Introduction

Bone ossification, or osteogenesis, is the process of bone formation. This process begins between the sixth and seventh weeks of embryonic development and continues until about age twenty-five; although this varies slightly based on the individual. There are two types of bone ossification, intramembranous and endochondral. Each of these processes begins with a mesenchymal tissue precursor, but how it transforms into bone differs. Intramembranous ossification directly converts the mesenchymal tissue to bone and forms the flat bones of the skull, clavicle, and most of the cranial bones. Endochondral ossification begins with mesenchymal tissue transforming into a cartilage intermediate, which is later replaced by bone and forms the remainder of the axial skeleton and the long bones.

Development

The development of the skeleton can be traced back to three derivatives[1]: cranial neural crest cells, somites, and the lateral plate mesoderm. Cranial neural crest cells form the flat bones of the skull, clavicle, and the cranial bones (excluding a portion of the temporal and occipital bones. Somites form the remainder of the axial skeleton. The lateral plate mesoderm forms the long bones

Bone formation requires a template for development. This template is mostly cartilage, derived from embryonic mesoderm, but also includes undifferentiated mesenchyme (fibrous membranes) in the case of intramembranous ossification. This framework determines where the bones will develop. By the time of birth, the majority of cartilage has undergone replacement by bone, but ossification will continue throughout growth and into the mid-twenties.   

Intramembranous Ossification

This process involves the direct conversion of mesenchyme to the bone. It begins when neural crest-derived mesenchymal cells differentiate into specialized, bone-forming cells called osteoblasts. Osteoblasts group into clusters and form an ossification center. Osteoblasts begin secreting osteoid, an unmineralized collagen-proteoglycan matrix that can bind calcium. The binding of calcium to osteoid results in the hardening of the matrix and entrapment of osteoblasts. This entrapment results in the transformation of osteoblasts to osteocytes. As osteoid continues to be secreted by osteoblasts, it surrounds blood vessels, forming trabecular/cancellous/spongy bone. These vessels will eventually form the red bone marrow. Mesenchymal cells on the surface of the bone form a membrane called the periosteum. Cells on the inner surface of the periosteum differentiate into osteoblasts and secrete osteoid parallel to that of the existing matrix, thus forming layers. These layers are collectively called the compact/cortical bone [2].

Five steps can summarize intramembranous ossification:

1. Mesenchymal cells differentiate into osteoblasts and group into ossification centers
2. Osteoblasts become entrapped by the osteoid they secrete, transforming them to osteocytes
3. Trabecular bone and periosteum form
4. Cortical bone forms superficially to the trabecular bone
5. Blood vessels form the red marrow

Endochondral Ossification

This process involves the replacement of hyaline cartilage with bone. It begins when mesoderm-derived mesenchymal cells differentiate into chondrocytes. Chondrocytes proliferate rapidly and secrete an extracellular matrix to form the cartilage model for bone. The cartilage model includes hyaline cartilage resembling the shape of the future bone as well as a surrounding membrane called the perichondrium. Chondrocytes near the center of the bony model begin to undergo hypertrophy and start adding collagen X and more fibronectin to the matrix that they produce; this altered matrix allows for calcification. The calcification of the extracellular matrix prevents nutrients from reaching the chondrocytes and causes them to undergo apoptosis. The resulting cell death creates voids in the cartilage template and allows blood vessels to invade. Blood vessels further enlarge the spaces, which eventually combine and become the medullary cavity; they also carry in osteogenic cells and trigger the transformation of perichondrium to the periosteum. Osteoblasts then create a thickened region of compact bone in the diaphyseal region of the periosteum, called the periosteal collar. It is here that the primary ossification center forms. While bone is replacing cartilage in the diaphysis, cartilage continues to proliferate at the ends of the bone, increasing bone length. These proliferative areas become the epiphyseal plates (physeal plates/growth plates), which provide longitudinal growth of bones after birth and into early adulthood. After birth, this entire process repeats itself in the epiphyseal region; this is where the secondary ossification center forms [3].

The physeal growth plate is separated into various sections based on pathologic characteristics. 

• Reserve Zone
  • Storage site for lipids, glycogen, proteoglycan 
• Proliferative Zone
  • Proliferating chondrocytes leading to longitudinal growth
• Hypertrophic Zone
  • Site of chondrocyte maturation
  • Within the hypertrophic zone, the chondrocytes go through a transformation process. The chondrocyte mature and prepare a matrix for calcification; then they degenerate which allows calcium release for calcification of the matrix. 
• Primary Spongiosa
  • Site for mineralization to form woven bone
  • Vascular invasion occurs
• Secondary Spongiosa
  • Internal modeling with the replacement of fiber bone with lamellar bone
  • External modeling with funnelization

Five steps can summarize endochondral ossification:

1. Mesenchymal cells differentiate into chondrocytes and form the cartilage model for bone
2. Chondrocytes near the center of the cartilage model undergo hypertrophy and alter the contents of the matrix they secrete, enabling mineralization
3. Chondrocytes undergo apoptosis due to decreased nutrient availability; blood vessels invade and bring osteogenic cells
4. Primary ossification center forms in the diaphyseal region of the periosteum called the periosteal collar
5. Secondary ossification centers develop in the epiphyseal region after birth

Cellular

Osteochondroprogenitor Cells

Osteochondroprogenitor cells are mesenchymal stem cells that can differentiate into chondrocytes or osteoblasts. The expression of the transcription factors CBFA1/RUNX2 and OSX induce osteoblast differentiation.[4] The expression of transcription factors SOX9, L-SOX5, and SOX6 are necessary for chondrocyte differentiation.

Osteoblasts

Osteoblasts are responsible for bone deposition. They also regulate osteoclasts. They derive from mesenchymal stem cells. During the embryonic period, they secrete osteoid, an unmineralized matrix, which is subsequently calcified and forms bone. Osteoblasts have a crucial role in maintaining the balance of bone formation and resorption. Osteoblasts secrete RANK ligand (RANKL), which binds to the RANK receptor on pre-osteoclasts and thus induces their differentiation. Osteoblasts also secrete osteoprotegerin (OPG), which prevents RANK/RANKL interaction by binding to RANKL; this prevents osteoclast differentiation. Thus, the balance between RANKL/OPG production by osteoblasts determines osteoclast activity.[5].

Osteoclasts

Osteoclasts are multinucleated cells that function in bone resorption.[6][7] They are derived from macrophages and enter the bone through blood vessels. Each osteoclast has numerous processes that extend into the matrix and secrete hydrogen ions, causing acidification and break down of bone. Osteoclast function is under tight control; overactivity results in osteoporosis while decreased activity results in osteopetrosis.

Osteocytes

Osteocytes are the most numerous cells present in bone. They form from osteoblasts trapped in osteoid.[8] Their primary function is mechanosensation. Osteocytes connect to each other and their environment via cytoplasmic processes. This communication with each other and the surrounding environment allows them to detect stress and deformation of the bone. Based on this information, osteocytes orchestrate the remodeling of bone. 

Molecular Level

Several transcription factors are involved in the process of endochondral bone formation. Sox-9 regulates chondrogenesis of several collagen types includes II, IV, and XI. PTHrP delays chondrocyte differentiation in the zone of hypertrophy. 

Intramembranous bone formation is controlled by the canonical Wnt and Hedgehog signaling pathway. Beta-catenin enters cells to induce the formation of osteoblasts. Additional transcription factors involved in the process include CBFA1 (Runx2), osterix (OSX), and sclerostin (SOST).

Pathophysiology

Cleidocranial Dysplasia (CCD)[9]

CCD occurs due to a mutation in CBFA1/RUNX2 (runt-related transcription factor 2) gene, which directs osteoblast differentiation - CCD is an autosomal dominant condition resulting in short stature, patent fontanelles, and supernumerary teeth

Camptomelic Dysplasia (CMD)[10][11]

CMD occurs due to a mutation in SOX9 (SRY-box 9) gene, which directs chondrocyte differentiation - CMD is an autosomal dominant condition that results in the bowing of long bones, and this condition usually results in neonatal death due to respiratory failure

Osteogenesis Imperfecta (OI)[12]

OI occurs due to a mutation in COL1A1 (collagen type I alpha 1 chain) or COL1A2 (collagen type I alpha 2 chain) genes, which encode the major component of type 1 collagen; this is an autosomal dominant condition that results in very fragile bones

Achondroplasia[13]

Achondroplasia occurs due to a mutation in FGFR3 (fibroblast growth factor receptor 3) gene, which aids in the formation of collagen and plays a role in the ossification of bone - this mutation prevents adequate bone formation in utero and results in a shortened stature 

Acromegaly [14]

Acromegaly occurs due to an increased amount of growth hormone and insulin-like growth factor-1. Causes of acromegaly include pituitary tumors and McCune-Albright syndrome. These factors have anabolic effects on cartilage and bone metabolism. The increased factors both cause the increased growth of bone and degenerative changes to cartilage resulting in arthropathy. 

Rickets [15]

Rickets is most commonly caused by a vitamin D deficiency which leads to the softening and weakening of bones in children. The main mechanism is insufficient calcification at the growth plate during bone formation. Symptoms of Rickets disease include bowed legs, spinal curvatures, rachitic rosary, and craniotabes.  Rickets results in failure of apoptosis of the hypertrophic chondrocyte in the physeal plate. Eventually, this leads to a cupping appearance of the epiphyseal ends of the bones. 

Clinical Significance

Physeal Fractures

Salter-Harris fractures are fractures of the epiphyseal plate.[16] These types of fractures have the potential to impair bone ossification depending on the location.[17] Injury to the epiphyseal plate can result in decreased longitudinal growth, angular deformity, and altered joint mechanics.[18] The classification is as follows [17]:

• Type I: separation through the physis
• Type II: fracture enters in the plane of the physics and exits through the metaphysis
• Type III: fracture enters in the plane of the physics and exits through the epiphysis
• Type IV: fracture crosses the physics and extends from metaphysics to epiphysis
• Type V: fracture is a crush injury

Forensic Significance

Age estimation of the fetus is one of the primary objectives of the fetal autopsy.

Forensic fetal osteology:

• The embryological method is one of the procedures employed in estimating the gestational age of the fetus which is crucial in determining fetal viability postpartum in forensic practice

The forensic examination of fetal remains [19][20][21][22]:

• It is not uncommon for a forensic pathologist to be called on to develop the forensic profile of fetal remains in a variety of medicolegal contexts, including cases of criminal abortion/feticide and infanticide.
• In such medicolegal contexts, the presence or absence of ossification centers aids in the gestational age estimation of fetal remains.
• Dimensions of various ossification centers are also useful in estimating the age of the fetus (for example, linear measurements of the neural arch of the atlas, the diameter of the distal epiphysis of the femur).
• Postmortem computed tomography (PM-CT) and plain radiography are useful imaging techniques employed to assess physical maturation of fetal bones.