Bone 81 (2015) 112121

Contents lists available at ScienceDirect

Bone
journal homepage: www.elsevier.com/locate/bone

Review

Osteoinduction of bone grafting materials for bone repair

and regeneration
Elena Garca-Gareta a,, Melanie J. Coathup b, Gordon W. Blunn b
a

RAFT Institute of Plastic Surgery, Mount Vernon Hospital, Northwood HA6 2RN, UK
John Scales Centre for Biomedical Engineering, Institute of Orthopaedics and Musculoskeletal Science, Division of Surgery and Interventional Science, University College London,
Royal National Orthopaedic Hospital, Stanmore HA7 4LP, UK

a r t i c l e

i n f o

Article history:
Received 28 July 2014
Revised 3 July 2015
Accepted 6 July 2015
Available online 8 July 2015
Keywords:
Osteoinductivity
Bone biomaterials
Ectopic bone formation
Bone regeneration
Review

a b s t r a c t
Regeneration of bone defects caused by trauma, infection, tumours or inherent genetic disorders is a clinical
challenge that usually necessitates bone grafting materials. Autologous bone or autograft is still considered the
clinical gold standard and the most effective method for bone regeneration. However, limited bone supply
and donor site morbidity are the most important disadvantages of autografting. Improved biomaterials are needed to match the performance of autograft as this is still superior to that of synthetic bone grafts. Osteoinductive
materials would be the perfect candidates for achieving this task.
The aim of this article is to review the different groups of bone substitutes in terms of their most recently reported
osteoinductive properties. The different factors inuencing osteoinductivity by biomaterials as well as the mechanisms behind this phenomenon are also presented, showing that it is very limited compared to osteoinductivity
shown by bone morphogenetic proteins (BMPs). Therefore, a new term to describe osteoinductivity by biomaterials is proposed. Different strategies for adding osteoinductivity (BMPs, stem cells) to bone substitutes are also
discussed. The overall objective of this paper is to gather the current knowledge on osteoinductivity of bone
grafting materials for the effective development of new graft substitutes that enhance bone regeneration.
2015 Elsevier Inc. All rights reserved.

Contents
1.
2.

Introduction
. . . . . . . . . . . . . . . .
Polymers . . . . . . . . . . . . . . . . . .
2.1.
Natural polymers . . . . . . . . . . .
2.2.
Synthetic polymers . . . . . . . . . .
3.
Ceramics . . . . . . . . . . . . . . . . . .
3.1.
Calcium-phosphate ceramics . . . . . .
3.2.
Bioglasses and glass-ceramics
. . . . .
3.3.
Other ceramics . . . . . . . . . . . .
4.
Metals . . . . . . . . . . . . . . . . . . .
4.1.
Titanium and its alloys
. . . . . . . .
4.2.
Porous tantalum or trabecular metal . .
4.3.
Other metals . . . . . . . . . . . . .
5.
Composites . . . . . . . . . . . . . . . . .
6.
Adding osteoinductivity to bone grafting materials
6.1.
BMPs
. . . . . . . . . . . . . . . .
6.2.
Stem cells
. . . . . . . . . . . . . .
7.
Discussion and concluding remarks . . . . . .
Disclosure . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .

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Corresponding author at: RAFT, Leopold Muller Building, Mount Vernon Hospital, Northwood HA6 2RN, UK.
E-mail address: garciae@raft.ac.uk (E. Garca-Gareta).

http://dx.doi.org/10.1016/j.bone.2015.07.007
8756-3282/ 2015 Elsevier Inc. All rights reserved.

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E. Garca-Gareta et al. / Bone 81 (2015) 112121

1. Introduction
Regeneration of bone defects caused by trauma, infection,
tumours or inherent genetic disorders leading to abnormal skeletal
development is a clinical challenge. Regeneration of bone is also
required in spinal fusions and in defects caused by osteolysis adjacent to implants. Repair of such bone defects usually necessitates
bone grafting materials.
Autologous bone or autograft is still considered the clinical gold
standard and the most effective method for bone regeneration as it
promotes bone formation over its surface by direct bone bonding
(osteoconduction) and induces local stem cells to differentiate into
bone cells (osteoinduction) without any associated immune response
[1]. In autologous bone grafting fresh cortical or trabecular bone or a
combination of both, are transplanted from one site in the body,
such as the iliac crest, to another within the same patient. However,
autografting presents several disadvantages; with limited bone supply
and donor site morbidity being the major drawbacks [13]. These disadvantages can be overcome by using allograft, transplanted cortical/trabecular bone or demineralised bone matrix from a donor tissue into
the patient. Usually harvested from sections of the pelvis from cadavers
or from removed femoral heads at primary total hip replacements, allogenic bone graft possesses osteoconductivity and when used as fresh
frozen or in a demineralised form also presents limited osteoinductivity
[3,4]. Disadvantages associated with allogenic bone grafts are disease
transmission or bacterial infection. Differences in graft preparation
techniques lead to inconsistency, immune response, fracture and nonunion due to differences in the bone quality between the donor and
the patient [3,4].
The use of bone substitutes or synthetic grafts aims at overcoming the disadvantages of using autologous and allogenic bone
grafts. Natural bone serves as the model for the development of
bone substitutes, which try to mimic the mineral composition of
bone tissue or the structure of interconnected struts of trabecular
bone [5,6]. Table 1 summarises the different groups of bone substitutes: natural and synthetic biodegradable polymers, ceramics

113

including bioglasses, metals and composites. All of these materials

have advantages as well as disadvantages (Table 1). For example,
calcium-phosphate ceramics, the most popular bone substitutes
due to their chemical similarity with bone mineral generally have
good biocompatibility, osteoconductivity, low cost and are readily
available. However these materials are stiff when compared to
bone, brittle, present unpredictable dissolution rates in vivo and
have limited osteoinductivity [68].
Performance of autologous bone graft is still the gold standard
and preferentially used and therefore, improved biomaterials are
needed to match the performance of autograft. Osteoinductive
synthetic materials that induce differentiation down the osteogenic
lineage of local stem cells would be appropriate materials for replacing autograft [9].
The historical background and denitions of osteoinduction were
thoroughly reviewed by Barradas and co-authors in 2011 [10]. It is
generally accepted and well established that an osteoinductive
material should induce bone formation upon implantation in nonosseous sites, also known as heterotopic or ectopic sites [6,9,10].
The osteoinductive potential of new materials can be assessed
in vitro by studying the osteogenic differentiation of undifferentiated mesenchymal stem cells or osteoprogenitor cells in contact with
candidate bone graft substitutes [1114]. We believe that while
such in vitro tests give an indication of the potential osteoinductivity
of a given material, conrmation of in vivo bone formation in ectopic
sites is necessary before a material is classied as osteoinductive
[15].
The aim of this paper is to review the different groups of bone substitutes in terms of their most recently reported osteoinductive properties.
Different strategies for adding osteoinductivity to bone substitutes, e.g.
by adding bone morphogenetic proteins (BMPs) or stem cells, are also
discussed. The overall objective of this paper is to gather the current
knowledge on osteoinductivity of bone grafting materials for the effective development of new osteoinductive bone substitutes that match
the clinical performance of autologous bone graft, considered the
gold standard for bone repair and regeneration.

Table 1
Bone grafting materials used for bone repair and regeneration: examples, advantages and disadvantages.
Bone grafting materials

Examples

Advantages

Disadvantages

Refs

Polymers Natural

Protein: collagen, brin, gelatin, silk broin

Polysaccharides: hyaluronic acid, chondroitin
sulphate, cellulose, starch, alginate, agarose,
chitosan, pullulan, dextran

Biodegradability
Biocompatibility
Bioactivity
Unlimited source (some of
them)

Low mechanical
strength
High rates of
degradation
High batch to batch
variations
Low mechanical
strength
High local
concentration of acidic
degradation products
Brittleness
Low fracture strength
Degradation rates
difcult to predict

[7,8,12,1625]

Poly-glycolic acid (PGA)

Poly-lactic acid (PLA)
Poly-(-caprolactone) (PCL)
Poly-(lactide-co-glycolide) (PLGA)
Poly-hydroxyethylmethacrylate (poly-HEMA)
Calcium-phosphate Coralline or synthetic hydroxyapatite (HA)
Silicate-substituted HA
-Tricalcium phosphate (-TCP)
Dicalcium phosphate dehydrate (DCPD)
Bioglasses and
Silicate bioactive glasses
glass-ceramics
(45S5, 13-93)
Borate/borosilicate bioactive glasses
(13-93B2, 13-93B3, Pyrex)
Others
Alumina ceramic (Al2O3)
Titanium and its alloys
Tantalum
Stainless Steel
Magnesium and its alloys
Synthetic

Ceramics

Metals

Composites

Calcium-phosphate coatings on metals

HA/poly-(D,L-lactide)
HA/chitosan-gelatin

Biodegradability
Biocompatibility
Versatility

Biocompatibility
Biodegradability
Bioactivity
Osteoconductivity
Osteoinductivity (subject to
structural and chemical
properties)

Excellent mechanical properties

(high strength and wear
resistance, ductility)
Biocompatibility
Combination of the above

Lack of tissue
adherence
Corrosion
Risk of toxicity due to
release of metal ions
Combination of the
above

[7,8,16,17,2534]

[6,7,16,3656,5974,76,77]

[7,78103]

[7,104129]

114

E. Garca-Gareta et al. / Bone 81 (2015) 112121

2. Polymers

3.1. Calcium-phosphate ceramics

2.1. Natural polymers

Calcium-phosphate (CaP) ceramics resemble the bio-minerals

that are naturally found in the body as part of bone or teeth [5].
The main properties that these CaP materials offer are excellent
biocompatibility, biodegradability and osteoconductivity [6,7,16,36,
37,39]. CaP materials have been shown to be able to form a bioactive
apatite layer on their surfaces thus enhancing osteointegration [6,36,
40]. Another reason for the good osteointegration shown by these
materials in vivo is that natural cytokines and adhesive proteins
such as bronectin are able to bind to CaP materials. The proteins
and cytokines adsorbed to a scaffold surface and provide a matrix
for cell attachment [41,42].
Generally, CaP materials are regarded as not osteoinductive as
they are not able to form bone de novo [6]. However, Zhang et al.
demonstrated that more bone formation in non-osseous as well
as osseous sites was obtained using HA with a 75550 m pore
size and 60% porosity [43]. Similarly, Yuan and colleagues reported
bone formation in CaP materials with a micro-porous structure
when implanted into the muscles of dogs [44]. These studies,
among many others [4556], suggested that CaP materials can
show osteoinductive properties when they exhibit specic chemical and structural characteristics.
Indeed, the inuence of macrostructure (i.e. macro-porosity,
concavities) has been demonstrated in several studies [4451]
where bone formation always occurred in the inner pores and on
the concave surfaces and never on the convex surface. Interestingly, it has been shown in vitro that osteoblasts grown on grooved
surfaces on biocompatible materials mineralise preferentially
within the groove rather than on the at surface [57]. The inuence
of surface structure (i.e. micro-porosity, strut porosity, particle
size) on in vivo osteoinductivity has also been demonstrated in a
number of publications [5256,58]. As an example, the effect of
strut porosity, micro-pores within the struts of the material, has
been studied by Chan and colleagues and Coathup and colleagues
[54,56], that have shown that bone grafting materials with greater
strut porosity (30% versus 20% in the study by Coathup et al. and
46% compared to 22.5% in Chan et al.) are more osteoinductive. In
both studies, materials were studied in an ectopic ovine model,
inserted into the paraspinalis muscle.
Chemical composition also affects the osteoinductivity of calciumphosphate ceramics. Coathup and co-workers showed that silicate
substitution may increase osteoinduction [53,54] while Wang and
colleagues very recently reported that phase composition of calciumphosphate ceramics may regulate the amount of osteoinductive factors
in the local microenvironment of the implant [59]. Strontium, a trace
element in the human body of special interest in the treatment of osteoporosis, shows a role in both the stimulation of bone formation and the
reduction in bone resorption and has recently received attention for the
fabrication of strontium substituted calcium-phosphate ceramics. These
materials have shown higher bone formation in osseous sites than the
non-substituted calcium-phosphate ceramic controls [6062]. Whether
strontium substitution affects osteoinductivity or not has not been
reported so far.
Thus, it can be concluded that when CaP materials present
certain chemical compositions, specic surface structures, geometries or pore sizes they are osteoinductive [6,63]. Table 2 offers a
summary of the different factors that inuence osteoinductivity
by biomaterials and the proposed explanations for the observed
inuence.

Natural polymers such as collagen, brin, gelatin, starch, hyaluronic

acid or chitosan offer the advantage of good biocompatibility and biodegradability as they compose the structural native extracellular matrix of
tissues. Natural polymers are bioactive as they have the potential to
interact with the host's tissue. Some of them such as starch or chitosan offer the advantage of an almost unlimited source [7,8,1619].
Electrospinning of natural polymers to create extracellular matrix
analogues is a promising research area towards the development of
biomimetic biomaterials [20].
Collagen is one of the most useful biomaterials with many biomedical applications such as drug delivery systems, nanoparticles
for gene delivery and basic matrices for cell culture systems [12,
2123]. Osteoconductivity of collagen scaffolds was shown: anionic collagen matrices were able to heal bone defects in rats therefore
demonstrating bone formation [24]. However, osteoinductivity of
collagen or other natural polymers has not yet been reported. The
great disadvantages of natural polymers are insufcient mechanical strength and high rates of degradation. Thus, they are often
used in composites or are chemically modied to improve mechanical properties and degradation rates [7,8,16,25].
2.2. Synthetic polymers
Synthetic polymers offer great versatility as they can have different
porosities, pore sizes, degradation rates, mechanical properties and
forms [7,8,16,17,2527]. The most commonly used are poly-(hydroxyacids), such as poly-glycolic acid (PGA) and poly-lactic acid
(PLA), and poly-(-caprolactone). The degradation products of these
polymers are glycolic acid and lactic acid, which are naturally found in
the human body and therefore are removed by natural metabolic pathways. The use of poly-(lactide-co-glycolide) (PLGA), which is a copolymer
formed by PLA and PGA, for bone regeneration has been extensively studied and approved for human clinical use by the American Food and Drug
Administration [2830]. The main disadvantage of synthetic polymers is
their poor mechanical properties, even when they are in the form of
rods or solid screws, and have therefore been applied in low mechanical
stress applications [7]. Another potential disadvantage is high local concentrations of acidic degradation products that can affect cell differentiation on the scaffolds in vitro and could induce an inammatory response
in vivo. Moreover, dissolution of the polymer is often accompanied by
breakup into smaller particles which then dissolve inducing an inammatory reaction [31,32].
The only synthetic polymer to date to have shown osteoinductivity is
poly-hydroxyethylmethacrylate (poly-HEMA): subcutaneous bone formation upon implantation of poly-HEMA sponge in pigs was reported
by Winter and Simpson in 1969 [33]. A year later Winter described
the same osteoinductive effect by poly-HEMA sponge subcutaneously
implanted in rats [34]. Interestingly, in both cases the poly-HEMA sponge
calcied after implantation but before any cell differentiation into bone
could be identied, suggesting for the rst time that calcication and
therefore calcium-phosphates may be important for osteoinduction [9,
10,35].
3. Ceramics
Ceramics have been widely used in the biomedical engineering eld
and for clinical applications for many years. As biodegradable polymers
they can be from a natural or a synthetic origin and can be synthesized
to different forms, porosities, pore sizes or topographies. An example of
natural ceramics is coralline hydroxyapatite (HA) while synthetic HA or
-tricalcium phosphate (-TCP) are among the synthetic ceramics more
commonly used [7,16,3638].

3.2. Bioglasses and glass-ceramics

In spite of their brittleness, bioglasses and glass-ceramics present
unique properties. They are biodegradable and their degradation rate
can be controlled. As they degrade they release ions that can promote

E. Garca-Gareta et al. / Bone 81 (2015) 112121

115

Table 2
Factors that inuence osteoinduction by biomaterials: proposed explanations and examples.
Factors that inuence
osteoinduction by
biomaterials

Proposed explanations

Examples

Refs

Chemical composition

Dissolution/reprecipitation of relevant ions for bone formation

Formation of a bone-like apatite layer on the surface of the
biomaterial
Calcium-phosphate coatings and biomaterials direct
mesenchymal stem cells towards the osteogenic lineage

[15,3335,4356,5962,6474,98103,114118]

Macrostructure

Invasion of blood vessels: supply of nutrients and oxygen and

removal of waste products and metabolites
Inltration of cells and tissue
Protection against high mechanical forces or body uid shear
stress
Increased surface area for binding of proteins including BMPs,
dissolution/reprecipitation of ions, mineral deposition from body
uids
Nano-topography directs mesenchymal stem cells towards the
osteogenic lineage

Calcium-phosphate
Silicon substitution in
calcium-phosphate
ceramics
Strontium substitution in
calcium-phosphate
ceramics?
(only in vivo data on
osseous sites)
Phase composition of
calcium-phosphate
ceramics
Ta2O5 nano-tube lm
coating on pure tantalum?
(only in vitro data)
Magnesium materials?
(only in vivo data on
osseous sites)
Porosity: macro-pores
Porosity: interconnectivity
Geometry: concavities

Surface structure

osteogenesis and angiogenesis. More importantly, they convert to a biologically active carbonated HA material that rmly binds to both
hard and soft tissues [64,65]. Bioglasses exhibit an amorphous
structure while glass-ceramics are crystallised glasses. There is a
relationship between mechanical strength and bioactivity: while
the crystalline glass-ceramics are mechanically stronger they
possess greatly reduced bioactivity compared to the amorphous
bioglasses. Thus, amorphous bioglasses are also referred to as bioactive glasses [6467]. Different types of bioactive glasses include
silicate glasses such as 45S5 or 13-93, which have been shown to
support proliferation and differentiation of osteoblast precursor
cell lines and bone marrow stromal cells in vitro [68.69], or borate/borosilicate glasses such as 13-93B2, 13-93B3 or Pyrex. Cell
proliferation and differentiation in vitro is supported by borate
glasses [70,71] as well as in vivo tissue inltration [72].
Osteoinductivity of porous 45S5 bioactive glass was reported by
Yuan and colleagues who showed that in vivo bone formation in a
non-osseous site was preceded by calcication [73], as with polyHEMA sponge implants [33,34]. In vitro studies suggest that the osteogenic properties of 45S5 bioactive glass are due to their dissolution
products which stimulate osteoprogenitor cells at the genetic level
[67,69]. Interestingly, although 13-93B borate glasses have not been
shown to be osteoinductive, greater bone formation after 12 weeks in
critical-sized calvarial defects has been reported for 13-93B3 glass
than for 45S5 silicate glass. However, blood vessel area was signicantly
higher for 45S5. Both 45S5 and 13-93B3 glasses completely converted
to HA in vivo [74].
The only other bioglass that shows osteoinductivity is Pyrex, which
as early as in 1960, was shown to form bone 60 days after subcutaneous
implantation in rats of 30 mm diameter by 20 mm length Pyrex glass
tubes [75]. Histological analysis also revealed the presence of cartilage
and haematopoietic tissue along with bone inside the Pyrex glass
tubes.

Micro-porosity
Strut porosity
Grain size
Particle size of injectable
biomaterials
Nano-porosity
Nano-topography

[6,9,10,4550,90,123]

[15,4144,5256,90,114,124]

3.3. Other ceramics

Yuan and colleagues reported osteoinduction of porous alumina
ceramics [76], which are able to form an apatite layer in vitro when
immersed in a supersaturated calcium-phosphate solution [77].
4. Metals
Metals possess excellent mechanical properties that make them
ideal candidates for load-bearing orthopaedic applications [7]. Investigation into porous metals is very active as they provide bone ingrowth
potential that leads to early xation of the implant. These metals are
morphologically and mechanically similar to trabecular bone [78] and
disadvantages include lack of tissue adherence, which may result in implant loosening with a necessary second surgery to remove it [79], and a
risk of toxicity due to accumulation of metal ions from corrosion which
is increased due to the high surface area associated with porosity
[8082].
4.1. Titanium and its alloys
Because of their biocompatibility, strength, lightness and high resistance to corrosion titanium and its alloys are the metal materials more
commonly used for biomedical applications [83,84]. The biocompatibility of titanium materials is based on a thin titanium dioxide (TiO2) layer
formed on the surface of the bulk material. Titanium is a very reactive
element even at room temperature. Therefore a newly polished titanium surface will have a thin layer of TiO2. Coating of titanium implants
with a TiO2 improves cell adhesion and osseointegration [85,86]. Porous
titanium materials have been developed to achieve material properties
compared to bone [83,87,88] and new families of titanium alloys are
constantly under research [89]. Osteoinductivity of titanium was
demonstrated by Fujibayashi and colleagues when they implanted 4

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E. Garca-Gareta et al. / Bone 81 (2015) 112121

different types of titanium implants in the dorsal muscles of mature

beagle dogs: porous blocks and bre mesh cylinders that were chemically treated (immersion in aqueous 5 M NaOH solution at 60 C for
24 h), followed by a thermal treatment (heating to 600 C at a rate of
5 C/min, maintained at 600 C for 1 h and then allowed to cool down
to the natural rate of the furnace) to create a bioactive surface composed
of micro-pores. The non-treated implants displayed smooth surfaces.
Bone formation was only demonstrated on treated porous blocks after
12 months of implantation and was observed to appear within the
pores and extend throughout the porous network [90]. Only the treated
implants had shown formation of an apatite layer in vitro. The authors
concluded that the complex interconnective macro-porous structure
of the porous blocks, the 3D surface micro-porous structure and the
ionic concentration of calcium and phosphorus played an important
role in the displayed osteoinductivity of this material. This study
highlighted the importance of calcium-phosphate and structural characteristics on the osteoinductive potential of materials, already seen
for calcium-phosphate ceramics.
4.2. Porous tantalum or trabecular metal
Trabecular metal, a highly porous (80%) biomaterial made of porous
tantalum with structural and mechanical resemblance with trabecular
bone, was approved by the Foods and Drugs Administration for use in
acetabular cups in 1997 [78,91]. The ingrowth potential of this material
was demonstrated [92] and has been used in primary and in revision
total hip arthroplasty components with excellent early clinical results
[9396]. Although osteoinductivity of tantalum materials has not been
reported yet, Wang and colleagues showed that by modifying the tantalum surface with a coating of tantalum oxide (Ta2O5) nano-tube lms
improved the anticorrosion, biocompatibility and osteoinductive potential in vitro of pure tantalum [97], suggesting that by modifying pure
tantalum surfaces with Ta2O5 nano-coatings this metal could become
osteoinductive in vivo.

drying followed by ionic crosslinking with CaCl2, showed high load

bearing capacity without fractural deformation [107].
We have seen that few of the materials show osteoinductivity,
mostly CaP ceramics, when they present certain chemical compositions, macrostructure and surface characteristics (Table 2). Several
strategies have been developed to make composite materials with
potential osteoinductivity. Ceramic based coatings on metals are a
popular strategy for such means as these coatings increase the biocompatibility and osteointegration of metals [108116] and in
some cases are osteoinductive [117,118]. Combination of ceramics
with natural [119122], synthetic [15,123126] or both types of
polymers [127,128] is another promising strategy: PLA/HA composites have been shown to be osteoinductive by different groups [15,
123,124]. In two of these studies the HA was nano-sized [15,124]
while in another the composite had an average interconnective
pore diameter of 62 m [123]. Finally, Fricain and colleagues recently
reported osteoinduction of a nano-HA-pullulan/dextran polysaccharide composite with macro-porosity and 37% porosity after 30 and
60 days of subcutaneous implantation in mice and after 1 and 6
months of intramuscular implantation in goat [129]. In both models,
no ectopic bone formation was seen for the polysaccharide matrices
without nano-HA.
6. Adding osteoinductivity to bone grafting materials

Recently, magnesium and its alloys have been receiving great interest as biomaterials for orthopaedic applications. These materials possess
elastic moduli (4145 GPa) closer to those of cortical bone (1218 GPa)
than titanium materials (110117 GPa) and show good biocompatibility and biodegradability [98,99]. As magnesium is one of the reported
substituting ions (Mg2+) in bone mineral [5], the release of magnesium
ions from these materials could benet cell attachment, proliferation
and migration of bone cells [100]. Several in vivo studies have demonstrated that magnesium based materials promote bone formation
when implanted in osseous sites [101103]. However, bone formation
of magnesium-based materials in non-osseous sites has not yet been
shown.

So far we have reviewed the different groups of materials used for

bone repair in terms of their osteoinductive properties. We can draw
the conclusion that osteoinductivity by biomaterials is an isolated phenomenon heavily inuenced by chemistry and structure. In terms of
how the ectopic bone is formed with calcium phosphate materials the
mechanism for osteoinduction is usually intramembranous and seen
inside pores. The majority of the studies describing bone formation by
biomaterials in ectopic sites cited so far in this review use large animal
models where the ectopic bone formation is slow, often requiring
months (Table 3). For example, in the mentioned study by Chan et al
[56], osteoinduction was only identied after 8 weeks of implantation
and the maximal amount of bone formation within the silicate bone
graft substitute was 30.00% after 24 weeks. A way of reducing the
time for osteointegration and increasing the overall amount of bone
formation would be extremely clinically relevant. Moreover, after
implantation of hydroxyapatite specimens in the rectus abdominus of
adult rabbits, dogs and baboons Ripamonti reported minimal amounts
of new bone formation in rabbits and dogs while substantial bone formation occurred in baboons [47]. Combination of the different materials
into composites also shows limited potential for osteoinductivity.
Therefore, other strategies are necessary to add osteoinductivity to
bone grafting materials. Scientists have been focusing on two strategies
for this purpose: the use of BMPs and seeding of scaffolds with stem
cells to create bone tissue engineered constructs.

5. Composites

6.1. BMPs

Each individual material discussed so far in this review has its advantages and drawbacks as bone grafting materials for bone regeneration
and repair, which could be overcome by combining different materials.
For instance, Kasuga et al. fabricated a composite consisting of the synthetic polymer PLA and calcium carbonate. The resulting composite
showed no brittleness and an improved modulus of elasticity compared
to that of PLA alone. Moreover, the composite was able to form a bonelike apatite layer on its surface when soaked in simulated body uid,
thus showing bioactive and osteoconductive potential [104]. PLGA/HA
composites have been shown to be osteoconductive [105] and brin
based scaffolds with incorporated nano-crystalline HA supported bone
formation when used in a mouse calvarial defect model [106]. Recently
a combination of three natural polymers (gelatin, hyaluronic acid and
alginate) into a 3D porous composite scaffold, synthesized by freeze-

BMPs are grouped into the TGF- superfamily due to their similarities in protein structure and sequence with TGF-. Back in 1965, Urist
discovered that demineralised bone matrix (DBM) could induce bone
formation when implanted ectopically in subcutaneous tissue in rats
[130]. This capability was later attributed to a protein called bone morphogenetic protein, which was puried in 1984 based on its potential to
induce bone formation [131,132]. Unlike calcium phosphate materials,
bone formation with BMPs usually occurs by endochondral ossication.
Growth factors, cytokines secreted by many cell types that function
as signalling molecules, promote and/or prevent cell adhesion,
proliferation, migration and differentiation by up-regulating or
down-regulating the synthesis of proteins, growth factors and receptors. They are essential for tissue formation and play an important
role in tissue repair [133136]. Bone tissue possesses a plethora of

4.3. Other metals

E. Garca-Gareta et al. / Bone 81 (2015) 112121

117

Table 3
Mechanism of osteoinduction by biomaterials and bone morphogenetic proteins (BMPs).
Mechanism of osteoinduction
Type of bone formation

Intramembranous
Endochondral

Animal model

Timeframe

Location of new ectopic bone

Small animals
(rodents)
Large animals
(dog, goat, sheep, baboon, rabbit)
Slow bone formation
(months)
Rapid bone formation
(2 to 3 weeks)
Periphery of implant/carrier
Inner pores of implant/carrier

6.2. Stem cells

Stem cells are undifferentiated cells, capable of self-renewal and
production of a large number of undifferentiated progeny. They have a
high proliferation capability and multi-lineage differentiation potential;

By bmps

Yes
(always)
No

Yes

Yes
(rarely)
Yes
(mostly)
Yes
(always)
No

In distant soft tissue from implant/carrier

growth factors, including BMPs, broblast growth factors (FGFs), insulin

growth factor I and II (IGF I/II) and platelet derived growth factor
(PDGF). The most heavily studied cytokines are BMPs [8,135]. In 1988,
Wozney and colleagues cloned these molecules and since then over 30
different BMPs have been identied with promising efcacy as therapeutic molecules for bone formation [134,137,138].
It is well established that bone formation by BMPs is easily induced
in small animals, as early as 2 to 3 weeks after ectopic implantation in
rodents, and that the new induced bone is observed on the periphery
of the carrier and even in the soft tissue distant from the surface of the
carrier (Table 3) [9,10,46,139142].
However, multiple questions regarding a suitable carrier for BMPs,
dosage, repeat exposure, carcinogenesis and long-term results have
hampered the potential benets these molecules could offer for bone
formation [143]. Moreover, there has been concern over the use of
BMPs for anterior cervical spinal fusion where inammation has
induced pressure on the trachea leading to death of patients in a number of instances [144]. This may be associated with the use of excessive
amounts of BMPs which lead to many fold increase in local concentrations compared with natural levels when commercial preparations are
used. Therefore, extensive research is being carried out on incorporating
BMPs into biomaterials as scaffolds and delivery systems [145154].
Another approach is gene therapy to engineer populations of
progenitor cells over-expressing the production of BMP, which offers
the advantage of continuous delivery of cytokines during a prolonged
period [134,135,138,155157].
Several studies have demonstrated that osteoinductivity can be
added to materials used for bone repair by immobilising BMPs
throughout their structure [147,149,151,158,159]. Lu and colleagues spatially immobilised BMP-4 in a collagen/PLGA composite
using a fusion BMP-4 that was composed of an additional collagenbinding domain derived from bronectin. The BMP-4-collagen/
PLGA material showed strong osteoinductivity in vivo [147]. Earlier, Kim and co-workers demonstrated ectopic and orthotopic bone
formation of BMP-2 protein immobilised in an absorbable collagen
sponge [149]. Recently Stenfelt et al. fabricated chemically crosslinked hyaluronan based hydrogels loaded with HA and BMP-2
that after implantation for 5 weeks in an ectopic site showed cancellous bone formation [151]. Studies such as the ones discussed
here show the potential for fabricating hybrid materials using
BMPs for added osteoinductivity.

By biomaterials

No
Yes
(always)
No

Yes
(mostly)
Yes
Yes
No
Yes
(always)
Yes
Yes
Yes

therefore they are involved in the regeneration of tissues [133,160].

Embryonic stem (ES) cells are pluripotent as they can differentiate
into a wide range of cell types, a plasticity that is essential in the early
development of the embryo [133,161,162], while adult stem cells are
found in the fully differentiated tissues and are responsible for the
regeneration of damaged tissue and the maintenance of tissue homeostasis. Adult stem cells have been found in the bone marrow, periosteum, muscle, fat, umbilical cord blood, placenta, brain or skin [133,160,
163]. In 2006 Takahashi and Yamanaka reported the seminal discovery
that differentiated cells can be reprogrammed to a pluripotent state by
introducing transcription factors that maintain the pluripotency in
both early embryos and ES cells [164]. Such reprogrammed cells are
named induced pluripotent stem cells (iPSCs) and offer exciting features for regenerative medicine such as autogenicity and ease of ethical
issues.
All of the above types of stem cells have been used in combination
with biomaterials to create tissue engineered constructs with great
potential for bone regeneration [165169]. Korda and colleagues
showed in 2010 that mesenchymal stem cells from bone marrow used
in combinations of allograft and HA and subjected to forces used during
impaction allografting enhanced ectopic bone formation in a ovine
model [168]. Recently, a study by He et al. reported that osteogenically
induced bone marrow mesenchymal stem cells in combination with a
chitosan scaffold and implanted into SD rat thigh muscles showed
ectopic bone formation and scaffold biodegradation [167].
7. Discussion and concluding remarks
As stated in the introduction the objective of this review article is to
gather the current knowledge on osteoinductivity so effective bone substitutes that mimic the performance of autograft can be developed.
Osteoinductivity by biomaterials appears to be a local phenomenon
inuenced by chemical composition and structure [6,15,5156,6374,
90,123,124]. The level of osteoinductivity achieved with biomaterials
appears to be very limited when compared with BMPs [9,10,56]. Therefore, hybrid materials incorporating BMPs for added osteoinductivity
seems the obvious strategy to follow. Yet, questions over dosage and
long terms results remain unsolved for BMPs [143,144]. Alternatively
stem cells have also shown the potential of adding osteoinductivity to
a priori non-osteoinductive biomaterials [167,168]. However, the development of cell constructs is laborious and, in the case of autologous
cells, may need a preliminary additional procedure to obtain the cells.
Unless allogenic cells can be used, a biomaterial without added stem
cells would be ideal in terms of off the shelf availability. Understanding the mechanism of osteoinduction by biomaterials is key to
develop such bone substitutes. Unfortunately, the number of available studies showing ectopic bone formation by biomaterials is

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E. Garca-Gareta et al. / Bone 81 (2015) 112121

Fig. 1. In vitro model of osteoinductivity and factors to take into account: cell type, culture conditions and measurable output parameters.

limited and understanding the mechanism of bone induction in

these materials is unknown. Chemistry, porosity and shape of the
graft inuences osteoinduction. Studies to understand these mechanisms require in vivo work in large animals which take months to
complete and therefore progression in this area is slow. The development of a standard in vitro model to test osteoinductivity as applicable to clinical use would be greatly advantageous.
Several groups have attempted to develop an in vitro model of
osteoinductivity [10,170172]. Important factors to take into
account are cell type (stem cells, osteoprogenitor cells, fully differentiated cells, co-culture of different cell types), culture conditions
(media with or without osteogenic or angiogenic factors, calcium/
phosphate enriched media, static or dynamic culture, mechanical
stimulation, electrical stimulation, timeframe) and which output
parameters should be measured at the end or throughout the culture
period (expression of osteogenic markers such as ALP, Runx-2 or
osteocalcin, calcium and phosphate ions release, mineralisation,
expression of inammatory markers such as TNF- or IL-6, BMPs
binding/release, deposition of a bone-like extracellular matrix with
collagen type-I) (Fig. 1). Unfortunately, more work in this area
is needed to develop a reliable and consistent in vitro model of
osteoinductivity.
Finally, we have seen that the mechanisms behind osteoinductivity
by biomaterials or by BMPs are very different. It is important to realise
that bone formation induced by BMPs is much faster, and it is more
reproducible than that seen on the surface of CaP materials. The clinical
reliability associated with osteoinductivity for BMPs is therefore
much greater than other materials. Osteoinductivity by bone grafts
is limited, late and until now not proven to provide meaningful clinical osteoinduction. Osteoinductivity for CaP bone grafts substitutes
does occur although it is a bio-product associated with using these
materials as an osteoconductive graft. Even DBM does not show as
great osteoinductivity as BMPs and just as with CaP materials the
attractiveness of using DBM is also that structurally acts as a scaffold.
Even autograft, which contains osteoprogenitor cells, stem cells and
osteoblasts together with bone extracellular matrix that may contain
low levels of BMPs is not as osteoinductive as BMPs. The downside
with BMPs is the selection of an appropriate dose together with the
use of a scaffold that provides maximum osteoinduction.

The question therefore arises as to whether ectopic bone formation

by biomaterials should be called osteoinduction at all or a new term
should be coined to name this phenomenon. Since the end result is
the same in both processes, ectopic bone formation, we believe that a
more descriptive expression such as limited osteoinduction or delayed osteoinduction should be used when describing osteoinductivity
by biomaterials.
The current knowledge on osteoinductivity by biomaterials should
be a starting point from which the continuous investigation of this
clinically important area should progress. Recent advances in fabrication techniques and additive manufacturing (not reviewed here as
they are not within the scope of this paper; see references [173178]
for comprehensive reviews on these elds) will be key for tuning the
structural properties of the perfect bone graft substitute.
Disclosure
The authors have no conicts of interest to declare.
Acknowledgements
This work was supported by the Restoration of Appearance and
Function Trust (UK, registered charity number 299811) charitable
funds.
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