Osteogenesis

Events in intra-cartilaginous or enchondral bone formation

• 1: Formation of cartilage model : In the region where the bone is to grow within the embryo, a hyaline cartilage model is developed. The cartilaginous model is surrounded by a vascular condensed mesenchyme or perichondrium. For a period this model grows, both appositionally and interstitially.

• 2: Appearance of a primary center of ossification: The chondrocytes in the center of cartilage model hypertrophy, accumulate glycogen in their cytoplasm and become vacuolated.

• 3: Formation of primary areolae. Hypertrophy of the chondrocytes results in enlargement of their lacunae. and reduction in the intervening cartilage matrix septa, which becomes calcified. The chondrocytes die and their lacunae are now called primary areolae.

• 4: Formation of subperiosteal bone collar: The osteogenic cells in the deeper layer of perichondrium differentiate into osteoblasts and perichondrium is transformed into periosteum.

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Events in enchondral bone formation

• Into osteoblasts and overlying perichondrium is converted into periosteum. The osteoblasts lay down bone matrix around the cartilage model in the intramembranous way making a bone collar around the center of ossification.

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• Perichondrium becomes the periosteum.• The newly formed osteoblasts of periosteum secrete bone

matrix around the cartilage model, forming the subperiosteal bone collar by intramembranous bone formation.

• The bone collar prevents the diffusion of nutrients to the hypertrophied chondrocytes within the core of cartilage model, causing them to die.

• The dead chondrocytes leave empty lacunar spaces separated by matrix septa. Confluence of these spaces creates marrow cavity.

• 5: Formation of osteogenic bud: Holes are etched in the subperiosteal bone collar by osteoclasts.

• Through these holes a subperiosteal bud (osteogenic bud) composed of osteoprogenitor cells, osteoblasts, osteoclasts

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• , hemopoitic cells, and blood vessels, invades the primary center of ossification (enters the cavities within the cartilage model).

• 6: Secondary areolae and primitive marrow: The osteoclasts erode the walls of primary areolae which now make larger confluent cavities called secondary areolae foreshadowing the future medulary cavity. Haemopoitic tissue, blood vessels osteoblasts and osteoclast fill the secondary areolae.

• 7: Osteoid formation: osteoblasts attach themselves to the residual walls of the calcified cartilage, laying down osteoid which rapidly changes , firstly into patches and then into continuous bone lining.

• Further layers of bone are added, enclosing young osteoblasts in lacunae and narrowing the perivascular spaces. The osteoblasts enclosed in lacunae become osteocytes.

• 8 : Calcification. The osteoid is mineralised by deposition of hydroxy apatite crystals. In this way a woven fibred bone is formed consisting of atypical Haversian system or primary osteons. The process of bone formation moves from primary center of ossification to both end of bone ( from diaphysis towards the epiphysis). Osteoblasts elaborate bone matrix on the surface of calcified cartilage. The bone matrix becomes calcified to form a calcified cartilage/ calcified bone complex.

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• 9: Appositional bone deposition. In the region of the shafts more bone is laid down at the periphery by bone collar as compared to central bone formation (interstitial deposition) increases the girth of the bone.

10. Bone erosion and medullary cavity formation:The osteoclasts erode the bone in the centre, and bone is not reformed in the centre to make future medullary cavity

11. Secondary osteons or typical Haversian system: Osteoclast erode the woven fibered bone in the region of the shaft which is destined to become the compact bone. In that region the vessels get arranged parallel to the long axis of shaft with surrounding osteoblasts. The osteoid is laid down with its collagen fibres in concentric lamellae, thus making the typical Haversian system.these steps comprehensively explain the basic process of endochondral ossification.

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BONE GROWTH IN LENGTH (Growth plate)

• The continued lengthening of bone depends upon the epiphyseal plate in the process of endochondral bone formation. Proliferation occurs at the epiphyseal aspect of growth plate and replacement by bone takes place at the diaphyseal aspect of the growth plate.

• Histologically the epiphyseal plate is divided into five recognizable zones. These zones beginning at the epiphyseal side are:

1. Zone of reserve cartilage- chondrocytes randomly distributed throughout the matrix are mitotically active.

2. Zone of proliferation- chondrocytes, rapidly proliferating forming rows of isogenous cells that parallel the direction of the bone growth.

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3. Zone of maturation and hypertrophy- chondrocytes mature, hypertrophy, and accumulate glycogen in their cytoplasm. The matrix between their lacunae narrows with corresponding growth in the lacunae.

4. Zone of calcification- lacunae become confluent, hypertrophied chondrocytes die and cartilage matrix becomes calcified.

5. Zone of ossification- osteoprogenitor cells invade t6he area and differentiate into osteoblasts, which elaborate matrix that becomes calcified on the surface of the calcified cartilage. This is followed by resorption of the calcified cartilage/ calcified bone complex.

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• Cacification: The osteoid is mineralized by deposition of hydroxyapatite crystals. This results in the formation of woven bone with atypical haversian systems.i

• Appositional calcification: The osteoclasts resorb the calcified cartilage /calcified bone complex and so enlarge the marrow cavity.

• As this process continues the cartilage of the diaphysis is replaced by bone, except for the epiphyseal plate, which are responsible for the continued growth of the bone for 18-20 years.

• Secondary centers of ossification: Begin to form at the epiphysis at each end of the long bone by a process similar to that in diaphysis except that a bone collar is not formed.

• The osteoprogenitor cells invade the cartilage of the epiphysis, differentiate into osteoblasts, and begin secreting matrix on the cartilage scaffold. Gradually the cartilage of epiphysis is replaced by bone except at (1) articular cartilage and (2) the epiphyseal growth cartilage

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• The articular surface of the bone remains cartilaginous throughout the life.

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• As long as the rate of mitotic activity in the proliferative zone equals the rate of resorption in the zone of ossification, the epiphyseal plate remains the same width, the bone continues to grow longer. At about the 20th year of age, the rate of mitosis decreases in the zone of proliferation and the zone of ossification overtakes the zone of proliferation and cartilage reserve. The cartilage of the epiphyseal plate is replaced by a plate of calcified cartilage/calcified bone complex, which becomes resorbed by osteoclastic activity and marrow cavity of the diaphysis becomes confluent with the marrow cavity of the epiphysis. Once the growth plate is resorbed, the growth in length is no longer possible.

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• Bone growth in width: the growth of diaphysis in girth takes place by appositional growth. The osteogenic layer of the cells of the periosteum proliferate and differentiate into osteoblasts that elaborate bone matrix on the subperiosteal bone surface. This process occurs throughout the growth period of the bone So that in the mature long bone the shaft is built via subperiosteal intramembranous bone formation.

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Intramembranous ossification Intramembranous ossification is the process in which ossification starts

directly in a mesenchymal membrane without having passed through cartilaginous stage. This is direct formation of bone in highly

vascular cellular sheets of mesenchymal tissue containing osteoprogenitor cells. Most of the flat bones of skull like frontal, parietal temporal, occipital bone and mandible develop through intramembranous ossification. This process also contributes to the growth of short bones and the thickening of long bones. In the early embryonic life, the region in which a membrane bone is to develop, shows condensation of the mesenchymal cells. Blood vessels rapidly proliferate within the condensed area so that it becomes more vascular. Further steps are

1. Differentiation and proliferation of osteoprogenitor cells in the membrane..

2. Formation of osteoblasts from osteoprogenitor cells in the center of membrane to form primary ossification centre..

3. Secretion of osteoid by osteoblasts around themselves and capillaries. The osteoid consists of matrix and collagen fibres but latter are disposed off irregularly in conformity with irregular capillary network. 21

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4. Calcification of the osteoid: The collagen fibers of the osteoid are randomly oriented and so they form a network of spicules and trabeculae whose surfaces are populated by osteoblasts. Osteoid is calcified and islands of developing bone surround elongated cavities contaning capillaries and bone marrow cells.

5. Osteoblasts are entrapped within bone , now being called osteocytes and they occupy lacunae. Also they send out cytoplasmic processes which pass through canaliculi and via these all osteocytes maintain contact with each other.

6. Primary osteons or primary Haversian systemAs matrix secretion, calcification and enclosure of osteoblasts proceed, the trabeculae thicken and intervening spaces become narrower. Where bone remains trabecular, the process of calcification becomes slow and spaces between trabeculae are occupied by hemopoitic tissue. Where compact bone is forming, trabeculae continue to thicken and vascular spaces to narrow. The collagen fibers of the matrix secreted on the walls of narrowing spaces between trabeculae, become organized as parallel, spiral or longitudinal bundles around a central canal. The enclosed cells occupy concentric sequential rows. These irregular, interconnected

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7. FORMATION OF SECONDARY OSTEONS The woven bone is eroded by osteoclasts and replaced by regular lamellar bone in trabeculae and typical Haversian systems or secondary osteons in compact regions. The process involves: Removal of woven bone Rearrangement of blood vessels parallel each other and bone surface. Secretion of Osteoid around vessels Mineralization of Osteoid.

8. Marrow Space: bone is not replaced after removal by osteoclasts in between bony septa and these spaces get occupied by marrow.

9. The process extends in all directions in same steps i.e early formation of woven fibered bone and its replacement by the lamellar bone later.

10. Periosteum and bone deposition from periphery: As the ossification proceeds inside, on the surface highly vascular connective tissue mesenchymal membrane condenses to form periosteum, containing in its deep part the osteoprogenitor cells. These cells differentiate into osteoblasts and latter start laying down bone at the periphery and also send in fresh generations of osteoblasts.

11. On the inner aspect of flat bones of skull, condensation of mesenchyme forms endosteum. During remodelling a process meant for reshaping the bone and continued through out the life,.

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FACTORS AFFECTING THE OSSIFICATION REMODELING AND APPLIED ASPECTS

i. Growth hormone excess of this hormone leads to gigantism with specially large hands, feet and facial skeleton (acromegaly).less than normal secretion leads to dwarfism.

ii. Oestrogens high levels cause endosteal and trabecular bone deposition whereas decreased levels cause bone. Absropiton reflecting osteoporosis after menopause when estrogen levels fall.

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III. Androgens high levels lead to premature fusion of epi and diaphysis leading to short stature. in low levels (hypogonadism) fusion is delayed and is specially characterized by excessively long limbs.

iv. Parathyroid hormone:hyperparathyroidism leads to sub periosteal and endosteal bone absorption by osteoclasts, a condition being referred to as osteitis fibrosa cysteica.

V. Thyroid hormones T3 and T4 (tri-iodothyronin and tetra iodo-thyronin respectively) are important for remodeling to adult mature shape 29

B. DIETARY ELEMENTSi. CALCIUM:

Proper intake of calcium is of explicit significance but adequate intake does not necessarily means adequate availability as absorption of calcium is dependent upon availability of vit. D as well.

II. VITAMINS: I. Vit. D.Vit. D is not only important for Ca absorption but also for normal mineralization of bone. Prolonged deficiency (even if Ca intake is more than normal) leads to following defects (but this is to be remembered that these malformations can result either due to vit. D or calcium deficiency or both)

III Osteoporosis:In adults this is demineralization and loss of bone tissue leading to fragile bones and hence fracture are common in old age.

IV Osteomalacia:In this condition bones are fragile with regions of deformable, uncalcified osteoid.

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Rickets:During growth deficiency of Ca or Vit. D causes failure of calcification of cartilage and thickening of growth plates. Poorly calcified cartilage is partially absorbed by osteoclasts, osteoid fails to ossify specially in metaphysial regions and as the child starts walking the body weight and gravitational traction deforms the bones. Another sign is compression of costal cartilages, leading to "pigeon chest".

II. Vitamin CThis is essential for normal collagen and proteoglycans synthesis. In deficiency, growth plates thin out, ossification almost ceases and whatsoever bone is formed is thin and fragile. Fracture healing is also delayed. Scurvy is the name given to gross deficiency of this vitamin.

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III. Vitamin A:Correct deposition and removal of bone, both are dependant upon this vitamin. Deficiency retards growth specially at base of skull, with resultant narrowing of foramina, at times to the extent that there is pressure atrophy (degeneration) of emerging cranial nerves. Cranial cavity and vertebral canal fail to expand with CNS growth, impairing nervous functions. (Recall small headed imbecile beggars seen at times in streets.Excess this vitamin leads to erosion of growth plates which may be completely lost with cessation of longitudinal growth.

iii) PHOSPHATES, PROTEINS AND GENERAL DIET:Mention has been made of many minerals as trace elements in bone. Deficiency of trace elements is rare but at times does occur. Adequate protein intake is of utmost importance for collagen and other proteins' deposition in the bone, specially the "essential amino acids" must be taken for normal osteoid formation. 32

BONE INJURY AND HEALINGWhen a bone fractures, following four stages are seen in healing process:

– Inflammation– Soft callus formation – Hard callus formation – Remodelling

First step is characterized by swelling, haematoma formation and pain. As the dead tissue is absorbed by phagocytes, next stage follows.Second step is characterized by formation of soft tissue blastema consisting of glycoproteins, collagen, osteoblasts, fibroblasts and chondroblasts and the area is highly vascular. Soft callus helps to immobilize the broken ends.Third step involves formation of woven bone and its gradual replacement by lamellar bone.

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First 3 steps take 2 - 3 months.Remodelling starts after that and continues for several years.SIGNIFICANCE OF PERIOSTEAL SUPPLY:Role of periosteal supply is clear from the fact that fractures of lower end of tibia have delayed healing, as no muscle is attached to lower 1/3 of tibia, hence periosteal vessels are scanty. (Cunningham's manual of dissection, vol. I)However all those elements mentioned above for normal bone formation are operative in bone healing process as well.

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First 3 steps take 2 - 3 months.Remodelling starts after that and continues for several years.SIGNIFICANCE OF PERIOSTEAL SUPPLY:

• Role of periosteal supply is clear from the fact that fractures of lower end of tibia have delayed healing, as no muscle is attached to lower 1/3 of tibia, hence periosteal vessels are scanty. (Cunningham's manual of dissection, vol. I)

• However all those elements mentioned above for normal bone formation are operative in bone healing process as well.

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Arthrology

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