Bone repair

(bone fracture – socket healing)

PhD ( spring 2021)

Presented by : Sara Mohamed Abd El Rahman

contents

Introduction to bone healing

Types of bone healing ( direct / indirect bone healing)

Histological events of bone healing

Socket healing.
Introduction

• A fracture is defined as a break in the structural

continuity of bone.
• Bone fractures are a common injury and the healing process is
complex.

• Bone is one of a few tissues that is able to heal without

forming a fibrous scar.

Bone healing refers to complex and sequential events that takes

place to restore injured bone to pre- injury state
 Based on bone histology
there are two types of
bone healing

Indirect (secondary)
Direct (primary) healing
healing

(without callus formation)

(through callus formation)
Primary (direct ) bone healing

• In order for primary bone repair to occur, the

fracture fragments must be reduced to anatomic
position. (surgeon closely reapproximates the
fractured ends of a bone)

• This is usually accomplished through rigid internal

fixation. (It does not commonly occur in the natural
process of fracture healing)

• Direct bone healing can occur by direct remodeling

of lamellar bone, the Haversian canals and blood
vessels without callus formation.
 Depending on the species, it
usually takes from a few
months to a few years, before
complete healing is achieved.
Direct bone
healing Primary healing of fractures
can either occur through
contact healing or gap
healing.
Contact healing
• When the gap between the
bone ends is less than 0.01 mm.
• Osteoclasts form across the
fracture lines, generating cavities
at a rate of 50–100 μm/day.
• Osteoblasts fill up the cavities
with the Haversian system.
• This causes the formation of
lamellar bone parallel to the
Gap healing
• If the fracture gap is 800 μm to 1
mm.
• The fracture is filled by osteoclasts
and then by lamellar bone oriented
perpendicular to the axis of the
bone.
• Perpendicular orientation of
lamellar bone is weak, thus a
secondary osteonal reconstruction
is required to re-orient the lamellar
bone longitudinally
Secondary (Indirect) bone healing
• Fractures left untreated or those that are treated
with external or by sling or cast immobilization heal
by secondary bone repair without rigid fixation.

• Indirect (secondary) fracture healing is the most

common form of fracture healing, consists of both
endochondral and intramembranous bone healing.

• Indirect bone healing depends on callus formation

 1) Haematoma and inflammatory phase

Stages of 2) Recruitment of mesenchymal stem cells ( MSCs).

secondary 3) formation of a Cartilaginous and a Periosteal Bony

Callus

bone 4) Revascularization and Neoangiogenesis at the

Fracture Site.

healing 5) Resorption of the Cartilaginous Callus and

replacement by hard callus (woven bone)

6) Bone remodeling
• 1) Haematoma and inflammatory phase

-This stage begins immediately following the fracture

-The blood vessels supplying the bone and periosteum
are ruptured during the fracture, causing a hematoma
to form around the fracture site.

>> hematoma forms at the site of fracture serves to

immobilize the area (via a fibrin network), and also
provides a source of signaling molecules that initiate
cellular events necessary for fracture repair.
- Highly regulated secretion of proinflammatory molecules following the injury is
critical for tissue regeneration.
- The initial proinflammatory response involves secretion of tumor necrosis factor-α
(TNF-α), interleukin-1 (IL-1), IL-6, IL-11 and IL-18.

- These factors recruit inflammatory cells (attracting macrophages, monocytes, and

lymphocytes).

- These cells act together to remove damaged, necrotic tissue and secrete cytokines
like vascular endothelial growth factor (VEGF) to stimulate healing at the site.

- The acute inflammatory response peaks within the first 24h and is complete after 7
days. (1-7 days)
• 2) Recruitment of mesenchymal stem cells ( MSCs).

-In order for bone to regenerate, specific mesenchymal stem cells (MSCs)
have to be recruited.

- Mesenchymal stem cells recruited to the area begin to differentiate to

fibroblasts, chondroblasts, and osteoblasts.

Exactly where these cells come from is not fully understood. Although most
data indicate that these MSCs are derived from surrounding soft tissues
(periosteum) and bone marrow, recent data demonstrate that a systemic
recruitment of circulating MSCs to the injured site might be of great
importance for an optimal healing response.
• Which molecular events mediate this recruitment is still under debate.
>> It has long been suggested that BMP-2 has an important role in this
recruitment.

>> Current data suggest that stromal cell-derived factor-1(SDF-1) is a key

regulator of recruiting and homing specific MSCs to the site of trauma.

SDF1 is released by the injured periosteum and drives mobilization and homing
of MSCs
• 3) formation of a Cartilaginous and a Periosteal Bony Callus

Although indirect fracture healing consists of both intramembranous and

endochondral ossification, the formation of a cartilaginous callus which
later undergoes mineralization, resorption and is then replaced with bone is
its key feature of this process.
• Following the formation of the primary hematoma, a fibrin-rich
granulation tissue forms. Within this tissue, endochondral
formation occurs in between the fracture ends >> (internal
callus) .

• At the same time, adjacent to the periosteal layers, a layer of

woven bone is laid down by the osteoprogenitor cells
(intramembranous ossification) >> (External callus)
• In this process the transforming growth factor-beta (TGF-β)
superfamily members have been shown to be of great
importance. TGF-β2 and-β3 are involved in chondrogenesis and
endochondral ossification.

• whereas BMP-5 and -6 have been suggested to induce cell

proliferation in intramembranous ossification at periosteal
sites.
• 4) Revascularization and Neoangiogenesis at the Fracture Site.

>> Fracture healing requires a blood supply and revascularization is

essential for successful bone repair.

The vascularization process is mainly regulated by two molecular

pathways, an angiopoietin-dependent pathway, and a vascular
endothelial growth factor (VEGF)-dependent pathway.
 Angiopoietins pathway
• The angiopoietins, primarily
angiopoetin-1 and 2, are vascular
morphogenetic proteins.
• Their expression is induced early in
the healing cascade, suggesting that
they promote an initial vascular in-
growth from existing vessels in the
periosteum.
VEGF pathway
• The VEGF pathway is considered
to be the key regulator of
vascular regeneration.
• VEGF promotes both :
• 1) Vasculogenesis, (de novo
formation of blood vessels) i.e.
aggregation and proliferation of
endothelial mesenchymal stem
cells into a vascular plexus
• 2) Angiogenesis, i.e. growth of
new vessels from already
existing ones.
• 5) Resorption of the soft Cartilaginous Callus and replacement by hard
callus (woven bone) :

• In order for bone regeneration to progress, the primary soft

cartilaginous callus needs to be resorbed and replaced by a hard
bony callus.

• A cascade orchestrated by macrophage colony-stimulating factor

(M-CSF), RANKL, osteoprotegerin (OPG) and TNF-α initiates the
resorption of this mineralized cartilage
• During this process M-CSF, RANKL and OPG are also thought to
help recruit bone cells to form woven bone.

• TNF-α further promotes the recruitment MSC with osteogenic

potential but its most important role may be to initiate
chondrocyte apoptosis.

• As the hard callus formation progresses and the calcified

cartilage is replaced with woven bone, the callus becomes
more solid and mechanically rigid.
• 6) Bone remodeling

• Although the hard callus provides biomechanical stability, it does

not fully restore the biomechanical properties of normal bone.

• In order to achieve this, the fracture healing cascade initiates a

second resorptive phase, this time to remodel the hard callus
into a lamellar bone structure with a central medullary cavity.
• The remodeling process is carried out by a balance of hard
callus resorption by osteoclasts, and lamellar bone deposition
by osteoblasts.

• Eventually, the fracture callus is remodeled into a new shape

which closely duplicates the bone's original shape and strength.

• Although the process is initiated as early as 3–4 weeks in animal

and human models, the remodeling may take years to be
completed to achieve a fully regenerated bone structure.
Histological stages of bone healing. Histological progression of
bone healing in rats. Representative photomicrographs showing
the least to the most advanced stages of bone healing from d 4
(A), 11 (B), and 18 (C), with regions of hematoma (HE),
granulation tissue (GT), IM woven bone (IWB), and cortical
bone (CB) labeled. Sections were stained with H&E, and images
were acquired at 10 magnification
Schematic diagram of callus
fracture repair in endochondral
bone.
Factors affecting bone healing

• Multiple factors affect fracture healing, which can broadly categorize into local
and systemic categories

• Local factors:

• Fracture characteristics - excessive movement, misalignment, extensive damage

and soft tissues caught within fracture ends can lead to delayed or non-union
• Infection - it can lead to poor healing and delayed or non-union.
• Blood supply - reduced blood supply to the fracture site can lead to delayed or
non-union
• Systemic factors (the presence of any of these factors predisposes to poor healing):

• Advanced age
• Obesity
• Anemia
• Endocrine conditions - Diabetes mellitus, Parathyroid disease, and Menopause
• Steroid administration
• Malnutrition
• Smoking
 Socket healing
• One of the most common oral wounds is an
extraction socket after tooth removal.

• The socket-healing process may be divided into

three consecutive, and frequently over-lapping,
phases

Inflammatory

Proliferative

Modelling / Remodelling
 Inflammatory phase
• The inflammatory phase may be subdivided into
two parts:
• 1) Blood clot formation
• 2) Inflammatory cell migration (wound cleansing)

• Blood clot formation >>

• Immediately after tooth extraction, hemorrhage
takes place and the socket is filled with blood.
• Both intrinsic and extrinsic pathways of the clotting
cascade are activated to produce blood clot .
• The blood clot plugs the injured vessels to stop
bleeding.
Histologic view of the extraction sockets in rat at day 0
after tooth extraction (H&E, ×40) . BC: blood
coagulum, RP: remaining periodontium.
• Inflammatory cell migration and wound cleansing>>

• Within 2–3 days, huge numbers of inflammatory cells

(neutrophils and macrophages) migrate to the wound
in order to “clean” the site before new tissue can start
forming.

• This migration occurs according to an established

chemokine concentration gradient - mainly governed
by complement system molecules C3a and C5a, TGF- β
and bacteria released products.

• Neutrophils enroll an active phagocytosis, removing Transmission electron

the clot structure, bacteria and eventual foreign microscopy (TEM) of
material, through the release of free radical species a human neutrophil.
and proteolytic enzymes.
• Macrophages are following recruited,
from circulating monocytes that
experience phenotypic maturation,
being responsible for the continuation
of phagocytosis and further providing
the release of effective growth factors
- Transforming growth factor-alfa
- Transforming growth factor-beta
- Fibroblast growth factor
- Epidermal growth factor
Macrophage. Transmission electron micrograph
( that activate subsequently recruited (TEM)
fibroblasts and osteoblasts .)
• The combination of inflammatory cells, vascular buds and immature
fibroblasts forms

Granulation tissue
Proliferative phase

• The proliferative phase may also be subdivided into two parts:

• 1)Fibroplasia
• 2) Woven bone formation

• This phase is characterized by intense and rapid tissue formation.

• Fibroplasia >>

After the site becomes sterilized, the granulation tissue is gradually replaced with a
provisional connective tissue matrix that is rich in collagen fibers and cells.

At this time, there is an intense fibroblast migration and proliferation, as well as an

increase in collagen synthesis and other extracellular matrix proteins (TGF-β1 and FGF-
2 seem to modulate activation and proliferation of fibroblasts)

Subsequently, the provisional matrix is penetrated by several new vessels and bone
forming cells.

The newly formed vessels provide the oxygen and nutrients that are needed for
increasing the number of cells that are essential in the new tissue formation.
•This microscopic picture shows a healing socket after one week.
Large amounts of provisional matrix (PM) are present within the
buccal (B) and lingual (L) wall of the sockets. And, in the center
of the socket, a residual blood clot (C) is still present.
• Woven bone formation >>
• The transition of the provisional connective tissue into bone tissue occurs along
the vascular structures.

• The mesenchymal stem cells migrate and differentiate into osteoblasts that
produce a matrix of collagen fibers which takes on a woven pattern.

• The process of mineralization is initiated within the osteoid. The osteoblasts

continue to lay down osteoid and occasionally such cells are trapped in the
matrix and become osteocytes.

• The newly formed bone is called woven bone which is the first type of bone to
be formed and is characterized but its rapid deposition as a finger like projections
along the vessels.
Eventually, the projections completely surround a vessel and the primary
osteon is formed.

Woven bone can be detected in the healing socket as early as 2 weeks

after tooth extraction and remains for several weeks.

Woven bone is a provisional type of bone without any load bearing

capacity and therefore needs to be replaced with mature lamellar bone .
At two weeks the provisional matrix (PM) is still present in the
most coronal and in the central part of the socket. However,
At four weeks the socket is almost completely filled
woven bone (WB) is rapidly forming and is already filling the
with woven bone.
apical portion of the socket. A layer of woven bone is also
covering the lingual (L) and buccal (B) walls.
Modelling / Remodeling phase
• The final phase of the socket healing process involves changes in the
structure of the bone tissue, which can occur with modification of the
architecture and shape (modelling), or without changes in
architecture and shape (remodeling).

• In socket healing :
• The substitution of woven bone with lamellar bone tissue/bone
marrow is regarded as remodeling
• While the resulting dimensional alterations of the alveolar ridge
comprise bone modelling
The modelling/remodeling process results from the active interplay
between osteoblasts and osteoclasts, being highly modulated by
the presence of factors such as macrophage colony stimulating
factor (M-CSF), RANK, RANKL and osteoprotegerin (OPG).

Although bone remodeling may start early during socket healing , it

will take several months until all woven bone in the extraction
socket has been replaced with lamellar bone and bone marrow.
 Mucosal healing (re-epithelialization of socket)

• Within 24 hours after tooth extraction, epithelial cells (basal cell layer) at the
margin of the wound dissolve their hemi-desmosomal adhesions and show
first signs of migration.(leading edge)

• In 48 hours, proliferation starts behind the leading edge, seeding more cells
into the wound site. Epithelial cells migrate through provisional matrix until
they contact the front of leading cells coming from the other side of the
wound.
• Epidermal growth factor and EGF receptor play a critical role in the
regulation of epithelial migration
Currently ,two different models have been
proposed to explain the migration of keratinocytes
over the wound.

1) One of the models proposes that basal

keratinocytes creep over the wound provisional
matrix as a sheet.

2) On the contrary, it has been proposed that

suprabasal keratinocytes leap over the basal
keratinocytes and attach to the wound matrix
forming a new migration front.