the diaphysis, the greater portion of the embryonic cancellous bone is also absorbed (by osteoclasts); the Haversian spaces unite to form a part of the marrow space of the shaft of the bone.

2. Intramembranous Bone.-This, the simpler type of ossification, occurs in bone developed from a connective-tissue foundation, and is exemplified in the formation of the bones of the

Fig. 84.-Longitudinal section through epiphysis of arm bone of sheep embryo; X 12. a, b, Primary marrow spaces and bone lamellæ of the diaphysis.

cranial vault and the greater number of the bones of the face, and also in bone developed from the periosteum (perichondrium) surrounding the cartilage fundaments of endochondral bone. fibrous-tissue bone is developed in the same way.

All

The intramembranous bone-development begins by an approximation and more regular arrangement of the osteoblasts of the osteogenetic layer of the periosteum about small fibrous-tissue bundles. The osteoblasts then become engaged in the formation of the osseous tissue which envelops the fibrous-tissue bundles. In this way a spongy bone with large meshes is formed, consisting of irregular osseous trabeculæ, surrounding primary marrow spaces. These latter are filled by embryonic marrow and blood-vessels developed from the tissue elements of the periosteum not engaged in the formation of bone.

Intramembranous bone first appears in the form of a thin lamella of bone, which increases in size and thickness by the formation of trabeculæ about the edges and surfaces of that previously formed and in the manner above described. A layer of intramembranous bone thus surrounds the endochondral bone in bones preformed in hyaline cartilage. The two modes of ossification may, therefore, be observed in either a cross or a longitudinal section of a developing bone preformed in hyaline cartilage. In such preparations the endochondral bone can be readily distinguished from the intra

Fig. 85.-Section through the lower jaw of an embryo sheep (decalcified with picric acid); X 300. At a and immediately below are seen the fibers of a primitive marrow cavity lying close together and engaged in the formation of the ground-substance of the bone, while the cells of the marrow cavity, with their processes, arrange themselves on either side of the newly formed lamella and functionate as osteoblasts.

membranous bone by reason of the fact that remnants of calcified cartilage matrix may be observed in the osseous trabeculæ of the former. It will be remembered that these osseous trabeculæ develop about the calcified cartilage matrix remaining after the disappearance of the cartilage-cells. In figure 86, which shows a cross-section of a bone from the leg of a human embryo, these facts are clearly shown. A study of this figure shows the endochondral bone, with the remnants of the cartilage matrix (shaded more

deeply) inclosed in osseous tissue, making up the greater portion of the section and surrounded by the intramembranous bone.

In figure 87, more highly magnified, the relations of endochondral to intramembranous bone and the details of their mode of development are shown; also the structure of the periosteum.

As was stated in the previous section, soon after the formation of the endochondral bone, this is again absorbed; the process of endochondral bone-formation and absorption extending from the center of ossification toward the ends of the diaphysis. Before the absorption of the endochondral bone, the intramembranous bone has attained an appreciable thickness and surrounds the marrow cavity formed on the absorption of the endochondral bone. Before,

however, the marrow cavity can attain its full dimensions, much of the intramembranous bone must also undergo absorption. While intramembranous bone is being developed from the periosteum and thus added to the outer surface of that already formed, osteoclasts are constantly engaged in its removal from the inner surface of the intramembranous bone. The marrow cavity is thus enlarged, the process continuing until the shaft attains its full size.

The compact bone of the shaft is developed from the primary spongy intramembranous bone after the following manner: The primary marrow spaces are enlarged by an absorption, through the agency of osteoclasts, of many of the smaller trabeculæ of osse

ous tissue and by a partial absorption of the larger ones, the primary marrow spaces thus becoming secondary marrow spaces, or Haversian spaces. The osteoblasts now arrange themselves in layers

Connective

tissue.

Outer fibrous --

laver of periosteum.

Osteogeneticlaver of periosteum.

Osteoblasts..

Marrow

space.

Blood-vessel.

Osteoblasts.

Remnants of cartilage

matrix.

Bone-cells.

Osseous
matrix.

Osteoblasts.

Fig. 87. From a cross-section of a shaft (tibia of a sheep); X 550. In the lower part of the figure is endochondral bone formation (the black cords are the remains of the cartilaginous matrix); in the upper portion is bone developed from the periosteum.

about the walls of the Haversian spaces and deposit lamella after lamella of bone matrix, concentrically arranged, until the large Haversian spaces have been reduced to Haversian canals. During

this process many of the osteoblasts become inclosed in bone matrix, forming bone-cells and the blood-vessels of the Haversian spaces remain as the vessels found in the Haversian canals. The spongy intramembranous bone not absorbed at the commencement of the formation of the system of concentric lamellæ, remains between the concentric systems as interstitial lamellæ. The circumferential lamellæ are those last formed by the periosteum. Calcificaation of the osseous matrix takes place after its formation by the osteoblasts.

From what has been stated it may be seen that the shafts of the long bones and bones not preformed in cartilage develop by the process of intramembranous bone-formation, while the cancellous bone in the ends of the diaphysis and in the epiphyses is endochondral bone. Further, that long bones grow in length by endochondral bone-development, and in thickness by the formation of intramembranous bone. In the development of the smaller irregular bones, both processes may be engaged; the resulting bone can not, however, be so clearly defined.

TECHNIC.

133. One of the methods for examining connective-tissue cells and fibers is that recommended by Ranvier (89); it is as follows: The skin of a recently killed dog or rabbit is carefully raised, and a 0.1% aqueous solution of nitrate of silver injected subcutaneously by means of a glass syringe. The result is an edematous swelling in which the connectivetissue cells and fibers (the latter somewhat stretched) come into immediate contact with the fixing fluid and are consequently preserved in their original condition. In about three-quarters of an hour the whole elevation should be cut out (it will not now collapse) and small fragments placed upon a slide and carefully teased. Isolated connective-tissue cells with processes of different shapes, having the most varied relations to those from adjacent cells, are seen. The fibers themselves either consist of several fibrils, or, if thicker, are often surrounded by a spirally encircling fibril. By this method numerous elastic fibers and fat-cells are also brought out. If a drop of picrocarmin be added to such a teased preparation and the whole allowed to remain for twelve hours in a moist chamber, and formic glycerin (a solution of 1 part formic acid in 100 parts glycerin) be then substituted for twenty-four hours, the following instructive picture is obtained: All nuclei are colored red, the white fibrous connective-tissue fibers pink, the fibrils encircling the latter brownishred, and the elastic fibers canary yellow. The peripheral protoplasm of the fat-cells is particularly well preserved, a condition hardly obtainable by any other method.

134. Connective tissue with a parallel arrangement of its fibers is best studied in tendon, those in the tails of rats and mice being particularly well adapted to this purpose. If one of the distal vertebræ of the tail be loosened and pulled away from its neighbor, the attached tendons will become separated from the muscles at the root of the tail and appear as thin glistening threads. These are easily teased on a slide into fibers and