Bone regeneration Biomaterials as local delivery systems with improved

Materials Science and Engineering C 82 (2018) 363–371
Contents lists available at ScienceDirect
Materials Science and Engineering C
journal homepage: www.elsevier.com/locate/msec
Review
Bone regeneration: Biomaterials as local delivery systems with improved
osteoinductive properties
Victor Martin, Ana Bettencourt ⁎
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Portugal
a r t i c l e
i n f o
Article history:
Received 28 January 2017
Received in revised form 5 April 2017
Accepted 6 April 2017
Available online 11 April 2017
Keywords:
Scaffolds
Local-drug-release
Growth factors
Resveratrol
Alendronate
Tetracyclines
a b s t r a c t
Bone is a mineralized conjunctive tissue, with a unique trauma healing capability. However, the replacement or
regeneration of lost bone is not always successful and becomes more difficult the wider the bone defect. A
significant growth in the demand for orthopedic and maxillofacial surgical procedures as a result of population
aging and increase in chronic diseases as diabetes is a fact and successful approaches for bone regeneration are
still needed. Until today, autogenous bone graft continues to be the best solution even with important limitations,
as quantity and the requirement of a donator area. Alternatively, local delivery systems combining an
osteoconductive biomaterial with osteoinductive compounds as hormones, growth factors or drugs is a popular
approach aiming to replace the need for autogenous bone grafts. Nevertheless, in spite of the intense research in
the area, presently there is no system that can mimic all the biological functions of the autogenous bone grafts. In
this context, the present work provides an overview of the most recent advances in the field of synthetic bone
grafts. The opportunities and limitations are detailed along with the remaining gaps in the research that are
still preventing the successful translation of more products into the market able to be a valuable option in comparison to the autogenous bone grafts.
© 2017 Elsevier B.V. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Current strategies and research on synthetic grafts as local delivery systems
2.1.
Growth factors . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Platelet lysate . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Hormones and phytohormones . . . . . . . . . . . . . . . . .
2.4.
Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Alendronate . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.
Simvastatin . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.
Raloxifene . . . . . . . . . . . . . . . . . . . . . . . . . .
3.
Concluding remarks and future directives . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Bone is a mineralized conjunctive tissue, with a unique trauma
healing capability [1]. Osteoblasts, osteocytes and osteoclasts are the
specialized bone cells (Fig. 1).
⁎ Corresponding author at: Research Institute for Medicines (iMed.ULisboa), Faculty of
Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
E-mail address: asimao@ff.ul.pt (A. Bettencourt).
http://dx.doi.org/10.1016/j.msec.2017.04.038
0928-4931/© 2017 Elsevier B.V. All rights reserved.
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When a bone defect occurs due to injury, cells are supplied from the
periosteum and the regeneration occurs [1,2]. However, satisfactory regeneration becomes more difficult the wider the bone defect. That is
why even with this property of healing in many situations bone graft
is necessary [3]. Orthopedic procedures, like pseudo-arthrosis
chirurgical treatment, hip or knee arthroplasty, arthrodesis, and tumor
removal commonly require bone graft [4]. Furthermore, oral and maxillofacial procedures often demands replacing the missing bone (Fig. 2).
For example, to allow the correct implant insertion, based on the
364
V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
Fig. 1. The specialized bone tissue cells: origin and characteristics. Abbreviations: alkaline phosphatase – ALP, cathepsin K – Cat-k, matrix metalloproteinase – MMP, mesenchymal stem cell
– MSCs, osteoprotegerin – OPG, receptor activator of nuclear factor κB ligand – RANKL, Runt-related transcription factor 2 - RUNX2, semaphorin-4D – SEMA 4D and transforming growth
factor beta - TGF-β.
prosthesis ideal position when the residual bone is not enough, to optimize functional/biomechanics results and to improve gingival and facial
aesthetic [3].
Also important is the expected population aging along with the increasing on chronic diseases as obesity and diabetes, meaning that the
number of patients undergoing orthopedic and/or dental procedures
will raise and, consequently, the need for optimal bone grafts will greatly increase.
To date, the main approach for replacing missing bone is by using
grafts. Bone grafts may be autogenous, homogenous, heterogeneous or
synthetic. Autogenous means that the bone is removed from the
patient's own body, often from the iliac crest, skullcap, mandible or
tibia. To the present it is considered the gold standard (Fig. 3) once it
contains growth factors for osteoinduction (i.e. to promote the differentiation of cells into active osteoblasts), cells for the osteogenesis and the
framework for osteoconduction (meaning bone growth on the surface)
[5]. However, it presents fast remodelation and limited source, once a
donator area is required. In addition, autogenous grafts are often associated with high surgical risks and morbidity [3].
Fig. 2. Example of bone draft demand in oral surgery. Maxillary sinus pneumatization
caused by loss of tooth, where the installation of dental implant is not possible by the
lack of bone height. Radiograph assigned by Victor Z Martin DDs.
The second most common type of bone grafts is homogenous or allograft, which is a graft removed from human cadavers and it is available in bone banks [5]. Heterogeneous or xenograft is a graft obtained
from species other than human, as bovine bone [3]. These types of
bone grafts often requires sterilization and deactivation of proteins, remaining only the mineral matrix. Homogenous or heterogeneous grafts
are only osteoconductive, meaning that these grafts are often combined
with patient's own stem cells (also termed mesenchymal stem cells) or
growth factors [6]. That is, osteogenic as bone marrow aspirate (BMA)
[7] or osteoinductive compounds as bone morphogenetic proteins
(BMPs) or platelet rich plasma (PRP) [8,9] must be also used in the procedures (Fig. 3).
Alternatively, research has been focusing on finding, safer, less expensive and easier to use synthetic bone grafts. These bone substitutes
can be created from biomaterials as hydroxyapatite (HA), tricalcium
phosphate (TCP), bioactive glass, ceramics and polymers [5]. To date,
those types of grafts can provide only osteoconduction, limiting its
usage in bone reconstruction [3].
To improve biomaterials clinical outcome as bone grafts, researchers
are trying to combine different compounds as hormones, growth factors
and drugs with the materials. Synthetic scaffolds are being developed to
deliver different compounds in the target area, aiming to achieve a high
local concentration with minimum side effects (Fig. 4), besides maintaining the space, so that defects can be adequately replaced by newly
formed bone. These grafts can facilitate the chirurgical procedure, reduce the time of the surgery, as well as the morbidity and provide an unlimited source.
With the ongoing intensive research in the field, there is a need to
review and update the current advances on the novel approaches in
terms of biological and synthetic compounds release by local scaffolds,
designed to provide osteoinduction in osteoconductive materials, favoring the enhancement of bone regeneration, in the attempt of replacing
the autologous grafts.
Up to date, published reviews are mainly focused on the biomaterial
as a bone graft or growth factors role, either without exploring or comparing the enormous potential of local delivery systems in the context of
bone regeneration. With that in mind, the present review aims to emphasize the current strategies, including their opportunities and limitations, which explore the role of biomaterials as local delivery systems to
improve bone regeneration.
V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
365
Fig. 3. The most used current therapies for bone repair and regeneration. Big gap between the gold standard and the current graft alternatives, presenting only one of three capabilities of
the autogenous type. Osteoinduction is the stimulation of osteoprogenitor cells to differentiate into osteoblasts; Osteoconduction is the capability of the material of serving as a scaffold for
the cells and blood capillary; Osteogenesis is vital osteoblasts that contribute to the growth of new bone along with bone formation. Abbreviations: Bone morphogenetic protein 2 – BMP-2,
bone marrow aspirate – BMA and platelet rich plasma – PRP.
2. Current strategies and research on synthetic grafts as local delivery systems
Current studies exploring different types of biomaterials (metallic,
polymeric, ceramic or composites) loaded with biological compounds
(growth factors, platelet lysates, hormones, phytohormones) or different types of drugs (antibiotics, alendronate, simvastatin, raloxifene)
will be next detailed.
Fig. 4. Biomaterials as local drug delivery systems. The released compounds from scaffolds
are in close contact with the osteoblasts.
2.1. Growth factors
BMP is a generic name given for specific proteins extracted from
bone matrix [3]. These proteins are members of the transforming
growth factor-beta (TGF-β) superfamily and the most studied are
BMP-2, BMP-3, BMP-4 and BMP-7 [11]. As function mechanism, BMP2 binds to BMP receptor (BMPr) and participates in the regulation of
Runx2 gene expression resulting in osteoblastogenesis activity [12].
Other independent pathways are also reported, involving P38/MAPK
pathway and miRNAs, as the enhancement of alkaline phosphatase
(ALP) activity [13], as illustrated in Fig. 5. However, BMP-2 usage is associated with severe edemas, undesired ectopic bone formation, delayed bone formation and possibly increased cancer risk [14]. One of
the possible reasons for these undesirable side effects is the overdoses
of the compound or a burst release, related with the loaded vehicle, as
observed with an absorbable collagen sponge (ACS) carrier [15]. Current
studies are trying to reach a minimal dosage and a controlled delivery
system capable of avoiding the initial high release related to the overdoses side effects (Table 1).
Various calcium phosphate based scaffolds are being tested as carriers of BMP-2. For example, in the study of Lee et al. [10] it was compared the bone regeneration of a scaffold composed with collagen
based biphasic calcium phosphate composite (BCPC) + BMP-2 and biphasic calcium phosphate (BCP) + BMP-2. In vitro results showed that
the release of BMP-2 was lower and more constant in the BCPC sample,
a desirable effect to avoid the burst release and to improve bone regeneration. The in vivo study showed that the BCPC with BMP-2 presented
more bone formation and more osteoblasts, including in the center of
the defect, when compared with the other groups in a two and eight
weeks period. It was also noticed that BCPC samples are clinically easier
to use, present more moldability and adaptability in comparison with
BCP samples.
Watanabe et al. [16] developed one scaffold made of honeycomb and
β-tricalcium phosphate (β-TCP) that could locally deliver BMP-2. The in
vivo results showed that the scaffolds containing porous between 300
366
V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
Fig. 5. Simplified BMP-2 signaling pathways, via SMADs and p38. miRNAs can be
associated with the BMP signaling. Abbreviations: Bone morphogenic protein – BMP,
Bone morphogenic.protein receptor – BMPr, distal-less homeobox 5 – DLX-5, mitogen
activated protein kinase – MKK/ERK, Osterix – OSX, Runt-related transcription factor 2 –
Runx2, phosphorylation – Pi, similar Mothers against Decapentaplegic – Smad, TGF beta
activated kinase – Tab/tak.
μm and 500 μm with 1000 ng of BMP-2 presented bone formation up to
the center of the scaffold and numerous osteoblasts were arrayed, with
large amount of capillaries. Additionally infiltration of inflammatory
cells was not observed in the scaffolds, showing high biocompatibility
of the material.
Other approaches, like particulate BCP loaded with low concentration of BMP-2 (0.005 mg/mL) were tested in a sinus lift procedure
[17]. The results showed superior mineralization and the formation of
an area with new bone. It was shown that, even in a very low concentration, BMP-2 able to increase bone regeneration with an osteoconductive
material in a non-scaffold shape. It also showed that procedures like
sinus lift, can present acceptable healing results even when only an
osteoconductive material is use in the reconstructive procedure.
Strategies using other type of carriers as liposomes are also being
tested. In the study of Crasto et al. [13] a liposome-based carrier system
composed by cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC) and polyethylene glycol loaded with BMP-2 was developed.
The liposomes were formulated to present sonodisruptable release capability. The in vivo assay compared the effects of an absorbable collagen
sponge (ACS) loaded with the regular BMP-2 product and the ACS loaded with the liposomes into biceps femoris muscle pouches in rats. The
results between the groups were comparable, suggesting that the liposomes can substitute the regular product, in a more secure way to
apply the BMP-2 with a trigger activation control. Unfortunately, this
system cannot provide the continuous delivery of the product like the
scaffolds, and, in a bone defect, it might be more difficult to activate
the liposomes by ultrasound, once these defects could be more profound than an intramuscular wound. Recently, poly-propylene fumarate (PPF) scaffolds were loaded with different amounts of poly(lacticco-glycolic acid) (PLGA) microspheres used as delivery vehicles of
BMP-2 (18). Results showed a sustained release over a period of
14 weeks and ectopic bone formation in a subcutaneous rat model. Interestingly, differences in the release kinetics did not correspond to
changes in the osteogenic efficacy of the several tested formulations.
More biomaterials should be tested in order to obtain a better understanding of the influence of BMP-2 release on bone formation.
Another possibility of working with BMP-2 in a lower dose exposure
is the combination with other growth factors. One of these experiences
was performed by Bhattacharjee et al. [19], combining BMP-2 and
transforming growth factor beta (TGF-β), which is a multifunctional cytokine responsible for regulating the transcription of different target
genes, related with inflammation, proliferation and differentiation of
cells [20]. A scaffold composed of poly (ε-caprolactone), nano-hydroxyapatite (25%) and non-mulberry silk fibroin was tested for the release of
100 ng BMP-2 and/or 4 ng TGF-β in vitro. Results showed that the combination of both GF presented superior cell viability, cell proliferation,
osteoblastogenesis gene expression and higher level of calcium deposits
without presenting a cytotoxic effect. Importantly, results suggested
that TGF-β could enhance the BMP-2 effects even in a very low dose delivery system.
Following this line of thinking, Liu et al. [22] studied the association
of BMP-2 and placental growth factor (PlGF), which is an angiogenic
growth factor [23]. Regarding bone regeneration, PIGF is also important,
enhancing markers of bone regeneration in osteoblasts and enhancing
Table 1
Overview of growth factors compound for bone regeneration.
Type
Active
compound
Carrier
Signaling
Limitations/systemic side effects
Reference
Growth
factors
BMP-2
Smads and P38/MAPK
pathways
- High cost
- Edema
- Cancer risks
BMP-7
Scaffolds: BCPC/BCP honeycomb +
β-TCP
PLC + HA + Silk
PPF + PLGA
Graft: BCP
Sponge: DSPC + ACS
Nanocomplexes: HTCC
Microparticles: Chitosan
[10]
[16]
[19]
[18]
[17]
[13]
[22]
[21]
TGF-β
Scaffold: PLC + HA + Silk
VEGF
Scaffold: β-TCP + PLGA
VEGF and BMP-2 pathways
PIGF-2
Nanocomplexes: HTCC
VEGF and Hypoxia pathways
- Less effective than BMP-2 with the same side
effects
- Inflammation
- ROS
- Cancer risks
- Unwanted vascularization
- Tumor growth
- Expressed by cancer cells
- Osteoclast recruitment and activation
[19]
[25]
[22]
Abbreviations:1,2-distearoyl-sn-glycero-3-phosphocholine – DSPC, Absorbable collagen sponge – ACS, Biphasic calcium phosphate – BCP, Biphasic calcium phosphate composite – BCPC,
Bone morphogenetic protein – BMP, hydroxyapatite – HA, Hyaluronic acid – HAc, N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride – HTCC, Placental growth factor – PlGF,
platelet-derived growth factor – PDGF, poly-caprolactone – PLC, poly(propylene fumarate) – PPF, poly(lactic-co-glycolic acid) - PLGA, reactive oxygen species – ROS, Transforming growth
factor beta - TGF-β, Tricalcium phosphate – TCP and vascular endothelial growth factor – VEGF.
V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
osteoclast migration and differentiation. While relatively high concentrations of PlGF enhance angiogenesis and osteoclast recruitment and
activation, low concentrations exhibit a direct osteogenic effect, related
to the activation of hypoxia-mediated pathways [24]. The performed research compared the effect of local delivery of 1 μg of BMP-2 and 0.5 μg
of PIGF-2 by nanocomplexes made with N-(2-hydroxyl)propyl-3trimethyl ammonium chitosan chloride (HTCC) and heparin in vitro. Results showed that the association of growth factors presented an enhancement of cell proliferation, ALP activity and mineral deposition.
As the association with BMP-2 and TGF-β, it can be the solution for
the side effects problems of higher dose of the BMP-2 alone, if in vivo assays confirm those advantages.
Other growth factor explored recently is the vascular endothelial
growth factor (VEGF), responsible for the promotion of vascular endothelial cells mitosis and the increase of vessels permeability [25]. It is
also known that VEGF can lead to an upregulation of BMP-2. Khojasteh
[25] fabricated a high porous β-TCP scaffold with controlled releasing of
VEGF encapsulated in PLGA on surface, and tested in vitro. Results
showed an increase of cell proliferation and matrix production, as well
as the upregulation of COL1 and Runx2 gene expression. One concern
regarding VEGF use is that it can promote vascularization in non-target
sites and enhance the risk of tumor growth in distant areas. In vivo studies should be performed to evaluate the risks of this growth factor [25].
2.2. Platelet lysate
Platelet Lysate (PL) is obtained by the activation of different batches
of platelet concentrate produced by plasma apheresis, buffy coat or PRP
with three cycles of freezing and thawing (Fig. 6) [26,6]. In other words,
platelets suffer lysis and release growth factors. In the study of Babo et
al. [26] an injectable calcium phosphate (CP) cement system incorporating hyaluronic acid (HAc) micro particles loaded with PL or PL directly
incorporated in calcium phosphate were developed. The results showed
that the incorporation of HAc and PL or PL alone in the CP system increased the porosity and the solubility of the cement. The compressive
strength was severely reduced in HAc samples, however no significant
changes in PL only sample were observed. Enhanced cell proliferation
and expression of osteoblastogenesis genes were present in both samples. The CP with PL resented the best results, without sacrificing the
mechanical properties of the cement. Future in vivo studies need to be
performed to prove those benefits.
At present, although PL represents a valuable opportunity as a coating agent for osteoconductive scaffolds as it contains many bioactive
factors essential for bone defect repair, some major limitations still persist as reviewed in detail by Altaie et al. [6]. For example, the exact
amounts and ratios between the bioactive molecules remain to be
established in order to achieve the desired and timely therapeutic effect
of bone repair; it is mandatory to establish standard protocols and
quality control procedures for PL production and clinical studies
367
investigating the benefits of PL-based products for improving bone regeneration are still lacking.
2.3. Hormones and phytohormones
One of the several functions of hormones in the organism is control
the blood levels of calcium and its homeostasis. Estrogens, growth hormones and thyroid hormones, as calcitonin and parathyroid hormone
(PTH) are some examples of hormones directly linked to bone metabolism. Current studies are trying to use some of these hormones to improve local bone regeneration (Table 2).
PTH is well known to stimulate bone remodeling; its usage is approved by FDA in the treatment of osteoporosis since 2002. Intermittent
administration can lead to bone formation, with an increase of osteoblasts activity by stimulating Wnt signaling pathway and the production of pro-angiogenesis growth factors like VEGF and angiopoetin1
[27]. However, in a continuous exposure, increases the osteoclasts activity [28]. In the study of Dang et al. [29], PTH was tested by an in vivo mice
model, for systemic and local use, with a local calvarial defect regeneration model and a biodegradable scaffold made with poly(l-lactic acid)
and a surface erosion polyanhydride (PA). The obtained results showed
that the local intermittent delivery of PTH presented the best regeneration in bone volume and bone area, without important systemic alterations and side effects. Subcutaneous PTH injection presented good
bone regeneration results, but also presented systemic side effects and
inferior results compared with local intermittent exposure in local
bone regeneration.
Estrogens, especially 17β-estradiol (E2), are responsible for the
maintenance of bone homeostasis in mammals. For instance, lack of
E2 results in bone resorption and the increase of fat tissue in bone marrow. E2, via membrane associated estrogen receptors (ERs) can increase
the differentiation of MSCs in osteoblasts instead of adipocytes, by activating MAPK and Erk1/2, increase their activity and inhibits osteoclasts
resorption function [30]. However, estrogens were related with increased risks of breast cancer, cardiovascular diseases and thromboembolisms [31].
Resveratrol is a phytoestrogen extracted from grapevine, peanut and
other plants when in contact with certain microorganisms. It has been
studied intensely for different purposes, like cancer treatment, anti-oxidation, cardio protection, anti-inflammatory and bone regeneration
without toxic effects even with long-term use [32]. In MSCs, resveratrol
can increase the osteogenic differentiation, the matrix mineralization
and decrease the adipogenic transcription factors in different pathways,
including ERs independent pathways, such as Wnt signaling and Sirtuin
1 signaling [33]. Furthermore, resveratrol can stimulate BMP-2 production by osteoblasts through Src kinase-dependent ER activation [34]. Regarding the prevention of alveolar bone loss, it is known that resveratrol
can inhibit the loss of this type of bone, reducing interleukin-17 in gingival tissue, activating Nrf2 pathway and decreasing oxidative stress
Fig. 6. Platelet lysate preparation methods. Adapted from [6]. Abbreviations: PRP: Platelet rich plasma; PL: platelet lysate.
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V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
Table 2
Overview of hormones with interest in local delivery applications.
Type
Active compound
Carrier
Signaling
Limitations/Systemic side effects
Reference
Hormones
PTH
Scaffold: PLLA + PA
Wnt signaling, VEGF Pathways and PTHr
[29]
Resveratrol
Scaffold: PLC + COOH
PAA
PLC + ALB
Systemic: Subcutaneous injection
ER, Wnt and Sirtuin 1 signaling
- High blood calcium levels
- Inhibition of bone regeneration
- Cancer risks
- Low solubility
- Short plasma half-life
- Low bioavailability
[38]
[40]
[39]
[35]
Abbreviations: albumin – ALB, carboxylic acid – COOH, estrogen receptor – ER, Parathyroid hormone – PTH, poly(l-lactic acid) – PLLA, poly-acrylic acid – PAA, polyanhydride – PA, polycaprolactone – PLC, and vascular endothelial growth factor – VEGF.
and pro-inflammatory cytokine production [35]. In osteoblasts, it is
known that resveratrol dose-dependently increased the DNA synthesis
and ALP activity [36]. However, resveratrol has low solubility and it is
quickly metabolized in human body, presenting an extremely short
plasma half-life and low bioavailability [37]. The systemic usage of resveratrol can avoid alveolar bone loss and help in local bone regeneration
[35].
Regarding studies focusing on local resveratrol use, the study of Li et
al. [38] evaluated a porous scaffold made with acrylic acid, poly-ϵcaprolactone (PLC), carboxylic acid and resveratrol, covalently bounded
with the carboxylic acid and tested in vitro and in vivo. The results
showed that the resveratrol scaffold could increase the ALP activity during 21 days showed more proteinaceous matrix and mineralized nodules. No significant difference was found regarding defect size when
compared with the control, however, the loaded scaffold presented significantly higher X-ray density and greater area of bone regeneration,
with bone-like only in the resveratrol scaffold.
Kamath et al. [39] performed another attempt, with a 3D porous
polycaprolactone (PCL) scaffold loaded with resveratrol albumin nanoparticles. The results showed that the samples presented adequate release properties. Cell viability, ALP activity and mineralization were
higher in the resveratrol samples, even when compared with the osteogenic medium.
Regarding the potential of resveratrol in reducing inflammation,
Wang et al. [40] tested a scaffold made of poly-acrylic acid (PAA) with
resveratrol in vivo. The results showed that cell viability increased and
the mRNA expression of IL-1β, MMP-13 and COX-2 was reduced,
which means reduction of inflammation. Furthermore, the levels of
mRNA of SOX-9, Coll II and Coll I were up regulated in the loaded
scaffold.
2.4. Antibiotics
Interestingly some antibiotics may have an effect on bone metabolism along with their antimicrobial activity (Table 3). An example is
the tetracyclines (TCs) and their derivatives. The effects include the direct interaction with matrix metalloproteinase enzymes (MMPs), tissue
inhibitors of MMPs, growth factors and cytokine [41,42]. Tetracyclines
bone effects are also related with the increase of collagen type 1 synthesis, preventing bone loss and increasing bone formation [43].
Among tetracyclines, minocycline and doxycycline are semi-synthetic antibiotics acting against gram positive and negative bacteria by
inhibiting bacterial protein synthesis, presenting longer half-life and
improved lipid solubility, in comparison with others from the same
class [44].
The study of Yoshinari et al. [45] demonstrated the effects of local
minocycline applications associated with guided tissue regeneration
(GTR) using a non-absorbable membrane in maxillary and mandibular
bone defects. The usage of minocycline increased the bone gain and decreased the number of bacteria in the tested samples, in comparison
with the control group, which received the GTR without the
minocycline. In this study, the antibiotic was not applied directly into
the wound, only in the membrane and the marginal area, exploring
only the antimicrobial effect of the minocycline, suggesting that the
presence of bacteria reduces the potential of bone regeneration.
On the other hand, the study of Gomes et al. [46] evaluated the in
vitro proliferation and activity of human bone marrow cells in an osteoblastic-inducing medium in different concentrations of doxycycline and
minocycline during 35 days. In a low dose regimen of 1 μg/mL of both
antibiotics, the functional activity of the cells was not altered, the proliferation was stimulated and mineral deposition was increased because
Table 3
Overview of antibiotics compounds with interest in local delivery applications.
Type
Active
compound
Antibiotics Tetracycline
Doxicycline
Carrier
Signaling
Limitations/systemic side
effects
Reference
Nanocomposite: HAP +
β-TCP
Inhibition of matrix metalloproteinases
- Normal ATB side effects
- Problems during tooth
development
- Antimicrobial Resistance
- Normal ATB side effects
- Antimicrobial resistance
- Normal ATB side effects
- Problems during tooth
development
- Dizziness
- Antimicrobial resistance
[49]
- Normal ATB side effects
- Antimicrobial resistance
[53]
Free form only
Minocycline
Free form
Nanocomposite: gelatin +
HA
absorbable membrane: COL
+ Chitosan
Cement: Acrylic + Lactose
Clarithromycin Microspheres: PLGA + BCP
Bind to 50S ribosomal subunit producing bone formation with the
reduction of inflammation
[46]
[46]
[47]
[48]
[50]
Abbreviations: Antibiotic – ATB, Biphasic calcium phosphate – BCP, calcium hydroxyapatite – HAP, collagen – COL, Hydroxyapatite – HA, poly (lactic-co-glycolic) acid – PLGA, Tricalcium
phosphate – TCP.
V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
of the higher number of cells with normal activity. Similar results, early
proliferation and the differentiation of the cells higher in the group exposed to minocycline, were obtained in the study of Dou et al. [47] with
a nanocomposite made with gelatin, hydroxyapatite and minocycline in
contact with BMSCs. As results, in 2016, Ma et al. [48] created an absorbable membrane made with collagen, chitosan and minocycline nanoparticles to be use in bone defects. Chitosan is a polymer composed by
glucosamine and it is structurally similar to glycosaminoglycan in the
extracellular matrix and can be loaded with minocycline nanoparticles.
This study investigated the antibacterial activity of that membrane, its
biodegradation, cytocompatibility and the bone regeneration in vivo.
The results showed a reduction of bacteria presence up to 95% and the
internal surface of the membrane presented satisfactory cell viability
and cytocompatibility. In terms of bone regeneration, it presented substantial new bone formation without significant inflammatory reactions, large number of small blood vessels. In addition, the study
showed that the membranes were almost degraded in 28 days, what
is an adequate length period for most of the orthopedic and dental procedures. The control group, without any kind of membrane, presented
loose connective tissue invasion into the bone defect area and suppressed the formation of new bone. Unfortunately, this study only presented the results of bone regeneration between the control group,
without membrane and the group with the collagen/chitosan/
minocycline membrane. A comparison between this membrane without the antibiotic and the membrane only loaded with the drug might
show the potential of the minocycline itself. In addition, the chitosan
nanoparticles toxicity should be tested including the risk assessment
and toxic kinetics evaluation.
Seeking a deeper effect regarding bone regeneration, Marycz et al.
[49] developed a nanocomposite composed of calcium hydroxyapatite
(HAP) and β-TCP with the capability of deliver tetracycline. The in
vitro study showed that the nanocomposite with 5% of TC presented
the best results, regarding cell proliferation and additional beneficial effects on the hAMSCs morphology, like extracellular connections with
well-developed cytoskeleton, which indicates their enhanced
cytophisiological activity. Regarding antibacterial effect, 5% of TC is
able to present bacteriostatic activity against E. coli and S. aureus. However, 80% of the TC was delivered up to 24 h, which could be a disadvantage for the bone repair.
Regarding acrylic bone cements (BC), Matos et al. [50] developed a
novel formulation composed with 2.5% of minocycline and 10% of lactose to be used in cemented arthroplasties. The results showed that
the full amount of the drug was released in seven days, presented a
higher concentration and higher antibacterial activity against all S. aureus evaluated strains when compared with the regular BC and standard
minocycline solution, without cytotoxicity and the biomechanical properties losses. The direct effect of minocycline in bone regeneration was
not evaluated in this particular study. Nevertheless, BC loaded with
minocycline might be effective in osteogenesis considering the welldocumented positive effects of the antibiotic in bone metabolism, [46].
Even with a non-absorbable product presenting insoluble characteristics, it is possible to create a local drug delivery system to improve the
chirurgical results.
Besides tetracyclines, other antibiotics may have potential to participate in bone metabolism as clarithromycin (CLT). The use of this
macrolide has been suggested to accelerate bone remodeling through
antibacterial and anti-inflammatory properties [51,52]. Moreover,
some studies reported that clarithromycin exhibits anti-inflammatory
properties [53]. Based on these anti-inflammatory properties, Alenezi
et al. [53] presented a study investigating the controlled release of CLT
using poly (lactic-co-glycolic) acid (PLGA) microspheres and BCP as a
carrier in vivo. The results suggested that PLGA microspheres loaded
with CLT group presented highest amount of bone formation, however
only 12 weeks after the procedure. No increase in bone formation was
observed in 2 and 4 weeks after the surgery, suggesting a long-term effect only.
369
2.5. Alendronate
Alendronate (ALN) is a drug that belongs to nitrogenous bisphosphonate class. It is used in the treatment of cancer, bone disorders
such as osteoporosis, fibrous dysplasia, and others diseases [54]. ALN
acts in osteoclast cells, altering the cytoskeleton, thus inhibiting its interaction with bone matrix. ALN prevents osteoclast recruitment, their
differentiation and causes apoptosis of these cells [55]. ALN also promotes osteoblastogenesis by initiating osteoblast precursor's formation,
mineralized nodules and inducing secretion of inhibitors of osteoclastmediated resorption by osteoblasts [56]. ALN promotes the enhancement of BMP-2, ALP, COL1 and osteocalcin (OCN) gene expression and
the increase of ALP activity [57,63]. As side effects, hypocalcemia, atrial
fibrillation and jaw necrosis are the most serious problems related with
the usage of ALN, mainly with high IV dose application [58]. Table 4 presents a summarized view of alendronate and other drugs regarding local
delivery.
In the study of Park et al. [59], a scaffold made with biphasic calcium
phosphate (BCP) and ALN was developed and able to release the drug
during 28 days. Results showed that the sample with 5 mg ALN presented an increase of ALP activity and calcium content, as well as mRNA expression of OCN and OPN. In vivo assay showed gain of bone volume and
revealed abundant mature bone formation with 5 mg ALN in 8 weeks.
Going further, Hur et al. [60] modified a clinically approved metallic
bone plate with 4-azidobenzoic acid-modified chitosan, loaded with
ALN aiming local delivery, and tested in vivo. The plate with ALN presented significant more bone formation in 4 and 8 weeks and showed
that the release of the ALN occurred in more than 60 days with 4.0 μg
of average rate release and no significant cytotoxicity. No alterations
in mechanical properties were observed in the modification process.
Combinations of ALN and platelet-rich fibrin (PRF) are also under investigation. PRF is a platelet concentrate, which incorporates platelets
and growth factors within fibrin membranes. Obtained from centrifugation of intravenous blood of the own patient, serves as a healing matrix
component in several chirurgical procedures [61]. Kanoriya [62] conducted one of these studies, performing a clinical assay with 72 patients
presenting mandibular degree II furcation defects and treated with
chirurgical periodontal techniques. Some patients received PRF + 1%
ANL gel made with polyacrylic acid (PAA) inside the wound. Patients
which received PRF plus ALN gel showed significant better results in
all clinical parameters in a 9 month following.
2.6. Simvastatin
Simvastatin (SV) is a small molecule drug that belongs to statin
group. It is known as coenzyme A reductase inhibitor that is mainly
used to decrease serum cholesterol levels and recently showed effects
on new bone formation through increasing the expression of the
BMP-2 gene in bone cells [64]. However, less than 5% of the drug reaches
the systemic circulation due to extensive first-pass metabolism in the
liver as SV presents high liposolubility [65].
In the study of Yue et al. [66] nanostructured lipid carriers loaded
with SV were tested in vivo with BioOss®. The release assay showed
that the nanoparticles extended the drug release in eight times when
compared with free SV release. The in vivo results showed that more
new bone area and neovascularization were found in the nanoparticles
group when compared with the other groups and the ALP activity were
augmented in SV groups. Unfortunately, the gene expression variations
and more in-depth in vitro assays were not performed.
2.7. Raloxifene
Raloxifene (RAL) is a nonsteroidal benzothiophen derivative that
acts as selective estrogen receptor modulator (SERM). It has estrogenic
actions on bone and anti-estrogenic actions on the uterus and breast
370
V. Martin, A. Bettencourt / Materials Science and Engineering C 82 (2018) 363–371
[67]. It is approved by FDA since 1997 for the treatment and prevention
of osteoporosis, and for reduction of breast cancer risks [67].
In the study of Zhang et al. [68] a scaffold made with chitosan, collagen, and β-TCP and loaded with PLGA microspheres containing RAL was
tested in vitro with the dosage of RAL ranged between 0.1 and 10 μg. The
results showed that the release rate of RAL from the microsphere-embedded scaffold was gradual and controlled, showing superior cell viability in all concentrations, significantly enhanced cell proliferation,
higher mineralization capacity and ALP activity. With promising in
vitro results, the next step is to perform in vivo assays to confirm RAL
as viable local bone regenerative drug.
3. Concluding remarks and future directives
Until today, the autogenous graft is still the gold standard to any procedure, only in some specific cases, with precise indication or small defects; biomaterials can be use with safety and efficiency.
Improvement of the osteoconductive properties of the biomaterials
associated with the controlled release of osteoinductive compounds
might replace the autogenous grafts in several clinical situations,
diminishing its usage that will result in faster, costly and easier surgeries, with less side effects (as edemas) and morbidity. That is, biomaterials basically have two functions as scaffolds for osteoprogenitor cells,
providing an interface for cells to attach, proliferate and differentiate,
and as drug delivery systems of substances with positive effects on
bone regeneration.
Several osteoinductive compounds were reviewed including wellknown products in the area as BMPs or newer ones as resveratrol. The
combination of two or more substances for local release can be beneficial, acting in different pathways and favoring bone regeneration. For instance, the release of BMP-2 and other growth factors as PIGF and TGF-β
can enhance bone regeneration with lower doses, which could avoid
BMP side effects. The combination of drugs/hormones and BMP-2 can
also presents interesting results, as it stimulates multiple bone regeneration pathways. Additionally, resveratrol is the only compound that can
be used in a systemic drug protocol for patients with indication for bone
surgical intervention, also presenting good results in bone loss prevention, with no side effects.
Along with intense research in the field, there are still major limitations in the use of synthetic grafts as local drug delivery systems mainly
related to the inadequate release profiles of the compounds from the
biomaterials. Although different approaches are being tested as the
use of liposomes and nanoparticles, a rapid burst release of the loaded
substances is still often presented in the studies. It means that the results at longer healing times will not be useful. Further, as certain substances could be more effective at later stages of bone regeneration, it
would be desirable that they were released by the biomaterials in a
sustained and controlled manner. Also, more biomaterials should be
tested in order to obtain a better understanding of the influence of
growth factors and drug release on bone formation.
Another gap in the field concerns materials testing and the successful translation of products from the lab to the clinics. In fact, researchers
are testing many materials and compositions, using different experimental methodologies. For example, different cell lines, incubation
times, and toxicity end points are described in the literature concerning
the in vitro biocompatibility assays. In addition, the composition of the
media to test materials bioactivity is under discussion [69]. Moreover,
pharmaceutical testing as drug release assays lack standardized methodology, resulting in difficulty for comparing the outcomes from different labs. Also, the type of material's treatment, which better mimetizes
the formation of the apatite interface layer prior to implantation that
should be evaluated before clinical application, is under discussion
[69]. In our opinion, a multidisciplinary approach involving the standardization of in vitro, ex-vivo and in vivo testing will assure fast clinical
trials of the novel materials taking bone regeneration to the next level.
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