Bone Formation - Lecture #15

Achondroplasia

(G. "without cartilage formation") is a genetic disorder of bone growth, that causes the most common type of dwarfism. This disorder occurs in all races and both sexes. It may be transmitted as 1) an autosomal dominant trait from one of the parents In this case, the abnormal gene is located on chromosome 4. If one parent is normal (does not carry the gene) and the other parent has the disorder (carries the defective gene), there is a 50% chance that a child will inherit the defective gene from the affected parent and be affected with the disease. OR 2) a spontaneous genetic mutation occurring in the sperm or oocyte In about 80% of cases, achondroplasia is not inherited but results from a new mutation. This means that both parents are normal (are not affected by the disease) and neither one carries the defective gene. In contrast, the gene mutation occurred spontaneously in either the sperm or oocyte.

Endochondral Ossification

(begins around the 12th week of gestation) • In endochondral ossification, a cartilage model of the bone-to-be is formed first, and then it becomes calcified, eroded and replaced by bone • Endochondral ossification is the mechanism by which long bones of the limbs and the vertebrae of the spine form

Zones in the epiphyseal plate

(starting at the epiphyseal side and progressing toward the diaphysis), are as follows: i. Zone of Reserve Cartilage a) this is the resting zone b) this is "typical hyaline cartilage" ii. Zone of Proliferation a) chondrocytes proliferate, form parallel columns of isogenous cells. Chondrocytes appear in stacks (like multiple stacks of pennies) that are parallel to the long axis of the bone b) chondrocytes produce organic matrix Note that cartilage here grows INTERSTIALLY

Endochondral Ossification (primary ossification center)

- in diaphysis The first step of endochondral ossification is the formation of a miniature version of a hyaline cartilage model of the bone-to-be. (Note that the hyaline cartilage model is derived from mesenchyme). Once cartilage forms, it grows both interstitially and appositionally, but maintains the "shape / outline of a bone" during its growth. The cartilage grows in length by interstitial growth and in width by appositional growth. The perichondrium becomes vascularized. This signals its chondrogenic cells in its inner cellular layer to differentiate into steoprogenitor cells. The osteoprogenitor cells differentiate into osteoblasts and the perichondrium converts into a periosteum. The new osteoblasts in the inner layer of the periosteum synthesize and secrete bone matrix to build the (sub)periosteal bony collar by intramembranous ossification. As the bony collar forms, the cartilage cells hypertrophy (they accumulate glycogen and form vacuoles). The hypertrophied cells produce alkaline phosphatase and the cartilage matrix becomes calcified. Since the cartilage matrix is now calcified, nutrients cannot diffuse across this medium and the chondrocytes die. (note: do not confuse "calcification of cartilage" which occurs here, with "mineralization of bone" discussed later). Osteoclasts form perforations on the bone collar. A periosteal bud consisting of blood vessels, osteoprogenitor cells, and hemopoietic stem cells passes into the cartilage model. Here osteoprogenitor cells become osteoblasts which form bone matrix (osteoid) on the remnants of the calcified cartilage.

Endochondral Ossification - Secondary Ossification Center

- in epiphysis A secondary center of ossification forms in one epiphysis shortly after birth. Another secondary ossification center forms in the other epiphysis shortly afterwards Bone forms in the epiphyses in a similar fashion as that of the diaphysis, but without a bone collar. The osteoprogenitor cells populate the calcified cartilage and differentiate into osteoblasts. The osteoblasts synthesize bone matrix and deposit it on the surface of the calcified cartilage remnants. Cartilage is replaced by bone, except at the a. articular surfaces of the epiphyses Cartilage here normally persists throughout life. If it wears down → osteoarthritis b. epiphyseal (growth) plates -Allow long bones to grow in length

Mineralization of Bone - the following occur in the osteoid:

1.Minerals are delivered by the blood vessels to the forming bone 2. Osteocalcin and other sialoproteins bind extracellular Ca2+ 3. The elevated concentration of Ca2+ activates the osteoblasts to release alkaline phosphatase (ALP), which in turn elevates the local extracellular concentration of PO4 ions. This further elevates the concentration of Ca2+ 4. The osteoblasts release matrix vesicles into the bone matrix (which now contains a high concentration of Ca2+ and PO4). These matrix vesicles regulate the initial site of mineral deposition in the osteoid 5. The vesicles membrane contains pumps that transport more and more minerals into the vesicle 6. Crystallization of calcium and phosphate occurs in the vesicles, forming hydroxyapatite crystals (CaPO4) 7. As the sharp crystals enlarge, they damage the vesicle's membrane and the crystals are deposited in the bone matrix

Bone Remodeling

A. In a young individual, bone growth occurs at a faster rate than bone resorption B. In adulthood, bone growth and bone resorption occur at about the same rate C.Surface remodeling by 1.bone deposition (addition) on the outer surface of bone 2. bone resorption (removal) on the inner surface of bone 3. bone responds to parathyroid hormone (PTH) and calcitonin

Histogenesis of Bone (Bone Formation)

Bone forms by two different mechanisms: intramembranous or endochondral ossification..

Bone Repair

Bone is repaired by intramembranous bone formation, cartilage formation and endochondral bone formation A. The following occur during the repair of a bone fracture 1. damage to bone matrix causes bone cells to die (disrupts nutrient delivery to cells) 2. damage to the periosteum and endosteum causes tearing of blood vessels 3. hemorrhage and blood clot formation. Then blood supply ceases 4. neutrophils arrive at the site of bone injury first 5. then macrophages arrive to clean up debris 6. blood vessels and fibroblasts from near-by CT grow into the blood clot. Fibroblasts form granulation tissue (a LCT). Later DCT and cartilage form.

Intramembranous Ossification (cont)

In addition to the organic substances mentioned above, osteoblasts also secrete bone osteonectin (which serves as "glue" between collagen and hydroxyapatite crystals), sialoproteins (osteopontin which binds cells to bone matrix, and sialoproteins I and II which also bind cells to bone matrix and initiate calcium phosphate formation during mineralization of bone matrix), and osteocalcin which traps calcium from the blood stream for matrix mineralization). The soft osteoid (organic, non-mineralized component) of primary (immature, woven bone) subsequently undergoes mineralization. As the bone matrix "sets" following deposition of minerals, osteoblasts become trapped in lacunae ("bubbles" in the bone matrix) and are then referred to as osteocytes. Their cytoplasmic processes enclosed in canaliculi, maintain contacts with the cytoplasmic processes of other nearby osteocytes.

Intramembranous Ossification (cont)

Mesenchymal cells continue to undergo mitosis forming an ongoing supply of osteoprogenitor cells which in turn differentiate into osteoblasts. They deposit bone matrix on the surface of existing spicules and trabeculae via appositional (bone) growth. Primary bone is later resorbed by osteoclasts and gradually replaced with secondary (mature, lamellar) bone, a new bone tissue, formed by osteoblasts. Newly formed bone spicules and trabeculae initially form spongy bone. The highly vascular CT surrounding the bone spicules and trabeculae differentiates into bone marrow. The spongy bone may remain spongy or become compact bone via a secondary deposition of bone tissue that fills-in the marrow spaces. Mesenchymal tissue also gives rise to the periosteum and endosteum.

Osteoclasts

Osteoclasts breakdown the calcified cartilage/mineralized bone complex, hollowing the bone marrow cavity The calcified cartilage of the diaphysis does NOT become bone, but instead, it is replaced by bone Note that the hyaline cartilage of the epiphyseal plates persists until about the age of 18 - 20 years

Intramembranous Ossification (cont)

The basophilic osteoblasts begin to synthesize and deposit the organic component of bone matrix, called osteoid (consisting of proteins such as collagen, proteoglycans, glycoproteins and GAGs). The first bone that is laid down is known as primary (immature, woven) bone. Its collagen fibers (mainly type I), appear "woven" and are randomly oriented. As primary bone is deposited, initially it takes the shape of eosinophilic spicules and trabeculae (pink "island-like" structures floating in a loose, pale-staining mesenchymal tissue in the background). The spicules and trabeculae form a 3-D network over time.

Achondroplasia (cont)

This disease is characterized by a decrease in the production of the cartilage cells in the epiphyseal growth plates of long bones. In these individuals, the cartilage in the growth plates is replaced by bone at a very slow rate resulting in short bones in the upper and lower limbs. Specifically, the zones of proliferation and hypertrophy are slender and disorderly. The zone of proliferation lacks stacks of chondrocytes and the chondrocytes present are enlarged and form clusters instead. Affected individuals display midface hypoplasia, large head with frontal bossing, nearly normal torso length, lumbar lordosis, short proximal upper and lower limb bones, bow legs, and short stature.

Bone deposition:

a) osteoblasts synthesize and release bone matrix b) bone matrix is mineralized on surface of the calcified cartilage. Cartilage is resorbed c) calcified cartilage remnants stain blue with hematoxylin d) newly formed bone stains pink with eosin NOTE: Newly formed bone is added here, on the diaphyseal side of the epiphyseal plate. When new cartilage matrix is formed (added) at the epiphyseal plate (in the zone of proliferation), the bone grows in length Around 20 years of age, the growth in the zone of proliferation slows down. In contrast, the rate of ossification outruns that of the zone of proliferation and the zone of reserve cartilage and eventually the entire epiphyseal plate is replaced by bone. This is referred to as epiphyseal closure e) a plate of calcified cartilage/mineralized bone complex replaces the cartilaginous epiphyseal plate f) osteoclasts resorb this complex g) the marrow cavity of the epiphysis becomes continuous with that of the diaphysis h) since the epiphyseal plate no longer exists, bone growth in length is not possible

Bone Growth in Length

a. Long bones grow in length by endochondral bone formation that occurs in the epiphyseal (growth) plates. That is, cartilage is continuously being replenished at the epiphyseal side of the growth plates and is concurrently replaced by bone at the diaphyseal side of the growth plates. Consequently i. the thickness of the growth plates remains about the same as the bone grows, and ii. the distance between growth plates gradually increases

Bone Growth in Width

a. bone grows in width by appositional growth b.osteoprogenitor cells of the osteogenic (inner cellular) layer of the periosteum differentiate into osteoblasts c.osteoblasts secrete bone matrix on the subperiosteal bone surface d. subperiosteal intramembranous bone formation i. osteoblasts: bone deposition on external surface of bone ii.osteoclasts: bone resorption on internal surface of bone iii. bone marrow cavity diameter increases iv. bone diameter increases

A calcified cartilage/mineralized bone complex forms = "mixed spicule" how you distinguish b/ calcified cartilage and mineralized bone With H&E

a. calcified cartilage = is basophilic (blue) i. does not contain cells (the cells have died) ii. will eventually become completely eliminated b. mineralized bone = is acidophilic (pink) i. contains living cells (it's normal for bone to be mineralized and contain living cells)

Zones in the epiphyseal plate (cont)

iii. Zone of Hypertrophy a) glycogen in chondrocytes b) enlarged lacunae iv. Zone of Calcified Cartilage a) lacunae coalesce b) calcification of cartilage matrix c) chondrocytes die v. Zone of Resorption and Ossification a) calcified cartilage remnants form long spicules in the direction of the diaphysis b) blood vessels bringing osteoprogenitor cells invade this zone c) osteoprogenitor cells emigrate to calcified cartilage matrix area, and differentiate into osteoblasts

Bone Repair (cont)

the granulation tissue and cartilage form a soft callus that covers the injured bone. The callus helps to form a bridge between the 2 bone fragments 8. osteoprogenitor cells in the periosteum divide and differentiate to form osteoblasts 9. the new osteoblasts synthesize and secrete osteoid on the outer surface of the bone. This begins at some distance from the fracture and progresses in the direction of the fracture. It continues until newly-formed bone forms a shell on the surface of the fibrocartilaginous callus 10. osteogenic buds (vessels and osteoprogenitor cells) grow into the callus 11. the cartilage calcifies, dies and is replaced by bone via endochondral ossification. New bone is deposited until the fibrous and cartilaginous callus is completely replaced with a bony callus 12. concurrently, endosteal proliferation results in bone spicules to grow toward the marrow cavity 13. spongy bone forms first 14. spongy bone is slowly replaced by compact bone and while this occurs, the bony callus is broken down by osteoclasts 15. bone is remodeled to its original shape

Intramembranous Ossification

• In intramembranous ossification, bone forms directly within a membrane of highly vascular mesenchyme •Intramembranous ossification is the mechanism by which flat bones of the skull, the face, mandible and clavicle form In intramembranous ossification, the initial site of osteogenesis is in a membrane of mesenchyme. Here mesenchymal cells differentiate into osteoprogenitor cells which in turn differentiate into osteoblasts.