BONE FORMATION AND HEALING

BY DR NSHIMIYIMANA ALEXIS/ ORTHOPEDIC

RESIDENT-UR
 outline

• MSK embryology
• Limb formation and growth
• Endomembranous and endochondral
• Bone healing and remodeling
• Non union
• bone grafting
• bone growth factors
• References
 MSK embr and limb growth
• Some of the major challenges in paediatric
orthopaedics relate to congenital or acquired
limb deformities.
• it is essential to have a comprehensive
understanding of both limb development in
utero and the pivotal role of the physis in
growth.
• The embryo develops in both a proximal-to
distal direction and a rostral-to-caudal
direction
• The upper-limb buds appear at 4 weeks
• lower-limb buds a few days later.
• A limb bud comprises mesoderm covered by a
thin surface of ectoderm.
• The mesoderm forms limb mesenchyme with
separate areas forming the somite and the
lateral mesodermal plate
• The lateral mesoderm forms the bone and
connective tissue
• The somatic mesoderm forms the muscle.
• The Homeobox (HOX) gene controls growth in the
distal direction of the condensing mesenchyme.
• The zone of proliferating activity (ZPA) in the
ectoderm, whose activity is mediated by the sonic
hedgehog (Shh) genes, directs development in the
anterior-to-posterior direction, producing the
apical ectodermal ridge(AER) at the tip of the limb
bud from which the digits form
• As embryonic development progresses, first the
mesenchyme condenses and then chondrocytes
differentiate within the mesenchyme
(chondrification)
• This chondrocyte formation and cartilage
differentiation first occurs in the diaphyseal
region of the humerus and the femur
• chondrocytes start to produce a matrix of
glycosaminoglycans
• The cartilaginous mesenchyme then undergoes
resorption and cavitation( segmentation)
• leads to the formation of the primitive joint
• This will later be followed by development of the
intra-articular structures.
• For example, a discoid meniscus results if the
meniscus fails to differentiate fully at this stage;
this happens in less than 1 per cent of knees and
occurs most commonly on the lateral side.
• primary ossification centre develops with
hypertrophy of middiaphyseal chondrocytes
• Invasion by blood vessels, which form a
primitive nutrient artery system.
• intramembranous ossification occurs
circumferentially from the periosteum around
this primary ossification centre.
.in the epiphyseal regions, the cartilage cells
become hypertrophic, and after vascular
invasion the process of enchondral ossification
commences.
• By the twelfth week, ossification centres are
present in all long bones.
• At around the ninth week, the limbs rotate.
• 90 degrees laterally and medially,
respectively.
• This results in a lateral thumb and a medial
hallux.
• Towards the end of fetal development, the
earliest secondary ossification centre
develops at the distal femur.
• By this time, a separate system of blood vessels
has grown into the epiphysis.
• Initially, these vascularized chondrocytes within
the epiphyseal cartilage hypertrophy to form a
central mass.
• This mass forms a spherical physis over its entire
surface, which later reshapes into a
hemispherical physeal outer surface and a discoid
non-physeal surface towards the metaphysis.
• The distal femoral secondary ossification
centre should be present after 36 weeks;
failure to see this centre on the radiograph of a
neonate implies prematurity or a skeletal
abnormality
• Other secondary centres appear throughout
early childhood, including the capital femoral
epiphysis that ossifies sometime after 3
months and should be seen before age 1 year.
• In childhood, the hemispherical physis of the
secondary ossification centre becomes less
active, with production of few new
chondrocytes.
• It thins until it is replaced by subchondral bone
and articular cartilage.
• As skeletal maturity is reached, the physis is
resorbed, allowing the metaphyseal and
epiphyseal blood systems to merge.
• If the normal sequence of events described
above is disturbed during the first trimester of
pregnancy, congenital limb deformities can
occur.
 Endomembranous
• Endomembranous ossification is the process
of bone development from fibrous
membranes.
• It is involved in the formation of the flat bones
of the skull, the mandible, pelvis and the
clavicles.
•  Ossification begins as mesenchymal cells form
a template of the future bone.
• One of the two essential processes during
fetal development bone formation and
fracture healing
• Also known as contact healing and haversian
remodeling
• Occurs without a cartilage model
• E.g: rigid fixation, IMN
 Steps
• aggregation of undifferentiated mesenchymal
cells
• osteoblast differentiation
• organic matrix deposition
 Regulation and signaling

• controlled by pathway called canonical Wnt and

Hedgehog signaling
• beta-catenin enters cells
  and induces cells to form
osteoblasts which then proceed with
intramembranous bone formation
• important transcription factors include CBFA1 (also
know as Runx2) and osterix (OSX)
• sclerostin, created by the SOST gene, decreases
bone mass by inhibiting the Wnt pathway 
 Endochondral
Occurs in:
• longitudinal physeal growth
• embryonic long bone formation
• non-rigid fracture healing (secondary healing)
 Cell biology

– enchondral bone formation occurs with a cartilage model

• chondrocytes produce cartilage which is absorbed by osteoclasts
• osteoblasts lay down bone on cartilaginous framework (bone
replaces cartilage, cartilage is not converted to bone)
• forms primary trabecular bone
• bone deposition occurs on metaphyseal side 
• type X collagen associated with enchondral ossification
 Molecular biology
• chondrocytes play a critical role in endochondral bone
formation throughout the formation of the cartilage
intermediate
• transcription factors involved in regulation of
chondrocytes include 
– Sox-9 
• considered a major regulator of chondrogenesis, regulates
several cartilage-specific genes during endochondral ossification,
including collagen types II, IV, and XI and aggrecan
– PTHrP
• delays differentiation of chondrocytes in the zone of hypertrophy 
 Longitudinal Physeal Growth
Physeal growth plate
Reserve Zone
• Cells store lipids, glycogen, and proteoglycan
aggregates for later growth and matrix
production
• Low oxygen tension
Proliferative Zone 
• Proliferation of chondrocytes with longitudinal
growth and stacking of chondrocytes.
• Highest rate of extracellular matrix production
• Increased oxygen tension in surroundings
inhibits calcification
Hypertrophic Zone
• Zone of chondrocyte maturation, chondrocyte hypertrophy, and
chondrocyte calcification.
• Three phases occur in the hypertrophic zone
– Maturation zone: preparation of matrix for calcification, chondrocyte growth
– Degenerative zone: further preparation of matrix for calcification, further
chondrocyte growth in size (5x)
– Provisional calcification zone: chondrocyte death allows calcium release,
allowing calcification of matrix
• Chondrocyte maturation regulated by local growth factors (parathyroid
related peptides, expession regulated by Indian hedgehog gene)
• Type X collagen produced by hypertrophic chondrocytes important for
mineralization 
Primary Spongiosa
(metaphysis) 
• Vascular invasion and resportion of transverse
septa.
• Osteoblasts align on cartilage bars
• Primary spongiosa mineralized to form woven
bone
• Then remodels to become secondary spongiosa
Physis periphery
• Groove of Ranvier
• During the first year of life, the zone spreads over the
adjacent metaphysis to form a fibrous circumferential
ring bridging from the epiphysis to the diaphysis. 
• This ring increases the mechanical strength of the
physis and is responsible for appositional bone
growths
– supplies chondrocytes to periphery
• Perichondrial fibrous ring of La Croix
• Dense fibrous tissue that is the primary
limiting membrane that anchors and supports
the physis through peripheral stability
Embryonic Long Bone Formation
– allows growth in width and length
– formed from mesenchymal anlage around 6th week in utero.
Steps of formation include
– vascularization
• vascular buds invade the mesenchymal model
– primary ossification centers form
• (at ~ 8 weeks) osteoprogenitor cells migrate through vascular buds and differentiate into
osteoblasts forming the primary ossification centers
– cartilage model forms
• grows through appositional (width) and interstitial (length) growth
– marrow forms
• marrow is formed by resorption of central portion of the cartilage anlage by myeloid precursor
cells that migrate in through the vascular buds
– secondary ossification centers form
• develop at bone ends and lead to epiphyseal ossification center (growth plate)
 Bone healing
PRIMARY (DIRECT CORTICAL, OSTEONAL OR
HAVERSIAN) BONE HEALING
I. anatomical reduction and interfragmentary
compression leading to absolute stability
II. The process is very intolerant of strain (movement) at
the fracture site.
III. Growing of new blood vessels
IV. Mesenchymal cells differentiate into osteoblasts,
laying down lamellar bone in small gaps and woven
bone in large gaps.
 Non rigid fracture

• Cell biology
– soft callus is the cartilage intermediate
– bone replaces callus via same chondrocyte
proliferation, chondrocyte hypertrophy, and finally chondrocyte
calcification
• Examples include casting and bracing
• intramedullary nailing
– allows for motion at the fracture site, which promotes bone
formation both directly (intramembranous ossification) and
through a cartilage intermediate (endochondral ossification)
 SECONDARY (CALLUS) BONE HEALING

• Periosteal bony callus (intramembranous

ossification): multipotent cells in the
periosteum differentiate into osteoprogenitor
cells, which produce bone directly without
first forming cartilage
• This hard callus forms early at the periphery of
the fracture site
• Fibrocartilaginous bridging
callus(endochondral ossification): this process
occurs simultaneously between the adjacent
bone ends and involves the formation of
fibrocartilage that becomes calcified and is
then replaced by osteoid or woven bone.
• This process also occurs within the
surrounding soft tissues.
• These processes are dependent on some
movement occurring at the fracture site
(strain).
• Rigid fixation inhibits the differentiation of
cells and the formation of callus.
 Stages of callus formation
1. hematoma and inflammation up to 1 week

• Haematoma from ruptured blood vessels forms fibrin clot.

• Damaged haematoma ,tissue and degranulated platelets release
signalling molecules, growth inflammation factors and cytokines.
• Migration of inflammatory cells into the haematoma occurs,
responding to local growth factors
• cytokines (IL-1, IL-6, TGF-β super-family including BMPs, PDGF, FGF,
IGF).
• Proliferation, differentiation and matrix synthesis as haematoma is
replaced by granulation tissue.
.
• Capillary in-growth (angiogenesis) and
recruitment of fibroblasts, mesenchymal cells
and osteoprogenitor cells.
• The periosteum plays an important role in this
process.
• Cell types involved include PMNs,macrophages
and then fibroblasts.
• bone resorption is mediated by osteoclasts
• removal of tissue debris by macrophages
2. Soft callus 1week to 1 month

• Increased cellularity, with proliferation, differentiation and

soft callus
• Callus is a combination of fibrous tissue, cartilage and woven
bone
• Intramembranous (bony/periosteal) callus = primary callus
response: type I collagen (osteoid) laid down from periosteal
osteoblasts as periosteal bony callus or woven bone.
• This is hard callus but it does not bridge the fracture.
• Endochondral (fibrocartilaginous/bridging) callus =
bridging external callus: multipotential cells differentiate
to form chondroblasts and fibroblasts within the
granulating callus, which produce the type II cartilaginous
and fibrous elements of the matrix (chondroid).
• Chondroblasts then calcify the chondroid matrix they
have produced, creating calcified fibrocartilage or soft
callus.
• Medullary callus: this is a later process and can slowly
unite the fracture if external callus fails.
3.Hard callus 1 to 4 months
• Calcified soft callus is resorbed by chondroclasts and invaded by
new hard callus blood vessels.
• These bring with them osteoblast precursors that produce the bony
(type I) elements of the matrix (osteoid) and then mineralize it to
form woven bone.
• Soft calcified chondroid callus becomes hard mineralized osteoid
callus.
• Bony bridging continues peripherally as subperiosteal new bone
formation.
• At this point the fracture is united, solid and pain-free to
movement.
4.Remodeling up to several years
• Once the fracture has united, the hard callus is remodelled from
woven remodelling several years bone to hard, dense lamellar
bone by a process of osteoclastic resorption followed by
osteoblastic bone formation.
• The medullary canal reforms at the end of this process.
• Bone assumes a configuration and shape based on stresses acting
upon it (Wolff’s law).
• Electric fields may play a role in Wolff’s law, with osteoclastic
activity being predominant on the electropositive tension side of
bone and osteoblastic activity on the electronegative compression
side.
 NON-UNION
• This is defined as lack of healing of a fracture within
the expected time, which varies with the bone
involved, e.g. distal radial fractures are expected to
heal by 6 weeks, scaphoid fractures by 8 weeks,
tibial fractures by 16 ± 4 weeks and femoral
fractures by 16 ± 4 weeks.
• The fracture is bridged by soft tissue, the
characteristics of which are defined by the local
blood supply and mechanical conditions (usually
cartilage and/or fibrous tissue).
• Clinical union is defined by the absence of
tenderness or motion at the fracture site with
no pain on loading, while radiological union is
defined as the presence of visible bridging
trabeculae on three out of four cortices on X-
rays.
 Hypertrophic non-union
• A good blood supply but excessive strain at
the fracture site prevents progression of the
callus to form bone.
• These usually require biomechanical
stabilization to allow callus progression to
bone to occur.
 Atrophic non-union
• A poor blood supply is caused by soft-tissue
damage, periosteal stripping and/or fracture
comminution, which may occur at the time of
injury or during the exposure for internal fixation.
• A fracture fixed with rigid fixation (zero strain)
and with the fragments distracted will also lack
stimulation of callus formation.
• Atrophic non-unions require stabilization and
biological enhancement in order to heal.
 Bone graft
• Bone grafting is the use of any implanted
material that alone or in combination with
other materials promotes a bone-healing
response by providing osteogenic,
osteoconductive or osteo-inductive activity to
a local site.
• Bone grafts have mechanical (providing
support) and biological (stimulus for bone
formation) functions.
 Indications
• Provision of structural stability: massive
proximal femoral grafting in revision total
hip arthroplasty for the reconstruction and/or
replacement of skeletal defects.
• Stimulation of bone formation: bone grafting
in spinal fusions where the graft improves
the fusion rate but does not provide any
mechanical support, even in the short term.
• Enhancement of fracture healing:
• Acute: for mechanical or biological
reasons, e.g. in comminuted fractures with
bone loss or during elevation of depressed
joint surface.
• Non-union: usually for biological reasons.
 PROPERTIES OF BONE GRAFTS
Osteogenicity
The graft contains living cells that are capable
of differentiation into bone.
Osteogenicity occurs independent of the host
bed, i.e. the graft may survive and incorporate
successfully
• OSTEOCONDUCTION
The graft provides a three-dimensional scaffold
that supports in-growth of capillaries, perivascular
tissues and osteogenic cell precursors.
The surface of the scaffold allows attachment,
division and differentiation of cells and ultimately
remodelling.
The graft does not have to be biological in order to
possess osteoconductive properties.
Osteo-induction
The graft provides a biological stimulus stimulates
mitosis differentiation of undifferentiated
mesenchymal cells into osteoprogenitor cells with
the capacity to form new bone.
The graft has the ability to promote
bone formation at non-skeletal sites
Note that demineralized bone graft has potent
osteoinductive.
 GENETICS OF BONE GRAFTS
Autografts
Tissue is harvested from and implanted into the
same individual.
Examples include cancellous, cortical and
vascularized grafts, and bone marrow.
 Allograft
• Tissue is harvested from one individual and implanted into another
individual of the same species.
• The host mounts an immune response to the cells of a fresh allograft
and, therefore, the graft is processed in order to remove
immunogenic cells, decreasing the risk of both immune response and
transmission of infection.
Allografts can be classified according to the following:
• Anatomy: cortical, cancellous, corticocancellous.
• Processing: fresh, frozen, freeze-dried, demineralized.
• Sterilization method: sterile processing, irradiated, ethylene oxide.
• Handling properties: powder, gel, paste/putty, chips, strips/blocks,
massive.
 xenograft
• Tissue is harvested from one species and implanted into
a different species.
• Unfortunately, a vigorous immune response precludes
the use of most preparations.
• Some currently used xenografts include Kiel
bone(defatted and deproteinated xenograft, which has a
decreased immune response), processed xenograft with
autologous bone marrow, and processed bovine collagen
(biocompatible flexible substrate material, which is a
component of several bone-graft preparations).
 Dangers of bone graft
• Autografts: donor site morbidity (scar,
haematoma, infection, pain).
• Allografts:
• Disease transmission: both microbiological
(e.g. two cases of human
immunodeficiency virus (HIV) per one
million grafts, two cases of hepatitis C per
one million grafts) and pathological (e.g.
8 per cent of femoral head allografts have
histological evidence of disease such as
malignant and benign tumours such as
osteomas).
• Immune sensitization
 GRAFT INCORPORATION
• This is the process by which invasion of the
graft by host bone occurs, such that the graft
is replaced partially or completely by host
bone.
 Cancelous graft incorporation

Autogenous non vascularized

• Phase 1: vascular in-growth, chemotaxis and invasion/differentiation
of multipotent stem cells.
• As revascularization is rapid, surface osteocytes
• survive.
• Phase 2: osteoblasts (whose formation is induced by factors within
the graft) lay down new bone on the scaffold of dead trabeculae,
with simultaneous osteoclastic resorption (creeping substitution).
• This leads to early increase in density on X-rays and an associated
transient increase in strength.
• Phase 3: osteoclast/osteoblast remodelling of the trabeculae along
lines of force, with associated decreased radiodensity.
Allogenic grafts
• undergo a similar process
• but with a more marked inflammatory phase
• less predictable and possibly incomplete
incorporation.
• Since there is prompt formation of new bone
on to the dead scaffold, cancellous grafts
achieve early structural strength.
 Cortical graft incorporation
Autogenous non-vascularized cortical grafts
• slow process of inflammation and
revascularization
• All donor bone has to be removed before
appositional bone formation occurs.
Allogenic grafts undergo a similar process
• more slowly and with less overall ingrowth.
• Rarely, allografts may undergo immunogenic
destruction following a massive inflammatory
response.
Vascularized grafts
are autogenous
• incorporate in a manner analogous to fracture repair
• The cells retain their viability.
 BONE GRAFT SUBSTITUTES
• Calcium phosphates:
• Bulk, e.g. tricalcium phosphate (which
undergoes partial conversion to
hydroxyapatite in vivo)
hydroxyapatite
combinations of the two.
These materials degrade at a very slow rate.
• Injectable, e.g. Norian SRS: injectable
paste of inorganic calcium and phosphate
used to fill bone voids after acute
fractures, which hardens in minutes and is
osteoconductive.
Eventually resorbed and replaced by host bone.
• Calcium carbonates, e.g. Biocora: chemically
unaltered marine coral that is resorbed and replaced by bone.
• Coralline hydroxyapatite, e.g. Pro-Osteon:
calcium carbonate skeleton undergoes a
thermo-exchange process to convert this into
calcium phosphate.
• Calcium sulphate, e.g. Osteoset:
osteoconductive calcium sulphate pellets.
• Silicon-based, e.g. bioactive glasses, glassionomer
cement: used as delivery systems for
osteo-inductive compounds.
• Synthetic polymers, e.g. polylactic acid and
polyglycolic acid: problems include the
production of acidic degradation products.
• Ceramic composites, e.g. Collagraft: calciumcollagen
graft material – an osteoconductive
composite of hydroxyapatite, tricalcium
phosphate and collagen used as a bone graft
substitute or expander mixed with
autologous bone marrow to provide cells and
growth factors.
 Bone growth factors
• Bone Morphogenetic Protein (BMP) & SMADs
• Overview 
– BMPs belong to the TGF-B superfamily
– BMP 2,4,6, and 7 all exhibit osteoinductive activity 
– BMP 3 does not exhibit osteoinductive activity 
– Mutations in BMP-4 are associated with Fibrodysplasia ossificans
progressiva  
• Mechanism
– osteoinductive
• leads to bone formation
• activates mesenchymal cells to transform into osteoblasts and produce bone 
• has been foung to increase chondrogenic phenotype and matrix synthesis in
intervertebral discs 
• Signaling Pathways and Cellular Targets
– BMP targets undifferentiated perivascular mesenchymal
cells
– activates a transmembrane serine/threonine kinase
receptor that leads to the activation of intracellular
signaling molecules called SMADs  
• SMADS are primary intracellular signaling mediators
• currently eight known SMADs, and the activation of different
SMADs within a cell leads to different cellular responses.
• Clinical applicationsFDA-approved uses   
– rhBMP-2 
• single-­level ALIF from L2 to ­S1 levels in degenerative disc disease
together with the lumbar tapered fusion device (LT Cage; Medtronic)
• open tibial shaft fractures stabilized with an IM nail and treated within 14
days of the initial injury
– rhBMP-7
• as an alternative to autograft in recalcitrant long bone nonunions where
use of autograft is unfeasible and alternative treatments have failed
• as an alternative to autograft in compromised patients (with osteoporosis,
smoking or diabetes) requiring revision posterolateral/intertransverse
lumbar fusion for whom autologous bone and bone marrow harvest are
not feasible or are not expected to promote fusion
Contraindications:
I. pregnancy
II. allergy to bovine type I collagen or
recombinant human rhBMP­-2
III. infection
IV. tumor
V. skeletal immaturity
 Transforming Growth Factor-B (TGF-B)
• Mechanism
– secreted in a paracrine fashion
– both osteoblast and osteoclasts synthesize and respond to TGF-B
– found in fracture hematomas and believed to regulate cartilage and bone
formation in fracture callus
– stimulates production of Type II collagen and proteoglycans
by mesenchymal cells.
– induces osteoblasts to synthesize collagen
• Signal Pathway & Cellular Targets
– signal mechanism involves transmembrane serine/threonine kinase
receptors
• Clinical applications
– TGF-B is used to coat porous coated implants to promote bone ingrowth
 Insulin-like Growth Factor 1 (IGF-1)
• Overview
– IGF-1, formerly known as somatomedin-C, possibly acts by both paracrine and
endocrine hormone pathways
– most abundant growth factor in bone
• Mechanism
– the products of the GH-IGF-1 system induce proliferation without maturation of the
growth plate and thus induce linear skeletal growth. 
– the action of the thyroid hormone axis is via an active metabolite that enters target
cells and signals a nuclear receptor to stimulate both proliferation and maturation of
the growth plate. Increased amounts of the active steroid hormone metabolite
promote proliferation and maturation of the growth plate
– IGF-1 may have a role in enhancing bone formation in defects that heal
via intramembranous ossification
• Signal Pathway & Cellular Targets
– signal mechanism involves tyrosine kinase receptors
 Insulin-like Growth Factor 2 (IGF-2)
• Overview
– more potent than IGF-1
• Mechanism
– stimulates type I collagen production
– stimulates cartilage matrix synthesis
– stimulates cellular proliferation
– stimulates bone formation
• Signal Pathway & Cellular Targets
– signal mechanism involves tyrosine kinase receptors
 Fibroblast growth factor(FGF)
• Overview
– FGF-1 and FGF-2 are most abundant
– promote growth and differentiation of a variety of cells
• epithelial cells
• myocytes
• osteoblasts
• chondrocytes
• Mechanism
– binds to membrane spanning tyrosine kinase
– associated with angiogenesis and chondrocyte and osteoblast
activation
– involved in early stages of fracture healing
 Platelet-derived growth factor(PDGF)

• Mechanism
– released from platelets and signals inflammatory
cells to migrate to fracture site
– role in fracture healing and bone repair has not
been clearly defined
• Signal Pathway & Cellular Targets
– signal mechanism involves tyrosine kinase
receptors
 Peroxisome proliferator activated receptor
gamma(PPAR-gamma)
• Overview
– key factor demonstrated in adipogenic differentiation of
mesenchymal precursor cells in vitro 
• Signal Pathway & Cellular Targets
– a nuclear binding receptor that binds to DNA and regulates
transcription of target genes
• Clinical and research applications
– utilized for adipogenic differentiation in vitro
– agonists are being utilized to attempt treatment of
hyperglycemia and hyperlipidemia
– targeted by thiazolidinediones for treatment of diabetes 
• Ramachandran stanmore guide basic scinence
• AAOS comprehensive orthopedic review