Endochondral Ossification

Endochondral ossification is the process by which bone tissue is formed in early fetal development.

From: Comprehensive Biomaterials , 2011

Pathogenesis of Osteochondrosis

Janet Douglas , in Diagnosis and Management of Lameness in the Horse (Second Edition), 2011

Endochondral Ossification

Endochondral ossification is the process by which growing cartilage is systematically replaced by bone to form the growing skeleton. 7 This process occurs at three main sites: the physis, the epiphysis, and the cuboidal bones of the carpus and tarsus. Chondrocytes in the physis can be divided into a series of layers or zones (Figure 54-3). The zone farthest from the metaphysis is the resting or reserve zone. Adjacent to this is the proliferative zone, in which chondrocytes divide. These cells progress to the hypertrophic zone, in which they enlarge and form ordered columns. During this stage the chondrocytes become surrounded by extracellular matrix that gradually becomes mineralized in the zone of provisional calcification. The chondrocyte columns are then invaded by metaphyseal blood vessels, and bone forms on the residual columns of calcified cartilage. This mixture of calcified cartilage and immature bone (primary spongiosa) is then gradually remodeled to produce the mature bone of the metaphysis.7 Endochondral ossification, which continues throughout the period of growth, also occurs in the AECC at the ends of long bones (Figure 54-4).8 The chondrocytes of the AECC that are closest to the articular surface produce articular cartilage, whereas those cells closer to the epiphysis participate in endochondral ossification in the same manner as occurs in the physis. It is generally accepted that the growth cartilages of both the physis and the AECC are susceptible to OC.1,3,8-11

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Diseases of the Bones, Joints, and Connective Tissues

Michelle C. Coleman , ... Scott A. Katzman , in Large Animal Internal Medicine (Sixth Edition), 2020

▪ Pathophysiology

Endochondral ossification is the process of bone formation in which cartilage scaffolds, arranged in zones, are gradually replaced by bone. It occurs at the articular/epiphyseal and metaphyseal growth plates and at secondary centers of ossification, such as the carpal and tarsal bones. Directly beneath the articular cartilage is a zone of resting chondrocytes that divide to form the next zone of proliferating chondrocytes. These proliferative cells divide rapidly, organizing into columns perpendicular to the long axis of growth. The cells swell in the hypertrophic zone, where the columns become more organized. The chondrocytes in this zone are surrounded by increasing amounts of extracellular matrix, which becomes mineralized in the zone of calcification. These columns of chondrocytes are vascularized by metaphyseal blood vessels supplying nutrients. It is on these calcified cartilage columns that bone forms, creating the primary spongiosa, which is subsequently remodeled into mature bone.

The exact pathogenesis of osteochondrosis is unclear. The traditional theory is that the process of endochondral ossification is disrupted, resulting in areas of thickened cartilage. The deeper layers of affected cartilage do not receive adequate nutrients, resulting in necrosis of the cells and failure of proper ossification. These retained cartilage "plugs" have less structural integrity than normal cartilage and are prone to damage. Specifically, shear forces acting on the abnormal cartilage can lead to fissure formation, which progress into fragments of cartilage and subchondral bone. When compressive forces predominate on an area of thickened cartilage, it is surmised that infolding of the cartilage plug occurs with normal endochondral ossification proceeding around it, possibly leading to the formation of a subchondral bone cyst.

The traditional theory of defective endochondral ossification may be a simplistic view of a more complex, multifactorial condition. Limited reparative responses of bone and cartilage make it difficult to determine whether the origin of a lesion is developmental or traumatic. One report failed to distinguish articular cartilage differences in naturally occurring osteochondrosis versus healing osteochondral fragments. 13

Computed tomography and magnetic resonance performed on fetuses and foals demonstrated that there was greater cartilage thickness in areas of joints that commonly develop OCD. More specifically, at 8 to 9 months of gestation, the lateral trochlear ridge of the femur, medial malleolus of the tibia, and distal intermediate ridge of the tibia, all OCD-susceptible sites, had the greatest percentage of cartilage compared to unsusceptible sites. Postpartum, the percentage of cartilage in the medial malleolus and distal intermediate ridge of the tibia remained high. These findings suggest that greater cartilage thickness at specific joint sites could play a role in the development of OCD. 14

Arthroscopic observations of normal-thickness cartilage defects and normal subchondral bone, as well as lesions occurring preferentially at single sites at the limits of articulation, suggest causative factors other than defective endochondral ossification. 15 The development and then spontaneous regression of osteochondrosis lesions in young animals suggest that the condition is a dynamic process that can be affected by numerous intrinsic and extrinsic factors, and a "window of susceptibility" may exist whereby lesions are constantly changing and result in the development of normal articular cartilage. Alternatively, these developmental lesions may not regress, leading to osteochondrosis. 16 It is currently impossible to predict how a lesion may behave while developing; thus treatment recommendations should be reserved until the lesion has been fully developed.

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Biologically Inspired and Biomolecular Materials

L. McNamara , in Comprehensive Biomaterials, 2011

2.210.3 Bone Formation

During embryonic development, bone formation occurs by two distinct mechanisms; either endochondral ossification or intramembranous ossification. Bone formation also persists throughout life to alter the geometry of bones and provide larger bones that are sufficient to withstand changes in mechanical loading (modeling), or to replace aged or damaged bone (remodeling) or fill fracture gaps ( Section 2.210.6 ). Bone formation is typically a two-step process whereby an organic matrix (osteoid/cartilage template) is initially laid down by osteoblasts, and then mineral crystals are precipitated and grow slowly over time to produce the composite material.

2.210.3.1 Endochondral Ossification

Endochondral ossification is the process by which bone tissue is formed in early fetal development. It begins when MSCs start to produce a cartilage template of long bones, such as the femur and the tibia, upon which bone morphogenesis occurs. 67 The process initiates when MSC cells differentiate to become chondroblast cells ( Figure 5(a) ) and form a membrane around the template known as the perichondrium. This template grows in length (interstitial growth) and thickness (appositional growth) when the chondroblasts proliferate or more chondroblasts are recruited from the perichondrium ( Figure 5(b) ). Together, these cells secrete an ECM comprised mainly of collagen and proteoglycans. Over time, these chondroblasts differentiate to become chondrocytes and begin to secrete alkaline phosphatase, which is an enzyme that acts as a nucleator for deposition of minerals on the template. They also secrete growth factors to promote the invasion of blood vessels into the perichondrium, which is known as vascularization. This process forms the outer membrane of the bone, which is primarily a dense irregular connective tissue known as the periosteum ( Figure 5(c) ). The periosteum is an important source for undifferentiated osteoprogenitor cells. 68 It is divided into an outer fibrous layer, which is a source for fibroblasts, and an inner osteogenic layer, which is a source for osteoprogenitor cells that develop into osteoblasts. The periosteum also provides sites for attachment for ligaments, tendons, and muscles. This process begins in the middle of the template, which is known as the primary center of ossification ( Figure 5(c) ). During mineralization, the chondrocytes undergo apoptosis and the cavities that remain are invaded by blood vessels from the perichondrium. These blood vessels are a source for hemopoietic cells that form the bone marrow and osteoprogenitor cells, which differentiate to become osteoblast cells and secrete bone proteins and minerals. Endothelial cells (ECs) on the lining of blood vessels produce essential growth factors that control the recruitment, proliferation, and differentiation of osteoblasts. 69 Therefore, vascularization is an essential requirement for bone formation. 68,70 A number of other factors regulate the formation of blood vessels, including oxygen tension, mechanical loading, nutrients, and growth factors. 71 Osteoclasts are also recruited during this time to remodel the template and form a cavity for bone marrow (medullary cavity), and together, these events provide the first bone tissue during fetal development. At birth, a secondary ossification center appears in the epiphyses of long bones, which is vascularized and forms a cartilage layer known as the growth plate ( Figure 5(d) ).

Figure 5. Schematic diagram of endochondral ossification.

The formation and growth of bones is ongoing throughout childhood and is regulated by the epiphyseal or growth plate ( Figure 5(d) ), which continues to produce new cartilage, which is replaced by bone, and thereby facilitates lengthening of bones. In adults, lengthening of bones stops and the growth plate fuses and is replaced by bone, known as the epiphyseal line. Bones can continue to grow in diameter around the diaphysis by deposition of bone by osteoblasts beneath the periosteum, and simultaneously osteoclasts on the interior surface (endosteum) resorb bone to maintain a lightweight structure. The coordinated process of endochondral ossification is essential to the development and growth of long bones of the body, but also regulates fracture repair, as is discussed in Section 2.210.6.3 .

2.210.3.2 Intramembranous Ossification

During embryonic development, bone formation also occurs by means of a process known as intramembranous ossification, which regulates the formation of nonlong bones such as the bones of the skull and clavicle. The primary difference between intramembranous and endochondral ossification is that the intramembranous process does not rely on the formation of a cartilage template. Instead, embryonic stem cells (MSCs) within mesenchymal tissue of the embryo, derived from primary tissue (germ layers), begin to proliferate and condense to form an aggregate of MSC cells. This nodule is surrounded by a membrane, and MSCs within the membrane begin to differentiate to first become osteoprogenitor cells and then osteoblasts. These osteoblasts line the nodule and secrete an ECM consisting of type I collagen fibrils within the center of the nodule. Some osteoblasts become embedded within the newly formed matrix, and in this environment, they differentiate and form interconnecting cytoplasmic processes to become osteocytes. The cells on the outer surface form a periosteum, and bone growth continues at the surface of the trabeculae. At this time, the nodule is mineralized to form rudimentary bone tissue that is populated by osteocytes and lined by active osteoblasts. 48 This tissue is known as a bone spicule and many spicules fuse to form trabeculae, known as primary spongiosa, which then fuse to form woven bone. Over time, this woven bone is remodeled to become lamellar bone, with concentric lamellae surrounding Haversian systems in what is known as an osteon. 48,72,73

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Mesenchymal Stem Cells as Regulators of the Bone Marrow and Bone Components

L.M. Martinez , ... N.A. Chasseing , in Mesenchymal Stromal Cells as Tumor Stromal Modulators, 2017

Osteoclasts in the Formation of the Hematopoietic Niche in the Bone Marrow/Bone

Endochondral ossification is required for the formation of the HSC niche, and it is a process that occurs before the appearance of HSCs in the bone marrow. HSCs and their progenitors, located in a region of active bone remodeling called endosteum, express receptors that are calcium sensitive and are involved in the retention of these cells near the endosteum, where osteoclasts and osteoblasts promote an increase in calcium levels. 106

Osteogenesis, osteoclastogenesis, and bone resorption processes are extremely regulated, involving complex cell interactions. Pharmacological treatments such as G-CSF or stress conditions induce HSC mobilization, with osteoclast involvement. 107 Stress-induced osteoclasts produce proteolytic enzymes such as metalloproteinases, which cleave factors involved in the regulation of the HSC niche, thus inducing cell mobilization. 108,109 Bone mineralization matrix growth factors such as IGF, basic FGF (bFGF), TGFβ, BMP, and PDGF are released during bone resorption and stimulate bone formation, 110 while active osteoclasts produce acid and proteases that activate growth factors such as TGFβ1, which in turn induce both the migration of MSCs to the resorptive places and their osteoblastic differentiation. 111 Osteoblasts and pericytes provide a vascular and endothelial niche where HSCs and progenitors are in close contact with their regulatory chemokines, cytokines, and growth factors. 20,25,31,36,39,106 For instance, both the membrane and soluble forms of CXCL12/SDF-1, which is produced by osteoblasts, are the major chemoattractant for hematopoietic progenitors and HSCs. 112 Osteoblasts and MSCs express osteopontin, which is a negative and positive regulator of HSCs, promoting, respectively, their proliferation and apoptosis. 106 These interactions point to a complex scenario of bone formation and resorption crosstalk, with the bone-remodeling processes osteogenesis, osteoclastogenesis, and bone resorption probably being involved in regulating the formation of the endosteal HSC niche.

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Volume I

Christa Maes , Henry M. Kronenberg , in Endocrinology: Adult and Pediatric (Seventh Edition), 2016

Endochondral Ossification

Endochondral ossification is the mechanism responsible for the formation of all long bones of the axial skeleton (vertebrae and ribs) and the appendicular skeleton (limbs). Most of the axial skeleton is derived from cells of the paraxial mesoderm that condense early in embryogenesis on both sides of the neural tube and the notochord. Some cells of this mesoderm form segmented structures called somites, portions of which later become the sclerotomes that will give rise to the vertebral bodies. The appendicular skeleton arises from the lateral plate mesoderm. The mechanisms underlying the early condensation, segmentation, differentiation, and patterning events define the precise arrangement of the individual anatomic elements and their patterning along the proximal-distal, dorsal-ventral, and posterior-anterior body axes. These mechanisms involve actions and cross-talk of several morphogens, including fibroblast growth factors (FGFs), sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnts, as well as control by Notch signaling and by transcription factors encoded by HOX, PAX1, and TBX genes. 9

As in intramembranous ossification, the development of the long bones proper starts with mesenchymal progenitor cells forming condensations at the sites where the bones will form. 10 Yet, in the mesenchymal condensations of endochondral bones, cells do not differentiate into osteoblasts but instead differentiate into chondrocytes that synthesize a characteristic extracellular matrix (ECM) rich in type 2 collagen and specific proteoglycans. As such, a cartilaginous model or anlage is established that prefigures the future bone. In mice, these differentiated cartilage structures appear around embryonic day 12, with the limb elements emerging in sequence along the proximodistal axis (i.e., hip to toes, shoulder to fingers). The sequential steps of the endochondral ossification process starting from this stage are illustrated in Figure 60-1. Initially, the cartilage further enlarges through chondrocyte proliferation and matrix production. Chondrocytes in the midportion of the bone model then stop proliferating, undergo further maturation, and ultimately become hypertrophic. These large hypertrophic chondrocytes secrete a distinct matrix, containing type X collagen, and then rapidly direct the calcification of the matrix. Concomitantly, the hypertrophic chondrocytes direct the cells surrounding the cartilage element called the perichondrium to differentiate into osteoblasts that deposit mineralized bone matrix—the "bone collar"—around the cartilage template. This bone collar forms the initiation site of the cortical bone, the dense outer envelope of compact, lamellar bone that provides the long bone with most of its strength and rigidity (see Fig. 60-1).

At this time in development, the cartilage model starts to become replaced by bone, vascular, and marrow elements: the primary ossification center (see Fig. 60-1). The transformation is initiated by the invasion of the hypertrophic cartilage core by blood vessels (around embryonic day 14 to 15 in mice). This process is accompanied by apoptosis of terminally differentiated hypertrophic chondrocytes, resorption of the calcified cartilage matrix by invading osteoclasts or related "chondroclasts," and deposition of mineralized bone matrix on the remnants of calcified cartilage by perichondrium/periosteum-derived osteoblasts. Recent studies have visualized the entry of osteoblast lineage cells into the primary ossification center at these early stages, showing a close temporal and spatial association between osteoprogenitors and blood vessels co-invading the developing long bones. 11

With the disappearance of the diaphyseal cartilage (see Fig. 60-1), the remaining chondrocytes, restricted to the opposing ends of the long bone, provide the engine for subsequent bone lengthening. This process is typified by precise temporal and spatial regulation of chondrocyte proliferation and differentiation, with the chondrocytes first flattening out and forming longitudinal columns of rapidly proliferating cells, and next, as they reach the ends of the columns closest to the center of the bones, maturing further to hypertrophic chondrocytes (Fig. 60-2). Finally, at the border with the metaphysis (see Fig. 60-1), the terminally differentiated chondrocytes are thought to mostly disappear through apoptosis, and the calcified hypertrophic cartilage matrix is progressively replaced with cancellous or trabecular bone (forming the primary spongiosa). This process of cartilage turnover and replacement by bone requires adequate neovascularization of the chondro-osseous junction by metaphyseal capillaries (see Fig. 60-1 and cellular details in Fig. 60-2). Thus, similar to the initial formation of the primary ossification center, endochondral bone formation at the growth cartilage involves rigorous coupling of vascular invasion with maturation and activity of chondrocytes, osteoclasts, and osteoblasts (for review, see reference 12).

At a certain time (around postnatal day 5 in mice), epiphyseal vessels (see Fig. 60-1), derived from the vascular network that overlays the cartilage tissue, invade the growth cartilage and initiate the formation of the secondary center of ossification. As a result, discrete layers of residual chondrocytes form true growth plates between the epiphyseal and metaphyseal ossification centers, mediating further postnatal longitudinal bone growth. Ultimately, at least in humans, the growth plates completely disappear (close) at the end of adolescence in a process that actively requires the action of estrogen in both boys and girls, and growth stops. Remodeling of existing bone, replacing the primary spongiosa with lamellar bone in the secondary spongiosa and renewing the cortical bone, takes place throughout adult life, ensuring optimal mechanical properties of the skeleton and contributing to mineral ion homeostasis. This continual bone turnover is accomplished through the balanced action of osteoclasts and osteoblasts (see later) and results in a dynamic organization of honeycomb platelike structures or trabeculae in the interior of the bone that are surrounded by blood vessels and bone marrow and housed within the cortical bone.

The mechanisms of embryonic bone development described here are largely recapitulated in the adult upon repair of bone defects. In contrast to soft tissues, which repair predominantly through the production of fibrous scar tissue at the site of injury, the skeleton possesses an astounding capacity to regenerate upon damage. As such, bone defects heal by forming new bone that is indistinguishable from adjacent, uninjured bone tissue. It has been appreciated for a long time that fracture repair in the adult bears close resemblance to fetal skeletal tissue development, with both intramembranous and/or endochondral bone formation processes occurring depending on the type of fracture. This close resemblance has been supported by genetic and molecular studies showing that similar cellular interactions and signaling pathways (see later) are at work in both settings, 8,13-15 although additionally, some molecules that are dispensable for development have been found to play essential roles in fracture repair. 16,17

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Skin and Musculoskeletal Pathology

Thomas C. King MD, PhD , in Elsevier's Integrated Pathology, 2007

Metabolic Bone Disease

Metabolic bone disease includes a variety of abnormalities in the endocrine function of the bone in calcium homeostasis as well as abnormalities of mineral deposition in newly formed or remodeled bone. Since bony remodeling is a constant process throughout life, even minor persistent abnormalities in bone mineralization can eventually result in marked changes in bone structure or loss of mineralized bone. Abnormalities of mineralization in growing children cause different clinical features with prominent deformity of long bones.

HISTOLOGY

Endochondral Ossification

Endochondral ossification is the normal process that forms all long bones. After a cartilaginous framework of a bone is formed, endochondral ossification results in the sequential conversion and resorption of calcified cartilage and its replacement by bone. Osteoblasts migrate to the ossification front and synthesize bone matrix. Endochondral ossification is a well-ordered process that has definite polarity with residual cartilage on one edge and newly formed osteoid on the other. Trailing osteoid is mineralized in an orderly fashion to form new mineralized bone.

In long bones, ossification begins in the diaphysis during embryonic development, whereas ossification of the epiphysis does not start until after birth. In neonates, the ossification fronts move toward each other until only the growth plates (epiphyseal plates) remain between the metaphysis and epiphysis. Continued endochondral ossification at the growth plates permits a linear increase in bone length until closure of the epiphyses in late adolescence or early adulthood.

Osteoporosis

Osteoporosis is an extremely common disease in United States and tends to be most severe in postmenopausal women. Progressive bone loss after menopause sets the stage for pathologic fractures in multiple bones. Normally, skeletal density increases during childhood and reaches a peak in young adulthood in all individuals, with men having greater bone density than women. There is a slow, progressive loss of bone mineral after young adulthood, and this loss can be accelerated by inactivity (mechanical stimulation is necessary to maintain bone mass), nutritional deficiency, and other factors. The extent of peak mineralization and the rate of bone mineral loss are the key determinants of when (and whether) individuals will develop symptomatic osteoporosis during their lifetime (and be at risk for pathologic fractures).

Most patients with osteoporosis appear to have an unequal balance between osteoblastic and osteoclastic activity. Over time, bony remodeling (in response to changing mechanical stress) accelerates age-dependent loss of bone mineral. It is now thought that the predominant defect in most patients with osteoporosis is decreased new bone formation. Osteoporosis affects bones with a large percentage of trabecular bone (metabolically active bone that modulates calcium homeostasis) most severely. For this reason, compression fractures of the vertebrae are very common in osteoporosis, and patients may experience decreasing height and nerve root compression as initial symptoms.

At menopause, there is a marked increase in bone mineral loss that is probably related to differences in cytokine secretion in bony tissue. For instance, increased TNF-α production by macrophages favors the differentiation of macrophages to osteoclasts and can enhance bone resorption. Estrogen replacement and calcium supplementation may be helpful in some patients but usually are not sufficient to reverse bone loss. Known genetic risk factors for osteoporosis include polymorphisms in the vitamin D receptor.

Hyperparathyroidism

Increased secretion of parathyroid hormone by the parathyroid glands results in increased osteoclastic activity in bone with increased resorption and release of free calcium into the circulation (Fig. 6-16). Hyperparathyroidism is characterized as primary if the abnormality resides in the parathyroid glands themselves (as a result of either hyperplasia or a functional adenoma). Secondary hyperparathyroidism results from abnormalities in free calcium and phosphorus levels in plasma that cause compensatory hyperplasia of the parathyroid glands, which then secrete large amounts of parathyroid hormone. This condition is common in patients with end-stage renal disease and contributes to the bony abnormalities in renal osteodystrophy (see below). Severe, prolonged hyperparathyroidism can result in the formation of so-called brown tumors, which are solid aggregates of osteoclasts stimulated by parathyroid hormone. Brown tumors are not true neoplasms, and removal of parathyroid hormone results in their regression. Hypercalcemia and bone mineral loss can also occur as a paraneoplastic syndrome, which may be mediated by parathyroid hormone-like peptides or osteoclast activating factor (OAF).

Osteomalacia

Osteomalacia means defective mineralization of newly formed osteoid. Osteomalacia can result from a deficiency of vitamin D (as occurs in rickets) or from resistance to vitamin D (e.g., abnormalities of the vitamin D receptor). In growing children, abnormal bone mineralization results in weakened long bones that tend to bow and curve, resulting in the characteristic skeletal deformities of rickets. Osteomalacia can also result from abnormal serum concentrations of calcium and phosphorus in patients with end-stage renal disease (so-called renal osteodystrophy) that prevent normal formation of hydroxyapatite.

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Clinical signs

Mike Davies BVetMed CertVR CertSAO FRCVS , in Clinical Signs in Humans and Animals Associated with Minerals, Trace Elements, and Rare Earth Elements, 2022

Angular deformity of long bones

Impaired endochondral ossification resulting in abnormal formation of long bones (bowed legs) often due to premature closure of growth plates (e.g., ulnar) with the continued growth of adjacent bone plate (e.g., radius)—common in dogs.

Element Species Comments
Calcium deficiency Birds, camels, cats, dogs, foxes, goats, horses, nonhuman primates, reptiles, squirrels Waterfowl: carpal rotation
Lemurs: (Tomson and Lotshaw, 1978).
Squirrels: limbs, spine, tail
Calcium toxicity Dogs Radius curvus
Copper deficiency Humans, camels, cats, cattle, pigs, poultry (chicks) Cats: Deformed carpi
Pigs: Crooked forelimbs
Cattle: Rickets
Poutry: Deformed metatarsals
Iodine deficiency Humans, dogs
Manganese deficiency Birds, dogs, goats, guinea pigs, pigs, rabbits, rats
Molybdenum toxicity Horses, rabbits, rats Horses: rickets
Phosphorus deficiency Birds, dogs, mink
Zinc deficiency Cattle Bowing of hindlegs

DD: trauma to growth plate(s) resulting in premature closure.

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Development of the Skeleton

Sylvain Provot , ... Henry Kronenberg , in Osteoporosis (Fourth Edition), 2013

Intramembranous Bone Formation

Both endochondral ossification and intramembranous ossification begin with formation of mesenchymal condensations. During endochondral ossification, these condensations form a cartilage matrix; during intramembranous ossification, mesenchymal condensations differentiate directly into osteoblasts without an intervening chondrogenic phase. In the calvaria, mesechymal blastemas prefigure sites of future skull bones, and calvarial sutures develop where two opposing bone fronts appose ( Fig. 6.7). The sutures are the predominant sites of bone growth, which must be carefully coordinated with enlargement of the underlying brain. The most actively proliferating cells are located at the edges of bone fronts, and this is where the differentiation of cells along the osteoblast lineage occurs. In the mammalian skull, the frontal and parietal bones, as well as portions of the temporal and occipital bones, are derived from the NC (see section patterning the skeleton).

FIGURE 6.7. Coronal suture at P1 in the mouse. This suture occurs at the border of the parietal (p) and frontal (f) bones. Arrows point to expression of the engrailed 1 gene in osteoprogenitors.

 Source: reprinted from Deckelbaum et al. (2006), with permission [276].

Although the molecular mechanisms underlying intramembranous ossification are not well understood, genetic mutations found in human syndromes have led to the identification of numerous important regulators. Mutations that affect intramembranous ossification are generally manifest as either craniosynostosis, resulting from premature fusion of sutures, or as enlarged fontanels, when two skull bones fail to appose correctly.

One of the first gene products in which mutations were identified was Msx2 [250]. Mutations in Msx2 result in Boston-type craniosynostosis [249], and lead to enhanced binding of Msx2 to target DNA sequences [101]. Conversely, haploinsufficiency of Msx2 leads to wide open fontanels in humans [251]. In mice, targeted deletion of Msx2 leads to an ossification defect of the frontal bone, with decreased osteoblast proliferation [100]. The mechanisms of action of Msx2 are unknown, but it may serve to inhibit expression of bone-specific genes such as collagen I [252] and osteocalcin [253] and direct precursors along the osteoblast lineage [103]. How, if at all, these actions contribute to the craniosynostosis phenotype is uncertain (see further discussion of Msx2 in section endochondral bone formation: osteoblasts).

Although Msx2 was the first mutated gene product linked to craniosynostosis, most craniosynostosis syndromes are associated with mutations in FGFRs. FGFs signal via four tyrosine kinase receptors, and craniosynostosis syndromes have been linked to mutations in FGFR1, FGFR2, and FGFR3. The majority of these syndromes are associated with mutations in FGFR1 and FGFR2, and, in fact, mice lacking FGFR3 do not have any apparent defects in cranial development [254,255]. Mutations in FGFR1 and FGFR2 associated with Crouzon, Pfeiffer, and Jackson-Weiss syndromes generally result in gain of function, for example by causing ligand-independent dimerization by stabilizing intermolecular disulfide bones [256–259]. Two specific missense mutations in FGFR2 lead to increased receptor signaling because the mutant receptors are activated by FGF ligands that do not normally activate the receptor [260].

Mice genetically manipulated to express the P250R mutant form of FGFR1, the ortholog of which causes Crouzon syndrome in humans, demonstrate premature fusion of cranial sutures accompanied by increased expression of the osteoblastic transcription factor, Runx2 [261]. Similarly, activating mutations of FGFR2 in mice result in coronal synostosis [262] reminiscent of Apert's syndrome.

The relevant FGF ligands involved in cranial development are being investigated. Multiple FGFs are expressed during intramembranous ossification, including FGF2, FGF4, FGF9, FGF18, and FGF20 [198]. Ectopic expression of FGF2 in mice leads to macrocephaly [263] and coronal synostosis [264]. In addition, retroviral insertion in the region between FGF3 and FGF4 leads to increased expression of both in cranial sutures and Crouzon-like craniosynostosis in mice [265]. In contrast, mice deficient in FGF18 have craniofacial defects and delayed ossification [202,203].

Twist 1 and Twist 2 are basic helix-loop-helix transcription factors that inhibit the actions of Runx2 in osteoblast development. Twist 1 is coexpressed with Runx2 in calvarial bones, while Twist 2 is expressed in the axial skeleton. As in the human craniosynostotic Saethre-Chotzen syndrome, caused by heterozygous inactivating mutations in Twist 1, haploinsufficiency of Twist 1 in mice leads to craniofacial abnormalities [266,267]. Furthermore, haploinsufficiency of Twist 1 can rescue the delayed fontanels seen with haploinsufficiency of Runx2, demonstrating a role for Twist 1 in inhibiting Runx2, through the interaction of the twist box of Twist with the runt domain of Runx2 [268].

In addition to FGF signaling, TGFβ signaling has been implicated in intramembranous bone formation in mice. Germline deletion of TGFβ2 results in mild defects in cranial formation and ossification [269], while double knockouts lacking both TGFβ2 and TGFβ3 have impaired formation of frontal and parietal bones [270]. A similar phenotype is seen in conditional knockout of the receptor Tgfβr2 in Wnt1Cre-expressing cells [271]. In addition, Prx1Cre-mediated deletion of Tgfβr2 also leads to defects in parietal and frontal bones, indicating a cell-autonomous requirement for TGFβRII-mediated signaling in intramembranous bone formation [272].

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Bone

Brian K. Hall , in Bones and Cartilage (Second Edition), 2015

Subperiosteal Ossification and Suppression of the Cartilage Phase

Intramembranous and endochondral ossification are usually regarded as separate and very different modes of ossification, which they are. This is so, even though much of the ossification in the long bones of many tetrapods occurs subperiosteally and so is essentially intramembranous; the membrane in this case is the perichondrium, which transforms into a periosteum (Kronenberg, 2007; Dirckx et al., 2013). Despite the strong stance taken by Patterson (1977), for whom exoskeletons and endoskeletons were absolutely separate, mixed bones consisting of fused dermal and chondral bone do exist; for example, in the skeleton of the striped bass, Morone saxatilis; in fusion between tooth-bearing dermal bones and the perichondral and endoskeletal bones of the visceral arches A in zebrafish and cichlids; and in fusion of the endochondral bone that replaces Meckel's cartilage (endoskeleton) and dermal mandibular bones in hamsters 20 .

We expect these patterns of ossification to be conserved phylogenetically – once an endochondral bone, always an endochondral bone – and, indeed, phylogenetic conservation is what we see almost all the time. There are examples, however, where an endochondral bone in an ancestral lineage has been replaced by a membrane bone in a descendant lineage. The simplest mechanisms would be suppression of the cartilaginous model and formation of bone de novo. Of course, in any such example, we have to be sure we are looking at the same elements in ancestor and descendant, and that one bone has not been replaced by another from a different position within the embryo and/or with a different phylogenetic history.

An oft-cited example is the orbitosphenoid of a South American limbless 'worm-lizard' (amphisbaenian), Leposternon microcephalum studied by Bellairs and Gans (1983). Although the orbitosphenoid is an endochondral bone in all other species studied, in L. microcephalum, it is a membrane bone with an associated cartilage nodule. This nodule is probably not a secondary cartilage for the two following reasons: (1) It does not lie in the periosteum of the membrane bone; and (2) secondary cartilages have not been reported from reptiles. Irwin and Ferguson (1986) investigated whether reptiles could form secondary cartilage by making incisions in parietal bones of three species of lizards and two species of snakes. Bony union typically occurred by 18 days after incision. Secondary cartilage was never seen; see Figure 5.4 for the phylogenetic distribution of secondary cartilage 21 . The cartilaginous nodule in L. microcephalum could be any of the following:

•

a remnant of the orbital cartilage, which is present but exceedingly small in this species;

•

a remnant of the cartilaginous model that forms the orbitosphenoid in other species; or

•

a neomorph.

A developmental (and phylogenetic?) series is required to resolve this issue.

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Bone Biology and Fracture Healing

Mandi J. Lopez , in Equine Surgery (Fifth Edition), 2019

Indirect Bone Healing

Healing by endochondral ossification involves sequential biological stages with overlapping transition phases. During the acute inflammatory response initiated by injury, a hematoma forms and is populated by cells from bone marrow as well as peripheral and intramedullary blood. Proinflammatory molecules such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1 and IL-6 from macrophages and other inflammatory cells peak within 24 hours of fracture to enhance extracellular matrix synthesis, stimulate angiogenesis, and recruit as well as direct other inflammatory cells and mesenchymal stem (progenitor) cells (MSCs). 40 The proteins remain active beyond the acute inflammatory phase that is usually complete by 7 days. Similarly, members of the transforming growth factor-β (TGF-β) superfamily, which include multiple bone morphogenetic proteins (BMPs), act in combinations to promote the various stages of intramembranous and endochondral bone formation. 41

At the end of the acute inflammatory phase, there is a highly cellular, fibrin-rich callus of loosely organized granulation tissue between the bone ends that is a template for the ensuing callus. Endochondral formation occurs in the matrix template and external to the periosteum. The cartilaginous tissue forms a soft callus around 7 to 9 days posttrauma. Concurrently, intramembranous ossification commences from solid bone beneath the periosteum on each fracture end. Angiogenesis during this early phase is regulated largely by angiopoietin-dependent and vascular endothelial growth factor (VEGF)-dependent pathways. 42 Vascular morphogenesis is driven by angiopoietins, primarily 1 and 2. The VEGF pathway supports both vasculogenesis (de novo vessel formation) and angiogenesis (generation of new vessels from existing). A transient blood supply to the callus originates from the surrounding soft tissues and is distinct from periosteal arteries. As healing progresses, the extraosseous blood supply diminishes.

The repair phase is typically associated with soft callus resorption and replacement with hard callus followed by woven bone. Fracture callus MSCs differentiate into chondroblasts that proliferate and secrete cartilage-specific matrix including collagens type II and III and proteoglycans. Chondroblasts mature into chondrocytes that become hypertrophic and surrounded by calcified extracellular matrix. Collagen type I replaces collagen types II and III, and calcium hydroxyapatite crystals cluster around the fibrils to create a hard callus by around day 14. M-CSF, RANKL, OPG, and TNF-α drive mineralized cartilage resorption and recruit bone cells to form woven bone. As vessels invade to unite fragment vasculature, hypertrophic chondrocytes are removed by chondroclasts and woven bone. Bony, bridging callus is formed as woven bone replaces calcified cartilage. This is considered the final step in the reparative phase of fracture healing and the point of clinical union.

The remodeling phase involves a second resorptive phase to remodel the hard callus into a bone structure with a central medullary cavity, and it is biochemically reliant on IL-1 and TNF-α. 40 Hard callus is resorbed by osteoclasts while osteoblasts simultaneously deposit lamellar bone. Mineralized collagen fiber sublayers that are variably oriented compose individual lamellar units that, together, form a complex, multilayered structure. Bone remodeling results from weight-bearing stresses that cause concave surfaces to become electronegatively and convex surfaces to become electropositively charged owing to polarity created when pressure is applied to a crystalline environment. Osteoblastic activity is enhanced on electronegative surfaces and osteoclast activity is higher on electropositive surfaces according to Wolff's law. Successful bone remodeling requires an adequate blood supply and gradual increase in mechanical stability. Without both, an atrophic, fibrous nonunion can result. With good vascularity but unstable fixation, a cartilaginous callus may form but progress to a hypertrophic nonunion or pseudoarthrosis. 43

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