Review Article

Hydroxyapatite–polymer biocomposites for bone regeneration: A

review of current trends

Niranjan Ramesh,1 Stephen C. Moratti,2 George J. Dias1

1
Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin 9054, New Zealand
2
Department of Chemistry, University of Otago, Dunedin 9054, New Zealand

Received 5 December 2016; revised 18 May 2017; accepted 15 June 2017

Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33950

Abstract: Bone tissue engineering has emerged as one of the
most indispensable approaches to address bone trauma in
the past few decades. This approach offers an efficient and a
risk-free alternative to autografts and allografts by employing
a combination of biomaterials and cells to promote bone
regeneration. Hydroxyapatite (HA) is a ceramic biomaterial
that mimics the mineral composition of bones and teeth in
vertebrates. HA, commonly produced via several synthetic
several polymers in the form of biocomposite implants to pri-
marily improve its mechanical properties and also enhance
the implants’ overall performance by simultaneously exploit-
ing the positive effects of both HA and the polymer involved
in making the biocomposite. This review article summarizes
the past and recent developments in the evolution of HA–
polymer biocomposite implants as an “ideal” biomaterial
scaffold for bone regeneration. V C 2017 Wiley Periodicals, Inc. J

routes over the years has been found to exhibit good bioac-
tivity, biocompatibility, and osteoconductivity under both in
vitro and in vivo conditions. However, the brittle nature of
HA restricts its usage for load bearing applications. To
address this problem, HA has been used in combination with
Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2017.
Key Words: bone, biomaterials, hydroxyapatite, polymers,
biocomposites

How to cite this article: Ramesh, N, Moratti, SC, Dias, GJ 2017. Hydroxyapatite–polymer biocomposites for bone regeneration:
A review of current trends. J Biomed Mater Res Part B 2015:00B:000–000.

INTRODUCTION requirements. This eliminates the need to harvest the bones

Traditional methods to address bone loss include autografts
and allografts. These alternatives are based on the simple
criteria of being physically able to replace the lost bone
while eliciting minimal immune response from the host.1
The use of autologous bone grafts offers sufficient osteo-
genic, osteoinductive, and osteoconductive properties, and
presents no immune complications as the donors them-
selves are the recipients.2 Main sources for autografts are
the iliac crest (for cancellous bones), fibula, and ribs (for
cortical bones). Although there are significant merits in the
use of autologous grafts such as no immune rejection, high
success rate, and low risk of disease transmission, the dis-
advantages of the use of autografts include low availability
of autologous bones, risk of fractures, and hematomas lead-
ing to postoperative donor site morbidity including chronic
pain.3,4
Allogeneic bones are a good alternative for bone grafts,
as there is no limitation of availability as in the case of auto-
grafts. Allograft bones are derived from cadavers and can be
shaped into different geometries and sizes according to the
from the hosts, thereby alleviating the problems of donor
site morbidity.5 However there is a prevalent risk of trans-
mission of infective agents from the bone to the host, which
impedes the widespread acceptance of allografts.6 Efforts to
address this issue by processing the allograft bones prior to
use are still met with skepticism as the processing was
observed to negatively affect the mechanical and biological
properties of the bone graft.7,8
BIOMATERIALS AND BONE TISSUE ENGINEERING
Although autografts and allografts offered a potential solu-
tion for surgeons to address bone trauma, the success rates
were restricted by their limitations.1 These outcomes led
the interest to analyze the bone at a microscopic level to
better understand its structure and composition to effec-
tively address bone damage. Such a need prompted the
researchers shift their focus on the development of biomate-
rials. Park and Lakes defined biomaterials as “any material
used to make devices to replace a part or a function of the
body in a safe, reliable, economical, and physiologically

Correspondence to: N. Ramesh; e-mail: niranjanr91@gmail.com

C 2017 WILEY PERIODICALS, INC.

V 1
acceptable manner.”9 The emergence of biomaterials turned
the spotlight toward tissue engineering and regenerative
medicine. Today, biomaterials are used in applications
including cardiovascular stents, ureteral stents, artificial
skin, nerve tissues, bones, cornea, and so forth.10–15
Bone tissue engineering typically involves the use of bio-
materials in the form of whole prostheses, scaffolds, and
hydrogels, and cells and growth factors to treat bone loss
due to fractures, osteoporosis, osteoarthritis, and neo-
plasms.16–19 The biomaterials were commonly administered
in the form of whole prostheses, scaffolds, or hydrogels.20–22
Turn of the twentieth century saw researchers develop and
test several biomaterials for potential applications in bone
tissue engineering. Biomaterials used included metals,
ceramics, and polymers.21,23,24
HYDROXYAPATITE
Hydroxyapatite (Ca10(PO4)6(OH)2) (HA) is a member of cal-
cium phosphate ceramics. This family also includes trical-
cium phosphates and bioglass ceramics and they are widely
used as bone-substitute materials. The preference of HA
over other calcium phosphates can be attributed to the
structural and functional similarity to mineral composition
present in bones and teeth. In the human bone HA consti-
tutes up to 70% along with 5% water and 25% organic
matter.25,26 Inherent bioactivity and biocompatibility of HA
and the ease of fabrication makes it suitable implant mate-
rial.27,28 HA can be fabricated in the form of solid scaffolds
with variable porosities, which are ideal for cell attachment,
migration, and bone formation. Moreover, HA can be ground
into nanoscale powders to be used in combination with
other biomaterials including polymers such as chitosan and
collagen for improved bone mineralization.29,30 There is also
evidence of HA coatings improving the integration of
implants such as titanium to the host bone.31,32
HA can be obtained from both synthetic and natural
sources.33,34 The synthetic route of preparation involves the
use of calcium and phosphate precursor salts such as cal-
cium oxide and phosphoric acid for example and combining
them using pH-controlled processing techniques.21 Although
the HA prepared through synthetic routes possesses similar
properties to that of natural bone and teeth, the
manufacturing costs involved are high.35 Xenograft bones
have been considered as a potential solution to address
bone loss for decades. The belief is based on the rationale
that bones of animals such as cow and sheep contain trace
elements in addition to HA that may accelerate the process
of bone regeneration.35 Although there are concerns about
the risk of disease transmission by xenogenous bone substi-
tutes, research has proven that such threat can be alleviated
by developing different multistage processing techniques
that screen for and eliminate any organic matter and patho-
gens if present.35 This has led to the extraction of HA from
a diverse range of sources such as mammalian bones, egg-
shells, marine mollusc shells, and even plants providing eco-
friendly and cost-effective bone substitutes.36–40
2 RAMESH ET AL. HYDROXYAPATITE/POLYMER BIOCOMPOSITES FOR BONE REGENERATION: A REVIEW
POLYMERS IN TISSUE ENGINEERING
With biomaterials and tissue engineering gaining promi-
nence, researchers are aiming to develop biomaterials that
would be easy to manufacture, offering desired properties
and improved healing rates without causing any adverse
effects. Polymeric materials offer a versatile alternative to
metallic and ceramic biomaterials which suffer from stress
shielding (reduction in density of the host bone due to
alteration or removal of the typical stress in the bones by
implants) effects that arise as a result of their high elastic
modulus.41 Polymeric materials are often biocompatible and
are much easier to fabricate; they can be molded into desir-
able shapes and sizes according to requirements. Their
mechanical properties and degradation characteristics can
also be controlled, enabling them to be tailor-made and tar-
geted toward a specific purpose once implanted in the host
tissue.42 The first successful clinical application of polymeric
materials dates back 50 years ago when Sir John Charnley
used polymethyl methacrylate (PMMA), an acrylic cement,
to attach a femoral head prothesis.20 Since then more
research has been performed on the behavior of different
natural and synthetic polymers widening their application
in tissue engineering scaffolds, biodegradable sutures, and
cardiovascular stents.43–45
Natural polymers are intrinsically biocompatible and
biodegradable and hence readily suited for tissue engineer-
ing applications. Natural polymers include polysaccharides
such as chitosan, proteins such as collagen and silk, and pol-
ynucleotides such as DNA and RNA.46 Synthetic polymers
are man-made polymers that might be biodegradable or
nonbiodegradable, bioactive, or bioinert. Nonetheless, they
have better longevity than natural polymers in terms of
shelf life and can be processed and fabricated according to
requirements in their properties. Commonly used synthetic
polymers include poly(lactic acid) (PLA), polycaprolactone
(PCL), and polyhydroxybutyrate (PHB).46
HA/POLYMER BIOCOMPOSITES
Though HA is osteogenic and osteoconductive, its brittle
nature makes it difficult to fabricate it into different shapes
and sizes.47 By combining HA with biodegradable polymers,
both natural and synthetic, best results can be achieved
with both the materials benefitting from each other’s prop-
erties.48 This section summarizes such biocomposite materi-
als made from combining HA with different polymers over
the years and their effect on bone regeneration. Some signif-
icant results are also presented in Table I.
Chitosan
Chitosan (CS) (Figure 1) is a versatile natural biopolymer
obtained from partial deacetylation of chitin under alkaline
conditions, and is a copolymer of glucosamine and N-acetyl
glucosamine forming a linear chain linked by b-1!4 link-
ages.49 The parent compound chitin is a polysaccharide rich
in nitrogen, commonly found in the hard exoskeletons of
crustaceans and other invertebrates. Chitin has been suc-
cessfully extracted from an array of marine sources and
 REVIEW ARTICLE

TABLE I. Summary of Hydroxyapatite/Polymer Biocomposites Used for Bone Regeneration

Type of Experimental
Biocomposite Study Materials/Models Results References

CS/HA In vitro L929 cells, goat bone Excellent proliferation and cell viability 75
marrow stromal cells (100%) of L929. GBMCs showed good
(GBMCs). cell viability, attachment, and prolifera-
tion. Increased ALP activity supported
chondrogeneic differentiation.
CS/HA/Glycerophosphate In vivo Male Wistar rats with 50% bone regeneration in 28 days. No 72
loaded with USSCs critical sized cranial inflammation and better quality bones
defects. than scaffolds without USSCs
Coll/HA (glutaraldehyde In vitro MC3T3 cells, human No cytotoxicity to MC3T3. Supported 91
crosslinked) STRO-1A cells adhesion, proliferation, and differentia-
tion of STRO-1A.
Coll/HA In vivo Beagles with tibial Bone defects were completely filled with 170
defects new bones 8 weeks postimplantation.
Alg/Coll/nano-HA In vitro Fibroblasts and rat cala- Good biocompatibilty to fibroblasts. Good 171
varial osteoblasts morphology, viabilty, attachment, and
proliferation of osteoblasts. ECM
formation observed on scaffolds.
Alg/CS/HA/BMP-2/MSCs In vivo Rat calavarial defects Higher bone mineral density and larger 172
bone formation than controls. Highly
osteogenic and complete closure of bone
defect.
HylA/HA/CaSO4/collagenase In vivo Rat alveolar bone New alveolar bone formation with good 120
defects biocompatibilty and mechanical strength
and accelerated bone remodeling
process.
PCL/CS/nano-ZnHA In vitro hAD-MSCs Increased biocompatibilty, cell adhesion, 173
enhanced proliferation, and protein
adsorption.
PCL/HA In vivo Mouse calavarial defects New bone formation after 6 weeks with 174
perceivable woven bone structure,
osteocytes and lacunae.
P3HB/HA In vivo Female Wistar rats and Biocompatible, enhanced osteoplastic 175
male chinchilla rabbits properties, and slow degradation rate
consistent with the repair of bone
defects.
PLA/HA In vivo Critical sized rat calava- Superior osteoconductivity. Bone formation 176
rial defects with little inflammation and high
resorption rate than b-TCP & DBM
scaffolds.
PLGA/HA In vitro MG63 cells Biocompatibilty, increased 177
osteoconductivity, and osteoblast
adhesion.
PLGA/b-TCP/HA In vivo Rabbit femoral defects Scaffolds were biocompatible, 178
osteoconductive, osteoinductive, and
biodegradable over a period of 12 weeks.

even from cell walls of fungi.50–56 Chitin and its derivatives
have been extensively used due to their biocompatibility,
biodegradability, and the intrinsic antibacterial activity
which are pivotal factors in tissue engineering applica-
tions.57–60 CS can be easily fabricated into porous scaffolds
or hydrogels and also offers the opportunity to improve its
intrinsic antibacterial activity upon chelation with different
metal ions.22,61,62
The earliest attempt at developing a CS/HA biocompo-
site material investigated the development of a bone filling
cement comprising of powdered HA/ZnO/CaO mixed into a
chitosan sol. The resultant paste was found to have a short
setting time, physiological pH, and high compressive
strength.63 Kawakami et al. studied the osteoconductive
potential of CS/HA self-hardening paste in rabbit models
observed the formation of bone-like layer at the site of
application of the bone paste.64 The conjugation between CS
and HA occurs through the interaction of the calcium ions
of HA with the amino groups of chitosan which serves as
the nucleation site for the growth of HA crystals making
them mechanically flexible.65 Researchers were able to
develop a CS/HA/gelatin biocomposite scaffold with a con-
trollable interconnected porous structure and a compressive
strength at par with cancellous bones.66 Under in vitro con-
ditions, CS/nano-HA biocomposite scaffolds seeded with
human fetal osteoblasts were found to secrete osteocalcin

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2017 VOL 00B, ISSUE 00 3

FIGURE 1. Structure of chitosan.
(a marker for bone matrix formation) in large amounts
within a short duration of time in comparison with plain CS
scaffolds demonstrating the benefits of combination with
HA.67 When silver-doped HA and magnetite nanoparticles
were combined with chitosan, the resultant biocomposite
scaffold demonstrated superior bioactivity, biocompatibility,
and antibacterial activity.68 CS/HA biocomposites showed
good integration and promoted bone tissue formation when
tested across different bone defects in rat models, demon-
strating their effectiveness under in vivo conditions as
well.69–71 Keeping in mind the benefits of CS/HA biocompo-
site systems, they also have been used as carriers to deliver
several drugs, stem cells and other growth factors into
hosts.72–77 CS/HA biocomposites have also been used as
coatings to improve the integration of other implant materi-
als such as titanium alloys, achieving success in establishing
bone formation in diabetic patients, where delayed osseoin-
tegration can pose several complications.78–82
Collagen
The concept of developing a collagen (Col)–HA biocomposite
is justified by the fact that they constitute the microarchitec-
ture of the bones.83 There are 28 major proteins that are
termed as collagens and they are divided across six major
groups namely, fibrillar, fibril associated collagens with
interrupted triple helices (FACIT), beaded filament, base-
ment membrane, short chain, and transmembrane collagens.
Type I collagen is the molecule of interest when it comes to
bone regeneration as it is abundantly found in the
bones.84,85. Traditionally collagen has been known to have
excellent biocompatibility, degradation characteristics, and
FIGURE 2. Structure of alginate (M: mannuronic acid; G: glucuronic acid).
4 RAMESH ET AL.
favorable interactions with the cells and other organic mac-
romolecules in our body.86 Col–HA biocomposites match the
cell migration and binding properties of collagen with HA’s
intrinsic bioactivity to promote osteogenic differentiation.87
The addition of collagen to sintered porous HA scaffolds
increased their mechanical strength by causing a reduction
in porosity.88 The increase in the mechanical strength is
attributed to the formation of intermolecular H-bonds
between collagen and HA which then increases the fracture
energy, thus creating a better resistance to failure.89
Col–HA biocomposites show enhanced cytocompatibility
over pure collagen scaffolds, proven by in vitro studies dem-
onstrating improved attachment and proliferation of differ-
ent cell lines such as MG63 osteosarcoma cells, MC3T3
osteoblast precursors, human osteoblasts, and L-929 fibro-
blasts when exposed to different HA content in the biocom-
posites.90–93 Increased torsional strength was observed in
rabbit models implanted with Col/HA biocomposites in tib-
ial defects when compared to ß-TCP controls highlighting
the positive effect of Col/HA biocomposites on the mechani-
cal properties of the bone.94 Col/HA scaffolds loaded with
human mesenchymal stem cells induced their differentiation
without the requirement of any specific growth factors,
making Col/HA biocomposites good option for delivering
biomolecules, drugs, and growth factors.95 The potential of
Col/HA biocomposites has been observed on implant coat-
ings and dental restorations, and products such as Collagraft
is commercially available.96–100
Alginate
Similar to chitosan, alginate (Alg) is a marine-sourced bio-
polymer, obtained from brown algae (Figure 2).101 Glucur-
onic acid and mannuronic acid copolymerize through a-
1,4-glycosidic linkages to form alginates.102 Alginates form
hydrogels that are partly soluble in water upon partial
reaction with divalent cations such as Na21 and Ca21 have
diverse applications in bone regeneration, wound healing,
and drug delivery.103,104 Alginates are biodegradable105
and biocompatible materials as noted by Becker et al., who
also studied their mechanical properties.106 The study
observed the correlation between biocompatibility and the
purity of alginates, where purified forms induced less
adverse response from tissues in comparison with their
less purified counterparts. Purified alginates at 40%
HYDROXYAPATITE/POLYMER BIOCOMPOSITES FOR BONE REGENERATION: A REVIEW
 REVIEW ARTICLE

Hyaluronic acid
Hyaluronic acid (HylA) is a hydrophilic linear unbranched
glycosaminoglycan made up of repeating disaccharide units
of N-acetyl glucosamine and glucuronic acid (Figure 3).111
First isolated from the vitreous humor of cows,112 HylA has
various biomedical applications including bone regeneration
due to its elasticity, biocompatibility, antimicrobial, and
osteoinductive properties.113–116 HylA is involved in key
pathways of cell signaling and plays a role in cell prolifera-
tion and differentiation.117 The effect of HylA on HA was
FIGURE 3. Structure of hyaluronic acid.
studied by Bakos et al. HA/Col composites conjugated with
HylA had a more compact structure and possessed a higher
bend strength than nonconjugated HA/Col composites
compression yielded a compressive strength of 22 kPa showing the positive influence of HylA on the mechanical
while retaining their elasticity, which was significantly properties of HA/Col scaffolds.118 Biocomposite scaffolds
higher than less purified alginates. It was also observed made of calcium sulphate/HA/HylA and encapsulated with
that hydrogels with high glucuronic acid content offered
collagenase were studied for their suitability to repair alveo-
better tensile strength and ductility properties as opposed
lar bone defects in rats. These scaffolds displayed excellent
to the ones with high mannuronic acid content.106,107 To
biocompatibility, compressive strength of 6.69 MPa (day 1)
further reinforce the mechanical properties of alginate,
decreasing to 3.57 MPa (day 28), fall within the range com-
ceramics such as HA can be included. Ca21 precursors in
parable to trabecular bones,119 and a sustained release of
the synthesis of HA bind to the carboxyl sites of sodium
the collagenase enzyme for up to 4 days. Histological analy-
alginates (SA) due to strong electrostatic bonds. The addi-
sis of the rat models revealed that defects filled with cal-
tion of the phosphate precursors to this calcium–alginate
cium sulphate/HA/HylA–collagenase scaffolds showed
complex causes the PO32 4 ions to interact with the calcium significant and uniform regeneration of the alveolar bones
in the complex leading to supersaturation and hence nucle-
8 weeks postimplantation verified by the increased number
ation of HA. The study also established that a moderate
of osteocytes observed on the defect site.120 However, rapid
increase in SA composition led to formation of biocompo-
enzymatic degradation of HylA must be taken into account
sites with increased density and hardness due to decreased
under in vivo conditions, which can deplete the mechanical
porosity and formations of strong bonds within the SA
properties of HylA based composites.121
making SA/HA composites improve mechanically when
compared to pure SA scaffolds.108
Poly-E-caprolactone
Biocomposites containing carbonated nano-HA with
Poly-E-caprolactone (PCL) is a synthetic, nontoxic, biocom-
strontium and sodium alginate (SrCHA) spheres and with-
patible polymer with easily alterable mechanical properties
out strontium (CHA) were subjected to in vitro and in vivo
(Figure 4).122,123 Though biodegradable, PCL (in its high
examinations. In vitro studies for cell viability conducted
molecular weight form) is known for its characteristic slow
by culturing MC3T3-E1 osteoblast precursor cells on the
degradation rate of up to 3 years to completely eliminate
CHA and SrCHA scaffolds revealed that both the scaffolds
itself from the host body.124 PCL is one of the polymers that
were cytocompatible but the SrCHA scaffolds demonstrated
have been approved by the U.S. Food and Drug Administra-
better metabolic activity, cell membrane integrity, and cell
tion to be used as an implantable material125 and PCL/HA
numbers compared to CHA scaffolds and positive control
composites have been prepared over the years using several
(1% SDS) and the results were at par with the negative
processing methodologies, reviewed in detail by Baji
control (cells untreated with scaffolds).109 In vivo study on
et al.126
rabbit models that underwent sinus lift procedures showed
The mechanical properties of PCL/HA scaffolds depend
lower amounts of SrCHA spheres at the end of 12 weeks
on the volume of HA in the composite. Solid PCL/HA scaf-
highlighting their enhanced biocompatibility, osteoconduc-
folds demonstrated an increase in their elastic modulus
tivity, and bioresorbability attributed to the presence of
from 299.3 to 498.3 MPa when the volume of HA was
strontium when compared to CHA spheres.109 Alg/Sr-HA
microspheres loaded with vancomycin showed better drug- raised from 0% to 30%, respectively. A similar trend was
loading efficacy and sustained drug release. These proper-
ties could be altered by increasing the pH of the buffer
solution making them a promising carrier for drug deliv-
ery.110 Although the mechanical properties of Alg/HA com-
posites are sufficient to fill critical sized defects (the
smallest sized tissue defect that does not normally heal by
itself over the lifetime of an animal) in animals, their suit-
ability to address long bone defects is something that
needs to be studied further. FIGURE 4. Structure of polycaprolactone.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2017 VOL 00B, ISSUE 00 5

FIGURE 5. Structure of poly(3-hydroxybutyrate).
observed in porous PCL/HA scaffolds, where scaffolds fabri-
cated with 30% volume HA showed a compressive modulus
that was 2.4 times higher than that of porous scaffolds
without any HA, indicating the improvement of PCL’s
mechanical properties with the addition of HA.127 The effect
of addition of HA to porous PCL influences the behavior of
the latter under in vitro conditions. Porous PCL/HA scaf-
folds showed to improve cell viability and proliferation of
primary fetal bovine osteoblasts than stock PCL scaffolds 11
days into the 21-day observation made by Shor et al. A
small decrease was observed in the cell numbers between
days 11 and 14, which the authors attributed to the inabil-
ity of alamar blue dye to penetrate and stain cells trapped
within the mineralized bone, it recovered its effects at the
end of 21 days. Cell differentiation measured by alkaline
phosphatase activity showed PCL-HA scaffolds clearly over-
shadowing the alkaline phosphatase activity of PCL scaffolds
throughout the observation period.128 PCL–HA matrices
implanted subcutaneously into white rats showed formation
of connective tissues after 7 days of implantation and vascu-
larization after 14 days evoking very low immune response
in comparison with the negative controls. The matrices pro-
ceeded to undergo biodegradation after 21 days proving
their efficacy and cytocompatibility inside in vivo models.129
However, PCL is highly hydrophobic in nature, offers limited
antimicrobial activity, and is known to have a slower degra-
dation rate than poly (L-lactic acid) (PLLA) and poly(gly-
colic) acid (PGA).130,131
Poly-3-hydroxybutyrate
Poly 3-hydroxybutyrate (P3HB) is a crystalline polyester
that belongs to the family of polyhydroxyalkanoates that
occur as a result of enzymatic synthesis by bacteria (Figure
5). P3HBs are accumulated as amorphous water-insoluble
aggregations inside the bacterial membrane and are recov-
ered using various extraction methods.132–135 P3HB make
highly biocompatible, nontoxic biomaterials as low-
molecular-weight P3HBs degrade into nontoxic D-3-
hydroxybutyrate in vivo, a by-product which is observed in
the human blood.136,137 A major drawback of P3HB is its
brittleness, hence it is often copolymerized with polyhydrox-
yvalerate (PHV) to form the copolymer PHBHV to improve
processability.138 P3HB scaffolds reinforced with HA nano-
particles showed varying effects on mechanical properties
of the composite. While a 5% weight of HA nanoparticles
added to P3HB matrix gave the composite maximum
mechanical strength and elastic modulus, nano-HA addition
above 10% showed a decline in the mechanical strength of
the composite, but lead to delayed failure due to the materi-
al’s viscoelasticity.139
6 RAMESH ET AL. HYDROXYAPATITE/POLYMER BIOCOMPOSITES FOR BONE REGENERATION: A REVIEW
P3HB/nano-HA scaffolds show better ability to promote
cell proliferation and differentiation of MG63 osteoblasts com-
pared to P3HB control scaffolds. Cell viability and proliferation
increased with time for both scaffolds but the P3HB/nano-HA
scaffold showed the best results. A similar trend was observed
with respect to alkaline phosphatase activity, where the
P3HB/nano-HA scaffolds showed maximum ALP synthesis; at
the end, a 3-week study indicating their enhanced ability to
support osteoblast differentiation.140 PHB/HA porous scaffolds
loaded with bone marrow cells showed promise under in vivo
conditions when tested subcutaneously on rat models. The
implants were covered with thin connective tissue layer 45
days after implantation. A healthy connective tissue in-growth
consisting of matured osteoblasts, macrophages, and capillaries
was observed in the pores of the scaffolds indicating the site
of active bone formation at the areas adjacent to the implant
site indicating their ability to support bone regeneration.141
However, the mechanical strength of the P3HB/HA biocompo-
sites is a concern as both P3HB and HA are brittle materi-
als.138,142 Hence, the above composite might not be the best
choice of implant owing to questions over their long-term
mechanical stability.
Poly(lactic acid)
Lactic acid undergoes polyesterification reaction to give rise
to poly(lactic acid) (PLA) (Figure 6). Owing to the chirality
of lactic acid, PLAs occur in 4 different forms: poly (L-lactic
acid) (PLLA), poly (D-lactic acid) (PDLA), Poly (D,L-lactic
acid) (PDLLA), and meso-poly(lactic acid).143,144 In general,
PLA exhibits high tensile strength and Young’s modulus.145
However, these parameters vary significantly within the dif-
ferent forms of PLA, which determines their applications.
For example, PLLA is a crystalline polymer that has a tensile
strength of 4.8 GPa and a slow degradation rate of up to 5
years in vivo.146 PLLA has applications in orthopedic fixation
devices and high strength sutures. PDLLA, on the other
hand, is amorphous and has a lower tensile strength of 1.9
GPa. However, PDLLA has a faster degradation rate when
compared to PLLA and hence finds its applications in drug
delivery.144,147 PLA/HA biocomposites are used for both as
scaffold materials and as carriers to deliver drugs and other
proteins into the host.148–150 The percentage of HA in the
PLA/HA biocomposites and the temperature at which they
are processed influences the mechanical properties of the
composite. Calcined PLA/HA composites containing about
80% HA possessed a Young’s modulus of 10 GPa, similar to
that of a cortical bone’s lower limit, and also showed com-
parable results for bending strength and fracture toughness.
However, when the composites were subjected to degrada-
tion in Hank’s Balanced Salt Solution (HBSS), the processing
FIGURE 6. Structure of polylactic acid.

FIGURE 7. Structure of polyglycolic acid.
temperatures became significant. The composites processed
at 1808C for 15 min fractured within 24 h while those proc-
essed at 2208C for 5 min, showed gradual degradation lead-
ing to a gradual decline in the Young’s modulus through a
period of 20 days. While samples with 80–85% HA showed
deterioration in their bending strength and fracture tough-
ness, composites containing 70% HA still managed to retain
their properties to a level comparable to the cortical bone
after a period of 20 days.151 PLA/HA biocomposites show
good biocompatibility toward a wide range of cell lines
including MG-63 osteosarcoma cells, L929 fibroblasts, and
MC3T3-E1 osteoblast precursors proven by the increase in
cell numbers, cell adhesion, expression of bone-specific
markers (osteocalcin), and promotion of osteoblast differen-
tiation by alkaline phosphatase activity.152–156 PLA/nano-
HA/collagen scaffolds seeded with rh-BMP2 (recombined
human bone morphogenic protein) into rabbits radius bone
defect showed promising signs of bone remodeling. The
implant showed cellular infiltration into its pores after
8 weeks and complete integration into the defect site after
12 weeks of implantation showing the formation new bony
(trabecular) tissues and replacement of the composite dem-
onstrating the composite’s biodegradability and bone regen-
erative potential.148
Poly(glycolic acid)
Poly(glycolic acid) (PGA) (Figure 7) is a semicrystalline poly-
mer with a high tensile modulus of 12.5 GPa and is insoluble
in most organic solvents.144 PLA and PGA were the first syn-
thetic biodegradable polymers to be approved by the U.S.
FDA to be used in the production of resorbable sutures.157
PGA has excellent mechanical properties and degrades faster
than PLA.130 A 1986 study found PGA implants to be biocom-
patible when implanted in cortical and cancellous bone
defects of rabbits. After 12 weeks of implantation, the
implants at the cancellous sites showed maximum degrada-
tion as opposed to partial degradation seen in the cortical
bones without producing an inflammatory response.158 How-
ever, more recent studies found that the degradation of PGA
produces glycolic acid under physiological conditions and
also some acidic by-products that are known to evoke a
strong inflammatory response.159 PGA/HA composite scaf-
folds showed good resorption in vitro and possessed the
FIGURE 8. Structure of PLGA (n: polylactic acid; m: polyglycolic acid).
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2017 VOL 00B, ISSUE 00
REVIEW ARTICLE
necessary porosity required for cellular infiltration and
attachment.160 However, the stiffness of PGA146 and the risk
of inflammation due to the acidic nature of its degradation
by-products159 have restricted their biomedical applications.
Poly(lactide-co-glycolide)
PLGA is known for its biocompatibility and favorable mechani-
cal properties.161,162 PLGA (Figure 8) is seen as the answer to
the disadvantages of both PLA and PGA. By copolymerizing
the two, researchers were able to rectify the problems of pre-
mature degradation by adjusting the ratio of the homopoly-
mers, which allowed some control over their degradation
rates. PLGA containing 75% PGA is known to be amorphous
and hydrolytically unstable, thereby degrading faster. Upon
degradation PLGA breaks down into lactic acid and glycolic
acid which are the usual by-products of human metabolism
and can be excreted by urine.144,159 Apart from drug delivery
where it is mainly used, PLGA systems are used in the areas
such as bone, skin, cartilage, and nerve regeneration.163–166
HA reinforces the mechanical properties of PLGA. A study by
Fisher et al. observed that addition of 30% nano-HA to PLGA
matrices formed composites that had three times the strength
of the polymer by itself and also a sixfold rise in the compressive
modulus was observed. The scaffolds were designed to be
injectable and their injection into femoral heads of porcine mod-
els improved the strength and compressive modulus of the tra-
becular bones from 3.5 to 5.9 MPa and 81 to 180 MPa,
respectively.167 PLGA/nano-HA composites present excellent in
vitro behavior. Addition of nano-HA (10 wt %) to PLGA
improved the mechanical properties and the water absorption
ability of the PLGA/HA composite. PLGA/(10 wt %) nano-HA
scaffolds cultured with mesenchymal stem cells showed higher
cell numbers, better cell adhesion, increased cell proliferation,
and better alkaline phosphatase activity compared to PLGA con-
trol scaffolds.168 PLGA/HA scaffolds implanted into mandibular
defects of rabbits showed the presence of bone trabeculae with-
out any ongoing osteoblast activity 21 days after implantation,
while PLGA control scaffolds presented high number of osteo-
blasts on their surface indicating increased cellular activity to
generate more bone trabeculae. PLGA/HA scaffolds showed
only scarce single bone trabeculae bordered by osteoblasts at
the end of 6 weeks, but transformed to mature bone tissues at
the end of 12 weeks signaling the end of bone forming activity.
This was in contrast to the PLGA control scaffolds that took 48
weeks to lay down mature bone tissues to fill the defect and
show no further signs of active bone formation. The PLGA/HA
scaffolds also tested positive for the bone markers osteonectin
and osteopontin at the end of 6 weeks indicating progressive
bone formation under in vivo conditions.169
CONCLUSION
By tapping the potential of biomaterials and cells, researchers
have been able to achieve bone regeneration and replace-
ment. It is evident from the above review that the design of
the “ideal” bone substitute greatly depends on factors such
as the intrinsic properties of the materials and the processing
methodology, which have a great influence on its
7
applications. HA/polymer biocomposites are seen as a step in
the right direction toward achieving it. However, research has
been restricted to small animal models with varying degrees
of success. It is to be seen whether the biocomposites can
fulfill their potential when implanted in long bone defects in
humans where they are subject constant heavy loads. How-
ever, with the constant advancements in the technology avail-
able to the researchers, biocomposites are expected to take a
central role in promoting bone regeneration.
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12 RAMESH ET AL. HYDROXYAPATITE/POLYMER BIOCOMPOSITES FOR BONE REGENERATION: A REVIEW