Jan Lindhe. Clinical Periodontology

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ALVEOLAR BONE FORMATION • 867

Fig. 38-1. Histological section illus

trating a bone multicellular unit

(

BMU). Note the presence of a re-

sorption front with osteoclast

(

OC) and a deposition front that

contains osteoblasts (OB), and

osteoid (OS). Vascular structures

(V) occupy the central area of the

BMU. RL = reserval line; LB =

lamellar bone.

progenitor cells are present in the bone marrow, in the

endosteum and in the periosteum that cover the sur-

faces of bone. Such cells possess an intrinsic capacity

to proliferate and differentiate into osteoblasts. Induc

ible osteogenic precursor cells, on the other hand,

represent mesenchymal cells present in other organs

and tissues (e.g. muscles) that may become bone-

forming cells when exposed to specific stimuli. As

osteogenesis is always closely related to the ingrowth

of vascular tissue, the stellate-shaped perivascular cell

(the

pericyte) is

considered to be the main osteopro-

genitor cell. The differentiation and development of

osteoblasts from osteoprogenitor cells are dependent

on the release of bone morphogenetic proteins (BMP)

and other growth factors such as insulin-growth factor

(IGF), platelet-derived growth factor (PDGF) and fi-

broblast growth factor (FGF).

The bone formation activity is consistently coupled

to bone resorption that is initiated and maintained by

osteoclasts.

Osteoclasts are multinucleated cells that

originate from hemopoietic precursor cells.

Modeling and remodeling

Once bone has formed, the new mineralized tissue

starts to be reshaped and renewed by processes of

resorption and apposition, i.e. through

modeling

and

remodeling.

Modeling represents a process that allows

a change in the initial bone architecture. It has been

suggested that external demands (such as load) on

bone tissue may initiate modeling. Remodeling, on

the

other hand, represents a change that occurs within

the

mineralized bone without a concomitant alteration of

the architecture of the tissue. The process of

remodeling is important (1) during bone formation,

and (2) when old bone is replaced with new bone.

During bone formation remodeling enables the sub-

stitution of the primary bone (woven bone), which has

low load bearing capacity, with lamellar bone which

more resistant to load.

The bone remodeling that occurs in order to allow

replacement of old bone with new bone involves two

processes: bone resorption and bone apposition (for-

mation). These processes are coupled in time and are

characterized by the presence of so called

bone mul-

ticellular units

(BMUs). A BMU (Fig. 38-1) is comprised

of (1) a front osteoclast residing on a surface of newly

resorbed bone – the resorption front, (2) a compart-

ment containing vessels and pericytes, and (3) a layer

of osteoblasts present on a newly formed organic

matrix – the deposition front. Local stimuli and release

of hormones, such as parathyroid hormone, growth

hormone, leptin and calcitonin, are involved in the

control of bone remodeling. Modeling and remodel

ing

occur throughout life to allow bone to adapt to

external and internal demands.

BONE HEALING — GENERAL

ASPECTS

Healing of an injured tissue usually leads to the for-

mation of a tissue that differs in morphology or func-

tion from the original tissue. This type of healing is

called

repair.

Tissue

regeneration,

on the other hand, is

term used to describe a healing that leads to com-

plete restoration of morphology and function.

The healing of bone tissue includes both regenera-

tion and repair phenomena depending on the charac-

ter of the injury. For example, a properly stabililized,

narrow bone fracture (e.g. green stick fracture) will

heal by regeneration, while a larger defect in the bone

will often heal with repair. There are certain factors

that may interfere with the bone tissue formation

following injury, such as:

failure of vessels to proliferate into the wound

improper stabilization of the coagulum and granu-

lation tissue in the defect

ingrowth of "non-osseous" tissues with a high pro-

liferative activity

bacterial contamination.

868 • CHAPTER 38

Fig. 38-2. Overall pattern of bone formation in an extraction socket. For details see text.

ALVEOLAR BONE FORMATION •

869

Fig. 38-3. Histologic section representing 1 day of healing (a). The socket is occupied with a blood clot that

contains

large numbers of erythrocytes (b) entrapped in a fibrin network, as well as platelets (blue cells in c).

The healing of a wound includes four phases:

blood clotting

wound cleansing

tissue formation

tissue modeling and remodeling.

These phases occur in an orderly sequence but, in a

given site, may overlap in such a way that in some

areas of the wound, tissue formation may be in pro-

gress while in other areas tissue modeling is the domi

nating event.

Model of bone tissue formation

The closure of an extraction socket may serve as a

model to describe tissue events that lead to bone

formation in a defect in the alveolar process. The

healing of such an extraction socket is described in Fig.

38-2. The mandibular premolars in a group of dogs

were extracted, and the healing of the extraction sites

was monitored in biopsies obtained at various time

intervals following tooth removal.

Overall pattern

of bone formation

The empty socket was first filled with blood and a

coagulum (clot) was formed (Fig. 38-2a). Inflamma-

tory cells migrated into the coagulum and the process

of wound cleansing was initiated (Fig. 38-2b). Vascu-

lar tissue and mesenchymal cells entered into the

coagulum and a granulation tissue was produced

(

Fig. 38-2c). This granulation tissue was gradually

replaced by a provisional connective tissue (Fig. 38-

2d) and the formation of new bone (woven bone)

started (Fig. 38-2d,e). The socket (identified by dotted

lines) was gradually filled with this woven bone (Fig.

38-2e) that later on was modeled and remodeled into

lamellar bone and marrow (Figs. 38-2f,g,h). Note the

dotted lines and the arrows which indicate the border

between the old bone and the newly formed bone

Important events in bone formation

Blood clotting:

Immediately after tooth extraction,

blood from the severed blood vessels will fill the

cavity. Proteins derived from vessels and damaged

cells initiate a series of events that lead to the forma-

tion of a fibrin network.

Platelets

form aggregates

(

platelet thrombi) and interact with the fibrin network

produce

blood clot

(a coagulum) that effectively

plugs

the severed vessels and stops the bleeding (Fig.

38-3).

The blood clot acts as a physical matrix that

directs

cellular movements and contains substances

that are

of importance for the continuation of the

healing

process. Thus, the clot contains substances

that (1)

influence mesenchymal cells (i.e.

growth fac

tors),

and (

2) affect inflammatory cells. These sub-

stances will

induce and amplify the migration of various types of

cells, as well as their proliferation, differ

entiation and

synthetic activity within the coagulum.

Although the blood clot is crucial in the initial

phase of wound healing, its removal is mandatory to

870 • CHAPTER 38

Fig. 38-4. Histologic section representing 3 days of healing (a). Note the presence of neutrophils and macrophages

that engaged in wound cleansing and the breakdown of the blood clot (b). Osteoclastic activity on the surface of

the walls of the old bone that lined the socket (c).

enable the formation of new tissue. Thus, within a few

days after the tooth extraction, the blood clot will start

to break down, i.e. "fibrinolysis" starts (Fig. 38-4).

Wound cleansing:

Neutrophils and macrophages mi-

grate into the wound, engulf bacteria and damaged

tissue (Fig. 38-4) and clean the site before tissue for-

mation starts. The neutrophils enter the wound early

while macrophages come into the scene somewhat

later. The macrophages are not only involved in the

cleaning of the wound but they also release several

growth factors and cytokines that further promote the

migration, proliferation and differentiation of me-

senchymal cells. Once the debris has been removed

and the wound has become "sterilized", the neutro-

phils undergo a programmed cell death (i.e.

apoptosis)

and are removed from the site through the action of

macrophages. The macrophages subsequently with-

draw from the wound.

In the extraction socket, a portion of the trauma-

tized bone facing the wound will undergo necrosis

and

will be removed by osteoclastic activity. Thus,

osteoclasts also may participate in the wound cleans-

ing phase of the bone healing.

Tissue formation:

mesenchymal, fibroblast-like cells

which migrate into the wound from, for example, the

bone marrow, start to proliferate and deposit matrix

components in an extracellular location (Fig. 38-5). In

this manner a new tissue, i.e.

granulation tissue,

will

gradually replace the blood clot. From a didactic point

of view the granulation tissue may be divided into two

portions: (1) early granulation tissue, and (2) late

granulation tissue. A large number of macrophages, a

few mesenchymal cells, small amounts of collagen

fibers and sprouts of vessels make up the early granu

lation tissue. The late granulation tissue contains few

macrophages, but a large number of fibroblast-like

cells and newly formed blood vessels present in a

connective matrix. The fibroblast-like cells continue

(

1) to release growth factors, (2) to proliferate, and (3)

to deposit a new extracellular matrix that guides the

ingrowth of new cells and the further differentiation

the tissue. The newly formed vessels provide the

oxygen and nutrients that are needed for the increas-

ing number of cells in the new tissue. The intense

synthesis of matrix components exhibited by these

mesenchymal cells is called

fibroplasia

while the for-

mation of new vessels is called

angiogenesis.

Through

the combined fibroplasia and angiogenesis

a provi-

sional connective tissue is

established (Fig. 38-6).

The transition of the provisional connective tissue

into bone tissue occurs along the vascular structures.

Thus, osteoprogenitor cells (e.g. pericytes) migrate

and

gather in the vicinity of the vessel. They differen

tiate

into osteoblasts that produce a matrix of collagen

fibers which takes on a woven pattern. The osteoid is

hereby formed and the process of mineralization is

initiated in its central portions. The osteoblasts con-

tinue to lay down osteoid and occasionally cells are

ALVEOLAR BONE FORMATION • 871

Fig. 38-5. Histologic section representing 7 days of healing (a). Note in the upper portion in the socket a richly vas-

cularized early granulation tissue with large numbers of inflammatory cells can be seen (b), while in more apical ar-

eas a tissue including large numbers of fibroblast-like cells is present, i.e. late granulation tissue (c).

Fig. 38-6. Histologic section representing 14 days of healing (a). In the marginal portion in the wound a provisional

connective tissue rich in fibroblast-like cells is formed (b). The formation of woven bone has at this time interval al-

ready begun in more apical regions of the bone defect (c).

872 • CHAPTER 38

Fig. 38-7. Histologic section representing 30 days of healing (a). The socket is filled with woven bone. This woven

bone contains a large number of cells and primary osteons (PO;b). The woven pattern of the collagen fibers of the

woven bone is illustrated in (c) (polarized light).

Fig. 38-8. Histologic section representing 60 days of healing (a). A large portion of the woven bone has been re-

placed with bone marrow through osteoclastic activity and subsequent bone marrow formation, i.e. modeling (b).

Note in (c) the large number of adipocytes that reside in a tissue that still contains portions of woven bone.

ALVEOLAR BONE FORMATION • 873

Fig. 38-9. Histologic section repre

senting a bone tissue after 60 days

of healing. Note in (a) the pres-

ence of osteoclasts (OC) on the

surface of a secondary osteon (SO)

within the bone trabeculae. (b)

The concentric pattern of the colla

gen fibers of the lamellar bone

within the secondary osteon (po-

larized light).

trapped in the matrix and become osteocytes. The

newly formed bone is called

woven

bone

(Fig. 38-7).

The woven bone is the first type of bone to be

formed and is characterized by (1) its rapid deposition

along the route of vessels, (2) the poorly organized

collagen matrix, (3) the large number of osteoblasts

that are trapped in its mineralized matrix, and (4) its

low load-bearing capacity. The woven bone forms as

finger-like projections along the newly formed ves-

sels. Trabeculae of woven bone are shaped and encir-

cle the vessel. The trabeculae become thicker through

the deposition of further woven bone, cells (osteocyts)

are entrapped and the first set of osteons, the

primary

osteons

are organized. The woven bone is occasionally

reinforced by the deposition of so called

parallel-fibered

bone

that has its collagen fibers organized not in a

woven but in a concentric pattern.

Tissue modeling and remodeling:

The initial bone forma-

tion is a fast process. Within a few weeks, the entire

extraction socket will be occupied with woven bone

as this tissue is also called,

primary bone spongiosa.

The

woven bone offers (1) a stable scaffold, (2) a solid

surface, (3) a source of osteoprogenitor cells, and (4)

ample blood supply for cell function and matrix min-

eralization.

The woven bone with its primary osteons is gradu-

ally replaced by lamellar bone and bone marrow (Figs.

38-8, 38-9) through the processes of modeling and

remodeling as described earlier (Fig. 38-10). In the

remodeling process the primary osteons are replaced

with secondary osteons. The woven bone is first

through osteoclastic activity resorbed to a certain

level. This level of the resorption front will establish

the so-called reversal line, which is also the starting

point for the new bone formation building up a sec-

ondary osteon. Although the modeling and remodel-

ing may start early it will take several months until all

woven bone in the extraction socket is replaced by

bone marrow and lamellar bone (Fig. 38-11).

In summary:

Following tooth extraction, the first 24

hours are characterized by the formation of a

blood clot

and the starting of

hemolysis

(Fig. 38-12a). Within 2-3

days the blood clot is contracting and is replaced by

the

formation of a granulation tissue

with the

blood vessels

and collagen fibers (Fig. 38-12b). After 3 days, an

increased density of fibroblasts is visible in the clot

and the

proliferation of epithelium

from the wound mar-

Fig. 38-10. Schematic drawing describing the transition between woven bone and lamellar bone, i.e. remodeling.

Woven bone with primary osteons (PO) is transformed into lamellar bone in a process that involves the presence of

BMUs. The BMU contains osteoclasts (OC), as well as vascular structures (V) and osteoblasts (OB). Thus, the

osteoblasts in the BMU produce bone tissue that has a concentric orientation around the vessel, and secondary

osteons (SO) within lamellar bone are hereby formed.

Fig. 38-11. After 6 months of healing the extraction site (within the area delineated by the vertical, dotted lines (a))

more or less fully healed. The site contains lamellar bone and marrow (b) and some remaining woven bone.

Through a process of modeling and remodeling the newly formed bone is now continuous with the "old bone"

(

OB (a)) of the neighboring areas.

gins is apparent. Remodeling of the sockets begins

with

the

presence of osteoclasts

inducing bone resorp

tion (

Fig. 38-12c). One week after extraction, the socket

characterized by granulation tissue consisting of a

vascular network,

young

connective tissue, osteoid forma

tion in the apical portion and epithelial coverage over

the

wound (Fig. 38-12d). One month following extrac

tion,

the socket is characterized by a

dense connective

tissue

overlying the residual sockets, which are now

filled with

granulation tissue.

A trabecular pattern of

bone starts emerging. Wound

coverage by epithelium is

complete (Fig. 38-12e). Two months following extrac-

tion, bone formation in the socket is complete. The

bony

height of the original sockets has not yet been

reached

and the trabecular pattern is still undergoing

remodeling (Fig. 38-12f).

874 • CHAPTER 38

Woven bone

BMU

Lamellar bone

ALVEOLAR BONE FORMATION • 875

Tooth extraction

48-72 h after extraction

96 h after extraction

Hemorrhagia,

Bleeding,

Blood clot

Blood clot,

Beginning of

granulation tissue formation

Residual blood clot,

Granulation tissue,

Epithelial proliferation

7 days after extraction

Young connective tissue,

Primary osteoid formation,

Epithelial proliferation

21 days after extraction

Connective tissue,

Osteoid start of mineralization,

Reepithelialization

6 weeks after extraction

Connective tissue,

Woven bone, trabeculae,

Reepithelialization

Fig. 38-12. Healing of extraction sockets (Amler 1969). (a) Bleeding and formation of a blood clot immediately after

tooth extraction. Blood vessels are closed by thrombi and a fibrin network is formed. (b) Already during the first 48

hours, neutrophil granulocytes, monocytes and fibroblasts begin to migrate along the fibrin network. (c) The blood

clot is slowly replaced by granulation tissue. (d) Granulation tissue forms predominantly in the apical third of the

alveolus. Increased density of fibroblasts. After 4 days, contraction of the clot and beginning proliferation of the

oral

epithelium. Osteoclasts are visible at the margins of the alveolus. Osteoblasts and osteoids seem to appear in

the

bottom of the alveolus. (e) Reorganization of the granulation tissue through formation of osteoid trabeculae.

Epithelial proliferation from the wound margins on top of the young connective tissue. Again, the formation of

osteoid trabeculae is evident from the wall of the alveolus in a coronal direction. After 3 weeks some of the trabecu-

lae start to mineralize. (f) Radiographically, bone formation may be visible. The soft tissue wound is closed and epi-

thelialized after 6 weeks. However, bone fill in the alveolus takes up to 4 months and does not seem to reach the

level of the neighboring teeth.

The placement of bone-grafting materials to favor

876 • CHAPTER 38

Fig. 38-13. Microphotograph (a) demonstrating bifurcation defect 3 weeks after grafting with autogenous cancel

lous jaw bone (G). New bone has invaded the defect, and the bone grafts have exerted an osteoconductive

function.

Epithelium (arrows) has migrated into one side of the defect. (b) Higher magnification of (a) showing

that new

bone has formed around the bone grafts (G), which have lost their vitality, indicated by the empty

osteocyte lacu

nae.

Bone grafting

Although bone tissue exhibits a large regeneration

potential and may restore its original structure and

function completely, bony defects may often fail to

heal with bone tissue. In order to facilitate and/or

promote healing, bone grafting materials have been

placed into bony defects. It is generally accepted that

the biologic mechanisms forming the basis for bone

grafting include three basic processes:

osteogenesis,

osteoconduction

and

osteoinduction.

Osteogenesis

occurs when viable osteoblasts and pre-

cursor osteoblasts are transplanted with the grafting

material into the defects, where they may establish

centers of bone formation. Autogenous iliac bone and

marrow grafts are examples of transplants with osteo

genic properties (see Chapter 33).

Osteoconduction

occurs when non-vital implant mate-

rial serves as a scaffold for the ingrowth of precursor

osteoblasts into the defect. This process is usually

followed by a gradual resorption of the implant ma-

terial. Autogenous cortical bone or banked bone allo-

grafts may be examples of grafting materials with

osteoconductive properties (Fig. 38-13). Such grafting

materials, as well as bone-derived or synthetic bone

substitutes, have similar osteoconductive properties.

However, degradation and substitution by viable bone

is often poor. If the implanted material is not

resorbable, which is the case for most porous hydroxy

lapatite implants, the incorporation is restricted to

bone apposition to the material surface, but no substi

tution occurs during the remodeling phase.

Osteoinduction

involves new bone formation by the

differentiation of local uncommitted connective tissue

cells into bone-forming cells under the influence of

one or more inducing agents.

Demineralized bone ma-

trix

(DMB) or

bone morphogenetic proteins

(BMP) are

examples of such grafting materials (Bowers et al.

1989a,b, Sigurdsson et al. 1994).

It often occurs that all three basic bone-forming

mechanisms are involved in bone regeneration. In

fact, osteogenesis without osteoconduction and

osteoinduction is unlikely to occur, since almost none

of the transmitted cells of autogenous cancellous bone

grafts survive the transplantation. Thus, the grafting

material predominantly functions as a scaffold for

invading cells of the host. In addition, the osteoblasts

and osteocytes of the surrounding bone lack the abil-

ity to migrate and divide which, in turn, means that

the transplant is invaded by uncommitted mesenchy-

mal cells that later differentiate into osteoblasts.

On that basis it is appropriate to define three basic

conditions as prerequisites for bone regeneration:

the

supply of bone forming cells

or cells with the

capacity to differentiate into bone forming cells

the presence of

osteoinductive stimuli

to initiate the

differentiation of mesenchymal cells into osteo-

blasts

the presence of an

osteoconductive environment

form

ing a scaffold upon which invading tissue can pro-

liferate and in which the stimulated osteoprogeni-

tor cells can differentiate into osteoblasts and form

bone.