The Biologic Aspects of Children’s Fractures

At birth, each epiphysis (except the distal femur)
consists of a completely cartilaginous structure at the end of each
long bone (Fig. 2-1), the chondroepiphysis. At
a time characteristic for each of these chondroepiphyses, a secondary
center of ossification forms and gradually enlarges until the
cartilaginous area has been almost completely replaced by bone at
skeletal maturity. This chondro-osseous transformation is
vascular-dependent (Fig. 2-2). Only articular cartilage remains at maturity.

As the ossification center expands, it undergoes
structural modifications. The region adjacent to the physis forms a
distinct subchondral plate parallel to the metaphysis, creating the
radiographically characteristic lucent physeal line. The appearance of
the ossification centers differ in certain chondroepiphyses, a factor
that must be considered when diagnosing fractures of these regions. The
ossification center imparts increasing rigidity to the more resilient
epiphyseal cartilage as the secondary osseous tissue expands.²⁰³

The external surface of an epiphysis is composed of either articular cartilage or perichondrium (Fig. 2-3).
Muscle fibers, tendons, and ligaments may attach directly to the
perichondrium, which is densely contiguous with the underlying hyaline
cartilage. The perichondrium contributes to the continued centrifugal
enlargement of the epiphysis. It also blends imperceptibly into the
periosteum. This perichondrial/periosteal tissue continuity contributes
to the biomechanical strength of the epiphyseal/metaphyseal junction at
the zone of Ranvier.

When the hyaline cartilage of the chondroepiphysis first
forms, there are no easily demonstrable histologic differences between
the cells of the joint surface and the rest of the epiphyseal
cartilage. However, at some point, a finite cell population becomes
stabilized and physiologically different from the remaining epiphyseal
cartilage. McKibbin¹²⁰ established
that these two cartilage types are different physiologically and
biochemically. If a contiguous core of articular and hyaline cartilage
is removed, turned 180°, and reinserted, the transposed hyaline
cartilage eventually will form bone at the joint surface, whereas the
transposed articular cartilage remains cartilaginous and becomes
surrounded by the enlarging secondary ossification center. Normally,
articular cartilage does not appear capable of

calcification
and ossification. As skeletal maturity is reached, a tide mark
progressively develops as a demarcation between the articular and
calcified epiphyseal hyaline cartilage.

An important aspect of McKibbin’s experiment was an
explanation of nonunion of certain fractures in which the fragment may
be rotated, causing the articular surface to lie against metaphyseal
and epiphyseal bone. Union is unlikely in such a situation because the
articular surface is incapable of a reparative osteogenic response, an
essential component of bone healing.

The growth plate, or physis, is the essential structure adding bone through endochondral ossification.¹³⁹,¹⁴³,¹⁴⁸,¹⁹⁴
The primary function of the physis is rapid, integrated longitudinal
and latitudinal growth. Injuries to this component are unique to
skeletally immature patients.

Because the physeal cartilage remains radiolucent,
except for the final stages of physiologic epiphysiodesis, its exact
location must be inferred from the metaphyseal contour, which follows
the physeal contour. The changing size of the secondary ossification
center more effectively demarcates the physeal contour on the
epiphyseal (germinal layer) side. As this center of ossification
enlarges centrifugally to approach the physis, the original spherical
shape of the ossification center flattens and gradually develops a
contour paralleling the metaphyseal contour. Similar contouring also
occurs as the ossification center approaches the lateral and
subarticular regions of the epiphysis (Fig. 2-4).
The region of the ossification center juxtaposed to the physis forms a
discrete subchondral bone plate that the essential epiphyseal blood
vessels must penetrate to reach the physeal germinal zone (Fig. 2-5). Damage to this osseous plate in a fracture may cause localized physeal ischemia.

If a segment of the epiphyseal vasculature is
compromised, whether temporarily or permanently, the zones of cellular
growth associated with these particular vessels cannot undergo
appropriate cell division. In contrast, unaffected regions of the
physis continue longitudinal and latitudinal growth, leaving the
affected region behind (Figs. 2-6 and 2-7).
The growth rates of the cells directly adjacent to the affected area
are more mechanically compromised than cellular areas farther away. The
differential rather than uniform growth results in an angular or
longitudinal growth deformity, or both.²⁴,¹⁵⁰

Interruption of the metaphyseal circulation has no
effect on chondrogenesis within the germinal zone or the sequential
cartilage maturation within the hypertrophic zone of the physis (see

Fig. 2-6). However, the subsequent
transformation of cartilage to bone (primary spongiosa) is blocked.
This causes widening of the affected area, because more cartilage is
added to the cell columns but none is replaced by invasive metaphyseal
vessels and bone. Once the disrupted metaphyseal circulation is
reestablished, this widened, calcified region of the physis is rapidly
penetrated and ossified, returning the physis to its normal width. This
is the mechanism seen in physeal and metaphyseal fractures. The
metaphyseal blood supply is temporarily blocked by separation or
impaction and requires 3 to 4 weeks for restoration. If the circulatory
compromise has been caused by a metaphyseal fracture, there also may be
a temporary halt to bone formation in the transiently ischemic portion
of the metaphysis. This leads to an apparent sclerosis when the bone is
compared with the adjacent vascularized metaphysis, which undergoes a
relative disuse osteoporosis. Compromise of the metaphyseal circulation
has minimal, if any, effect on physeal development, particularly when
compared with the major detrimental effects of epiphyseal circulatory
compromise.

The effects of physeal ischemia have been studied extensively by Trueta and coworkers.²⁰⁹,²¹¹,²¹²
Disrupting the epiphyseal circulation leads to either partial or
complete cessation of growth. The central region seems more sensitive
to ischemia than the periphery, which may have a variable capacity to
recover through continued latitudinal growth.¹²⁸,¹³⁸ Ischemic compromise leads to different rates of growth across the affected physis and significant changes in physeal contour.¹⁹ Some changes may be caused by venous stasis rather than arterial damage.⁸⁶

The metaphysis is a variably contoured flare at each end
of the diaphysis. Its major characteristics are decreased thickness of
the cortical bone and increased trabecular bone in the secondary
spongiosa. Extensive endochondral modeling centrally and peripherally
initially forms the primary spongiosa, which then is remodeled into the
more mature secondary spongiosa, a process that involves osteoclastic
and osteoblastic activity. Therefore, the metaphyses exhibit
considerable bone turnover compared with other regions of the bone, and
this factor is responsible for the increased uptake of radionuclides in
technetium 99m bone scans.⁸⁰

The metaphyseal cortex also changes with time. Compared
with the confluent diaphysis, the metaphyseal cortex is thinner and is
more porous (trabecular fenestration; Fig. 2-8).
These cortical fenestrations contain fibrovascular soft-tissue elements
that connect the metaphyseal marrow spaces with the subperiosteal
region. The metaphyseal cortex exhibits greater fenestration near the
physis than in the diaphysis, with which it gradually blends as an
increasingly thicker, dense bone (Fig. 2-9). As
longitudinal growth continues, cortical fenestration becomes a less
dominant feature, and the overall width of the cortex increases,
creating a greater morphologic transition between the juxtaphyseal and
juxtadiaphyseal cortices. The metaphyseal region does not develop
extensive secondary and tertiary Haversian systems until the late
stages of skeletal maturation. These microscopic anatomic changes
appear to be directly correlated with changing fracture patterns and
are the reason why torus (buckle) fractures are more likely to occur
than complete metaphyseal or epiphyseal/physeal fractures.

Another microscopic anatomic variation in the metaphysis
occurs at the junction of the primary spongiosa and the hypertrophic
region of the physis. In most rapidly growing bones, the trabeculae
tend to be longitudinally oriented. However, in shorter growing bones,
such as the metacarpals and phalanges,

trabecular
formation is predominantly horizontal. As growth decelerates in
adolescence, a similar horizontal orientation may be seen in the major
long bones. These variations in trabecular orientation affect the
responsiveness of metaphyseal and physeal regions to abnormal stress
and predispose to certain fracture modes.

Although the periosteum is attached relatively loosely
to the diaphysis, it is firmly fixed to the metaphysis because of the
increasingly complex continuity of fibrous tissue through the
metaphyseal fenestrations. Such intermingling of endosteal and
interosseous fibrous tissues with the periosteal tissue imparts
additional biomechanical strength to the region.¹⁹⁸
The periosteum subsequently attaches densely into the peripheral
physis, blending into the zone of Ranvier as well as the epiphyseal
perichondrium. The fenestrated metaphyseal cortex extends to the physis
as the thin osseous ring of Lacroix.

The metaphysis is the site of extensive osseous modeling and remodeling, both peripherally and centrally (Fig. 2-10).
The metaphyseal cortex is fenestrated, modified trabecular bone on
which the periosteum deposits membranous bone to thicken the cortex
progressively. Similar endosteal bone formation occurs. As this
metaphyseal region thickens, the trabecular bone is progressively
invaded by diaphyseal osteon systems, not unlike osteons traversing the
fracture site in primary bone healing. This converts peripheral
trabecular (woven or fiber) bone to lamellar (osteonal) bone, which has
different biomechanical capacities, and thus progressively transforms
metaphyseal cortex into diaphyseal cortex as longitudinal growth
continues. A torus (buckle) fracture is most likely to occur in a
metaphyseal region with a trabecular, fenestrated, compressible cortex.

As in the diaphysis, there are no significant direct muscle attachments to the metaphyseal bone. Instead, muscle fibers

primarily blend into the periosteum. The medial distal femoral
attachment of the adductor muscles is a significant exception. Because
of extensive remodeling and insertion of muscle and tendon in this
area, the bone often appears irregular and may be misinterpreted as
showing chronic trauma (i.e., a stress fracture), infection, or a tumor.

The diaphysis constitutes the major portion of each long
bone. It is principally a product of periosteal, membranous osseous
tissue apposition on the original endochondral model. This leads to the
gradual replacement of the endochondrally derived primary ossification
center and primary spongiosa; the latter is replaced by secondary
spongiosa in the metaphyseal region. At birth, the diaphysis is
composed of laminar (fetal, woven) bone that characteristically lacks
Haversian systems. The neonatal femoral diaphysis appears to be the
only area exhibiting any significant change from this fetal osseous
state to a more mature bone with osteon systems (lamellar bone) before
birth (Fig. 2-12).

Periosteum-mediated, membranous, appositional bone
formation with concomitant endosteal remodeling leads to enlargement of
the overall diameter of the shaft, variably increased width of the
diaphyseal cortices, and formation of the marrow cavity. Mature,
lamellar bone with intrinsic but constantly remodeling osteonal
patterns progressively becomes the dominant feature (Fig. 2-13).

The developing diaphyseal bone in a neonate or young
child is extremely vascular. When analyzed in cross section, it appears
much less dense than the maturing bone of older children, adolescents,
and adults. Subsequent growth leads to increased complexity of the
Haversian (osteonal) systems and the formation of increasing amounts of
extracellular matrix, causing a relative decrease in cross-sectional
porosity and an increase in hardness, factors that constantly change
the child’s susceptibility to different fracture patterns. Certain
bones, especially the tibia, exhibit a significant decrease in
vascularity as the bone matures; this factor affects the rate of
healing and risk of nonunion.

The vascularity of the developing skeleton constantly
changes. In experimental studies, significant chronobiologic changes in
flow patterns were found in the developing canine tibia and femur.¹⁰²,¹⁰³,¹⁰⁴,¹²¹,¹⁸⁷,¹⁸⁸,²²² In particular, there was a dramatic decrease in tibial circulation with increasing skeletal maturation.¹⁸⁸
This also occurs in humans, which helps to explain the increasing delay
in fracture healing and the increased incidence of nonunion of the
tibia in adolescents and adults. A poor vascular response could impair
the early, crucial stages of callus formation.

Other researchers have suggested that adequate vascularity was a major factor in fracture healing,¹⁰⁷,¹⁷⁷,¹⁷⁸,²¹³,²²¹,²²⁷ but they did not consider chronobiologic changes in blood flow patterns.

A child’s periosteum is thicker, is more readily
elevated from the diaphyseal and metaphyseal bone, and exhibits greater
osteogenic

potential than that of an adult.¹³⁹
The periosteum is loosely attached to much of the shaft of the bone,
but it attaches densely into the physeal periphery (the zone of
Ranvier; Fig. 2-14) through intricate collagen meshwork, thereby playing a role in fracture mechanics and treatment of growth mechanism injuries.¹⁹⁸
The thicker, stronger, more biologically active periosteum affects
fracture displacement, reduction, and the rate of subperiosteal callus
formation. It also may serve as an effective internal restraint in
closed reductions.

Because of its contiguity with the underlying bone, the
periosteum is usually injured to some extent in all fractures in
children. However, because the periosteum more easily separates from
the bone in children, there is much less likelihood of complete
circumferential rupture. A significant portion of the periosteum
usually remains intact on the concave (compression) side of an injury.
This intact periosteal hinge or sleeve may lessen the extent of
displacement of the fracture fragments, and it also can be used to
assist in the reduction, because the intact portion contributes to the
intrinsic stability. Because the periosteum allows some tissue
continuity across the fracture, the subperiosteal new bone that forms
quickly bridges the fracture gap and leads to more rapid long-term
stability. The periosteum may be specifically damaged, with or without
concomitant injury to the contiguous bone. Such avulsion injuries may
lead to the formation of ectopic bone.¹⁴⁷
In contrast, severe disruption of the periosteum, as in an open injury,
may impair the fracture healing response. Complete loss of a bone
segment, with the periosteal sleeve reasonably intact, may be followed
by complete reformation of the missing bone.¹⁵

Histologically, periosteum comprises two tissue layers.
While the outer fibroblast layer provides fibrous attachment to
subcutaneous connective tissue, muscles, tendons, and ligaments, the
inner cambium layer contains a pool of undifferentiated mesenchymal
cells that support bone formation and repair.¹⁹³
During embryonic and postnatal bone growth, mesenchymal osteoprogenitor
cells at the inner layer differentiate directly into bone-forming cells
(osteoblasts) and form

periosteal bone collar by the intramembranous method.¹⁶⁴
Formation of new periosteal bone keeps pace with formation of new
endochondral bone. During fracture healing, the mesenchymal cells at
the cambium layer undergo both intramembranous ossification and
chondrogenic differentiation with subsequent endochondral ossification.¹⁷⁰
Due to the osteochondrogenic potential of these cells from the inner
periosteal layer, there has been a lot of interest surrounding the use
periosteum as graft tissues or as sources of osteochondroprogenitor
cells for repairing cartilage/bone defects or for tissue engineering.¹³⁰,¹³⁴,¹³⁶,¹⁹⁹,²²⁶

The periosteum, rather than the bone itself, serves as
the origin for most muscle fibers along the metaphysis and diaphysis.
This mechanism allows coordinated growth of bone and muscle units; this
would be impossible if all the muscle tissue attached directly to the
developing bone or cartilage. Exceptions include the attachment of
muscle fibers near the linea aspera and into the medial distal femoral
metaphysis. The latter pattern of direct metaphyseal osseous attachment
may be associated with significant irregularity of cortical and
trabecular bone. Radiographs of this area often are misinterpreted as
showing a neoplastic, osteomyelitic, or traumatic response, even though
they exhibit only a variation of skeletal development.

Because of the differing histologic composition of the tibial tuberosity (fibrocartilage instead of columnar cartilage; Fig. 2-15),
failure patterns differ from those in other physes. This area develops
primarily as a tensile-responsive structure (i.e., an apophysis).
However, the introduction of an osseous secondary ossification center,
initially in the distal tuberosity, interposes osseous tissue, which
tends to fail in tension and thus may lead to avulsion of part of this
ossification center (Fig. 2-16). Healing of the
displaced fragment to the underlying undisplaced secondary center
creates the symptomatic reactive overgrowth known as an
Osgood-Schlatter lesion.¹⁴⁶,¹⁵¹ Similarly, in adolescents, excessive tensile stress may avulse the entire tuberosity during the late stages of closure.¹⁵²

Endochondral ossification is the process by which bone
forms via a cartilaginous intermediate. The physis (or the growth
plate) best reflects this process. Physes are temporary cartilaginous
tissue situated between the primary and secondary ossification centers
of all long bones. From 9 to 10 weeks’ gestational age to skeletal
maturity at 15 to 17 years, they are responsible for the longitudinal
growth of bone. The physis can be divided into at least three zones.
The reserve or resting zone is situated on the epiphyseal side and
contains small, spherical cells in groups of two or three cells
randomly distributed throughout the zone. These cells are stem
cell-like, responsible for generating new chondrocytes of the physis.
In the adjacent proliferative zone, chondrocytes undergo mitosis and
are organized into columns running parallel to the axis of bone growth.
Cells in the proliferative zone mature and eventually increase to five
to ten times their volume in the hypertrophic region. Matrix vesicles
are also deposited within the longitudinal septa of the physis. Matrix
vesicles are membraneencapsulated structures that are thought to
concentrate calcium and phosphate. Enzymes such as alkaline phosphatase
convert organic phosphates to inorganic phosphates. The longitudinal
septum around the terminal hypertrophic chondrocytes mineralizes, and
this mineralized matrix forms the template for new bone deposition in
the metaphysis (Fig. 2-20).

Associated with these changes in cellular arrangement
and volume, the matrix in the physis also undergoes a continual
modification in content. The two major macromolecules of cartilage
matrix produced by the chondrocytes are the proteoglycans
(predominantly aggrecan and with lesser amounts of decorin, biglycan,
and fibromodulin) and the collagens (types II, IX, X, and XI). The
major change in physeal proteoglycan structure occurs as chondrocytes
organize into columns in the proliferative zone. Additional variation
occurs in the hypertrophic region, where the glycosaminoglycan
sulfation pattern demonstrates

differences
between the pericellular and extracellular spaces and the appearance of
a unique collagen (type X) is observed. The small
proteoglycans—decorin, biglycan, and fibromodulin —are also
differentially expressed across the physis, although detailed studies
of these proteoglycans have not been done (see Table 2-1).

The normal process of endochondral bone formation is
tightly regulated by endocrine/paracrine/autocrine factors, such as
hormones, vitamins, transcriptional factors, and growth factors, and
involves coordinated and sequential expression of growthregulatory
factors. Although growth hormone (GH) in the circulation has a global
effect on physeal function throughout the body, many locally produced
growth factors act locally. Many hormones (such as GH, thyroid hormone,
estrogen, glucocorticoids, calcitonin), vitamins (vitamin D3,
ascorbate, retinoic acid), morphorgens (Indian hedgehog [IHH], BMPs),
growth factors (IGFs, BMPs, FGFs, parathyroid hormone-related peptide
[PTHrP], PDGF, TGF-β, VEGF) and their binding proteins (such as IGFBPs, chordin, noggin), and cytokines (tumor necrosis factor-α,
interleukin IL-1, and others) have now been shown to have important
roles in regulating various processes of the endochondral ossification (Fig. 2-21).⁵
Cellular response is determined by parallel processing of the
intracellular signals that are induced by a number of active growth
factors binding

to
their specific receptors. Presented below is an outline of the likely
actions of a number of key growth factors on endochondral ossification.

It is well known that growth hormone (GH) and IGF-1 are
two major factors regulating postnatal growth. In skeletal tissues,
both chondrocytes and osteoblasts synthesize IGF-1, and GH modulates
its synthesis in both cell populations. According to the original
somatomedin hypothesis, GH stimulates skeletal growth through IGF-1
that is produced in the liver under the influence of GH and secreted
into the circulation.³⁹ Upon
reaching target tissues, IGF-1 interacts with its receptors and induces
a cellular growth signal. However, normal postnatal growth achieved in
liver-IGF-1 null mice (in which hepatic IGF-1 expression was abolished
specifically) suggests the importance of extrahepatic IGF-1 expression
and an autocrine-paracrine role for IGF-1 in normal skeletal growth.¹⁹⁶
Therefore, both circulating and locally expressed IGF-1 play important
roles in longitudinal bone growth and the maintenance of bone mass, and
IGF-1 plays an essential role in longitudinal bone growth in response
to GH exposure.¹⁹⁵

GH controls stem cell maturation and this action is at
least partially mediated via local production and action of IGF-1 at
the stem cell zone.¹⁷⁵ GH receptor has been localized in the physeal chondrocytes particularly in the proliferative zone,⁶,⁴¹,²²³ and it is generally accepted that GH acts at both the stem and proliferative phases of chondrocyte differentiation.⁷⁵
At present, apart from the IGF-1-mediated effects on the stem cells of
the physis, GH also stimulates proliferation of the prechondrocytes in
the resting zone¹⁵³ and has some
priming effect on these stem cells to promote their differentiation
independently of IGF-1, as proposed by the dual effector theory. In
addition, there is genetic evidence that supports this dual,
IGF-1-independent and IGF-1-dependent roles for GH in promoting
longitudinal bone growth.²²⁰

Apart from IGF-1 and GH, BMP-2 and BMP-7 also promote
proliferation, differentiation, and matrix synthesis in
undifferentiated chondrocytes in the resting zone.⁴⁵,⁹⁰
It is believed that once the chondrocytes start differentiating, the
expression of noggin inhibits the continual outgrowth of the
undifferentiated chondrocytes.¹⁸ Once the chondrocyte has lost its resting phenotype, IGF-1 may act as a stimulator of proliferation and differentiation.¹³⁵,²⁰³ Epidermal growth factor (EGF) can augment IGF stimulation by increasing the expression of the IGF-1 receptor.¹³ Although the chondrocytes synthesize large quantities of matrix molecules, they also synthesize FGF-1, FGF-2, TGF-β, VEGF, and a number of the BMPs.¹⁵,²⁶,³²
These molecules can act in an autocrine or paracrine manner, but many
are sequestered into the newly formed cartilage matrix. While FGF-2 in
low doses is mitogenic for the chondrocytes,¹¹⁰
as occurs in achondroplasia, constant activation of FGF receptor
(FGFR3) inhibits chondrocyte proliferation and accelerates terminal
differentiation of chondrocytes.⁹³,⁹⁹,¹¹¹
FGF/heparin sulfate interaction is probable in the differentiation of
the physeal chondrocytes because the continuous exposure of FGF-2
inhibits chondrocyte differentiation in vitro and inhibitors of
glycosaminoglycan sulfation (including heparin sulfate) restore the
differentiation process. Additional sulfate permits glycosaminoglycan
sulfation and returns the effect of FGF-2.³³

Parathyroid hormone (PTH) and parathyroid
hormone-related protein (PTHrP) act to maintain the proliferative state
of and inhibit the maturation of chondrocytes. It is postulated that
two negative feedback loops involving actions of PTHrP regulate the
pace of chondrocyte differentiation in the postnatal growth plate.²¹⁵
The first loop is confined to the proliferativehypertrophic transition
zone and early hypertrophic chondrocytes, which express morphogen
Indian hedgehog (IHH), its receptor Patched, and PTH/PTHrP receptor.
IHH, which stimulates chondrocyte proliferation and inhibits
hypertrophic differentiation, binds Patched in the hypertrophic zone
resulting in a stimulated production of PTHrP. PTHrP then acts on its
receptor at the proliferating chondrocytes to keep them proliferating
and, thereby to delay the production of IHH, thus closing the IHH-PTHrP
feedback loop (Fig. 2-22). In the second
feedback loop, IHH can bind Patched in the resting stem cell zone, and
this may stimulate PTHrP production, which then diffuses to its
receptor leading to IHH down-regulation. These two IHH-PTHrP signaling
cascade feedback loops limit maturation of proliferative chondrocytes
to hypertrophic form.⁹⁴,²¹⁵,²¹⁷

Studies have shown that several signaling pathways
regulating chondrocyte proliferation and hypertrophic differentiation
interact with the IHH-PTHrP feedback loops (Fig. 2-22).
Apart from inhibiting chondrocyte proliferation, part of the effects of
FGF signaling in positively affecting chondrocyte maturation is
mediated by the suppression of IHH expression.¹²⁵ However, FGF effects on chondrocytes can occur independently of PTHrP/IHH action.¹⁸⁵ In the physis, expression of the mRNA for BMP-2 and BMP-6 peaks in hypertrophic chondrocytes before mineralization.²⁶
Studies have demonstrated that BMPs act on chondrocytes to induce
proliferation through the induction of IHH expression by
prehypertrophic chondrocytes, suggesting that BMP signaling modulates
the IHH/PTHrP signaling pathway that regulates the rate of chondrocyte
differentiation.⁹³,²³⁴ In addition, in vitro studies have suggested that during embryonic development, signaling of TGF-β2 may act as a critical

signal relay between IHH and PTHrP. It mediates the effects of IHH
inhibiting hypertrophic differentiation and induces PTHrP expression,
maintaining the chondrocytes in proliferative state and slowing down
the pace of their maturation.²

Although the chondrocytes of the physis will proliferate
and form a cartilaginous matrix with only the epiphyseal vascular
supply, the metaphyseal vessels are critical for the mineralization
process.²¹² Metaphyseal vascular
invasion occurs at the hypertrophic-metaphyseal interface. The
endothelial cells invade most likely as a consequence of angiogenic
factors present in the matrix or secreted by the chondrocytes
themselves. VEGF, TGF-β, and FGFs are known
to be present in the physeal cartilage matrix and are angiogenic. It is
interesting that an oversupply of FGF-2 infused into the physis induces
vascular invasion from the metaphysis only; even if the FGF-2 is
present at the epiphyseal side of the physis, the epiphyseal vessel
will not invade.⁷ Apart from
providing the necessary nutrients for the mineralization process, the
metaphyseal vessels also bring in osteoblasts, osteoclasts, and other
cell types. The osteoclasts degrade the mineralized cartilage matrix
while osteoblasts lay down new bone that is also rich in growth factors
such as TGF-β, FGF-2, IGF-1, and BMPs.

All axial and appendicular skeletal elements are
involved in secondary membranous ossification. The diaphyseal cortex of
developing tubular bone is progressively formed (modeled) by the
periosteum and modified (remodeled) by the re-formation of osteons.
This peripheral periosteal process of membrane-derived ossification is
extensive and rapid in fracture healing in infants and young children.
The replacement process also may be seen when portions of the
developing metaphysis or diaphysis are removed for use as bone grafts.

Intramembranous ossification occurs when osteoprogenitor
cells are formed from the overlying tissue, the inner cambium layer of
the periosteum¹⁹³ (see Periosteum
section earlier). The osteoprogenitor cells continue to differentiate
into osteoblasts, which produce and add new bone matrix peripherally
that later undergoes mineralization.

The first bone to be laid down either from the physis at
the metaphysis or in the fracture callus is woven bone, which is
remodeled to lamellar bone. At the metaphysis, the trabeculae of
bone-covered calcified cartilage (primary spongiosa) are resorbed by
osteoclasts and the calcified cartilage template is replaced by
lamellar bone and remodeled into more mature bone trabeculae (secondary
spongiosa). The deposited secondary bone trabeculae at the
metaphyseal-diaphyseal junction is further remodeled and incorporated
into the diaphysis, in a process in which osteoclasts remove bone from
the periphery of the metaphysis and new bone is formed at the endosteal
surfaces. Although cancellous bone can be remodeled and obtain its
nutrients from the surface, cortical bone is remodeled into a complex
structure of osteons that together form the cortical bone. Osteons are
tubular structures that interconnect. They consist of layers of ordered
lamellar bone around a central canal. The central canal contains blood
vessels, lymphatics, and in some cases, nerves.²⁰

Bone is constantly remodeled by osteoclasts and
osteoblasts. The bone is encapsulated by bone-lining cells that have
the potential to become activated osteoblasts. The bone-lining cells
have slender cellular processes that make contact with the osteocytes
within the mineralized bone. Osteocytes are thought to arise from
osteoblasts that have become entrapped during bone formation. It has
been proposed that the bone-lining cells need to erode the osteoid that
covers the underling bone for osteoclasts to bind.¹²²,¹²³
Osteoclasts are bone-degrading cells that are produced from the
hematopoietic pathway. Upon activation, they bind to the surface of the
bone and secrete enzymes into the space beneath. The space is acidic
and contains many proteolytic and bone degrading enzymes.¹¹⁴ The acidic pH and proteases are thought to release and activate the sequestered growth factors IGF-1 and TGF-β,
resulting in the recruitment, proliferation, differentiation, and
activation of the stromal osteoprogenitor cells and bone-lining cells
to become active osteoblasts, synthesizing bone matrix and increasing
their survival (Fig. 2-23).⁴⁶,⁴⁷,¹¹⁶,¹¹⁸ The newly laid osteoid by osteoblasts is subsequently mineralized to become bone.

It is now generally accepted that the osteoblast
activity and osteoclast activity are linked during the bone remodeling.
On the one hand, osteoclastic activity releases growth factors IGF-1
and TGF-β stimulating osteoblastic differentiation and activity as described above (Fig. 2-23),
and on the other, cells of the osteoblast lineage provide factors
essential for the differentiation of osteoclasts. The discovery of the
interaction between the receptor activator of NF-kappaB (RANK) ligand
(RANKL) expressed by osteoblasts and its receptor RANK expressed on
osteoclast precursors confirms the well-known hypothesis that
osteoblasts play an essential role in osteoclast differentiation (Fig. 2-23).²⁰¹
It is now known that two hematopoietic factors, namely RANKL and
macrophage colony-stimulating factor (M-CSF), together are necessary
and sufficient for osteoclast formation.¹⁶
Several factors have now been identified that can modulate RANK-induced
osteoclastogenesis. Although vitamin D3 metabolite, PTH, PTHrP, PGE2,
cytokines IL-1, IL-6, TNF, LIF, and IL-11, and corticosteroids have
been shown to induce the expression of RANKL in stromal/osteoblastic
cells and thus stimulate osteoclast formation, osteoprotegerin (OPG), a
decoy receptor for RANKL, blocks the RANKL-RANK interaction and
inhibits osteoclastogenesis.¹⁶,¹¹² Recent studies have also shown that lipopolysaccharide and inflammatory cytokines such as TNF-α
and IL-1 can also directly regulate osteoclast differentiation and
function through a mechanism independent of the RANKL-RANK interaction.⁹² TGF-β superfamily members and interferon-gamma (INF-γ) are also shown to be important regulators in osteoclastogenesis.⁸⁴ Estrogens, calcitonin, BMP2/4, TGF-β, IL-17, PDGF, and calcium have been shown to be anabolic or inhibit osteoclastogenesis¹⁶ (Fig. 2-21). In addition, EGF receptor signaling has been shown to be important for the secretion of matrix metalloproteases (MMPs)¹²⁴ and maintenance of osteoclast activity,²⁹ both of which are important for the bone remodeling.

The osteocytes may serve as the mechanosensory system
that may be associated with bone remodeling. Osteocytes also possess
cellular processes that connect osteocytes to one another and to the
bone-lining cells above.³⁶ It is
possible that the osteocytes are responsible for sensing bone stress;
if undue stress is detected, they favor bone deposition, whereas if a
lack of stress is detected, they favor bone resorption.

The progressive changes of the normal process of osseous
fracture healing, whether in the diaphysis, metaphysis, or epiphyseal
ossification center, can be grouped conveniently into a series of
phases that occur in a reasonably chronologic sequence.¹²⁰,¹⁸⁰ Several factors that influence bone healing have been identified from clinical observation as well as experimental work¹⁶⁰,²¹⁴
and must be taken into account when treating childhood fractures on a
rational basis. However, certain areas of the developing skeleton,
particularly the physis and epiphyseal hyaline cartilage, probably do
not heal by classic callus formation. In fact, when this type of
osseous (callus) repair occurs in these cartilaginous regions,
significant growth deformities may result due to formation of an
osseous bridge between the secondary ossification center and the
metaphysis (see “Physeal Healing Patterns”).

As in adults, there are three basic mechanisms of
fracture repair: primary osteonal, secondary osteonal, and nonosteonal.
Primary osteonal fracture healing occurs when cortical bone is laid
down without any intermediate, and therefore hardly any callus forms;
it is only possible if cortical bone is repositioned and fixed in close
proximity. Secondary osteonal union occurs if cortical bone is laid
down between two segments of fractured cortical bone before callus
formation. Nonosteonal union occurs through endosteal and periosteal
callus formation.¹⁸³

Fracture repair by callus production in the immature
skeleton can be divided into three closely integrated, but sequential,
phases: the inflammatory phase, the reparative phase, and the
remodeling phase (Fig. 2-24). In children, the
remodeling phase is temporally much more extensive and physiologically
more active (depending on the child’s age) than the comparable phase in
adults. The remodeling phase is further modified by the effects of the
physis responding to changing joint reaction forces and biologic
stresses to alter angular growth dynamics. This occurs even when the
fracture is mid-diaphyseal.

The physis has a limited ability to repair; it primarily
heals by increased endochondral bone and cartilage formation, and
gradual reinvasion by the disrupted metaphyseal vessels to replace the
temporarily widened physis eventually. Very little experimental work,
mostly in rats, has been directed at assessing the post-traumatic
cellular response patterns of the physis.¹⁷,⁹⁷,²²⁸

Depending on the level of cellular injury within the physis, three types of chondro-osseous healing
may occur. First, when the fracture occurs through the cell columns,
healing occurs primarily by continued, relatively rapid increases in
the number of cells within the columns, causing moderate widening of
the physis. Because there are some small epiphyseal vessels in this
region, some damaged tissue may be resorbed early in the healing
process. These vessels also exhibit a hyperemic response, increasing
cellular proliferation rates, especially in the peripheral zone of
Ranvier. The metaphyseal response parallels this, in that an increased
rate of bone replacement of the hypertrophic cartilage also occurs.
Once the level of fracture fibrosis and debris within the physis is
encountered, the vessels rapidly invade to reach the rest of the
maturing cell columns. These cellular response patterns lead to
restoration of normal anatomy within 3 to 4 weeks.¹⁷⁹

Second, when the fracture occurs through the transition
of hypertrophic cells to primary spongiosa (the most commonly involved
cellular level), there may be marked separation, with the gap filled by
hemorrhagic and fibroblastic tissue. This region may progressively form
disorganized cartilaginous tissue, which is similar to the initial,
disorganized cartilaginous callus around a diaphyseal fracture.
Meanwhile, cellular proliferation, cell column formation, hypertrophy,
and calcification continue on the epiphyseal side of the disorganized
callus, leading to widening of the physis. Vascular invasion of the
remnants of hypertrophic, calcified cartilage also rapidly occurs on
the metaphyseal side of the fracture. However, once metaphyseal vessel
invasion reaches the disorganized cartilaginous callus,
vascular-mediated bone replacement is temporarily slowed, because there
is no pattern of cell columns to invade in an organized fashion. As the
callus cartilage matures and calcifies, the metaphyseal vessels begin
to invade and replace the cartilage with bone irregularly.²¹
This callus may be variably thick, depending on the degree of
longitudinal and lateral displacement and periosteal continuity with
the physeal periphery. The callus is replaced at different rates, and
the invading metaphyseal vessels reach the normal cell columns, which
have been maturing in a normal sequence but without osseous
replacement. This widened physis is rapidly invaded by the vessels and
replaced by primary spongiosa, and normal physeal width is
progressively restored.

The callus in the subperiosteal region of the metaphysis
contributes to early stability. This region heals by vascular invasion
of the callus to form trabecular bone between the original metaphyseal
cortex and the subperiosteal membranous bone forming continuously
external to the metaphyseal cartilaginous callus. These three
microscopic bone regions progressively merge and remodel, strengthening
the bone. These initial cellular replacement processes in both
metaphyseal and physeal regions probably take 3 to 6 weeks. However,
remodeling may continue for months to years, and it enhances the
capacity for spontaneous correction of many residual deformities.

Third, when the injury extends across all cell layers of
the physis, the repair processes differ slightly. Fibrous tissue
initially fills the gap between separated physeal components, whereas
typical callus formation occurs in the contiguous metaphyseal spongiosa
or epiphyseal ossification center. If large surfaces of nonossified
epiphyseal cartilage also are involved, fibrous tissue initially forms
in the intervening region. The reparative response shows irregular
healing of the epiphyseal and physeal cartilage, with loss of normal
cellular architecture. Within the central physeal regions, diametric
expansion of cell columns is minimal, so closure of a large defect by
physeal cartilage is unlikely. The gap will remain fibrous, but with
the potential to ossify. Toward the physeal periphery, diametric
expansion is more likely, but still may not lead to closure of large
cartilage gaps by progressive replacement of fibrous tissue. This
replacement process essentially requires the germinal and hypertrophic
cell regions to diametrically expand by cell division, maturation, and
matrix expansion. The intervening fibrous tissue may disappear through
growth, but only if the gap is narrow. Because blood supply is minimal
in this region, the fibrous tissue similarly is not well vascularized,
and significant cell modulation, especially to osteoblastic tissue, is
less likely in the short term. However, the larger the gap filled with
fibrous tissue and the longer the time from fracture to skeletal
maturity, the greater the likelihood of developing sufficient
vascularity to commence an osteoblastic response and to form an osseous
bridge. Further, in young children with minimal epiphyseal
ossification, the blood supply to the physeal germinal region is not as
well defined, whereas once the ossification center expands and forms a
subchondral plate over the germinal region, microvascularity probably
increases and the chances for vascularization and ossification of the
fibrous region increase. This explains the delayed appearance of the
osseous bridge.

If accurate anatomic reduction is performed, a thin gap
should be present that should fill in with minimal fibrous tissue,
allowing progressive replacement of the tissue by diametric expansion
of the physis and contiguous epiphysis. However, if the fragment has
been partially or completely devascularized by either the initial
trauma or subsequent dissection to effect an open reduction, cellular
growth and diametric and longitudinal expansion may not occur. This
increases the chances of cellular disorganization, fibrosis, and
eventual osteoblastic response. Failure to correct anatomic
displacement, especially in Salter-Harris type IV growth mechanism
injuries, increases the possibility of apposition of the epiphyseal
ossification center and metaphyseal bone, and thereby enhances the risk
of forming an osseous bridge between the two regions. When the defect
was large enough and the fracture involved the whole width of the
physis extending from the metaphysis to the epiphysis, the injured
physis will have structural disorganization, formation of vertical
septa, and finally formation of a bone bridge. When the bone bridge is
large enough, particularly in Salter-Harris types III and IV injuries,
the defect will result in a growth arrest. While growth arrest at the
peripheral portions of the physis results in angular deformities,
centrally located lesions may cause longitudinal shortening.¹⁴¹

The cellular and molecular mechanisms for the bone
bridge formation at the site of physeal injury site remain largely
unknown. Using a proximal tibial drill-hole transphyseal injury model
in rats, a study from the authors’ laboratory characterized the
injury-induced responses and cellular mechanisms for the

bone bridge formation (Fig. 2-25).²²⁸
At the physeal injury site, this study demonstrated an early acute
inflammatory response (up to day 3 postinjury). Straight after this
inflammatory response, the injury site was filled by mesenchymal
infiltrate (days 3 to 14) and subsequently was repaired by bone bridge
formation (starting from day 7). Histologically, bony bridge trabeculae
appeared on day 7 (Fig. 2-25A) and became well-constructed on day 14 with marrow (Fig. 2-25B). Before and during physeal bar formation, there were no new cartilage formation, no collagen-X synthesis at the injury site (Fig. 2-25C), and no expansion of chondrocyte proliferation from adjacent physeal cartilage (Fig. 2-25D),
suggesting that the bone bridge formation did not involve endochondral
ossification in this rat model. These results are consistent with an
earlier study that reported a lack of increased expression of IHH and
collagen-2, two molecules typically involved in endochondral
ossification.⁹⁸ Furthermore, Xian et al’s²²⁸ study also demonstrated infiltration of marrow-derived fibroblast-like mesenchymal cells starting from day 3 (Fig. 2-25E),
presence of osteoblast precursor cells among the mesenchymal
infiltrates and their close proximity to bone bridge trabeculae (Fig. 2-25F, Fig. 2-25G), and production of bone matrix protein osteocalcin during formation

of bone bridge trabeculae (Fig. 2-25H).
These results suggested that bone bridge formation after physeal injury
in this rat model occurs directly via intramembranous ossification
through recruitment of marrow-derived osteoprogenitor cells.²²⁸

In a growing child, the normal process of bone
remodeling in the diaphysis and metaphysis (particularly the latter)
may realign initially malunited fragments, making anatomic reduction
less important than in a comparable injury in an adult. However,
although some residual angular deformities undergo spontaneous
correction, accurate anatomic reduction should be the goal whenever
possible.⁵⁸,¹⁴²,¹⁴⁹
Bone and cartilage generally remodel in response to normal stresses of
body weight, muscle action, and joint reaction forces, as well as
intrinsic control mechanisms such as the periosteum. The potential for
spontaneous, complete correction is greater if the child is younger,
the fracture site is closer to the physis, and there is relative
alignment of the angulation in the normal plane of motion of the joint.
This is particularly evident in fractures involving hinge joints such
as the knee, ankle, elbow, or wrist, in which corrections are
relatively rapid if the angulation is in the normal plane of motion.
However, spontaneous correction of angular deformities is unlikely in
other directions, such as a cubitus varus deformity following a
supracondylar fracture of the humerus. Similarly, rotational
deformities usually do not correct spontaneously.

Specific growth factors have been targeted for their
ability to promote bone formation. Due to their ability to stimulate
proliferation and differentiation of mesenchymal and osteoprogenitor
cells, two bone morphogenic proteins (BMP-2 and BMP-7) have shown great
promise and acceptance for their ability to promote fracture repair.⁹⁰,⁹¹,¹⁰³,¹⁰⁴,¹¹³,²⁰²,²²⁴
BMP-2 promotes bone formation and repair in critical size defects,
fractures and spinal fusions in human, and has been recently approved
for clinical use in fracture repair (Wyeth Pharmaceutical) and spinal
fusion (Medtronic Sofamor Danek, Memphis, TN). Like BMP-2, BMP-7 (also
called osteogenic protein-1, OP-1)

induces ectopic bone formation in vivo and enhances bone repair in preclinical models and clinical studies.²⁵
Clinically, OP-1, delivered with a type-1 collagen carrier, induced
bone repair, which was found to be equivalent to autogenous bone graft
in a clinical trial of patients with tibial nonunions.⁵⁴ OP-1 has now been approved for use in the treatment of established nonunions (Stryker Biotech).

A number of other growth factors, such as TGF-β, IGF-1, PDGFs, and FGF-2, also may prove to be useful. TGF-β plays a major role in fracture repair by promoting proliferation and differentiation of the mesenchymal cells. Exogenous TGF-β administration can initiate the repair process and callus formation in uninjured bone.⁸² The addition of TGF-β to fractures results in a larger, stronger callus.⁸²
It also may be of use in promoting repair in nonhealing bone defects.
PDGF also increases callus size but does not improve the fracture
mechanically.¹³¹ Growth hormone and
IGF-1 have also been tested to determine their effects on fracture
repair. Although growth hormone produces inconsistent results,³ the administration of IGF-1 increases intramembranous bone formation.²⁰⁵
The FGFs stimulate mitogenesis of mesenchymal cells and osteoblasts,
increase the callus size and mineral content, and improve mechanical
stability at early stages of fracture repair.⁷⁷,⁸⁵,²¹⁸ FGF-2 also increases osteoclastic bone remodeling.¹³¹ In addition, it is possible that the effects of FGFs²²
and of a number of the other growth factors are a result of the
angiogenic properties of such growth factors, as most osteogenic
factors also stimulate angiogenesis, if not directly, then indirectly,
through production of angiogenic molecules, such as VEGF. The potential
synergism between potent proangiogenic factors (such as VEGF) and
strong osteoinductive factors (such as BMPs) suggest that combination
therapies might produce optimal results, particularly for individuals
at risk of delayed repair or nonunions.²⁵

Although there have been many studies and reviews on the use of growth factors for fracture repair²⁵,⁴³,⁴⁴,¹⁰³,¹⁰⁴,¹⁷³,²⁰⁴
and some successful clinical applications of BMP-2 and OP-1 in inducing
bone formation and repair, more research is required to establish the
most effective delivery devices for these growth factors.⁷⁶
Apart from the requirements of biocompatibility and effectiveness, the
growth factor carriers or delivery matrix systems or devices need to
make the growth factor delivery cost-effective and practical in their
clinical applications to induce bone formation in vivo, which should
allow the application of relatively low doses of growth factors for
optimal bone or cartilage regeneration in clinical contexts.

In the past decade, there has been an increasing
interest in tissue engineering and mesenchymal stem cells for bone
reconstruction of nonunion defects and for articular cartilage
regeneration. Using tissue-engineering technologies, it is now possible
to enhance bone repair and/or replace bone defect; and using the
patient’s own articular chondrocytes retrieved during arthroscopy and
expanded in vitro, it is now possible to repair full-thickness
articular cartilage defects with satisfactory clinical results.¹³⁷
Researchers worldwide are working to prepare the three fundamental
components for the successful orthopaedic tissue engineering: (a)
appropriate biological or artificial carriers or delivery systems or
extracellular matrix scaffolds, and (b) the right set of viable
responding cells (such as mesenchymal stem cells) in combination with
(c) appropriate soluble inductive signal molecules or growth factors
that, once transplanted, will ensure bone repair and/or cartilage
regeneration.¹⁷³

In particular, due to the capacity of self expansion in
vitro, the less stringent ethical and regulatory issues and the lack of
immunologic implications, mesenchymal stem cells derived from
autologous bone marrow stroma have been the research focus for their
potential applications, which have offered a new perspective for bone
and cartilage tissue engineering. There have been some excellent
advances in understanding the stem cells, their interactions with their
matrix, particularly stimuli or signal molecules controlling their
proliferation (such as LIF, FGF-2, HGF, Wnt and Dickkopt-1), osteogenic
differentiation (such as IHH, Notch-1 and PPARgamma), and chondrogenic
differentiation (such as BMP-4 and TGF-β3).¹⁵⁷
However, preclinical and clinical evaluations of the stromal stem cells
or their derived osteoblasts or chondrocytes or the engineered bone or
cartilage tissues will yet have to prove their efficacy,
biocompatibility, safety, practicality, and reproducibility in bone and
cartilage regeneration.¹⁰⁸

Genetic engineering or gene transfer technology has also
opened novel treatment avenues for the regeneration or repair of
damaged articular cartilage, as gene transfer provides the capability
to deliver bioactive proteins or gene products to sites of tissue
damage locally and in a sustained manner. Previous research has already
convincingly demonstrated the principle of gene delivery to synovium,
chondrocytes and mesenchymal progenitor cells, and efficacy studies
provide optimism that this gene transfer approach can be used to
enhance articular cartilage repair.²⁰⁶

Biological regeneration of physeal cartilage remains a great challenge. Foster and colleagues⁵³ have used a sheep tibial physeal injury model to investigate therapeutic potential of transplanted chondrocytes or periosteum,²²⁶ and more recently growth factor OP-1.⁸¹
They unfortunately found that these treatments under the experimental
conditions were not preventative of bony bridge formation. Similarly,
in a rat tibial physeal fracture model, Gruber et al⁶³
found that interposed periosteum at the fracture site could not enhance
healing of the fractured physis, and in a rabbit tibial physeal injury
model, Lee et al⁹⁷ observed that
BMP-2 gene therapy delivered via adenoviral vectors within muscle
implant caused increased osteogenic activity in the injured physis.
Previous work has demonstrated that implantation of cultured
chondrocytes embedded in agarose into physeal defects resulted in a
partial correction of angular deformity and a significant reduction in
growth arrest in a rabbit model,⁹⁶ and muscle-based gene therapy with adenoviral vectors encoding for IGF-1 restored the injured physeal cartilage.⁹⁷
More recently, direct transfer of periosteum-derived mesenchymal stem
cells embedded in agarose into the tibial physeal defect resulted in
regeneration of the physis, preventing growth arrest or angular
deformity of the tibia.³¹,¹⁰¹ The search for the optimal treatment options to achieve correction of angular deformity and to prevent

limb length discrepancy using tissue engineering will continue and is not clinically applicable at this time.