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Current Protein and Peptide Science, 2017, 18, 850-863

REVIEW ARTICLE
ISSN: 1389-2037
eISSN: 1875-5550

Bioactive Molecule-loaded Drug Delivery Systems to Optimize Impact

Factor:
2.441

Bone Tissue Repair

BENTHAM
SCIENCE

João Augusto Oshiro Jr.1, Mariana Rillo Sato1, Cassio Rocha Scardueli2 , Guilherme José Pimentel
Lopes de Oliveira2, Marina Paiva Abuçafy1 and Marlus Chorilli1,*

1
Faculdade de Ciências Farmacêuticas, UNESP-Univ Estadual Paulista, Araraquara-Jaú, Km 1, Araraquara, 14800-
903 SP, Brazil; 2Faculdade de Odontologia, Universidade Estadual Paulista (UNESP), Humaitá, 1680, Araraquara,
14801-385 SP, Brazil

ARTICLE HISTORY
Received: February 13, 2017
Revised: March 17, 2017
Accepted: March 21, 2017
Current Protein & Peptide Science
Abstract: Bioactive molecules such as peptides and proteins can optimize the repair of bone tissue;
however, the results are often unpredictable when administered alone, owing to their short biological
half-life and instability. Thus, the development of bioactive molecule-loaded drug delivery systems
(DDS) to repair bone tissue has been the subject of intense research. DDS can optimize the repair of
bone tissue owing to their physicochemical properties, which improve cellular interactions and enable
the incorporation and prolonged release of bioactive molecules. These characteristics are fundamental to
favor bone tissue homeostasis, since the biological activity of these factors depends on how accessible
they are to the cell. Considering the importance of these DDS, this review aims to present relevant in-

DOI:
10.2174/1389203718666170328111605
formation on DDS when loaded with osteogenic growth peptide and bone morphogenetic protein. These
are bioactive molecules that are capable of modulating the differentiation and proliferation of mesen-
chymal cells in bone tissue cells. Moreover, we will present different approaches using these peptide and
protein-loaded DDS, such as synthetic membranes and scaffolds for bone regeneration, synthetic grafts,
bone cements, liposomes, and micelles, which aim at improving the therapeutic effectiveness, and we
will compare their advantages with commercial systems.

Keywords: Bioactive molecules, drug delivery systems, osteogenic growth peptide, bone morphogenetic protein, tissue repair.

1. INTRODUCTION treatments with greater predictability of bone formation in

Bone healing is a complex process and difficult stage of
reconstruction in medicine [1]. Although this tissue has the
potential to self repair, this process can be committed in ma-
jor defects that are associated with the absence of bone walls
[2]. Critical defects are bone losses that do not heal sponta-
neously, due to the inability of bone progenitors to migrate
long distances and difficulty in obtaining a complete local
revascularization, impairing tissue maintenance through lack
of nutrients and oxygen. Therefore, successful bone healing
is intimately connected with vascularization, stability at the
defect site, and with the presence of bone cells. However,
critical defects require biomaterials with properties that help
the bone formation and can be expressed by different
mechanisms of action (osteoinduction, osteocondution, and
osteogenesis) [3].
Several studies show different strategies to help bone
formation, with diverse biomaterials and techniques, and
consequently, miscellaneous results [4]. The search for
*Address correspondence to this author at the Faculdade de Ciências Far-
macêuticas, UNESP-Univ Estadual Paulista, Araraquara-Jaú, Km 1, Arara-
quara, 14800-903 SP, Brazil; Tel: +551633016998; Fax: +551633016900;
E-mail: chorilli@fcfar.unesp.br
critical defects is the target of new studies [4, 5]. Three es-
sential elements of bone regeneration (osteogenesis, osteoin-
duction, and osteoconduction) are necessary for successful
bone healing, acting separately or in synergy. The presence
of all these biological elements involved in bone formation
permits higher success rates [2].
The osteogenic characteristic of progenitor bone cells
(osteoblasts and osteocytes) shows a higher activity for bone
formation, with viable bone cells directly linked to produc-
tion or maintenance of bone matrix [6]. The autogenous bone
is the single graft that presets this property of bone formation
at the moment [7]. Similarly, some molecules exhibit an ac-
tivity that stimulates the differentiation of mesenchymal or
undifferentiated cells into osteogenic cells, which are capa-
ble of producing bone tissue (osteoinduction) [3]. These
molecules can be found in autogenous bone, or isolated and
incorporated in different bone substitutes [8]. Furthermore,
many grafts or biomaterials possess only the characteristics
for maintenance of skeleton for bone formation, allowing the
presence of blood (osteoconduction) and consequently cells
and nutrients. Osteoconduction examples are inorganic bone
and scaffolds (without inductive molecules) [2].

1875-5550/17 $58.00+.00 © 2017 Bentham Science Publishers

Bioactive Molecule-loaded Drug Delivery Systems
In bone grafting in medical or dentistry areas, the autoge-
nous bone is considered the gold standard [9], as it shows all
properties related to bone formation. However, due to bone
removal from a donor area associated with greater morbi-
dity, limited tissue availability, additional surgical pain, and
advance of biotechnology, some biomaterials can be of inte-
rest, especially when associated with bioactive molecules,
leading to similar properties and results to the autogenous
bone [10-12].
The use of bioactive molecules for bone formation has
arisen and gained attention; when associated with their bin-
ding proteins, they regulate cell functions (proliferation, mi-
gration, and differentiation) of bone cell lineages. Thus, se-
veral molecular mediators, such as proteins and peptides, can
optimize the bone formation process [13, 14]; among them,
bone morphogenetic proteins (BMPs) and osteogenic growth
peptide (OGP) stand out.
1.1. Bone Morphogenetic Protein
Proteins extracted from bone tissue can be used to induce
the formation of bone and cartilage. In 1965, Urist identified
an osteoinductive protein by demonstrating that mineralized
bone matrix implanted in muscular tissues induces the for-
mation of cartilage and bone tissue [15]. These findings con-
firmed the presence of bioactive factors in demineralized
bone, as referred to BMPs [16]. However, it was only in
1988 that Wang et al. reported the isolation of BMP from
bovine bone tissue [17]. Consequently, its DNA was cloned,
and by using peptide or amino acid sequence homology to
human database, it was considered a new member of the
transforming growth factor β (TGF-β) family [18, 19].
BMPs are released during repair and remodeling processes,
due to degradation of inorganic bone by osteoclasts. There-
fore, in vivo studies with animals showed promising results
on the role of BMP in bone synthesis without association
with osteoconductive biomaterials [20]. So far, 20 different
types of BMPs with distinct characteristics and roles have
been described; nevertheless, they all share osteoinductive
characteristics that induce differentiation of mesenchymal
BMP
B MP

P
P
P

Smad P Osteoblast

P Smad
DNA
Mesenchymal undifferentiated cell
Fig. (1). Possible mechanism of action of BMPs in bone formation.
Current Protein and Peptide Science, 2017, Vol. 18, No. 8 851
cells into osteoblasts [21]. However, only BMP-2 and BMP-
7 have been approved for clinical use [15].
Although BMPs are multifunctional proteins, recombi-
nant human BMP-2 (rhBMP-2) plays a major role in the re-
pair of fractures, and the absence of this protein causes de-
velopment abnormalities in bone and impairs the healing of
fractures [22]. BMP-7 stimulates the synthesis of erythro-
poietin, a hormone that controls the production of erythro-
cytes from precursors in the bone marrow. However, so far,
it is still not totally understood how BMPs regulate the for-
mation and repair of tissues [15]. It is known that, being
soluble molecules and acting as local signaling proteins,
BMPs are involved in inducing specific markers of os-
teoblast cell differentiation [23] and can induce a differenti-
ated phenotype in some types of cells [24]. Therefore, BMPs
can regulate growth, differentiation, chemotaxis, and apopto-
sis, and play pivotal roles in the morphogenesis of a variety
of tissues and organs. Nonetheless, the activity of BMPs can
be controlled at different levels, intracellularly through Smad
proteins (specific receptors at the surface of the cell that
transduce signals to transcription factors that activate spe-
cific genes) [25], and tissue-specific transcription factor (ba-
sic helix-loop-helix factor and its binding sequence (E-box)).
Extracellularly, many protein inhibitors can bind BMPs and
inhibit their binding to cell surface receptors [25, 26]. All
these mechanisms act in a controlled and limited manner,
and BMP expression is only observed in localized site
(Fig. 1).
In vivo studies in animals showed that BMPs were able to
induce bone and cartilage, when implanted in ectopic tissues
[18], or improve bone formation [27, 28]. Although BMP
potential to induce bone formation has been demonstrated in
preclinical studies, the findings in clinical trials are limited
but show positive results for bone formation [29-33]. How-
ever, although positive, many factors are uncertain and limit
the clinical use of BMPs, such as fast degradation, high
costs, high doses, osteolysis, high inflammation, and ectopic
bone formation. To limit fast liberation and consequently
lesser action, BMP's can be associated to release vehicle that

P
P Smad
852 Current Protein and Peptide Science, 2017, Vol. 18, No. 8
difficult the degradation and can be released slowly, increas-
ing its biological activity [34, 35]. Currently, the use of high
doses is to compensate BMP short half-life [36]. Thus, some
side effects may be present, such as bone reabsorption (oste-
olysis), edema, and ectopic bone formation that need extra
medical treatments [37, 38].
The beneficial effects of BMPs in regeneration or repara-
tion of bone defects are known; however, the mechanisms
and their potential collateral effects remain undiscovered,
thus cautious use and more studies are needed.
1.2. Osteogenic Growth Peptide
OGP is a sequence of 14 well-preserved amino acids
(Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-
Gly). Its sequence is similar to C-terminal histone H4 (89-
102), and was isolated from the blood during bone remode-
ling or bone marrow regeneration [14]. The pentapeptide in
its cleaved form (OGP 10-14) has demonstrated an even
greater potential for bone synthesis. An analysis of the struc-
ture indicates that its activity is related to the phenolic group
of tyrosine (Tyr10) and the aromatic ring of phenylalanine
(Phe12) [39].
OGP is present physiologically in mammalian serum in
micromolar concentrations; its small size and linearity make
it susceptible to proteolysis, and the low concentrations of
OGP in vivo suggest the presence of a circulating protein
binding partner for OGP. This specific or non-specific pro-
tein provides protection against proteolysis and acts as a
“system” that releases low concentrations of OGP into the
circulation [40-44]. It is believed that the protein binding
partner of OGP at higher concentration is α2-microglobulin
(α2M). After cleavage of complex α2M-OGP, the peptide is
cleaved proteolytically [41, 42].
Similar to BMPs, OGP has been shown to be a potent os-
teoinducer due the stimulation of the differentiation of undif-
ferentiated mesenchymal cells into osteoblasts, preventing
the formation of condroblasts and adipocytes [40]. This os-
teoinductive effect was demonstrated in studies with mesen-
chymal stem cells derived from bone marrow with OGP, as
well as OGP (10-14) [41-44].
The mechanisms by which OGP induces osteoblast dif-
ferentiation and increases osteoblastic activity are unknown.
Different studies demonstrate several mechanisms of action
of OGP on bone formation (Fig. 2). It has been suggested
that the stimulation of osteoblastic differentiation may occur
via the RhoA/ROCK pathway [45] and CDK2/cyclin A [46].
The effects of OGP on osteoblastic activity may be due to
the upregulation of MAPK and ERK 1/2 pathways [47].
OGP regulates the expression of various TGF-β such as
TGF-β1, TGF-β2, and TGF-β3 [48]. In vitro studies have
shown that OGP regulates cell proliferation and alkaline
phosphatase (ALP) activity in osteoblastic cell lines through
self-regulatory feedback mechanism [49, 50]. Another possi-
ble mechanism of action of OGP is the induction of osteo-
protegerin (OPG) production by osteoblasts [51]. This action
interferes with the RANK/RANKL/OPG pathway, involved
in the formation of osteoclasts.
Preclinical studies have shown that OGP improved the
healing of bone fractures [52] and the osteodistraction of
Oshiro Jr. et al.
long bones [53]. Furthermore, it was also demonstrated that
OGP promoted the maintenance of bone tissue structure in
ovariectomized rats, indicating a possible use of this peptide
in osteoporosis therapy [54-57].
2. APPLICATIONS OF BIOACTIVE MOLECULE-
LOADED DRUG DELIVERY SYSTEMS TO OPTI-
MIZE BONE TISSUE REPAIR
Despite the promising results of isolated administrations
of bioactive molecules, they are often unpredictable because
their biological half-life is short, and they show low long-
term stability, are high cost treatment, and have low tissue
specificity, requiring administration of high doses, which in
some cases may exceed the potential carcinogenicity dose
[58].
These disadvantages led researchers to seek new strate-
gies for the administration of these substances. One solution
to these problems is the development of vectorization sys-
tems capable of transporting the active substance, improving
its distribution in specific tissues, and limiting the side ef-
fects by preserving the efficacy of the active compound [51,
55].
Recently, in medical and other fields, the interest in new
technologies for drug delivery has increased. Thus, the com-
bination of bioactive molecules with DDS presents further
advantages compared with conventional devices, such as
decreased toxicity, prolonged half-life in the bloodstream,
gradual and controlled release of the drug, safe (no local
inflammatory response), and adequate (small dose require-
ment) administration, targeting specific sites, and the ability
to incorporate lipophilic and hydrophilic substances [58-60].
The functionalities of these DDS (protection against degra-
dation, targeting, controlled release, easier cell penetration)
are due to their internal structure and surface properties that
give them better physicochemical properties, such as shape,
composition, molecular weight, identity, purity, stability, and
solubility [59], allow an increased therapeutic efficacy of
many drugs by modifying the pharmacokinetics or biodis-
tribution of the active molecules, have lower costs, and
maintain the concentration of these factors within the thera-
peutic range over a longer period of time [60]. Thus, the os-
teoprogenitor cells migrate to the target site and differentiate
into osteoblasts, resulting in a major impact on current thera-
pies for bone tissue regeneration [20, 58].
These physicochemical properties allow the development
of DDS to be used in different approaches in clinical prac-
tice, such as synthetic membranes and scaffolds, synthetic
grafts, bone cements, liposomes, and micelles. Therefore, in
this section, we mainly introduce different approaches of
DDS with OGP and BMP aiming at therapeutic effectiveness
in bone formation.
2.1. Synthetic Membranes and Scaffolds
The regeneration of a tissue needs to be oriented to result
in a new and functional physiology. The guided bone rege-
neration (GBR) technique assists the restoration of bone tis-
sue by preventing competition between bone tissue cells and
soft tissue cells [61].
Bioactive Molecule-loaded Drug Delivery Systems Current Protein and Peptide Science, 2017, Vol. 18, No. 8 853

Osteogenic
Osteogenic Growth Peptide
Growth Peptide

Alkaline
Alkaline
Regulates
Regulatesexpression
expression Phosphatase Osteoprotegerin
Active
Active Phosphatase Osteoprotegerin
TGF-b
TGF-b ERK1/2 (OPG)
MAPKand
MAPK and ERK1/2 (OPG)

Mesenchymal
PO 3434-- OO OPOP OO RANKL
RANKL
MesenchymalCell
Cell
Regulation
Regulation transcription
transcription OO
factors
factorsof
of osteoprogenitory
osteoprogenitory
cells
cells OPG
OPG
RANK
RANK
Mineralization
Mineralizationofof
the bone
bone matrix
matrix
Osteoprogenitor
Osteoprogenitor
Osteoprogenitorcell
Osteoprogenitor cell cell
cell

Increasednumber
Increased numberofof Osteoclasts
Osteoclasts
Osteoblasts
Osteoblasts

Bone Formation
Bone Formation

Fig. (2). Possible mechanisms of action of OGP in bone formation.

The principle was first described in 1959 by Hurley and
contributors in an experimental treatment that aimed for a
mechanical barrier, to isolate the tissues in a spinal fusion
surgery [62]. However, this pioneering study did not imme-
diately lead to a broad clinical application in patients. The
clinical potential of GBR was only recognized in the early
1980s when Karring et al. evaluated synthetic membranes
for periodontal regeneration in several clinical trials [63].
A few years later, based on the promising results of these
studies, Dahlin et al. tested a polytetrafluoroethylene mem-
brane to isolate the connective tissue of defects created in
bone tissue of rat mandibles, and defects without membrane
were used as control group. The results showed that the con-
trol group presented lower bone growth (2.2 mm) when
compared to the defects treated with the membrane (3.8
mm). Another factor observed in the control group was the
presence of soft tissue cells within the bone defects. These
results reveal the importance of the use of synthetic mem-
branes in the regeneration of bone tissue [64].
Commercially, synthetic membranes can be made of
polytetrafluoroethylene, which is classified as non-
resorbable, or polylactic acid, collagen, or polyglatin, which
are classified as resorbable [65].
The non-resorbable membranes require a second surgical
procedure to remove them, which may compromise the suc-
cess of the procedure, since the second surgical intervention
may disturb newly formed tissues [66]. In addition, it may
result in patient discomfort, increased cost, and post-surgical
infection [67]. Resorbable membranes, however, have the
advantage of eliminating this second surgical phase, as well
as the trauma to the neoformed tissues. Nevertheless, they
present invasion of soft tissue cells resulting from membrane
degradation and instability at the surgical site (non-fixation)
[65].
To overcome the disadvantages of resorbable and non-
resorbable membranes, researchers are looking to developing
membranes from new materials [68], with characteristics
such as physical barrier, cell affinity (osteoconduction), and
promoter of bone growth (osteoinduction) (Fig. 3) [51, 69-
71]. For this purpose, it is necessary that the membranes are
able to incorporate and release controlled bioactive mole-
cules that are capable of stimulating migration and differen-
tiation of mesenchymal cells, such as BMPs, fibroblast
growth factor, TGFs, and bone growth peptide among others
[71, 72]. Another advantage of these materials is the ability
to maintain the therapeutic concentration of these bioactive
substances, since BMPs have a low plasma half-life (2-4
hours) when administered alone, they must however be on
site for a period of 4 weeks to induce a successful bone re-
generation process [72].
Bioactive electrospun fibers based on poly(lactide-co-
glycolic acid) (PLGA) by immobilizing bone-forming pep-
tide 1 (BFP1) derived from the immature region of BMP-7
were developed by Lee et al. in order to achieve an ideal
854 Current Protein and Peptide Science, 2017, Vol. 18, No. 8
  / + 
+ #*
&'
Fig. (3). Synthetic membrane able to incorporate and release bioactive molecules acting as: physical barrier and as promoter of bone growth
(osteoinduction) [69].
barrier synthetic membrane [73]. The membrane was tested
in a 4 mm critical-sized calvarial bone defect mouse model.
The authors observed that after 8 weeks, the bone volume
formed in the defects with membrane was statistically higher
than that of the control group. The semi-quantification of
bone volume revealed that the area was approximately 20%
in the defect-only group and 57.59 ± 15.24% in the group
implanted with a membrane. The results suggest that mem-
branes are interesting substances for the treatment of critical-
sized bone defects [73].
Pigossi and co-workers [55] prepared bacterial cellulose-
hydroxyapatite (BC-HA) membranes associated with OGP
and OGP (10-14) at the concentration of 10-9 mol L-1 to treat
critical-size calvarial bone defects (4-mm diameter) in mice.
It was concluded that a high percentage of bone formation
was observed after 60 days; however, this effect was associ-
ated with BC-HA, and the addition of OGP had low effect
[55].
Policastro et al. [42] studied amino acid-based poly(ester
urea)s (PEU), degradable polymers functionalized by tether-
ing OGP to tyrosine-based monomer subunits to improve
repair of bone defects in rats. The authors observed that after
4 weeks, PEU-OGP resulted in matrix mineralization with-
out inflammatory response. Moreover, migration of blood
vessel within the material was observed, demonstrating the
osteogenic and angiogenic capacity of these materials [42].
Lopes et al. [74] studied the effect of laser light in bone
defects (diameter = 5 mm) filled with BMP on a commercial
membrane. The results showed that the association of these
techniques improved significantly improved bone formation
when compared to the defects without laser therapy [74].
Oshiro Jr. et al.
+ 
+ 
&)' &'
+  
Among membranes for bone regeneration, scaffolds have
emerged as the new generation membranes with features
suitable for tissue regeneration. These biomaterials have the
ability to act as a template that allows the cells to replace it
by newly formed tissues. One of the strategies used in the
development of these materials is the immobilization of bio-
active molecules with osteoinductive capacity in order to get
better results [75, 76].
Therefore, to establish more effective treatments of large
bone defects, Kolambkar et al. [76] reported the use of an
alginate-based hybrid system with rhBMP-2. These authors
analyzed the release of bioactive BMP-2 (500 ng) from this
system. The results demonstrated that 71.2 ± 3.8 ng was re-
leased within 21 days; however, 98.6% of BMP-2 was re-
leased with 7 days. Critical-sized (bilateral 8 mm segmental
defects) femoral segmental defect in a rat model was used to
verified bone formation. They observed that after 12 weeks,
the hybrid system with bioactive rhBMP-2 (37.65 ± 2.22
mm3) had significantly more bone volume relative to the
system alone (3.96 ±1.40 mm3). These results indicate that
these systems can be used for bone repair in a GBR proce-
dure [76].
Lee et al. [77] bound BMP-2 with heparin-binding pep-
tide amphiphile (HBPA) nanofibers, which were introduced
in an absorbable collagen material, aiming for bone regen-
eration in a rat critical-size femoral defect model (5 mm). It
was verified that a decrease in the release of BMP-2 oc-
curred when nanofibers contained heparan sulfate (HS) com-
plex. The amount released after 8 days reached approxi-
mately 84.0 ± 18.9% of rhBMP-2-HBPA without HS com-
plex, while the release of protein reached 34.5 ± 8.1% with
Bioactive Molecule-loaded Drug Delivery Systems
HBPA-HS. The in vivo bone formation was evaluated after 6
weeks and showed a significant increase in the bone volume
in defects treated with HBPA-HS-BMP-2-collagen (20.3 ±
4.2 mm3) relative to the control group of untreated animals
(2.4 ± 0.1 mm3), those treated with collagen scaffolds with
BMP-2 (8.2 ± 1.7 mm3) or without (3.5 ± 0.8 mm3) it [77].
Aiming to improve the chemical conjugation methods be-
tween polymer and bioactive molecules (requiring multistep
and complicated procedures), Ko et al. [78] conducted a
study in which BMP-2 was efficiently immobilized onto
PLGA scaffolds by a single step of polydopamine-mediated
coating. The system was transplanted into a mouse model of
calvarial bone defect (diameter: 4 mm). The results revealed
that after 8 weeks, the group treated with PLGA-
polydopamine-BMP-2 exhibited enhanced bone regeneration
(~ 60%) while the bone formation was ~ 38% with no treat-
ment [78].
Other studies tested OGP as an integral part of tissue en-
gineering and showed conflicting results. The use of OGP
and OGP (10-14) in coating of biocellulose membranes did
not promote greater formation of bone tissue relative to the
control membranes, despite the induction of greater expres-
sion of bone formation biomarkers [55]. OGP associated
with crosslinked PEU polymer-based scaffolds showed bio-
compatibility and enhanced mechanical properties and bioac-
tivity (reabsorption and revascularization) when these scaf-
folds were inserted in the dorsum of rats [56]. It also showed
that the addition of OGP in PLGA scaffolds improved the
healing of segmental bone defects in long bones [57]. Fur-
ther studies are needed to identify the best parameters of
using OGP as a coating of membranes and associated with
biomaterials. The concentration of this peptide, the ideal type
of scaffold or membrane, and the type and concentration of
cells that should be used with OGP as a component of tissue
engineering remained to be determined. After obtention of
these data, clinical trials that evaluate the effect of OGP in
bone repair can be performed.
However, the three-dimensional (3D) printing technology
represents an alternative to fabrication of GBR membranes,
which traditionally used solvent casting and it is difficult to
make a freeform membrane with a given thickness, pore size,
and external shape, factors that contribute to the amount of
bone formed, since they influence the release of the active
substance and the adhesion/penetration of cells into the ma-
terial [79]. Thus, Shim et al. [80] investigated a 3D printing-
based PCL/PLGA/β-TCPGBR membrane that incorporated
BMP-2 (5 µg mL−1). This system can be used to control drug
release. In vitro studies demonstrated that BMP-2 release
reached ~ 25.5% in 24 h and was sustained for 28 days. To
evaluate bone formation, the membrane was tested in an 8-
mm critical-sized calvarial bone defect rabbit model. The
results showed that after 8 weeks, the bone volume formed in
the defects with membrane loaded with BMP-2 represented~
60%, which was statistically higher than that of the control
group (15.24%) [80]. These results demonstrated that better
conditions in the manufacturing process (3D printer use) can
help the development of biomaterials with optimized specif-
ics (thickness, external shape and number of pores standard-
ized), sustained release of bioactive molecules and high ca-
pacity to bone formation.
Current Protein and Peptide Science, 2017, Vol. 18, No. 8 855
2.2. Synthetic Bone Grafts
One of the most significant advances in biomaterials over
the last few years has been in the field of bone synthetic graft
substitutes. They have been extensively studied in order to
be substituted to autografts, due to some disadvantages such
as poor bone quality, inadequate amount of bone available,
and a possible immunogenicity that limits the use of these
grafts. Thus, synthetic bone graft has become a potential
material for clinical applications in different medical areas.
There are characteristics that synthetic bone grafts should
have, such as being of various shapes and sizes with suitable
mechanical properties to be used in different sites, biocom-
patible, osteconductive, and preferably being resorbable and
replaced by new bone formation [81] (Fig. 4).
Synthetic bone grafts should act as scaffolds for bone
cells, providing bone growth inside the material. Therefore,
the scaffold must have adequate mechanical properties and
be highly porous, and these pores have to be interconnected.
Several physicochemical features are considered fundamen-
tal for successful tissue engineering such as surface chemis-
try and roughness, mechanical properties, topography, and
interfacial free energy [82, 83]. Reabsorption of biomaterial
is linked to several factors, such as particle size, porosity,
and crystallinity [84]. Particles with nanometric size are re-
absorbed faster than micrometric size ones, and highly crys-
talline structures are more resistant to resorption than amor-
phous ones.
Bone growth proteins, such as OGP and BMP, marked
the history because they were two of the first materials to be
produced by recombinant genetic technology. These materi-
als have given rise to a new generation of synthetic and bio-
active bone graft substitutes.
Shuqiang et al. [57] evaluated the effect of locally ap-
plied OGP incorporated into a synthetic bone graft, PLGA
scaffolds, in healing bone defects in rabbits (1.5 cm segment
defect was made in the right radius using a small saw). OGP
was incorporated into PLGA, and was released during 7
days. The in vitro result revealed that 90% of peptide total
quantity was released after a week. After 12 weeks, the rate
of development of bone bridging was significantly higher in
the group with OGP than in the control group. The experi-
ments concluded that OGP release from the synthetic bone
graft at the site of bone defect resulted in faster and more
complete bone healing, and suggested that the porous struc-
ture and pore size of material were appropriate for carrying
OGP [57].
Song et al. [85] studied a vancomycin-bearing synthetic
bone graft that delivers rhBMP-2 in rats. They developed a
poly(2-hydroxyapatite methacrylate)-nanocrystalline hy-
droxyapatite (pHEMA-nHA) synthetic bone graft with 3 µg
rhBMP-2 preabsorbed per graft. The experiments were done
with 5-mm femoral defect in SASCO-SD male rats (289-300
g of body weight). They observed that after 12 weeks,
pHEMA-nHA-vancomycin without rhBMP-2 resulted in
partial bridging of defect, and full bridging with mineralized
restoration was achieved with rhBMP-2. The complete com-
posite repaired the 5-nm rat femoral segmental defects. The
success of this strategy requires that pHEMA-nHA effec-
tively retains and releases vancomycin and rhBMP-2 without
856 Current Protein and Peptide Science, 2017, Vol. 18, No. 8
-0 +%- 1 % +%- -(%-
1 +  *
4 #**   5
+ 
&%  +'
Fig. (4). Advantages of the bone grafts with bioactive molecules to bone regeneration.
compromising the structural integrity of the graft or its abi-
lity to promote bone cure [85].
Smith et al. [86] analyzed an animal evaluation of syn-
thetic bone graft, a paste of chitosan glutamate and hy-
droxyapatite. Cranial defects of 8-mm diameter were made
in rat calvaria, and the defect evaluation was studied with
pure paste and paste with 40 µg of BMP-2. Rats were sacri-
ficed at intervals during 18 weeks. Calvaria containing a
defect were harvested, and bone mineral density was deter-
mined by energy X-ray absorptiometry. This study demon-
strated that a paste of chitosan glutamate and hydroxyapatite
could be used to deliver BMP-2, and the bone mineral den-
sity and histology data confirmed that this paste containing
BMP-2 formed mineralized bone and showed osteoblastic
activity [86].
Tagil et al. [87] investigated a synthetic bone graft com-
posed of a tricalcium phosphate hydroxyapatite scaffold that
can be used with BMP-7 (25 µg). They performed a 6-mm
femoral nailing, and the defects were grafted after 4 week.
After 11 weeks, the bones were explanted for evaluation
with radiography, micro-CT, and infrared spectroscopy. Iso-
lated scaffolds without BMP-7 did not heal any defects,
whereas treatment with BMP-7 had greater volume of highly
mineralized bone and higher volume of fraction. It was con-
cluded that synthetic scaffold with BMP-7 can heal a critical
size defect [87].
2.3. Bone Cement
Cement is a compound made of two materials, a liquid
and a powder form, which when mixed make a moldable
paste that can be adapted to variable surfaces of imperfect
bone structures [88, 89].
Oshiro Jr. et al.
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Cement has several attractive advantages compared to
other materials, such as the ability to promote an adjustable
fit in the fixation of the prosthesis, easy handling, adherence
to the hard tissue, hypoallergenic or non-carcinogenic char-
acteristics and increased resistance to loading by uniformly
distributing the tensions between the prosthesis and the bone
[90-92]. Moreover, it is able to induce osseointegration and
act as a release system of several molecules, which have a
synergistic effect that increases the effectiveness of the mate-
rial in bone repair (Fig. 5) [93-95].
One of the most promising molecules that can be carried
by cement is the osteogenic peptide, such as OGP and BMP,
which are used as an interesting alternative because they
have a broad spectrum of activity and binding to specific
sites of extracellular matrix proteins and can be transported
with other drugs [96].
Studies have evaluated the in vivo behavior of OGP and
BMP in different routes of administration, such as systemic
administration [96] and topical administration [57]. This
analysis showed an important result for the route of admini-
stration of these molecules, revealing a more effective thera-
peutic effect when applied locally, which highlights the im-
portance of materials such as bone cement, which can be
used as carrier of the biomolecule, allowing its retention in
the defect site for prolonged periods of time with better re-
sults than via the intravenous route [42].
Kroese-Deutman et al. [97] studied the osteoinductive
capability of bone calcium phosphate (Ca-P) cement loaded
with rhBMP-2. The cement discs were loaded with 30% of
rhBMP-2 in vitro and implanted subcutaneously in rabbits
for 2 and 10 weeks. Thereafter, the histological analysis re-
vealed bone formation in rhBMP-2-loaded cement discs with
Bioactive Molecule-loaded Drug Delivery Systems
+ 
&.;# 4 /# 4 +  '
  
#)  
Fig. (5). Advantages of the bone cement with bioactive molecules to bone formation.
pores filled at around 18% after 10 weeks. The fraction of
rhBMP-2 retained in the discs in subcutaneous sites was less
than 10% after 10 days, probably too low to induce bone
formation. This suggests that although rhBMP-2 is a power-
ful osteoinductive material in other locations, it is less sui-
table for subcutaneous administration. The pattern of fluo-
rescent labeling showed that bone formation started at the
cement surface and then proceeded into the center of pores.
The scaffold was stable for 10 weeks, which indicates that
the material is a suitable carrier material for bone formation
[97].
The in vivo release kinetics of rhBMP-2-loaded
PLGA/Ca-P cement was studied by Ruhe et al. [92]. rhBMP-
2 was radio labeled and encapsulated or adsorbed onto the
surface of PLGA microparticles. PLGA microparticles were
prepared at high and low molecular weight (HMW PLGA
and LMW PLGA, respectively). The adsorption efficiency
was 93% for LMW and HMW microparticles, and the frac-
tion of rhBMP-2-loaded microparticles in cement was 54%.
Release of rhBMP-2-loaded composites was assessed by
scintigraphic imaging during 4 weeks, and the in vivo release
kinetics showed release without initial burst and displayed a
linear release from 3 to 28 days. The retention of rhBMP-2
was 16% higher for each formulation and time period than
that measured by conventional ex vivo counting. The authors
affirmed that rhBMP-2 release from the composites was
slowed down by an interaction of rhBMP-2 with cement
after its release from PLGA microparticles. Therefore, this
system can be considered as a sustained slow release vehicle,
and the retention of rhBMP-2 can be changed according to
need [92].
Another group of researchers [98] worked with rhBMP-
2-loaded porous cement that was pretreated with albumin
Current Protein and Peptide Science, 2017, Vol. 18, No. 8 857
  #
7    #: ) *  
*  *     7
7 (   *     
#    ) * 
#  )    )/
    #  )7
7      *   *
 6     
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    )   7
and studied the release in vitro and in vivo. They thought that
the pretreatment of the ceramic with albumin prior to
rhBMP-2 loading would result in weaker binding between
the peptide and the cement and enhanced rhBMP-2 release.
These researchers chose albumin for pretreatment because it
is the most abundant body protein and its limited specific
activity is known. Therefore, the pretreatment of albumin
would result in release of the growth factor without changing
the properties of the material. Each side of the discs was
carefully loaded with 12.5 µL of rhBMP-2. Albumin-
pretreated cement discs showed a 30% release after 24 h and
22% without albumin. The release kinetics in the rat ectopic
model showed 20-30% retention after 4 weeks. Although
albumin pretreatment of the cement resulted in an increased
initial release of rhBMP-2, the in vivo release kinetics was
similar with non-pretreated cement.
2.4. Liposomes
Liposomes are formed by spherical structures consisting
of one or more phospholipid bilayers (natural or synthetic
polymers) around the inner aqueous compartment, which
diameter can range from 20 nm up to 1 µm [99-103].
Liposomes are widely used as drug carriers because they
promote controlled drug delivery, improve their pharma-
cokinetic properties and bioavailability, minimize the ad-
verse effects observed in conventional therapies, allow the
incorporation of hydrophilic drugs into the inner compart-
ment and hydrophobic ones in the lipid membranes, are bio-
degradable, biocompatible, and non-toxic, and can be pro-
duced on a large scale [104]. Furthermore, the similarity
between plasma membrane and phospholipid bilayer struc-
tures of liposomes allow a better interaction of the particles
with cells and tissues of the organism [105]. Fig. (6) shows
858 Current Protein and Peptide Science, 2017, Vol. 18, No. 8
   
  
  

)
 
  & '
)  
   

"   
  
Fig. (6). Schematic representation demonstrated by Lombardo et al. [106] of three types of liposomal drug delivery systems: Charge and
polymer stabilized (A), targeted (B), and theranostic (C) liposomes [106].
the schematic representation demonstrated by Lombardo et
al. [106] of three types of liposomal drug delivery systems.
With the commercialization of Doxil® [107], the first
nanostructured drug approved in 1995 by the US Food and
Drug Administration (FDA), feasible strategies have been
developed with liposomes in the cosmetic and pharmaceuti-
cal industries, mainly for the delivery of cytotoxic drugs,
genes, and vaccines [108], as well as bone regeneration
[109].
In bone regeneration, bone growth peptide and proteins
OGP and BMP have been the targets of investigation for
therapeutic intervention, as they have shown promising re-
sults in vivo and in clinical studies. Thus, the aim of these
peptide and proteins loaded liposomes was to develop effec-
tive bone regeneration [110].
Park et al. [111] compared liposome-mediated and ade-
noviral gene transfer for the generation of autologous BMP-
2-producing bone marrow stromal cells that aim to treat
critical size defect of rat femur (diameter of 6 mm). In the 6
weeks after transfer of the BMP-2 gene, the liposome group
showed a complete healing of the critical size bone defects,
whereas the adenoviral-mediated transfer of BMP-2 cDNA
showed bone healing within 4 weeks, showing that both are
suitable methods for the healing of critical size bone defects
in rats. None of the control groups revealed bone healing,
even after 8 weeks. However, considering the ease of prepa-
ration, no limitation DNA size, lower immunological and
safety problems, liposomes have proven to be more advanta-
geous than any other vector [111]. Matsuo et al. [112] pre-
pared magnetic liposomes with incorporated rhBMP-2 and
Oshiro Jr. et al.
(  
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(  
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(  
#! 
% 
 
$!  
#!
  

 
    ! 
evaluated bone formation in a bone defect model in rats (5
mm segment). The results revealed that this system was
more effective for treatment of segmental bone defects in
rats when compared with control groups, as a unique topical
application of magnetic rhBMP-2 liposomes (3.0 µg) incor-
porated under magnetic induction immediately after surgery
showed effective new bone formation. In addition, radio-
graphic assessment over 9 weeks showed significantly higher
values (4.8 ± 0.4) and higher bone formation area (12.93 ±
1.80 mm2) than in the control group. This is possibly due to
the magnetic induction that enhanced retention of the drug at
the implantation site [112].
Ono et al. [113] combined porous hydroxyapatite (HAP)
with BMP-2 cDNA plasmid in cationic liposomes as a vector
to treat 12-mm diameter bone defects located on rabbit cra-
nium. Bone formation on the cranial defects of four groups
of rabbits was histologically evaluated at 3, 6, and 9 weeks
after the surgery. The group that received the BMP-2 plas-
mid with liposomes without HAP showed a marked osseous
formation 3 weeks after the operation and a complete bone
formation in the cranial defect at 9 weeks, whereas the BMP-
2 plasmid plus HAP group presented on bone formation after
9 weeks [113].
In a pilot study, Kroczek et al. [114] analyzed the effi-
ciency of four morphogenetic and mitotic osteoinductive
proteins, BMP-2, BMP-7, TGF-β, IGF-1, and a liposome-
mediated gene transfer system on bone regeneration by con-
tinuous osteodistraction using Goettingen minipigs. In the
histological and radiological analyzes, a complete bone for-
mation was observed after osteoinduction with BMP-2 and
Bioactive Molecule-loaded Drug Delivery Systems Current Protein and Peptide Science, 2017, Vol. 18, No. 8 859

Table 1. Data on bone formation in different critical bone defects using different bioactive molecule-loaded DDS.

DDS Bioactive Molecule “In Vivo” Bone Formation Comments References

Bioactive electrospun fibers BMP-7 57.59 ± 15.24% of bone formation after The membrane was tested in 4- [73]
based on poly(lactide-co-glycolic 8 weeks mm critical-sized defect.
acid) (PLGA)

Alginate-based hybrid system BMP-2 37.65 ± 2.22 mm3 of bone formation The membrane was tested in [76]
after 12 weeks bilateral 8-mm segmental de-
fects.

Heparin-binding peptide am- BMP-2 The membrane was tested in critical- The amount released after 8 [77]
phiphile (HBPA) nanofibers sized defect model (5mm) and showed days reached 34.5 ± 8.1%.
20.3 ± 4.2 mm3 of bone formation after 6
weeks.

PLGA scaffolds BMP-2 High bone regeneration (~ 60%) after 8 The DDS was transplantated [78]
weeks. into a calvarial bone defect (4
mm)

Printing-based PCL/PLGA/β- BMP-2 8-mm defects presented ~ 60% bone ~ 25.5% BMP-2 release in vitro [80]
TCP guided bone regeneration formation after 8 weeks. at 24 hr and a sustained rate for
(GBR) membrane 28 days

PLGA OGP A 1.5-cm segment defect was made, and 90% of peptide released in vitro [57]
the results showed a rate of development after 1 week.
of bone bridging significantly higher in
the group with OGP than in the control
group after 12 weeks.

Bone calcium phosphate (Ca-P) BMP-2 Histological analysis revealed bone Stability of the material during [97]
cement formation in rhBMP-2-loaded cement 10 weeks, which indicates that
discs with pores filled at 18% after 10 this is a suitable material. More
weeks of implantation. studies are necessary.

Liposomes BMP-2 6 weeks after transfer of the BMP-2 Liposomes were tested in criti- [109]
gene, the liposome group showed a cal size defect of rat femur
complete healing of critical size bone model (diameter of 6 mm).
defects.

Magnetic liposomes BMP-2 Statistical significance of radiographic The liposome was tested in a [110]
values after 9 weeks (4.8 ± 0.4) and bone defect model in rats (5-mm
higher bone formation area (12.93 ± 1.80 segment).
mm2) were observed.

INFUSE® Bone Graft, Medtronic
Spinal and Biologics, Memphis,
TN, USA (commercially avail-
able in the concentration of 1.5
mg/cc)
BMP-2 12 weeks after implantation, 22.8 ±
10.1% of the defect treated with IN-
FUSE®was filled with new bone, and
Ca-P ceramics showed better results with
new bone covering 33.9 ± 6.8% of the
defects.
INFUSE® Bone Graft used as [121]
positive control in in vivo study
of bone formation in a bilateral
iliac wing defect with a critical-
size diameter of 17 mm in
sheep.

BMP-7, with a mean bone formation of 50.1% and 61.0%,
respectively. Application of TGF-β, IGF-1, and the liposo-
mal vector had restricted effects on bone regeneration, with
quantitative values of 26%, 36% and 30%, respectively. This
suggests that the association of osteodistraction with os-
teoinduction could reduce consolidation and treatment times.
2.5. Micelles
Structurally, micelles are defined as thermodynamically
stable aggregates of globular amphiphilic molecules or am-
phiphilic block copolymers, with a size between 10 and 100
nm [115, 116].
For applications in the clinical and pharmaceutical fields,
polymeric micelles or reverse micelles are particularly rele-
vant DDS [117, 118]. Thus, due to their nanometer size and
core-shell structure, micelles have the following advantages:
prolonged drug release, incorporation of hydrophilic and
hydrophobic drugs, increased bioavailability and solubility,
low toxicity, targeting of the specific site of action in a pas-
sive or active mechanism, reduced dosage and frequency of
860 Current Protein and Peptide Science, 2017, Vol. 18, No. 8
administration, and lower side effects when compared with
conventional drugs, as well as large scale production [119].
Despite these advantages, there are few reports of the use of
micelles with bioactive molecules for bone regeneration.
Ratanavaraporn et al. [120] investigated local suppres-
sion of pro-inflammatory cytokines, such as interleukin IL-6
and IL-10, and the effects in BMP-2-induced bone regenera-
tion. Gelatin hydrogels were prepared by incorporating 2.5,
5.0, or 10 mg of immunosuppressive triptolide-loaded mi-
celles and 5 µg of BMP-2 for in vitro and in vivo release
studies. The results of in vitro release of triptolide-loaded
micelles and BMP-2 from the hydrogels demonstrated a
saturated pattern within 7 days. The in vivo studies showed
that triptolide and BMP-2 were released simultaneously from
the hydrogels over 7 days. When implanted into a critical-
sized bone defect in rats (6-mm length), the hydrogels incor-
porating mixed 2.5 or 5.0 mg of triptolide-loaded micelles
and BMP-2 showed a significantly lower number of neutro-
phils, lymphocytes, macrophages, dendritic cells, or mast
cells infiltrated into the defect, and lower expression level of
cytokines than those incorporating BMP-2 without triptolide-
loaded micelles (IL-6: ~900-fold and TNF-α genes: ~80-
fold). The authors concluded that a lower number of inflam-
matory cells in defects implanted with the hydrogels incor-
porating mixed 2.5 or 5.0 mg of triptolide-loaded micelles
and BMP-2 enhanced bone regeneration, due to the adequate
local modulation of inflammatory responses [120].
3. FINAL CONSIDERATIONS
This review of current research present an account of the
innovation that these bioactive molecule-loaded DDS repre-
sent in bone repair. Table 1 presents some data of bone for-
mation rate in different critical bone defects, and these re-
sults depended on the composition of the bioactive molecule-
loaded DDS.
These results highlight that DDS may in the future be an
innovative approach to mainly replace the use of autogenous
graft, which is considered the “gold standard” but has many
disadvantages (see introduction), and have the potential to
overcome the bone formation capacity of the current treat-
ment with osteoinductive autograft replacement based on
rhBMP-2 on a collage sponge (INFUSE® Bone Graft, Med-
tronic Spinal and Biologics, Memphis, TN, USA) approved
by the FDA (see results obtained by Yuan et al. [121] in Ta-
ble 1), as DDS are able to increase the half-life of bioactive
molecules, release them in specific sites, and can be used by
consumer/patients resulting in greater amount of bone
formed and in less time with a lower cost, benefiting both the
surgeon and patient.
Therefore, DDS represent a new alternative to conven-
tional treatment of bone defects. The versatility of these ma-
terials can be used for the controlled release of bioactive
molecules, such as OGP and BMP, offers many advantages
such as protection against degradation, targeting, controlled
release, easier cell penetration, better therapeutic efficacy by
modifying the pharmacokinetics or biodistribution of the
active molecules, lower cost and maintaining the concentra-
tion of these factors within the therapeutic range over a
longer period of time, allowing the osteoprogenitor cells to
migrate to the target site and differentiate into osteoblasts,
Oshiro Jr. et al.
resulting in a major impact on current therapies for bone
tissue regeneration.
AUTHORS’ CONTRIBUTIONS
João Augusto Oshiro Junior was involved in conceiving
ideas and overall management, data collection and evalua-
tion, publication and writing. Mariana Rillo Sato, Cassio
Rocha Scardueli, Guilherme José Pimentel Lopes de
Oliveira, Marina Paiva Abuçafy was involved in data collec-
tion and evaluation and publication writing. Marlus Chorilli
was involved in conceiving ideas and overall management.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
This study was supported by Coordination for the Im-
provement of Higher Education Personnel (CAPES), by São
Paulo Research Foundation (FAPESP, Brazil), National
Counsel of Technological and Scientific Development
(CNPq) and the Programa de Apoio ao Desenvolvimento
Científico (PADC) of the School of Pharmaceutical Sci-
ences/UNESP, Brazil.
REFERENCES
[1] Cuthbert, R.J.; Churchman, S.M.; Tan, H.B.; Mcgonagle, D.; Jones,
E.; Giannoudis, P.V. Induced periosteum a complex cellular scaf-
fold for the treatment of large bone defects. Bone, 2013, 57(2),
484-492.
[2] Verdonk, R.; Goubau, Y.; Almqvist, F.K.; Verdonk, P. Biological
methods to enchance healing and fracture repair. Arthroscopy,
2015, 31(4), 715-718.
[3] Giannoudis, P.V.; Einhorn, T.A.; Marsh, D. Fracture healing: the
diamond concept. Injury, 2007, 38(4), S3-S6.
[4] Nasr, H.F.; Aichelmann-Reidy, M.E.; Yukna, R.A. Bone and bone
substitutes. Periodontol, 1999, 19, 74-86.
[5] Giannoudis, P.V.; Dinopoulos, H.; Tsiridis, E. Bone Substitutes: an
update. Injury, 2005, 36(3), S20-S27.
[6] Heslop, B.F.; Zeiss, I.M.; Nisbet, N.W. Studies on transference of
bone. I. A comparison of autologous and homologous bone im-
plants with reference to osteocyte survival, osteogenesis and host
reaction. Braz. J. Exp. Pathol., 1960, 41, 269-287.
[7] Misch, C.E. In: Implantes dentais contemporaneos. 3rd ed.; Elsevi-
er: Rio de Janeiro, 2001.
[8] Beaman, F.D.; Bancroft, L.W.; Peterson, J.J.; Kransdorf, M.J. Bone
graft materials and synthetic substitutes. Radiol. Clin. North Am.,
2006, 44(3), 451-461.
[9] Oshiro-Junior, J.A.; Mortari, G.R.; Moreno-Freitas, R.; Marcanto-
nio-Junior, E.; Lopes, L.; Spolidorio, L.C.; Marcantonio, R.A.C.;
Chiavacci, L.A. Assessment of biocompatibility of ureasil-
polyether hybrid membranes for future use in implantodontology.
Int. J. Polym. Mater. Polym. Biomater., 2016, 65(13), 647-652.
[10] Anderson, J.M.; Patterson, J.L.; Vines, J.B.; Javed, A.; Gilbert,
S.R.; Jun, H.W. Biphasic peptide amphiphile nanomatrix embedded
with hydroxyapatite nanoparticles for stimulated osteoinductive re-
sponse. ACS Nano, 2011, 5(12), 9463-9479.
[11] Vallet-Regí, M.; Ruiz-Hernández, E. Bioceramics: from bone re-
generation to cancer nanomedicine. Adv. Mater., 2011, 23(44),
5177-5218.
[12] Tang W.; Lin, D.; Yu, Y.; Niu, H.; Guo, H.; Yuan, Y.; Liu, C.
Bioinspired trimodal macro/micro/nano-porous scaffolds loading
rhBMP-2 for complete regeneration of critical size bone defect.
Acta Biomaterialia, 2016, 32, 309-323.
[13] Katagiri, T.; Watabe, T. Bone morphogenetic proteins. Cold Spring
Harb. Perspect. Biol., 2016, 8(6), pii: a021899.
Bioactive Molecule-loaded Drug Delivery Systems
[14] Pigossi, S.C.; Medeiros, M.C.; Saska, S.; Cirelli, J.A.; Scarel-
Caminaga, R.M. Role of osteogenic growth peptide (OGP) and
OGP(10-14) in bone regeneration: a review. Int. J. Mol. Sci., 2016,
17(11), pii: E1885.
[15] Urist, M.R. Bone: Formation by autoinduction. Science, 1965,
150(3698), 893-899.
[16] Urist, M.R.; Strates, B.S. Bone morphogenetic protein. J. Dent.
Res., 1971, 50(6), 1392-1406.
[17] Wang, E.A.; Rosen, V.; Cordes, P.; Hewick, R.M.; Kriz, M.J.;
Luxenberg, D.P.; Sibley, B.S.; Wozney, J.M. Purification and char-
acterization of other distinct bone-inducing factors. Proc. Natl.
Acad. Sci. USA, 1988, 85(24), 9484-9488.
[18] Wozney, J.M.; Rosen, V.; Celeste, A.J.; Mitsock, L.M.; Whitters,
M.J.; Kriz, R.W.; Hewick, R.M.; Wang, E.A. 1988.
Novel regulators of bone formation: molecular clones and activi-
ties. Science, 1988, 242(4885), 1528-1534.
[19] Ozkaynak, E.; Schnegelsberg, P.N.; Jin, D.F.; Clifford, G.M.; War-
ren, F.D.; Drier, E.A.; Oppermann, H. Osteogenic protein-2. A new
member of the transforming growth factor beta superfamily ex-
pressed early in embryogenesis. J. Biol. Chem., 1992, 267(35),
25220-25227.
[20] Faßbender, M.; Minkwitz, S.; Strobel, C.; Schmidmaier, G.;
Wildemann, B. Stimulation of bone healing by sustained bone
morphogenetic protein 2(BMP-2) delivery. Int. J. Mol. Sci., 2014,
15(5), 8539-8552.
[21] Xiong, L.; Zeng, J.; Yao, A.; Qiquan Tu, Q.; Li, J.; Yan L.; Tang Z.
BMP2-loaded hollow hydroxyapatite microspheres exhibit en-
hanced osteoinduction and osteogenicity in large bone defects. Int.
J. Nanomed., 2015, 10, 517-526.
[22] Tsuji, K.; Bandyopadhyay, A.; Harfe, B.D.; Cox, K.; Kakar, S.;
Gerstenfeld, L.; Einhorn, T.; Tabin, C.J.; Rosen, V. BMP2 activity,
although dispensable for bone formation, is required for the initia-
tion of fracture healing. Nat. Genet., 2006, 38(12), 1424-1429.
[23] Yamaguchi, A.; Katagiri, T.; Ikeda, T.; Wozney, J.M.; Rosen, V.;
Wang, E.A.; Kahn, A.J.; Suda, T.; Yoshiki, S. Recombinant human
bone morphogenetic protein-2 stimulates osteoblastic maturation
and inhibits myogenic differentiation in vitro. J. Cell Biol., 1991,
113(3), 681-687.
[24] Katagiri, T.; Yamaguchi, A.; Komaki, M.; Abe, E.; Takahashi, N.;
Ikeda, T.; Rosen, V.; Wozney, J.M.; Fujisawa-Sehara, A.; Suda, T.
Bone morphogenetic protein-2 converts the differentiation pathway
of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol., 1994,
127(6), 1755-1766.
[25] Ebara, S.; Nakayama, K. Mechanism for the action of bone
morphogenetic proteins and regulation of their activity. Spine J.,
2002, 27(16), S10-S15.
[26] Termaat, M.F.; Den Boer, F.C.; Bakker, F.C.; Patka, P.; Haarman,
H.J.T.M. Development and clinical efficacy in the treatment of
fractures and bone defects. J. Bone Joint Surg., 2005, 87(6), 1367-
1378.
[27] Boden, S.D.; Kang, J.; Sandhu, H.; Heller, J.G. Use of recombinant
human bone morphogenetic protein-2 to achieve posterolateral
lumbar spine fusion in humans: a prospective, randomized clinical
pilot trial: 2002 Volvo Award in clinical studies. Spine J., 2002,
27(23), 2662-2673.
[28] Faria, M.L.; Lu, Y.; Heaney, K.; Uthamanthil, R.K.; Muir, P.;
Markel, M.D. Recombinant human bone morphogenetic protein-
2 in absorbable collagen sponge enhances bone healing of tibial os-
teotomies in dogs. Veter. Surg., 2007, 36(2),122-131.
[29] de Freitas, R.M.; Susin, C.; Spin-Neto, R.; Marcantonio, C.;
Wikesjö, U.M.; Pereira, L.A.; Marcantonio E Jr. Horizontal ridge
augmentation of the atrophic anterior maxilla using rhBMP-2/ACS
or autogenous bone grafts: a proof-of-concept randomized clinical
trial. J. Clin. Periodontol., 2013, 40(10), 968-975.
[30] Pimenta, L.; Marchi, L.; Oliveira, L.; Coutinho, E.; Amaral, R. A
prospective, randomized, controlled trial comparing radiographic
and clinical outcomes between stand-alone lateral interbody lumbar
fusion with either silicate calcium phosphate or rh-BMP2. J. Neu-
rol. Surg. A Cent. Eur. Neurosurg., 2013, 74(6), 343-350.
[31] Schmal, H.; Niemeyer, P.; Zwingmann, J.; Stoffel, F.; Südkamp,
N.P.; Mehlhorn, A.T. Association between expression of the bone
morphogenetic proteins 2 and 7 in the repair of circumscribed carti-
lage lesions with clinical outcome. BMC Musculoskelet. Disord.,
2010, 11, 170.
[32] Triplett, R.G.; Nevins, M.; Marx, R.E.; Spagnoli, D.B.; Oates,
T.W.; Moy, P.K.; Boyne, P.J. Pivotal, randomized, parallel evalua-
Current Protein and Peptide Science, 2017, Vol. 18, No. 8 861
tion of recombinant human bone morphogenetic protein-
2/absorbable collagen sponge and autogenous bone graft for maxil-
lary sinus floor augmentation. J. Oral Maxillofac. Surg., 2009,
67(9), 1947-1960.
[33] Zimmermann, G.; Wagner, C.; Schmeckenbecher, K.; Wentzensen,
A.; Moghaddam, A. Treatment of tibial shaft non-unions: bone
morphogenetic proteins versus autologous bone graft. Injury, 2009,
40(3), S50-S53.
[34] Oryan, A.; Alidadi, S.; Moshiri, A.; Bigham-Sadegh, A. Bone
morphogenetic proteins: a powerful osteoinductive compound with
non-negligible side effects and limitations. Biofactors, 2014, 40(5),
459-481.
[35] Khanna-Jain, R.; Agata, H.; Vuorinen, A.; Sándor, G.K.; Suuronen,
R.; Miettinen, S. Addition of BMP-2 or BMP-6 to dexamethasone,
ascorbic acid, and β-glycerophosphate may not enhance osteogenic
differentiation of human periodontal ligament cells. Growth Fac-
tors, 2010, 28(6), 437-446.
[36] Suliman, S.; Xing, Z.; Wu, X.; Xue, Y.; Pedersen, T.O.; Sun, Y.;
Døskeland, A.P.; Nickel, J.; Waag, T.; Lygre, H.; Finne-Wistrand,
A.; Steinmüller-Nethl, D.; Krueger, A.; Mustafa, K. Release and
bioactivity of bone morphogenetic protein-2 are affected by scaf-
fold binding techniques in vitro and in vivo. J. Control Release,
2015, 197, 148-157.
[37] Singh, K.; Nandyala, S.V.; Marquez-Lara, A.; Cha, T.D.; Khan,
S.N.; Fineberg, S.J.; Pelton, M.A. Clinical sequelae after rhBMP-2
use in a minimally invasive transforaminal lumbar interbody fu-
sion. Spine J., 2013, 13(9), 1118-1125.
[38] Tannoury, C.A.; An, H.S. Complications with the use of bone
morphogenetic protein 2 (BMP-2) in spine surgery. Spine J., 2014,
14(3), 552-559.
[39] Gabarin, N.; Gavish, H.; Muhlrad, A.; Chen, Y.C.; Namdar-Attar,
M.; Nissenson, R.A.; Chorev, M.; Bab, I. Mitogenic G(i) protein-
MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: Ac-
tivation by C-terminal pentapeptide of osteogenic growth peptide
[OGP(10-14)] and attenuation of activation by cAMP. J. Cell. Bio-
chem., 2001, 81(4), 594-603.
[40] Bab, I.; Chorev, M. Osteogenic growth peptide: from concept to
drug design. Biopolymers, 2002, 66(1), 33-48.
[41] Li, H.; Zhang, Z.; Chen, Z.; Zhang, D. Osteogenic growth peptide
promotes osteogenic differentiation of mesenchymal stem cells
mediated by LncRNA AK141205-induced upregulation of
CXCL13. Biochem. Biophys. Res. Commun., 2015, 466(1), 82-88.
[42] Policastro, G.M.; Lin, F.; Smith Callahan, L.A.; Esterle, A.; Gra-
ham, M.; Sloan Stakleff, K.; Becker, M.L. OGP functionalized
phenylalanine-based poly(ester urea) for enhancing osteoinductive
potential of human mesenchymal stem cells. Biomacromolecules,
2015, 16(4), 1358-1371.
[43] Spreafico, A.; Frediani, B.; Capperucci, C.; Leonini, A.; Gambera,
D.; Ferrata, P.; Rosini, S.; Di Stefano, A.; Galeazzi, M.; Mar-
colongo, R. Osteogenic growth peptide effects on primary human
osteoblast cultures: potential relevance for the treatment of gluco-
corticoid-induced osteoporosis. J. Cell. Biochem., 2006, 98(4),
1007-1020.
[44] Moore, N.M.; Lin, N.J.; Gallant, N.D.; Becker, M.L. The use of
immobilized osteogenic growth peptide on gradient substrates syn-
thesized via click chemistry to enhance MC3T3-E1 osteoblast pro-
liferation. Biomaterials, 2010, 31(7), 1604-1611.
[45] Chen, Z.; Wang, X.; Shao, Y.; Shi, D.; Chen, T.; Cui, D.; Jiang, X.
Synthetic osteogenic growth peptide promotes differentiation of
human bone marrow mesenchymal stem cells to osteoblasts via
RhoA/ROCK pathway. Mol. Cell. Biochem., 2011, 358(1-2), 221-
227.
[46] Fei, Q.; Guo, C.; Xu, X.; Gao, J.; Zhang, J.; Chen, T.; Cui, D. Os-
teogenic growth peptide enhances the proliferation of bone marrow
mesenchymal stem cells from osteoprotegerin-deficient mice by
DK2/cyclin A. Acta Biochim. Biophys. Sinica, 2010, 42(11), 801-
806.
[47] Miguel, S.M.; Namdar-Attar, M.; Noh, T.; Frenkel, B.; Bab I.
ERK1/2-activated de novo Mapkapk2 synthesis is essential for os-
teogenic growth peptide mitogenic signaling in osteoblastic cells. J.
Biol. Chem., 2005, 280(45), 37495-37502.
[48] Brager, M.A.; Patterson, M.J.; Connolly, J.F.; Nevo, Z. Osteogenic
growth peptide normally stimulated by blood loss and marrow ab-
lation has local and systemic effects on fracture healing in rats. J.
Orthoped. Res., 2000, 18, 133-139.
862 Current Protein and Peptide Science, 2017, Vol. 18, No. 8
[49] Matti, L.; Fazzi, F.; Moscato, S.; Segnani, C.; Pacini, S.; Galim-
berti, S.; D-Alessandro, D.; Bernardini, N.; Petrin, M. Carboxy-
terminal fragment of osteogenic growth peptide regulates myeloid
differentiation through RhoA. J. Cell. Biochem., 2004, 93, 1231-
1241.
[50] Chen, C.; Kong, X.; Zhang, S.; Lee, I. Characterization and in vitro
biological evaluation of mineral/osteogenic growth peptide nano-
composites synthesized biomimetically on titanium. Appl. Surf.
Sci., 2015, 334, 62-68.
[51] Saska, S.; Scarel-carminaga, R.G.; Teixeira, L.N.; Franchi, L.P.;
Santos, R.A.; Gaspar-Minarelli, A.M.; Oliveira, P.T.; Rosa, A.R.;
Takahashi, C.T.; Messaddeq, Y.; Ribeiro, S.J.L.; Marchetoo, R.
Characterization and in vitro evaluation of bacterial cellulose
membranes functionalized with osteogenic growth peptide for bone
tissue engineering. J. Mater. Sci. Mater. Med., 2012, 23, 2253-
2266.
[52] Bab, I.; Gazit, D.; Chorev, M.; Muhlrad, A.; Shteyer, A.; Green-
berg, Z.; Namdar, M.; Kahn, A. Histone H4-related osteogenic
growth peptide (OGP): a novel circulating stimulator of osteoblas-
tic activity. EMBO J., 1992, 11(5), 1867-1873.
[53] Lavini, F.; Dall'Oca, C.; Bartolozzi, P. Bone transport and com-
pression-distraction in the treatment of bone loss of the lower
limbs. Injury, 2010, 41(11), 1191-1195.
[54] Chen, Y.C.; Bab, I.; Mansur, N.; Muhlrad, A.; Shteyer, A.; Nam-
dar-Attar, M.; Gavish, H.; Vidson, M.; Chorev, M. Structure-
bioactivity of C-terminal pentapeptide of osteogenic growth pep-
tide [OGP(10-14)]. J. Peptide Res., 2000, 56(3), 147-156.
[55] Pigossi, S.C.; de Oliveira, G.J.; Finoti, L.S.; Nepomuceno, R.;
Spolidorio, L.C.; Rossa, C. Jr.; Ribeiro, S.J.; Saska, S.; Scarel-
Caminaga, R.M. Bacterial cellulose-hydroxyapatite composites
with osteogenic growth peptide (OGP) or pentapeptide OGP on
bone regeneration in critical-size calvarial defect model. J. Biomed.
Mater. Res. A., 2015, 103(10), 3397-3406.
[56] Stakleff, K.S.; Lin, F.; Smith Callahan, L.A.; Wade, M.B.; Esterle,
A.; Miller, J.; Graham, M.; Becker, M.L. Resorbable, amino acid-
based poly(ester urea)s crosslinked with osteogenic growth peptide
with enhanced mechanical properties and bioactivity. Acta Bioma-
terialia, 2013, 9(2), 5132-5142.
[57] Shuqiang, M.; Kunzheng, W.; Xiaoqiang, D.; Wei, W.; Mingyu, Z.;
Daocheng, W. Osteogenic growth peptide incorporated into PLGA
scaffolds accelerates healing of segmental long bone defects in
rabbits. J. Plast. Reconstr. Aesthet. Surg., 2008, 61(12), 1558-1560.
[58] Lee, S.H.; Shin, H. Matrices and scaffolds for delivery of bioactive
molecules in bone and cartilage tissue engineering. Adv. Drug De-
liv. Rev., 2007, 54, 339-359.
[59] Calixto, G.M.F.; Garcia, M.H.; Cilli, E.M.; Chiavacci, L.A.;
Chorilli, M. Design and characterization of a novel p1025 peptide-
loaded liquid crystalline system for the treatment of dental caries.
Molecules, 2016, 21, 158.
[60] Bernegossi, J.; Calixto, G.M.F. ; Santos, B.F.; Aida, K.L.; Negrini,
T.C.; Duque, C.; Gremião, M.P.D.; Chorilli, M. Highlights in pep-
tide nanoparticle carriers intended to oral diseases. Curr. Top. Med.
Chem., 2015, 15, 345-355.
[61] Wechsler, S.; Fehr, D.; Moleberg, A.; Raeber, G.; Schense, J.C.;
Weber, F.E. A novel, tissue occlusive poly(ethylene glycol) hydro-
gel material. J. Biomed. Mater. Res. A., 2008, 85, 285-292.
[62] Hurley, L.; Stinchfield, F.; Bassett, A.; Lyon, W. The role of soft
tissues in osteogenesis. An experimental study of canine spine fu-
sions. J. Bone Joint. Surg. Am., 1959, 41, 1243-1254.
[63] Karring, T.; Nyman, S.; Lindhe, J. Healing following implantation
of periodontitis affected roots into bone tissue. J. Clin. Periodon-
tol., 1980, 7(2), 96-105.
[64] Dahlin, C.; Linde, A.; Gottow, J.; Nyman, S. Healing of bone de-
fects by guided tissue regeneration. Plast. Reconstr. Surg., 1988,
81(5), 672-676.
[65] Fujihara, K.; Kotaki, M.; Ramakrisna, S. Guided bone regeneration
membrane made of polycaprolactone/calcium carbonate composite
nano-fibers. Biomaterials, 2005, 26, 4139-4147.
[66] Pereira, N.S.; Souza, L.R.B.; Soares, L.C.; Santos, I.M.S.P.; Arau-
jo, K.S. Regeneração óssea guiada utilizando membrana reabsorví-
vel fixada com etilcianocrilato. Rerista Brasileira de Odontologia,
2011, 68(2), 233-237.
[67] Kim, K.; Jeong, L.; Park, H.; Shin, S.; Park, W.; Lee, S.; Kim, T.;
Park, Y.; Seol, Y.; Lee, Y.; Ku, Y.; Rhyu, I.; Han, S.; Chung, C.
Biological efficacy of silk fibroin nanofiber membranes forguided
bone regeneration. J. Biotechnol., 2005, 120, 327-339.
Oshiro Jr. et al.
[68] Wang, J.; Wang, L.; Zhou, Z.; Lai, H.; Xu, P.; Liao, L.; Wei, J.
Biodegradable polymer membranes applied in guided bone/tissue
regeneration: A review. Polymers, 2016, 8, 2016.
[69] Oshiro-Junior, J.A.; Paiva-Abuçafy, M.; Berbel-Manaia, E.; da
Silva, B.L.; Chiari-Andréo, B.G.; Aparecida-Chiavacci, L. Drug
delivery systems obtained from silica based organic-inorganic hy-
brids. Polymers, 2016, 8, 91.
[70] Oshiro-Junior, J.A.; Scardueli, C.R.; Oliveira, G.J.P.L.; Marcanto-
nio, R.A.C.; Chiavacci, L.A. Development of ureasil-polyether
membranes for guided bone regeneration. Biomed. Phys. Eng. Ex-
press, 2017, 3(1), 1-9.
[71] Izquierdo-Barba, I.; Salinas, A.J.; Vallet-Regi, M. Bioactive
Glasses: From Macro to Nano. Int. J. Appl. Glass Sci., 2013, 4(2),
1-13.
[72] Park, Y.J.; Kim, K.H.; Lee, J.Y.; Ku, Y.; Lee, S.J.; Min, B.M.;
Chung, C.P. Immobilization of bone morphogenetic protein-2 on
ananofibrous chitosan membrane for enhanced guidedbone regen-
eration. Biotechnol. Appl. Biochem., 2006, 43(1), 17-24.
[73] Lee, Y.J.; Lee, J-H.; Cho, H-J.; Kim, H.K.; Yoon, T.T.; Shin, H.
Electrospun fibersimmobilized with bone forming peptide-1 de-
rivedfrom BMP7 for guided bone regeneration. Biomaterials, 2013,
34(21), 5059-5069.
[74] Lopes, C.B.; Pachecob, M.T.T.; Silveira-Jr, L.; Duarte, J.; Can-
gussúc, M.C.T.; Pinheiro, A.L.B. The effect of the association of
NIR laser therapy BMPs, and guided bone regeneration on tibial
fractures treated with wire osteosynthesis: Raman spectroscopy
study. J. Photochem. Photobiol. B Biol., 2007, 89(2-3), 125-130.
[75] Brien, F.J.O. Biomaterials & scaffolds for tissue engineering. Ma-
ter. Today, 2011, 14(3), 88-95.
[76] Kolambkar, M.Y.; Dupont, K.M.; Boerckel, J.D.; Huebsch, N.;
Mooney, D.J.; Hutmacher, D.W.; Guldberg, W.E. An alginate-
based hybrid system for growth factor delivery in the functional re-
pair of large bone defects. Biomaterials, 2011, 32(2011), 65-74.
[77] Lee, S.S.; Huang, B.J.; Kaltz, S.R.; Sur, S.; Newcomb, C.J.; Stock,
S.R.; Shah, R.N.; Stupp, S.I. Bone regeneration with low dose
BMP-2 amplified by biomimetic supramolecular nanofibers within
collagen scaffolds. Biomaterial, 2013, 34(2), 452-459.
[78] Ko, E.; Yang, K.; Shin, J.; Cho, S.W. Polydopamine-assisted os-
teoinductive peptide immobilization of polymer scaffolds for en-
hanced bone regeneration by human adipose-derived stem cells.
Biomacromolecules, 2013, 14(9), 3202-3213.
[79] Shim, J.H.; Huh, J.B.; Park, J.Y.; Jeon, Y.C.; Kang, S.S.; Kim,
J.Y.; Rhie, J.W.; Cho, D.W. Fabrication of blended polycaprolac-
tone/poly(lactic-co-glycolic acid)/β-tricalcium phosphate thin
membrane using solid freeform fabrication technology for guided
bone regeneration. Tissue Eng. A., 2013, 19(3), 317-328.
[80] Shim, J.H.; Yoon, M.C.; Jeong, C.M.; Jang, J.; Jeong, S.I.; Cho,
D.W.; Huh, J.B. Efficacy of rhBMP-2 loaded PCL/PLGA/β-TCP
guided bone regeneration membrane fabricated by 3D printing
technology for reconstruction of calvaria defects in rabbit. Biomed.
Mater., 2014, 9(6), 1-9.
[81] Moore, W.R.; Graves, S.E.; Bain, G.I. Synthetic bone graft substi-
tutes. ANZ J. Surg., 2001, 71(6), 354-361.
[82] Liu, H.; Webster, T.J. Nanomedicine for implants: a review of
studies and necessary experimental tools. Biomaterials, 2007,
28(2), 354-369.
[83] Li, P. Biomimetic nano-­‐apatite coating capable of promoting bone
ingrowth. J. Biomed. Mater. Res. A., 2003, 66(1), 79-85.
[84] Kovaleva, E.S.; Kuznetsov, A.V.; Soin, A.V.; Veresov, A.G.; Put-
lyaev, V.I.; Tret'Yakov, Y.D. In: Study of materials bioactivity with
the use of model media, 2005; Springer: pp. 213-216.
[85] Skelly, J.D.; Lange, J.; Filion, T.M.; Li, X.; Ayers, D.C.; Song, J.
Vancomycin-bearing synthetic bone graft delivers rhBMP-2 and
promotes healing of critical rat femoral segmental defects. Clin.
Orthopaed. Relat. Res., 2014, 472(12), 4015-4023.
[86] Mukherjee, D.P.; Tunkle, A.S.; Roberts, R.A.; Clavenna, A.;
Rogers, S.; Smith, D. An animal evaluation of a paste of chitosan
glutamate and hydroxyapatite as a synthetic bone graft material. J.
Biomed. Mater. Res. B Appl. Biomater., 2003, 67(1), 603-609.
[87] Bosemark, P.; Perdikouri, C.; Pelkonen, M.; Isaksson, H.; Tägil, M.
The masquelet induced membrane technique with BMP and a syn-
thetic scaffold can heal a rat femoral critical size defect. J. Ortho-
paed. Res., 2015, 33(4), 488-495.
[88] Ginebra, M.P.; Fernandez, E.; De Maeyer, E.A.P.; Verbeeck,
R.M.H.; Boltong, M.G.; Ginebra, J.; Driessens, F.C.M.; Planell,
Bioactive Molecule-loaded Drug Delivery Systems
J.A. Setting reaction and hardening of an apatitic calcium phos-
phate cement. J. Dental Res., 1997, 76(4), 905-912.
[89] Lewis, G. Properties of acrylic bone cement: State of the art re-
view. J. Biomed. Mater. Res., 1997, 38(2), 155-182.
[90] Pascual, B.; Vázquez, B.; Gurrachaga, M.; Goni, I.; Ginebra, M.P.;
Gil, F.J.; Planell, J.A.; Levenfeld, B.; San Román, J. New aspects
of the effect of size and size distribution on the setting parameters
and mechanical properties of acrylic bone cements. Biomaterials,
1996, 17(5), 509-516.
[91] Lemaitre, J.; Mirtchi, A.; Mortier, A. Calcium phosphate cements
for medical use: state of the art and perspectives of development.
Silicates Industriels, 1987, 52(9-10), 141-146.
[92] Ruhe, P.Q.; Boerman, O.C.; Russel, F.G.M.; Spauwen, P.H.M.;
Mikos, A.G.; Jansen, J.A. Controlled release of rhBMP-2 loaded
poly (dl-lactic-co-glycolic acid)/calcium phosphate cement com-
posites in vivo. J. Control. Release, 2005, 106(1), 162-171.
[93] Åberg, J.; Pankotai, E.; Hulsart Billström, G.; Weszl, M.; Larsson,
S.; Forster-Horváth, C.; Lacza, Z.; Engqvist, H. In vivo evaluation
of an injectable premixed radiopaque calcium phosphate cement.
Int. J. Biomater., 2011, 2011, 232574.
[94] Wang, J.; Qiao, P.; Dong, L.; Li, F.; Xu, T.; Xie, Q. Microencapsu-
lated rBMMSCs/calcium phosphate cement for bone formation in
vivo. Biomed. Mater. Eng., 2014, 24(1), 835-843.
[95] Woo, B.H.; Fink, B.F.; Page, R.; Schrier, J.A.; Jo, Y.W.; Jiang, G.;
DeLuca, M.; Vasconez, H.C.; DeLuca, P.P. Enhancement of bone
growth by sustained delivery of recombinant human bone morpho-
genetic protein-2 in a polymeric matrix. Pharm. Res., 2001, 18(12),
1747-1753.
[96] Gabet, Y.; Müller, R.; Regev, E.; Sela, J.; Shteyer, A.; Salisbury,
K.; Chorev, M.; Bab, I. Osteogenic growth peptide modulates frac-
ture callus structural and mechanical properties. Bone, 2004, 35(1),
65-73.
[97] Kroese-Deutman, H.C.; Ruhe, P.Q.; Spauwen, P.H.M.; Jansen, J.A.
Bone inductive properties of rhBMP-2 loaded porous calcium
phosphate cement implants inserted at an ectopic site in rabbits.
Biomaterials, 2005, 26(10), 1131-1138.
[98] Ruhe, P.Q.; Boerman, O.C.; Russel, F.G.M.; Mikos, A.G.; Spau-
wen, P.H.M.; Jansen, J.A. In vivo release of rhBMP-2 loaded po-
rous calcium phosphate cement pretreated with albumin. J. Mater.
Sci. Mater. Med., 2006, 17(10), 919-927.
[99] Fonseca-Santos, B.; Gremião, M.P.D.; Chorilli, M. Nanotechnol-
ogy-based drug delivery systems for the treatment of Alzheimer ’s
disease. Int. J. Nanomed., 2015, 10, 4981-5003.
[100] Silva, P.B.; Ramos, M.A.S.; Bonifácio, B.V.; Negri, K.M.S.; Sato,
M.R.; Bauab, T.M.; Chorilli, M. Nanotechnological Strategies for
Vaginal Administration of Drugs-A Review. J. Biomed. Nanotech-
nol., 2014, 10, 2218-2243.
[101] Maherani, B.; Arab-Tehrany, E.; Mozafari, M.R.; Gaiani, C.;
Linder, M. Liposomes: a review of manufacturing techniques and
targeting strategies. Curr. Nanosci., 2011, 7, 436-452.
[102] Achiêta-Júnior, J.J.L.; Lima, H.R.S.; Cavalcante, I.M.F.; Leite,
J.R.S.A.; Magalhães, N.S.S.; Rolim, H.M.L. Nanoencapsulação de
um peptídeo isolado de anuros: citotoxicidade in vitro em células
tumorais humanas. Revista de Ciências Farmacêuticas Básica e
Aplicada, 2014, 35(1), 119-125.
[103] Allen, T.M.; Cullis, P.R. Liposomal drug delivery systems: from
concept to clinical applications. Adv. Drug Deliv, Rev., 2013, 65,
36-48.
[104] Azadi, A.; Ashraf, H. Cell organelle-shaped liposomes: a novel
approach to present the stable intracellular drug delivery systems.
Trends Phramaceut. Sci., 2016, 2(2), 85-88.
Current Protein and Peptide Science, 2017, Vol. 18, No. 8 863
[105] Wilczewska, A.Z.; Niemirowicz, K.; Markiewicz, K.H.; Car, H.
Nanoparticles as drug delivery systems. Pharmacol. Rep., 2012, 64,
1020-1037.
[106] Lombardo, D.; Calandra, P.; Barreca, D.; Magazú, S.; Kiselev,
M.A. Soft interaction in liposome nanocarriers fortherapeutic drug
delivery. Nanomaterials, 2016, 6, 1-26.
[107] Barenholz, Y.C. Doxil®-the first FDA-approved nano-drug: les-
sons learned. J. Control. Release, 2012, 160, 117-134.
[108] Pimentel, L.F.; Jácome-Júnior, A.T.; Mosqueira, V.C.F.; Maga-
lhães, N.S.S. Nanotecnologia farmacêutica aplicada ao tratamento
da malária. Revista Brasileira de Ciências Farmacêuticas, 2007,
43(4), 503-514.
[109] Leucht, P.; Helms, J.Á. Wnt signaling: an emerging target for bone
regeneration. J. Am. Acad. Orthop. Surg., 2015, 23(1), 67-68.
[110] Haidar, Z.S.; Hamdy, R.C.; Tabrizian, M. Delivery of recombinant
bone morphogenetic proteinsfor bone regeneration and repair. part
A: current challenges in BMP delivery. Biotechnol. Lett., 2009, 31,
1817-1824.
[111] Park, J.; Ries, J.; Gelse, K.; Kloss, F.; Mark, K.; Wiltfang, J.; Neu-
kam, F.W.; Schneider, H. Bone regeneration in critical size defects
by cellmediated BMP-2 gene transfer: a comparison of adenoviral
vectors and liposomes. Gene Therapy, 2003, 10, 1089-1098.
[112] Matsuo, T.; Sugita, T.; Kubo, T.; Yasunaga, Y.; Ochi, M.; Mura-
kami, T. Injectable magnetic liposomes as a novel carrier of re-
combinant human BMP-2 for bone formation in a rat bone-defect
model. J. Biomed. Mater. Res., 2003, 66(4), 747-754.
[113] Ono, I.; Yamashita, T.; Jin, H.Y.; Ito, Y.; Hamada, H.; Akasaka,
Y.; Nakasu, M.; Ogawa, T.; Jimbow, K. Combination of porous
hydroxyapatite and cationic liposomes as a vector for BMP-2 gene
therapy. Biomaterials, 2004, 25, 4709-4718.
[114] Kroczek, A.; Park, J.; Birkholz, T.; Neukam, F.W.; Wiltfang, J.;
Kessler, P. Effects of osteoinduction on bone regeneration in dis-
traction: Results of a pilot study. J. Craniomaxillofac. Surg., 2010,
38, 334-344.
[115] Figueiras, A.R.R.; Coimbra, A.B.; Veiga, F.J.B. Nanotecnologia na
saúde: aplicações e perspectivas. Boletim Informativo Geum, 2014,
5(2), 14-26.
[116] Torchilin, V.P. Structure and design of polymeric surfactant-based
drugdelivery systems. J. Control. Release, 2001, 73, 137-172.
[117] Muller-Goymann, C.C. Physicochemical characterization of colloi-
dal drug delivery systems such as reverse micelles, vesicles, liquid
crystals andnanoparticles for topical administration. Eur. J. Phar-
maceut. Biopharmaceut., 2004, 58, 343-356.
[118] Husseini, G.A.; Pitt, W.G. Micelles and nanoparticles for ultrasonic
drug and gene delivery. Adv. Drug Deliv. Rev., 2008, 60(10), 1137-
1152.
[119] Devalapally, H.; Chakilam, A.; Amiji, M.M. Role of Nanotechnol-
ogy in pharmaceutical product development. J. Pharmaceut. Sci.,
2007, 96(10), 2547-2565.
[120] Ratanavaraporn, J.; Furuya, H.; Tabata, Y. Local suppression of
pro-inflammatory cytokines and the effects in BMP- 2-induced
bone regeneration. Biomaterials, 2012, 33(1), 304-316.
[121] Yuan, H.; Fernandes, H.; Habibovic, P.; Boerb, J.; Barradas,
A.M.C.; Ruiterc, A.; Walshd, W.R.;Van Blitterswijkb, C.A.; Brui-
jna, J.D. Osteoinductive ceramics as a synthetic alternative to
autologous bone grafting. Eng. Med. Sci., 2010, 107(31), 13614-
13619.