Cartilage develops when mesenchyme cells on the proximal, central core of the limb bud transform into chondroblasts and condense. This transformation is independent of the condensation, and these cells can from this point on differentiate only into cartilage even if transplanted. This condensation is prevented in the distal end of the limb bud by an unknown inhibiting factor secreted by the ectoderm, [A]. Upon transformation, the chondroblasts begin to secrete an extensive extracellular matrix of Type II collagen fibrils embedded in a fibrous connective tissue with a high concentration of proteoglycan molecules. Because the connective tissue is composed of molecules with negatively charged ends, it holds water between the molecules, much like a mop. The cartilage in an embryo is different from the cartilage that forms the large skeletons of adult sharks and rays in that it has a higher ratio of cells to matrix and is not calcified, which would make it more rigid. Because chondrocytes (mature chondroblasts) have a low metabolic rate, vascularization is minimal, and tends to occur near the surface in the fibrous perichondrium, which covers the free surfaces of cartilages [D].

Picture: [D]

Cartilage grows in two ways:

1. On the surface, by recruitment of chondroblasts.

2. Interstitially, by mitotic divisions of the chondrocytes, which separate and continue to synthesize matrix.

Because it grows so easily and quickly without requiring any involved remodeling, cartilage is an excellent material for a template, allowing the "bones" of the embryo to grow at the same rate as the embryo while the slower process of ossification makes them structurally sound for life after the womb. [D]

The next step in forming the appendicular skeleton is the ossification of the cartilage template, or the laying down of bone. Bone is a hard, highly vascularized tissue with both a mineral component which gives it strength and a connective tissue component which gives it a degree of flexibility. Most important though is the ability of bone to remodel and to change in structure as the forces on an individual's body change throughout life. [D]

The initial ossification in long bones occurs around the daphysis or shaft of the bone. Undifferentiated cells in the perichondrium become osteoblasts, and the perichondrium is now the periosteum. Next, the chondrocytes within the diaphysis enlarge and line up in rows, reabsorbing some of the matrix and depositing calcium in what remains of it. This mineralization cuts the chondrocytes off from the vascularized periosteum, denying them nutrients and gas exchange, whch causes them to die. In the honeycombed spaces left by the perishing chondrocytes, vascular tissue from the periosteum begins to invade, bringing with it cells that differentiate into osteoblasts. These osteoblasts line up along the remnants of the matrix and begin to form a bony matrix on the surface of the diaphysis. This surface ossification is known as perichondral ossification, and is very ancient, found in both early jawless vertebrates and gnathostomes. [D]

Picture: [D]

After the chondral template has been invaded by cells that differentiate into osteoblasts as well as the vasculature required to support them, ossification continues from the center of the bone in what is called endochondral ossification. This begins at the center of the bone shaft and grows outward toward the end of the bone, where the cartilage remains as a growth plate that aids the bone in elongation as the animal matures. The osteoblasts produce a matrix of Type I collagen fibers which at this point exist in randomly organized bundles. This matrix is calcified with calcium phosphate, which strengthens it and traps the osteocytes (mature osteoblasts) in the matrix they have created. Later in life, these bundles of collagen fibers are organized into parallel layers called lamella with rows of osteocytes between them. These layers are organized with the fibers running in different directions which adds to the strength of the bone. Also, the degree of calcification varies depending on the function of the bone: for example a femur, which is highly load bearing, has a high mineral content, whereas a deer antler bears no weight but must be more flexible and is therefore less mineralized.[D]

Picture: [D]

Bone growth cannot occur interstitally as cartilage growth does, because its rigid, mineralized matrix traps osteocytes and prevents them from dividing mitotically as chondrocytes do. Growth occurs in two directions:

1. Length, by maintenance and growth of a plate of cartilage near the end of the bone. Because the natural properties of cartilage suit it for growth, these epiphyseal plates allow a bone to expand lengthwise.

2. Diameter, by continuous formation of bone around the periphery of the diaphysis.

As this growth occurs, bone is continuously remodeled through the action of osteoclasts. Osteoclasts secrete a strong acid that dissolves the mineral components of bone and enzymes that digest the collagen. In reptiles, birds, and mammals, these osteoclasts tunnel into bone, making new paths for vascularization with brings in osteoblasts, which line up on the inner wall of the tunnel. This causes the concentric deposit of bone to form columns or osteons which run vertically through the bone. At the center of the bone is a marrow cavity, which also expands as the bone grows. The dense, load bearing bone tends to lie around the periphery and is known as compact bone. [D]