Bones are one of the most important types of connective tissue found in vertebrates as they provide a framework to the body, a definite shape, protect internal organs and the foundation of movement and locomotion. The process of bone formation is called osteogenesis and that of their calcification is termed as ossification. In humans, the process of bone formation begins during 6-8 weeks of embryonic development . The blastula differentiates into three germ layers; ectoderm, mesoderm and the endoderm. Out of the three germ layers mesoderm contributes towards the formation of bones in the body. Mesenchymal cells are the representatives of mesoderm and were first identified by Charles Minot in 1879. Later, in 1881, Oscar and Richard Hertwig found that these mesenchymal cells are of mesodermal origin.
During the period 1966-1987, Alexander Friedenstein showed that these mesenchymal cells were actually multipotent stem cells that could differentiate into osteocytes (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells) . These informatories marked a golden period to understand the concept of bone formation and ossification.
Bones are formed by either of the two pathways – Intramembranous ossification and Endochondral ossification, Let us study these two in detail:
Intramembranous ossification, as the name suggests, is the process of bone formation that takes place within the mesenchyme membrane. It is a relatively less time consuming process of bone formation that results in the formation of flat bones, facial bones, mandible and medial clavicle. The process involves following steps:
1-Formation of ossification center: As a response to embryonic development, mesenchymal cells accumulate at the area where bone has to be formed. They form a cluster of a definite shape which determines the shape of future bone. The mesenchymal cells differentiate into osteoblasts which secrete osteiods, the extracellular matrix comprising collagen.
2-Calcification: Osteoblasts surrounded by extracellular matrix of collagen are now known as osteocytes, the bone cells. These osteocytes extend their cytoplasm in various directions and form inter-osteocyte network via structures called canaliculi. Later, as minerals like calcium, amino acids and hydroxyapatite [Ca10(PO4)6(OH)2], magnesium etc. get deposited in extracellular matrix as a result of nutrients supply by capillaries, it hardens.
3-Trabaculae or spongy bone: Once the extracellular matrix calcifies, osteoclasts dissolve some of the calcified matrix in the inner region and form empty spaces which get interconnected and surround the blood vessels. Spaces in trabeculae are filled with bone marrow giving some sponginess to the inner tissue, this gives rise to the spongy bone found in the core of flat bones, sandwiches between two layers of compact tissue.
4-The Periosteum: Apart from the formation of compact and spongy tissue at the center, mesenchymal cells arrange themselves at the periphery and form the outer membrane of the bone called the periosteum.
Picture 1: Showing various stages of intramembranous ossification. a) formation of ossification center, b) calcification, c) trabeculae formation and d) formation of periosteum
Picture Reference : Source
Endochondral ossification is the process of bone formation that takes place inside a cartilage model. In this type of ossification, bones are formed using cartilage as a template and is a more time consuming process as compared to intramembranous ossification . Long bones of the body like those of the limbs and ribs are formed by endochondral ossification.
1-Formation of the Cartilage Model (hyaline cartilage): When the germ layers start differentiating into various tissues and organs, mesenchymal cells from mesoderm arrive at the site of bone formation due to a chemical stimulus. Mesenchymal cells arrange themselves in the shape of future bone and differentiate into chondroblasts. These chondroblasts secret extracellular matrix comprising hyaline cartilage surrounded by a network of mesenchymal cells at the periphery called perichondrium, the outer membrane of the cartilage model.
2-Growth of cartilage template: Chondroblasts surrounded by extracellular matrix are called are called chondrocytes, the structural and functional unit of a cartilage. The chondrocytes have a capability to divide and simultaneously secrete extracellular matrix which increases the length of bone (interstitial or endogenous growth). On the contrary, chondroblasts that differentiate from perichondrium deposit more and more extracellular matrix on the cartilage template cause an increase in the girth of the template (appositional or exogenous growth). As the cartilage grows in size, chondrocytes present at the center enlarge and the extracellular matrix begins to calcify.
3-Primary ossification center: Due to calcification of the extracellular matrix, chondrocytes present at the center are unable to receive the minimal nutrition required for their survival and begin to die. Dead chondrocytes disintegrate and form empty spaces called lacunae. These empty spaces provide entrance sites for blood vessels which carry osteoblasts to the desired places, at the same time, mesenchymal cells of the perichondrium differentiate as osteoblasts. The perichondrium is now known as periosteum. This process at first occurs at the center of the cartilage template where osteoblasts secrete osteiods comprising collagen which gets deposited over the disintegrated remnants of chondrocytes forming trabeculae or spongy bone at the center. This area of activity at the center is called primary ossification center.
4-Formation of medullary cavity: The shaft of long bones has a hollow cavity at the center called the medullary cavity. It is formed by the activity of osteoclasts at the center which resorp much of the spongy bone tissue at the center forming a hollow cavity which tapers towards the epiphyseal ends of the long bones. The medullary cavity is guarded at the vertical peripheries of the bone by a compact tissue formed by osteoblasts.
5-Formation of secondary ossification center: The ossification centers present at the epiphyseal ends of bones are called secondary ossification centers. These are formed by the invasion of epiphyseal arteries (arteries invading the epiphyses of bones) in a way similar to which results in the formation of primary ossification center. However, there is no osteoclast activity in this region and hence spongy bone is present.
6-Articular cartilage and epiphyseal growth plate: Since the formation of secondary ossification center radiates outwards, the remnant hyaline cartilage is pushed towards the surface of epiphyseal ends of the bone and is known as articular cartilage as it is present at the articulating end of bones. However, up to a certain age, up to which the bones increase in length, hyaline cartilage remains between the epiphyses and diaphysis of the bone in the form of epiphyseal growth plate which is responsible for increase in length of bones during adolescence.
Picture 2: Showing endochondral ossification. 1)formation of cartilage model, 2) growth of cartilage, 3) formation of primary ossification center, 4) formation of medullary cavity, 5) secondary ossification center and 6) epiphyseal plate and articular cartilage.
Picture Reference: Source
3-Gerard Tortora and Bryan Derrickson, Principles of Anatomy and Physiology, Chapter 6 (pg.no. 182-192)