BONE HEALING

Anatomy
 Bone is generated by osteoblasts which manufacture the collagen fibres and
proteoglycans that make up the bony matrix
 Osteoblasts become completely surrounded by the matrix they produce. After the
matrix ossifies the cells are trapped in the lacunar space and once entrapped in
bone these cells become smaller and are called osteocytes
 Osteoclasts are derived from a monocytic-macrophage system and are responsible
for bone resorption. They are multinucleated cells with fine fingerlike cytoplasmic
processes and are rich in lysosomes containing tartrate-resistant acid phosphatise.
Osteoclasts lie in resorption craters known as Howship lacunae on bone surfaces or
in deep resorption cavities called cutting cones. They can only resorb mineralized
bone matrix.
Bone matrix
 Bone matrix consists of organic(30%) and mineral(70%) components. 8% is water
o Organic matrix
1) fibres: (collagen, reticulin, etc)
2) ground substance: (mucopolysaccharide, proteoglycan [especially,
chondroitin sul¬phate], glycoprotein, etc)
3) cells: (osteoblasts, osteocytes, osteoclasts)
o Inorganic matrix
 Imparts the quality of hardness and strength to the bone.
 Mineral content is mainly calcium and phosphorus as hydroxyapatite,
as well as smaller amounts of bicarbonate, citrate, magnesium,
potassium, and sodium.
 In demineralised bone allgrafts – cells and minerals are removed. Only matrix left
which contain growth factors.
 Osteoid is uncalcified organic matrix.
 Apart from its tendency to calcify, bone is similar to cartilage.

 The stimulus to calcification is unknown.

Layers of bone

1) Periosteum
 The periosteum consisits of
i. inner cambium layer immediately adjacent to the bone surface
 contains blood vessels and osteoprogenitor cells
ii. outer layer.
 dense fibrous layer with fibroblasts
 The cambium layer consists of osteoprogenitor cells, which are flat and
spindle shaped and are capable of differentiating into osteoblasts and
forming bones in response to various stimulations.
 The collagen fibers in the outer layer are contiguous with the joint capsule,
ligament, and tendons.
 Also functions to carry blood supply to the outer 1/3 of the cortex
 It is somewhat anchored to the cortex by Sharpey fibers that penetrate into
the bone.
2) Endosteum
 covers the inner aspect of bone.
 thin layer of reticular cells that lines the walls of bone marrow cavities and
the haversian canal system.
 Has both haematopoietic and osteogenic potential.
 Both periosteum and endosteum are active in the healing of fractures.
3) Bone marrow
 contains many of the cellular elements of loose connective tissue that are
absent from compact bone.
 Haematopoietic function, but also active in osteogenesis.
 The reticular bone marrow cells readily transform into cells of bone.
Marrow is richly vascular.

Classification of bone

By embryology
 1. Endochondral bone
 Occurs at:
1. the end of long bones(epiphysis)
2. petrous, occipital, ethmoid, mastoid, and sphenoid.
 Requires a cartilage model.
 Active in extending bone length.
 The germinal layer of the cartilage is on the epiphysis and derives nutrition
from the epiphyseal vessels. Cartilage cells grow from the epiphysis
towards the metaphysis, forming columns of cells that degenerate,
fragment, and undergo hypertrophy. The fragments of cells mineralize.
This is the zone of provisional calcification forming the metaphyseal
border, and is not bone. Note that no circulation exists in the cartilage
zone.
 Neovascularization occurs from the metaphysis towards the epiphysis.
Endothelial cells transform into osteoblasts and use the degenerate cell
debris to form primary immature bone. This immature bone progressively
is remodeled to mature woven bone and further is remodeled by cutting
cones to form mature haversian system bone. Damage to either epiphyseal
or metaphyseal vascular supply disrupts bone growth; however, damage to
the layer of cartilage may not be significant if the surfaces are reapposed,
and vascular supply to the growing cartilage is not permanently
interrupted.
 Physis has 5 zones, starting from the epiphyseal side of cartilage, as
follows:
1. Resting zone - This zone consists of small chondrocytes.
2. Proliferative zone - The proliferative zone consists of rapidly
dividing chondrocytes in columns parallel to the long axis of the
bone, resulting in interstitial growth of cartilage. The chondroid
matrix is laid down, and mitotic figures may be detected.
3. Hypertrophic zone - This zone consists of large chondrocytes
containing abundant cytoplasmic glycogen. In the hypertrophic
zone, chondrocytes are maturing and degenerating, with associated
chondroid matrix resorption.
4. Calcified cartilage zone (zone of provisional calcification) - This
zone is where chondrocytes die. Chondrocyte death is followed by
blood vessel invasion and bone deposition on the calcified
cartilage.
5. Ossification zone - The ossification zone is where primary
spongiosa forms by rapidly mineralized osteoid laid down on the
calcified cartilage septa .

Zone Description Hallmarks Epiphysis

Zone of Reserve Randomly arranged Cells most sparse
Cartilage chondrocytes Appears like “normal”
No proliferation cartilage
Source of bone-destined Closest to distal edge of
chondrocytes epiphyseal plate

Zone of Chondrocytes undergo division Look for cells of “normal” size

Proliferation and are organized in distinct
columns (stacks of poker chips)
Actively producing matrix
Zone of Chondrocytes and lacunae are
Hypertrophy enlarged
that have increased in number
& appear to stack.
Clear cytoplasm from
glycogen accumulation
Matrix compressed between
columns of large cells.

Zone of Matrix begins to mineralize Huge dying cells

Calcification Cuts chondrocytes from Empty lacunae
nutrients Lacunae invaded by blood
Chondrocyte death vessels.

Zone of Osteoblasts deposit osteoid on Look for layer of osteoblasts.

Ossification exposed cartilage
Zone of Nearest diaphysis Osteoclasts Cells look irregular, warped
Resorption absorb oldest bone on spicules Osteoclasts present
Look for bone marrow nearby. Diaphysis

2. Membranous bone
 flat bones of the skull, face, and mandible (the mandible has some
endochondral component with Meckel's cartilage origin)
 Pre-existing mesenchymal cells differentiate into osteoblasts which lay
down osteoid directly without cartilaginous intermediates.
 becomes hard bone after undergoing mineralization by calcium phosphate.

Endochondral vs membranous bone grafts

 the literature points to membranous bone with greatest survival(less resorption)
(Kusiak 1985)

By Type
Cortical bone
 Osteocytes with the osteocytes with the primary matrix are primarily arranged in
concentric lamellae around haversian canals which contain blood vessels
 The osteocytes intercommunicate with the haversian systems by fine canaliculi
that persist in the bony matrix
 Additional lamellae are circumferentially around the periphery of long bones
underlying the periosteal blood supply

Cancellous bone
 Cancellous bone is characterised by large units of bone called trabeculae and
smaller units called spicules. Complete osteons are present only in the thickened
trabeculae. The surface of the trabeculae are covered with resting osteoblasts.
 The axis of the trabeculae and spicules is generally perpendicular to muscular and
gravitational forces
 These units also consist of osteocytes surrounded by osseous matrix although the
bone is not as compact and organized as cortical bone
By Histology
1) Woven Bone: Formed during rapid osteogenesis. Irregular collagen and
osteocytes. Fine trabeculae of coarse collagen fibres embedded in generous
amounts of matrix.

2) Lamella Bone: Normally replaces previously formed cartilage or woven

bone. Collagen is arranged in parallel sheets as haversian systems or flat
plates.

Healing

Bone healing involves osteo conduction and osteoinduction

Osteoinduction (Huggins, 1931; Urist and Reddi, two of Huggin’s students)
 Osteoinduction is the inducement of undifferentiated cells (connective tissue)
to differentiate into bone.
 Seen with bone healing and using demineralised bone matrix graft
 Undifferentiated mesenchymal cells are transformed into bone forming cells in
response to an inducing substance. The sources of the undifferentiated
mesenchymal cells are endosteal, periosteal, from the BM and from
connective tissue. In addition, host osteoblasts are activated. The inducing
substance may be
1. hypoxia or acidosis
2. electronegativity
3. cytokines or GFs (inductive proteins) - BMP important(BMP 1-8)
4. specific surface properties (micropore size)

 The phases of osteoinduction are:

o chemotaxis,
o mitosis
o and differentiation of cells
 BMP irreversibly induces differentiation of peri-vascular mesenchymal type
cells into osteoprogenitor cells. Fibronectin also plays a role, especially in
chemotaxis.
 With osteoinduction, the bone that forms usually goes through an intermediate
cartilage stage, even membranous bone.
 Cancellous bone contains less BMP than does cortical bone, but cancellous
bone may contain more synergistic GFs.
 Autogenous and banked bone contains BMP which aids in the take by
osteoinduction.
 Hydroxyapatite implants and bone grafts can be impregnated with BMP to
allow osteoinduction and enhance take.
 The correct dose, carrier substance, effect on different sets of target cells, local
environment influences, importance of associated bone GFs and mass
production still needs to be fully evaluated.

Osteoconduction (Creeping Substitution)

  dead bone acting as a scaffold for the ingrowth of vessels, followed by the
resorption of the scaffold and deposition of the new bone derived from
adjacent skeleton
 The graft framework provides a scaffolding into which capillaries,
perivascular tissue and osteoprogenitor cells can grow and deposit new bone.
This method of incorporation is common to autografts, allografts and
biological materials used to simulate bone.
 Cortical (non demineralised) grafts heal by osteoconduction. A slow process

Note also:
1) Osteogenesis - Formation of new bone de novo from progenitor cells
2) Osseointegration - the stable anchorage of an implant achieved by direct
bone-to-implant contact.

Bone injury
 Healing can occur in one of several ways
 The response depends on
1) proximity of fracture fragments
2) vascularity of bone
3) immobilization of the ends
4) if wide displacement and poorly immobilized and vascularized = collagenous
scar -> fibrous union

Healing of bone
 When endochondral bone are held in reasonable proximity and immobilized the
healing process results in new bone formation
 Unique in that there is reconstruction of the original tissue rather than healing with
scar formation as in other tissues.

Secondary Healing
 occurs where there is relative instability of the reduced fracture and mobility of the
bone ends
 Despite the intermediate stages of tissue differentiation associated with secondary
healing, secondary bone healing is no slower than primary bone healing.

Stage I - Haematoma and inflammation

 Fracture causes soft tissue injury and ruptured vessels
 Formation of Fracture haematoma
 Necrotic tissue leads to inflammatory response with increased blood flow
 Increased cell division evident within the first 8 hours reaching a maximum in
some 24 hours

Stage II - Soft Callus

 Begins at 3-4 days and continues for few weeks
 Organisation of haematoma occurs = primary callus formation
 osteoclasts and macrophages remove debris and resorb hamatoma
 growth factor release
 Endothelial and smooth muscle migrate into area and contribute to
neovascularization
  Pluripotential mesenchymal cells from cambian layer periosteum migrate
into area and differentiate into fibroblasts, chondroblast and osteoblasts and
then proliferate and synthesize collagen, cartilage and osteoid that makes
immature woven bone or soft callus
 Callus forms externally along the shaft and internally in marrow cavity
 Primary component of soft callus is unmineralized cartilage
 Type II collagen makes up 40-60% of the collagen found in immature healing
bone and this fibro cartilaginous union limits motion at the fracture site
 Micro environment is acidic and electronegative
 Cartilage forms particularly in the periphery of the callus in regions of low O2
tension and in areas of increased movement

Stage III - Hard callus

 Mineralization of soft callus
 Begins at 3-4 weeks and continues until union
 Osteoclasts continue their removal of damaged bone
 Endochondral ossification occurs
 Collagen changes from Type II to I
 Vascular supply improves as result of both periosteal and endosteal
contributions. The new blood vessels run through the interstices of the new
bone formed
 More osteogenisc cells diff into osteoblasts and lay down more immature bone
which forms a network of fine trabeculae which contributes to the hard callus
formation
 Hard callus consists of woven bone following lines of capillary ingrowth

Stage IV- Reshaping

 Consists of modelling and remodelling
 Modelling
o cellular interaction that results in normalization of bony macro
structure such that orientation reflects lines of stretch
o The ability for bone to reorientate its fibres along lines of stress is
known as Wolff’s Law
 Remodelling
o Cell mediated breakdown and formation of bone leading to a stable
orientation of bony infrastructure
o The woven fibrous bone in hard callus is replaced by successive layers
of mature lamellar bone under the influence of functional stress
resulting in cortical callus being replaced by dense compact bone
 Mineralization occurs with restoration of the normal structure of mature bone
and reestablishment of a marrow cavity and haversian system

Primary Bone healing (Contact healing)

 Healing without cartilaginous intermediates and callus formation
 Main type of bone healing in membranous bone and endochondreal bone only
when the fragments well vascularized and rigidly fixed
 New bone bridges the fracture site during the early phases of healing and
 modelling and remodelling immediately occurs without callus formation
 Osteoclasts form spear heads at the ends of the haversian canals and advance
across the fracture site creating new haversian like canals in the space between
the immobilized bone segments
 Osteoblasts follow osteoclasts and generate new osteons with lamellae of bone
surrounding the new haversian canals
 No intermediate phase occurs and collagen type I seen from beginning

Cytokines involved in bone healing

 BMP irreversibly induces differentiation of peri-vascular mesenchymal
type cells into osteoprogenitor cells. Fibronectin also plays a role, especially
in chemotaxis.
 BMP is thus considered to be morphogen and differentiatating factor. It is a
protein with a number of side arm proteins. The mw is 17 500. It is also
known as osteogenin (BMP-3, mw = 22 000) and osteoinductive protein.
 A number of different isoforms have been found (8 in SRPS).
 BMP induces perivasc connective tissue cells to transform into chondroblasts
and osteoprogenitor cells and stimulate the proliferation of these cells and
produce new bone elements
 TGFβ, PDGF and FGF are competence/regulatory/mitogenic factors which
regulate cell cycles and promoted cell division. These factors ready the cells
for the next stage, which is differentiation under the influence of BMP.
 Cancellous bone contains less BMP than does cortical bone, but cancellous
bone may contain more synergistic GFs. BMP.

Factors affecting fracture healing:

Local wound
1. Soft tissue injury and local blood supply
2. Excessive compression ( more than 30lbs) inhibits enchondral ossification but
cyclic compression is beneficial
3. Intermittent shear stresses promotes cartilage formation
4. High shear stresses promotes fibrous tissue formation
Systemic
1. Radiation, chemical or thermal burns
2. Infection, anaemia or hypoxia
3. Corticosteroids inhibit osteoblast differentiation = slow healing
4. Growth hormone increases fracture healing (only if deficient)
5. Denervation retards fracture healing
6. Exercise increases fracture healing
7. Head injury promotes fracture healing by a humoral mechanism
8. Vitamin C is required for normal collagen matrix formation

Bone Grafting

Autografts heal by
1) Inflammation
2) Revascularisation – twice as slow in cortical grafts due to decreased porosity
3) Osteogenesis – with cancellous grafts
 4) osteoinduction – less with cortical grafts
5) osteoconduction– less with cortical grafts
6) remodelling

Allografts heal by creeping substitution (osteoconduction) and a small degree of

osteoinduction (presence of BMP)

Healing in Autografts
 Living bone transplants take by all 3 methods: osteoconduction, osteogenesis and
osteoinduction, but in cancellous bone, osteogenesis is the prime event, while in
cortical bone, osteoconduction is the principle occurrence.
 Revascularisation occurs by micro-reanastomosis of graft with host vessels.
 Allografts re-vascularise by invasion of capillary sprouts from the host bed while
resorption of the matrix occurs. Osteoconduction and osteoinduction occur, but not
osteogenesis (as no living cells are present in the implant).
 For the first 2 weeks, the process is the same for cortical and cancellous bone:
inflammatory response, influx of cells and infiltrating vascular buds; increased
osteoclastic activity; phagocytosis of necrotic tissue by macrophages.
 After 2 weeks, bone graft healing is different in cancellous and cortical bone:

Cancellous bone
 Cancellous bone is coarse, open and trabeculated. Found between the cortical
surfaces of flat bone and in the metaphysis of long bones. Cancellous bone is
characterised by large units of bone called trabeculae and smaller units called
spicules. Complete osteons are present only in the thickened trabeculae. The
surface of the trabeculae are covered with resting osteoblasts and the bone is thus
osteoblast-rich compared with cortical bone.
 In cancellous bone, vascularisation occurs rapidly due to inosculation and end to
end microanastomoses. The process is usually complete by the end of 2 weeks.
 Osteogenesis is the primary process in cancellous bone. It is rapid with
mesenchymal cell invasion, rapid differentiation to osteoblasts and laying down of
osteoid around a central core of necrotic bone. Some degree of osteoinduction
occurs.
 Sources of osteoblasts are from the endosteum, periosteum, marrow and primitive
undifferentiated cells in the host bed and graft.
 There is rapid and complete resorption of necrotic bone and replacement of the
graft with new bone resulting in a rapid completion of the repair.
 Cancellous bone continues to strengthen as bone is laid down.

Cortical bone
 Cortical bone is dense and better able to withstand mechanical stress. The
surface is penetrated by Volkmann’s canals which carry blood vessels that
anastomose with the haversian canals. Covered by periosteum. Forms the outer
surface of cylindrical bones and both surfaces of flat bones. Most often used as
onlay grafts.
 Vascularisation in cortical bone is poor and takes much longer, usually only
starting towards the end of the first week and reaching completion after 1-2
months. Angiogenesis has to follow pre-existing Volkmann’s and haversian
channels.
 Resorption of necrotic material by osteoclasts is the first event and necessary
to allow bone ingrowth. Resorption continues for up to a year following cortical
bone grafting.
 Osteoconduction is the main process in cortical bone. A delayed creeping
substitution and ingrowth of bone occurs. Osteoblasts also grow into the bone
resulting in a degree of osteogenesis.
 In cortical bone grafts, often the graft is left as a mixture of necrotic and viable
bone.
 Cortical bone grafts weaken considerably during the 1st 8 weeks d/t resorption
after which they gradually start to strengthen over a period of months.
 There is thus poorer graft incorporation, viability, and volume maintenance.

Cancellous Cortical

Structure Open, Dense, compact

trabeculated

Vascularisation Rapid (2 weeks) Slow (2 months)

Healing Osteogenesis Osteoconduction

Result Complete healing Admixed necrotic and viable

bone

Strength es progressively es initially. Then slow 

CLINICAL “RULES” FOR BONE GRAFTING

Kazanjian elucidated 4 clinical rules for bone grafting of the mandible. These are
clinical guidelines for all bone graft procedures. To these 4, McC adds a 5th.
1) The recipient site must have adequate blood supply to ensure survival of
surface cells.
2) The recipient bed must be healthy and not infected.
3) Bone to bone contact must be obtained to facilitate creeping substitution.
4) Rigid fixation must be maintained to allow continued creeping substitution.
5) The graft must be handled with care and respect to preserve the living cells.

FACTORS AFFECTING BONE GRAFT SURVIVAL

The graft
1) Cancellous better than cortical.
2) Membranous better than endochondrial (early revascularisation and less
resorption of membranous).
3) Preservation of the periosteum is associated with better survival.
Revascularisation is quicker and osteoblasts on the surface remain viable.
 Periosteum enhances new bone formation.
4) Small grafts survive better than large. Maximum thickness (excluding cortex)
should be < 5 mm.
5) Graft orientation: Cancellous surface deep in contact with recipient bone,
cortex superficial have less resorption than if bone placed the other way
around. Direct cancellous to cancellous grafting is best (burr the recipient
area).
6) Graft state: fresh autografts exhibit the least resorption. Chemically treated,
freeze dried or autoclaved bone resorbed far more.

The recipient bed and site

1) Must be well vascularised and non-infected and non-irradiated bed
2) Better placed on a depository surface rather than a resorptive surface
3) Placed in heterotopic sites (eg, soft tissue pocket), bone is replaced by fibrous
tissue. Better in orthotopically placed sites, ie, sites where bone is normally
found.
4) Local factors/mechanical stress influence differentiation of mesenchymal
cells. Compression causes differentiation into osteoblasts, low O2 tension into
chondrocytes, soft tissue pocket into fibroblasts. Inlay thus better than onlay.

Placement and fixation

1. Direct bone to bone contact. Burring of recipient bed increases survival: removes
infection, exposes vasculature.
2. Rigid fixation aids graft take and survival. It allows vessel ingrowth.
3. Good surgical technique with avoidance of dead space, haematoma, etc.

The patient
1) Age
2) Disease

INDICATIONS FOR BONE GRAFTING

1) Treatment of non unions
2) Arthrodesis of joints
3) Filling of bone defects secondary to infections, trauma or tumour
4) Replacement of joint surfaces

VASCULARISED BONE TRANSFERS

Vascularised bone grafts should be considered in
1) segmental defects > 6 cm in stress bearing sites.
2) when growth of the transferred segment is important
3) when the host bed is compromised by scarring, infection or irradiation
4) when rapid healing is preferred to creeping substitution

The advantages of vascularised bone include

1. cellular viability maintained
2. rapid union of bone which heals similar to #s and does not require creeping
substitution of dead bone matrix
3. more rapid stabilisation can be achieved and thus sooner mobilisation
4. undergo less resorption than free grafts, therefore stronger
5. larger pieces of bone can be transferred
6. the bone graft can be placed in a poor bed (poorly vascularised, irradiated) as it
brings in its own blood supply
7. fewer fatigue #s
8. rapid hypertrophy of bone

 Muscle frequently used as a carrier when pedicled, in which case donor sites
limited.
 Free transfers have broadened the scope of vascularised bone transfers and allowed
a wide variety of donor sites to be used and transferred to any recipient location.
 Transferred vascularised bone that contains an epiphysis will grow. This may be of
use in 2nd MT to mandible transfers in hemifacial microsomia. Vascularised
membranous bone has also been shown to grow.
 Long bone receives its vascular supply via the following sources:
1) Endosteal via the nutrient artery
2) Periosteal via muscular and ligamentous attachments of the diaphysis, of
the epiphysis and metaphysic
 The nutrient artery is the principle blood supply to long bone. It enters in the mid-
diaphysis and supplies ascending and descending branches that run in the marrow
cavity. The nutrient artery supplies the inner 2/3-3/4 of the cortex. In bone
transfers, if the pedicle is interrupted by osteotomies, the part distal to the
osteotomy becomes devascularised if the nutrient artery is the only supply.
 The diaphyseal periosteal vessels supply the outer cortex of the diaphysis directly.
These vessels supply the outer 1/3-1/2 of the cortex.
 The periosteal vessels of the epiphysis and metaphysis are related to the supply of
the epiphysis during growth and development. The periosteum contains a vascular
ring around the growth plate. Unlike the diaphyseal periosteal vessels, these
vessels penetrate deeply into the cortex. After growth has ceased, these vessels
anastomose with those of the nutrient artery and can be used as a pedicle on which
to base the bone flap. Prior to epiphyseal fusion, however, these vessels do not
cross the growth plate.
 In the adult, bone can therefore be pedicled on either the periosteum or the nutrient
artery (endosteal supply) and most of the bone will survive the transfer. No
differences are noted clinically.
 In flat bones and membranous bones, although nutrient arteries are present, they
are difficult to use as vascular pedicles and it is far more usual to transfer these
bones on their periosteal supply. The periosteal vessels fortunately penetrate flat
and membranous bones better than they do long bones. The periosteal supply is
advantageous in that it allows osteotomies to be made in the bone.

NON-AUTOGENOUS BONE
 Allograft and xenograft bone has the advantage of (almost) unlimited supply.
 Rejection involves the cellular elements of the graft, and in xenografts, the
collagen and ground substance. As with other tissue, antigenicity can result in cell
mediated rejection (and humoral), poor take and possible extrusion.
 Antigenicity is predominantly a feature of the cells, the collagen being minimally
antigenetically active.
 Multiple attempts have been made to reduce antigenicity by (in order of
 successfulness): freeze-drying, freezing, decalfiying, irradiation, deproteinating,
etc. Freeze drying is probably the best.
 Cortical bone, because it has fewer cells and more collagen than cancellous bone is
the preferred bone allograft material. Osteocytes provoke minimal rejection
response and are well shielded.
 Prone to non union, resorption, collapse and fractures. Requires prolonged
immobilisation.