NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-.

Embryology, Bone Ossification

Grant Breeland; Margaret A. Sinkler; Ritesh G. Menezes.

Author Information and Affiliations

Last Update: May 1, 2023.

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.

Review Questions

Access free multiple choice questions on this topic.

Comment on this article.

References
1. Jin SW, Sim KB, Kim SD. Development and Growth of the Normal Cranial Vault : An
Embryologic Review. J Korean Neurosurg Soc. 2016 May;59(3):192-6. [PMC free article:
PMC4877539] [PubMed: 27226848]
2. Percival CJ, Richtsmeier JT. Angiogenesis and intramembranous osteogenesis. Dev Dyn.
2013 Aug;242(8):909-22. [PMC free article: PMC3803110] [PubMed: 23737393]
3. Ortega N, Behonick DJ, Werb Z. Matrix remodeling during endochondral ossification.
Trends Cell Biol. 2004 Feb;14(2):86-93. [PMC free article: PMC2779708] [PubMed:
15102440]
4. Wysokinski D, Pawlowska E, Blasiak J. RUNX2: A Master Bone Growth Regulator That
May Be Involved in the DNA Damage Response. DNA Cell Biol. 2015 May;34(5):305-15.
[PubMed: 25555110]
5. Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O'Brien CA. Matrix-embedded
cells control osteoclast formation. Nat Med. 2011 Sep 11;17(10):1235-41. [PMC free article:
PMC3192296] [PubMed: 21909103]
6. Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. 2008 Nov;3 Suppl
3(Suppl 3):S131-9. [PMC free article: PMC3152283] [PubMed: 18988698]
7. Bar-Shavit Z. The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing
osteoimmune cell. J Cell Biochem. 2007 Dec 01;102(5):1130-9. [PubMed: 17955494]
8. Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011 Feb;26(2):229-38. [PMC free
article: PMC3179345] [PubMed: 21254230]
9. Lo Muzio L, Tetè S, Mastrangelo F, Cazzolla AP, Lacaita MG, Margaglione M, Campisi G. A
novel mutation of gene CBFA1/RUNX2 in cleidocranial dysplasia. Ann Clin Lab Sci. 2007
Spring;37(2):115-20. [PubMed: 17522365]
10. Lefebvre V, Dvir-Ginzberg M. SOX9 and the many facets of its regulation in the
chondrocyte lineage. Connect Tissue Res. 2017 Jan;58(1):2-14. [PMC free article:
PMC5287363] [PubMed: 27128146]
11. Jain V, Sen B. Campomelic dysplasia. J Pediatr Orthop B. 2014 Sep;23(5):485-8. [PubMed:
24800790]
12. Rauch F, Glorieux FH. Osteogenesis imperfecta. Lancet. 2004 Apr 24;363(9418):1377-85.
[PubMed: 15110498]
13. Baujat G, Legeai-Mallet L, Finidori G, Cormier-Daire V, Le Merrer M. Achondroplasia.
Best Pract Res Clin Rheumatol. 2008 Mar;22(1):3-18. [PubMed: 18328977]
14. Lieberman SA, Björkengren AG, Hoffman AR. Rheumatologic and skeletal changes in
acromegaly. Endocrinol Metab Clin North Am. 1992 Sep;21(3):615-31. [PubMed: 1521515]
15. Ozkan B. Nutritional rickets. J Clin Res Pediatr Endocrinol. 2010;2(4):137-43. [PMC free
article: PMC3005686] [PubMed: 21274312]
16. Levine RH, Thomas A, Nezwek TA, Waseem M. StatPearls [Internet]. StatPearls
Publishing; Treasure Island (FL): Jan 15, 2023. Salter Harris Fractures. [PubMed:
28613461]
17. Cepela DJ, Tartaglione JP, Dooley TP, Patel PN. Classifications In Brief: Salter-Harris
Classification of Pediatric Physeal Fractures. Clin Orthop Relat Res. 2016
Nov;474(11):2531-2537. [PMC free article: PMC5052189] [PubMed: 27206505]
18. Caine D, DiFiori J, Maffulli N. Physeal injuries in children's and youth sports: reasons for
concern? Br J Sports Med. 2006 Sep;40(9):749-60. [PMC free article: PMC2564388]
[PubMed: 16807307]
19. Huxley AK, Angevine JB. Determination of gestational age from lunar age assessments in
human fetal remains. J Forensic Sci. 1998 Nov;43(6):1254-6. [PubMed: 9846409]
20. Huxley AK. Gestational age discrepancies due to acquisition artifact in the forensic fetal
osteology collection at the National Museum of Natural History, Smithsonian Institution,
USA. Am J Forensic Med Pathol. 2005 Sep;26(3):216-20. [PubMed: 16121075]
21. Castellana C, Kósa F. Estimation of fetal age from dimensions of atlas and axis ossification
centers. Forensic Sci Int. 2001 Mar 01;117(1-2):31-43. [PubMed: 11230944]
22. Sakurai T, Michiue T, Ishikawa T, Yoshida C, Sakoda S, Kano T, Oritani S, Maeda H.
Postmortem CT investigation of skeletal and dental maturation of the fetuses and newborn
infants: a serial case study. Forensic Sci Med Pathol. 2012 Dec;8(4):351-7. [PubMed:
22392019]
Disclosure: Grant Breeland declares no relevant financial relationships with ineligible companies.

Disclosure: Margaret Sinkler declares no relevant financial relationships with ineligible companies.

Disclosure: Ritesh Menezes declares no relevant financial relationships with ineligible companies.

Copyright © 2023, StatPearls Publishing LLC.

This book is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
(CC BY-NC-ND 4.0) ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ), which permits others to distribute the work,
provided that the article is not altered or used commercially. You are not required to obtain permission to distribute this article,
provided that you credit the author and journal.

Bookshelf ID: NBK539718 PMID: 30969540