Bone regeneration mediated by a bioactive and biodegradable ECM-like hydrogel based on elastin-like recombinamers

Abstract

The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel biomaterials that are able to promote the healing and regeneration of injured bones are needed in order to overcome the limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-to-batch consistency, amongst others. Furthermore, they can self-assemble into physically cross-linked hydrogels above a certain transition temperature, in this case body temperature, but are injectable below this temperature, thereby markedly reducing surgical invasiveness. Herein we have developed two bioactive hydrogel-forming ELRs, one including the osteogenic and osteoinductive BMP-2 and the other the RGD cell-adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand White rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neo-formation was confirmed using different techniques, such as radiography, computed tomography and histology. This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.

Full text

SPECIAL FOCUS: STRATEGIC DIRECTIONS IN OSTEOINDUCTION
AND BIOMIMETICS*
Bone Regeneration Mediated by a Bioactive
and Biodegradable Extracellular Matrix-Like Hydrogel
Based on Elastin-Like Recombinamers
Dante J. Coletta, MD,
1,
** Arturo Iba´n˜ ez-Fonseca, MSc,
2,
** Liliana R. Missana, PhD,
3,4,
** Marı´a V. Jammal, PhD,
3,4
Ezequiel J. Vitelli, MD,
1
Mariangeles Aimone, BSc,
1
Facundo Zabalza, BSc,
1
Joa˜o P. Mardegan Issa, PhD,
5
Matilde Alonso, PhD,
2
Jose´ Carlos Rodrı´guez-Cabello, PhD,
2
and Sara Feldman, PhD
1
The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel
biomaterials that are able to promote the healing and regeneration of injured bones are needed to overcome the
limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical
invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are
very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-
to-batch consistency, among others. Furthermore, they can self-assemble into physically crosslinked hydrogels
above a certain transition temperature, in this case body temperature, but are injectable below this temperature,
thereby markedly reducing surgical invasiveness. In this study, we have developed two bioactive hydrogel-forming
ELRs, one including the osteogenic and osteoinductive bone morphogenetic protein-2 (BMP-2) and the other the
Arg-Gly-Asp (RGD) cell adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded
extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules,
thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after
bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on
RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs
were implanted in a pilot study involving a femoral bone injury model in New Zealand white rabbits, showing
complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone
neoformation was confirmed using different techniques, such as radiography, computed tomography, and histology.
This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-
regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2,
which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.
Keywords: bone regeneration, elastin-like recombinamers, bioactive hydrogels, BMP-2
Introduction
It is well known that dental, maxillofacial, and other
orthopedic surgeries often require the use of different
biomaterials for the treatment of injuries and other diseases
through tissue engineering, including osteoporosis.
1–6
In
addition, changes of the age pyramid toward an older popu-
lation have led to an increasing number of bone fractures.
7–11
Despite the availability of numerous biomaterials for tissue
regeneration, autologous bone is usually the first option for
the replacement of injured bone tissue. However, a large
number of different types of biomaterials and bone grafts
1
LABOATEM, Osteoarticular Biology, Tissue Engineering and Emerging Therapies Laboratory, School of Medicine, National Rosario
University, Rosario, Argentina.
2
BIOFORGE Lab, University of Valladolid, CIBER-BBN, Valladolid, Spain.
3
Experimental Pathology and Tissue Engineering Laboratory, Dental School, National Tucuma
´n University, Tucuma
´n, Argentina.
4
Tissues Laboratory, Proimi-Biotechnology-Conicet, Tucuma
´n, Argentina.
5
Ribeira
˜o Preto School of Dentistry, University of Sa
˜o Paulo, Sa
˜o Paulo, Brazil.
**These authors contributed equally to this work.
*This article is part of a special focus issue on Strategic Directions in Osteoinduction and Biomimetics.
TISSUE ENGINEERING: Part A
Volume 23, Numbers 23 and 24, 2017
ªMary Ann Liebert, Inc.
DOI: 10.1089/ten.tea.2017.0047
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have been used to date for the healing of bone defects.
12
These biomaterials should be (1) osteoinductive, hence pro-
moting stem cell differentiation to osteoblasts, (2) osteo-
conductive, thereby inducing growth of the surrounding
healthy bone, and (3) osseointegrative, merging with the
nearby bone.
13–15
They should also stimulate an optimal cell
response and be liable to be replaced by de novo formed
tissue, acting as a provisional substitute.
1,16,17
Engineered biomaterials, in combination with growth
factors, have been shown to be an effective approach in
bone tissue engineering since they can act both as a scaffold
and as a drug-delivery system to promote bone repair and
regeneration.
18,19
For instance, the osteoinductive bone
morphogenetic protein-2 (BMP-2)
20,21
has been shown to
enhance the formation of bone tissue in situations that lead
to bone degradation, such as alcohol dependence
22
and os-
teometabolic diseases.
23
Due to the high cost and rapid re-
lease of BMP-2 when placed at the site of injury, it is often
associated with carrier matrices that act as drug-delivery
systems to increase its half-life and to avoid the adverse
effects associated with high doses of BMP-2.
24–27
On the contrary, protein-based recombinant biomaterials,
such as resilin-, silk-, collagen-, and elastin-like polypeptides,
have been developed over the last few decades with the aim
of improving the features of traditional biomaterials in terms
of ease of design and synthesis, biocompatibility, and bio-
activity.
28
As an example, elastin-like recombinamers
(ELRs), thus named due to their polymeric and recombinant
nature,
29
have been shown to be a potential tool for the de-
velopment of biomedical devices for regenerative medicine
due to their thermosensitivity. This smart behavior is a result
of their composition, which is based on repetitions of the Val-
Pro-Gly-X-Gly pentapeptide, in which X (guest residue) is
any amino acid except L-proline. Moreover, it is character-
ized by a transition temperature (T
t
), which itself depends on
the polarity of the side chain in the guest residue. Thus, in an
aqueous medium, the ELR chains remain soluble below their
T
t
while above that T
t
(e.g., physiologic temperature), the
ELR self-assembles hydrophobically, undergoing a phase
transition.
30
In this study, two different ELRs have been de-
veloped, based on a previously described hydrogel-forming
ELR.
31
Taking advantage of the recombinant nature of these
biomolecules, one of the novel ELRs designed in this work
has been genetically engineered to include Arg-Gly-Asp
(RGD) motifs to enhance cell adhesion via cell membrane
integrins,
32
whereas the other ELR was designed to include
BMP-2. Both ELRs also contain elastase-sensitive domains
resulting from repetition of the Val-Gly-Val-Ala-Pro-Gly
hexapeptide
33
to improve the enzymatic biodegradability of
the biomaterial (see Supplementary Fig. S1 for a schematic
representation of both ELRs; Supplementary Data are avail-
able online at www.liebertpub.com/tea).
To study the potential of these novel ELRs in bone re-
generation, we have used a previously developed model of
femoral bone injury (FBI) in New Zealand white rabbits.
This involves the creation of a defect 6 mm in diameter in a
femoral condyle 8 mm in diameter.
34,35
This animal model
allows the study of the defect by computed tomography
(CT) and by radiological studies given the size of the bone.
The aim of this work was to evaluate whether novel bio-
active ELRs are cytocompatible and degradable, while being
able to form extracellular matrix (ECM)-like hydrogels and
promoting bone regeneration after implantation into an FBI in
rabbits, as a preliminary step for their use in humans. For this
purpose, the cytocompatibility and biodegradation ability
were assessed in vitro, and a highly reproducible model was
subsequently used to carry out a pilot in vivo study.
Materials and Methods
Ethical approval
Experimental procedures regarding the use of animals
were approved by the Bioethics Committee of Rosario Na-
tional University (Resolution No. 150/2015). Its regulations
include well-established guidelines for animal care and
manipulation to decrease pain and suffering of the animal,
according to the 3Rs (replacement, reduction, and refine-
ment), and are in accordance with international laws con-
cerning the use of animals.
ELR biosynthesis and characterization
The genetic construction of the ELRs used in this work was
performed as described elsewhere.
36
Briefly, their DNA se-
quences were obtained by genetic engineering techniques and
cloned into a pET-25b(+) vector for expression in Escher-
ichia coli. ELRs were biosynthesized in a 15-L bioreactor and
purified by several cooling and heating purification cycles
(inverse transition cycling) taking advantage of the ability of
these recombinamers to precipitate above their T
t
. Further
centrifugation steps led to a pure product, which was dialyzed
against ultrapure water, filtered through 0.22-mm filters
(Nalgene; Thermo Fisher, USA) to obtain a sterile solution,
and freeze-dried before storage. The ELRs were found to
contain <2 endotoxin units per milligram of ELR, as deter-
mined using the limulus amebocyte lysate assay with the
Endosafe

-PTS system (Charles River Laboratories). This
process allowed the production of two different ELRs, both of
which were derived from a previously synthesized block
corecombinamer.
31
Further information can be obtained in
Supplementary Methods.
The characterization techniques used included sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-
PAGE) and matrix-assisted laser desorption/ionization time-
of-flight (MALDI-TOF) spectrometry for purity and molecular
weight (Mw) evaluation compared to the theoretical values
of 113,556 Da for ELR-Elastase-RGD (ELR-E-RGD) and
107,752 Da for ELR-Elastase-BMP-2 (ELR-E-BMP-2);
differential scanning calorimetry (DSC) to determine the T
t
(Supplementary Fig. S2); high-performance liquid chroma-
tography (HPLC) to determine the amino acid composition of
both ELRs (Supplementary Tables S1 and S2); and nuclear
magnetic resonance to provide recombinamer fingerprint data
(Supplementary Figs. S3 and S4; Supplementary Tables S3
and S4). The procedure for the measurement of the me-
chanical properties of ELR-based hydrogels is described in
Supplementary Methods.
Elastase-mediated cleavage of the ELR in solution
Different quantities (1.2, 1.8, and 2.4 U) of porcine pan-
creas elastase (4 mg/mL, 6.8 U/mg) (Sigma-Aldrich, USA)
were added to solutions of the mixture of both ELRs [98%
(w/w) ELR-E-RGD and 2% (w/w) ELR-E-BMP-2] at a final
concentration of 1 mg/mL dissolved in ultrapure water to
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evaluate the biodegradation rate for each quantity of en-
zyme. The quantity of elastase used was 2000-, 3000-, and
4000-times the amount needed to cleave the mixture of
ELRs used as substrate in 30 min, since preliminary exper-
iments showed that larger quantities than those calculated
are required to observe an actual effect of the enzyme
in vitro. Samples were incubated at 37C, collected at var-
ious time points (10, 20, 30, 45, 60, 90, and 120 min), and
then stored frozen at –20C until further use. A negative
control, namely an ELR molecule lacking elastase-sensitive
sequences but with the same elastin-like structure as the two
ELRs designed for this work,
31
was also treated with 1.2 U
of elastase for further comparisons. Methods concerning the
evaluation of the biodegradation are explained in Supple-
mentary Methods.
In vitro cell culture
Bone marrow-derived human mesenchymal stem cells
(hMSCs) were extracted and isolated as described else-
where
37
and were generously provided as a gift by Citospin
S.L. (Spain). They were cultured for expansion in Dulbecco’s
modified Eagle’s medium (DMEM) low glucose (1 g/L)
(Gibco, USA) supplemented with 10% fetal bovine serum
(Gibco) and 1% penicillin/streptomycin (Gibco).
All cells were used at passage 3–5 in subsequent exper-
iments. They were detached from the wells using a trypsin-
ethylenediaminetetraacetic acid (EDTA) solution (0.25%;
Gibco) and counted using a hematocytometer.
Cell viability
hMSCs were used to determine the in vitro viability using
the calcein AM assay (Molecular Probes, USA) when
cultured in DMEM supplemented with 10 mg/mL of the
different ELRs or the mixture of them [98% (w/w) ELR-E-
RGD and 2% (w/w) ELR-E-BMP-2] for 3 days. This assay
was performed in a black, 96-well plate with clear bottom
(Greiner Bio One, USA) according to the manufacturer’s
instructions and the fluorescence intensity measured at
530 nm using a plate reader (SpectraMax M2e; Molecular
Devices, USA). The intensity measured at this wavelength,
corresponding to live cells, was then used to calculate cell
numbers by using calibration curves obtained with different
known quantities of cells (from 1000 to 10,000 cells per
well) seeded on 96-well plates 24 h before the measurement.
Each condition was performed in triplicate, with four ex-
periments for each (n=4).
Cell adhesion on ELR-coated tissue culture plates
Ninety-six-well plates were used for the coating of dif-
ferent wells with both ELRs separately and combined [98%
(w/w) ELR-E-RGD and 2% (w/w) ELR-E-BMP-2]. Briefly,
a 5 mg/mL solution of the recombinamers in ultrapure water
was placed in the well and allowed to adsorb to the surface
for 24 h at 4C. The wells were washed twice with 1·
phosphate-buffered saline (PBS; Gibco), blocked with 1%
bovine serum albumin for 2 h at 37C, then rinsed again and,
finally, 3000 cells per well were seeded onto the modified
surfaces to study cell adhesion after 24 h. The number of
cells in each well was determined using the calcein AM
assay as described above.
Dissolution of the ELRs for the in vivo experiments
A mixture of both ELRs [98% (w/w) ELR-E-RGD and 2%
(w/w) ELR-E-BMP-2] was prepared and dissolved in sterile
tubes (1 per animal) at 300 mg/mL with 1·sterile PBS
(Gibco) by incubation at 4C for 24 h. A 2% (w/w) ELR-E-
BMP-2 solution at 300 mg/mL gives a similar amount of
BMP-2 in our device (5.57 $10
-5
M) as in INFUSE

Bone
Graft (5.77 $10
-5
M; Medtronic, USA).
38
This solution was
kept in an ice bath during surgery until implantation.
In vivo experiments
Adult female New Zealand white rabbits (n=7) with an
average weight of 3.5 kg were used for the creation and
treatment of bone defects. These animals were kept in in-
dividual cages with food (ACA Cooperativas, Argentina)
and water ad libitum.
Antibiotic prophylaxis, anesthetic treatment, and surgical
techniques were performed according to a previously de-
scribed procedure.
34,35
Further details regarding the surgical
procedure can be found in Supplementary Methods. Three
months postsurgery, the animals were euthanized using
three doses of anesthesia, as previously described.
34,39
The
femora were then collected to perform different experiments
to assess bone regeneration (see Multislice computed tomo-
graphy and Bone histopathology).
Multislice computed tomography
Multislice computed tomography (MSCT) was performed
on the seven right femurs of the rabbits using a Toshiba
Alexion apparatus with 16 detectors and a thickness of 0.5 mm.
Coronal, sagittal, and axial slices were obtained and the images
were processed using Alexion Advance Edition software with
the adaptive iterative dose reduction (AIDR 3D) algorithm,
thus obtaining the 3D reconstruction for every sample. All
images were analyzed together for an optimal comparison.
Bone histopathology
Femoral bone samples were evaluated by way of radio-
graphic studies using a conventional dental X-ray ma-
chine with dental occlusal films (Eastman Kodak, USA) to
determine the implant position to guide the histological
procedures. The femoral epiphysis was cut 4 cm below the
metaphysis using a carborundum disk cutter (Dochem, Chi-
na) attached to a dental drill under irrigation with distilled
water. The implanted area was marked with Indian ink. Two
samples were selected for decalcification using modified
Morse solution (Okayama University Dental School) and
embedded in paraffin following well-established protocols.
The samples were serially cut (7 mm thick) using a manual
rotary microtome (Micron-Zeiss, Germany), and stained with
hematoxylin and eosin. All specimens were examined by
light microscopy and evaluated by a single pathologist.
Subsequently, another pathologist (certified by the Argenti-
nean Ministry of Health No. 31455) performed an indepen-
dent review to verify microscopic observations. The reported
results reflect the mutually-agreed-upon diagnoses by both
pathologists. Photomicrographs were taken from slides of
each specimen using a Sony digital camera fitted to an
Olympus CH30 microscope with an Olympus stereo zoom
SZ51.
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Statistical analysis
Data for the in vitro experiments are reported as
mean –standard deviation (n=4). Statistical analysis of data
following a normal distribution was performed using a
one-way analysis of variance and the Holm–Sidak method.
Ap<0.05 was considered to be statistically significant,
while p>0.05 indicates no significant differences (n.s.d.).
*p<0.05, **p<0.01.
Results
ELR biosynthesis and characterization
Both ELRs were obtained as a lyophilized product in a
yield of *200 mg/L (ELR/culture volume). Their Mws and
purities were confirmed as satisfactory by SDS-PAGE and
MALDI-TOF (Fig. 1), while the T
t
calculated by DSC for
the ELRs dissolved in PBS (pH 7.4) was found to be 15.8C
and 15.3C for ELR-E-RGD and ELR-E-BMP-2, respec-
tively (Supplementary Fig. S2).
Regarding mechanical characterization, the storage mod-
ulus (G¢) of the ELR-based hydrogel at a concentration of
300 mg/mL (98% ELR-E-RGD and 2% ELR-E-BMP-2) was
found to be *1600 Pa at 37C (Supplementary Fig. S5). In
addition, hydrogels were formed above the T
t
, as observed
macroscopically (Supplementary Fig. S6).
Enzymatic cleavage of ELR molecules
by elastase digestion
Due to the incorporation of elastase-sensitive domains in
the ELR molecules designed for this work, we aimed to
verify whether elastase was able to cleave these ELRs.
Hence, a mixture of them (98% ELR-E-RGD and 2% ELR-
E-BMP-2) was dissolved at 1 mg/mL, and ELRs were found
to be cleaved in vitro in solution when different quantities of
elastase were added. As observed in Figure 2, and as ex-
pected, the ELRs are sensitive to the quantity of elastase,
and therefore, biodegradation was slower when only 1.2 U
of elastase was added to the ELR solution (Fig. 2a), whereas
an increase in the biodegradation rate was observed if 1.8 U
(Fig. 2b) or 2.4 U (Fig. 2c) of elastase was supplemented. In
contrast, no elastase-mediated cleavage was observed in the
negative control at any sample collection time (Fig. 2d).
The disappearance of the larger bands at 113.6 and
107.8 kDa was further studied by image analysis, and the
results are summarized in Figure 3. This figure clearly re-
inforces the statement made above regarding the biodegra-
dation rate, namely that biodegradation is faster as more
elastase is added to the solution.
Regarding the nascent bands observed by SDS-PAGE
(Fig. 2), we expected to obtain bands in three different Mw
ranges, namely 65.5–66.5, 46.7–48.2, and 12–12.9 kDa, as
by-products of ELR-E-RGD/BMP-2 digestion since there
FIG. 1. Molecular weight
and purity assessment by
SDS-PAGE and MALDI-
TOF mass spectrometry for
ELR-E-RGD and ELR-E-
BMP-2. MALDI-TOF spec-
tra represent nonquantitative
intensity (a.u.) against m/z
(mass divided by net charge
of the molecule) of the ELRs.
BMP-2, bone morphogenetic
protein-2; ELRs, elastin-like
recombinamers; ELR-E-
BMP-2, ELR-Elastase-BMP-
2; ELR-E-RGD, ELR-
Elastase-RGD; MALDI-
TOF, matrix-assisted laser
desorption/ionization time-
of-flight; Mw, molecular
weight; RGD, Arg-Gly-Asp;
SDS-PAGE, sodium dodecyl
sulfate–polyacrylamide gel
electrophoresis.
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are two different elastase-sensitive domains at different
points in the ELR-E-BMP-2 molecule. However, the Mw of
the higher bands was found to be 80.8 and 54.2 kDa, re-
spectively, while the band at 12–12.9 kDa could not be
observed due to the limitations of SDS-PAGE in terms of
resolution. Nevertheless, these results correlate well with
previous studies that reported a 20% increase in the apparent
Mw for different ELRs.
40,41
As such, we estimated that the
Mw plus 20% and the values showed good agreement with
those found empirically, with the experimental values for
the nascent bands being 80.8 and 54.2 kDa, while the ex-
pected values of Mw +20% were 78.6–79.8 and 56.0–57.8,
respectively (Supplementary Table S5).
hMSC viability and integrin-mediated cell adhesion
The viability of the cells after culture for 3 days in media
supplemented with the ELRs was found to be similar to that
for the negative control, that is, medium without supple-
mentation, as can be observed in Figure 4. Since no sig-
nificant differences were observed, we can conclude that
the ELRs alone, or the mixture thereof, do not affect cell
viability.
FIG. 2. SDS-PAGE images
showing the biodegradation of the
mixture of ELR-E-RGD (98%) and
ELR-E-BMP-2 (2%) in solution at
1 mg/mL mediated by (a) 1.2 U, (b)
1.8 U, and (c) 2.4 U of elastase, at
different sample collection times,
as indicated above each picture (0,
10, 20, 30, 45, 60, 90, and 120 min
after addition of the specific quan-
tity of elastase). Picture (d) shows
the lack of elastase-mediated bio-
degradation in the case of the
nonsensitive ELR. M represents the
protein molecular weight marker.
FIG. 3. Graph showing the elastase-mediated cleavage
rate of the highest molecular weight band with data obtained
from analysis of the SDS-PAGE gels from Figure 2. The net
intensity of this double band at 113.6 and 107.8 kDa is re-
presented at different sampling times.
FIG. 4. Graph showing hMSC viability results after 3 days
of culture in terms of cell number as measured using the
calcein AM assay for different ELR supplements in medium
at 10 mg/mL: ELR-E-RGD (represented as RGD), ELR-E-
BMP-2 (BMP-2), the mixture of both [98% (w/w) ELR-E-
RGD and 2% (w/w) ELR-E-BMP-2, RGD/BMP-2], and
supplement-free medium (medium only). No significant
differences ( p>0.05) were found in any case. hMSCs, hu-
man mesenchymal stem cells.
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Furthermore, the evaluation of cell adhesion in ELR-
coated tissue culture plates showed good results in the case
of ELR-E-RGD and the mixture of both. This finding was in
agreement with our expectations since the mixture contains
98% ELR-E-RGD. However, coating only with ELR-E-
BMP-2 led to statistically significantly lower levels of at-
tachment due to the lack of cell adhesion domains in the
recombinamer (Fig. 5).
Biochemical and clinical results
The welfare of the animals in the first 2 days post-
implantation was slightly affected, with disrupted walking,
as expected. After 7 days, treated animals behaved similarly
to their nonoperated control counterparts. The temperature
values, food intake, and all the biochemical parameters
measured were similar between animals from control groups
at every time point studied (n.s.d., p>0.05).
MSCT studies
MSCT studies showed bone healing in the defect area. A
closer examination of the distal metaepiphysis region, in the
medial cortical plane, showed total closure of the defect in
most of the coronal and sagittal slices for all of the samples
analyzed. However, it was possible to identify the persis-
tence of a small defect with a diameter of 1 mm in the
medial cortical plane of the lesion site in one of the femora
extracted, although only in one coronal slice and two axial
ones (Fig. 6a, b, white arrow). These results were confirmed
by radiological studies (Supplementary Fig. S7).
Bone restitution in the distal femoral metaphysis was also
observed in the 3D reconstructions of all samples, and the
created defect could not be detected (injury site indicated
with a black circle; Fig. 6c), even in the case of the sample
that showed a small defect remaining in the axial and
FIG. 5. Graph showing the number of hMSCs attached to
the ELR-coated well plates as measured using the calcein
AM assay for different ELR coatings absorbed at 5 mg/mL:
ELR-E-RGD (represented as RGD), ELR-E-BMP-2 (BMP-
2), the mixture of both [98% (w/w) ELR-E-RGD and 2%
(w/w) ELR-E-BMP-2, RGD/BMP-2], and non-coated TCP
(n=4). *p<0.05, **p<0.01. TCP, tissue culture plates.
FIG. 6. (a) Axial computed to-
mography of right rabbit femora
showing full cortical medial re-
generation in six out of seven
samples. The fourth sample from
the left shows persistence in the
cortical defect at the injury site
(white arrow). (b) Axial computed
tomography of right rabbit femora
showing a coronal slice of the
samples. From left to right, in the
fourth sample, the persistence of a
cortical defect *1 mm in size can
be observed in the distal meta-
physis (white arrow). (c) Medial
view of the 3D reconstruction of a
rabbit right femur. The restitution
of cortical bone at the distal meta-
physis can be observed (black cir-
cumference). (d) Medial view of
the 3D reconstruction of a rabbit
right femur. In this case, the sample
with the remaining partial defect
shows a small hollow with conti-
nuity (white arrow).
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coronal slices. This 3D reconstruction showed a tiny hollow
(white arrow), but the processed signal correlates to cortical
bone (Fig. 6d).
Histopathology results
The histological analytical results obtained for experi-
mental samples showed de novo bone formation in the ex-
perimental femoral injury (EFI) region. The new bone
formed was thick and comprised lamellar bone. In addition,
it showed numerous vascular channels of different calibers
and was surrounded by various osteoblast layers (Fig. 7a).
Each bone layer was deposited on the remaining ELR
hydrogel in a disorganized manner (Fig. 7b), resembling
pagetoid-like bone (Fig. 7c), in which cellular activity
produces a mosaic pattern rather than the normal linear la-
mellar pattern.
Remnants of nonbiodegraded ELR hydrogels were ob-
served inside the EFI center, showing a network or mesh
shape and surrounded by microhemorrhages and congestive
vessels. Rounded, triangular, rhomboid, or even amorphous
structures were found inside the network, showing an eo-
sinophil, granular, and mineralized pattern. These mineral-
ized structures were surrounded by osteoblast-like cells,
with osteocyte-like cells being found inside them, and an-
giogenesis could also be observed (Fig. 7d).
FIG. 7. Microphotographs taken from decalcified femoral bone sections stained with hematoxylin and eosin. (a) Thick
lamellar bone showing numerous vascular blood channels (black asterisk), remnants of the ELR hydrogel (blue asterisk),
hematopoietic bone marrow and osteoblast layers (green asterisk) are observed in the EFI region. Magnification 46.6 ·, scale
bar =1 mm. (b) The high magnification images show the interface between new lamellar bone (black asterisk) and the ELR
hydrogel (red asterisk), with a network aspect acting as a guide for cells. Magnification 233.4 ·, scale bar =500 mm. (c) At high
magnification, a few vascular channels are observed in new lamellar bone at the EFI. Each layer of bone is deposited in the form
of a ‘‘mosaic pattern,’’ resembling pagetoid-like bone (#). Magnification 700.2 ·, scale bar =250 mm. (d) At high magnifi-
cation, the ELR hydrogel shows mineralized amorphous regions surrounded by osteoblast-like cells (black arrows). Micro-
hemorrhage (red asterisk) and congestive vessels (gray asterisk) are also observed. Magnification 700.2 ·, scale bar =250 mm.
(e) At low magnification, a panoramic microphotography of the femoral epiphysis and metaphysis shows new bone formed in
the EFI region (black asterisk). This bone is surrounded by hematopoietic bone marrow (green asterisk) with a few trabeculae
(orange asterisk). Magnification 80 ·, scale bar =2 mm. (f ) A few bone nodules surrounding the EFI region are lined by several
layers of prominent osteoblasts (black arrows). Osteocytes (green arrows) are observed inside a nodule. Magnification 700.2 ·,
scale bar =250 mm. EFI, experimental femoral injury. Color images available online at www.liebertpub.com/tea
BONE REGENERATION MEDIATED BY ELR-BASED HYDROGELS 1367
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Hematopoietic bone marrow was observed surrounding
the newly formed bone in the EFI, with scattered, rounded,
nodule-like trabecular bone (Fig. 7e). This new trabecular
bone was covered by two, three, or even more layers of
prominent osteoblasts. Osteocyte cells were observed in the
inner region (Fig. 7f). Furthermore, several congestive
vessels were observed close to each trabecula.
Discussion
To address the main aim of this work, namely the re-
generation of an FBI in 3-year-old female New Zealand
white rabbits, two different bioactive ELRs have been de-
veloped and characterized to meet the requirements of novel
biomaterials commonly used for that purpose. These novel
ELRs were specifically designed to be osteoinductive, by
fusing BMP-2 to one of them, and osteoconductive, by
fusing RGD domains that promote cell adhesion, thus al-
lowing surrounding cells to interact with the hydrogel and
possibly promote bone formation even from inside the
scaffold.
Initially, it was shown that the T
t
is lower than body
temperature, which may permit the formation of hydrogels
once the ELR solution is injected into the body. In addition,
this T
t
is similar to that described previously for the non-
bioactive ELR, which was found to be 13.0C,
42
although an
increase of 2.8C and 2.3C was observed for ELR-E-RGD
and ELR-E-BMP-2, respectively. This can be explained by
the lower hydrophobicity of the ELR molecule when other
more hydrophilic peptides or proteins containing charged
residues are fused to it.
43,44
Regarding the rheological data, although this system is
intended to be used for bone regeneration and the storage
modulus is very low in comparison with bone tissue, this
hydrogel was designed to be able to promote cell invasion
and proliferation inside itself, acting as a temporary soft
tissue that promotes optimal regeneration in a manner
through which the implanted scaffold is substituted by host
tissue. As such, although it may not be useful on its own for
treating large bone defects, it has been shown to be very
suitable in the FBI model used in this work since the hy-
drogel remains free from significant mechanical stress.
45
Biodegradation of the ELR molecules in solution has
been confirmed in vitro, thus showing that this process can
also be controlled by varying the quantity of elastase used.
Although this fails to imitate in vivo conditions, it sheds
light onto the biodegradation kinetics. The use of elastase-
sensitive sequences should also allow the slow release of
BMP-2 from the ELR molecule to exert its biological effect.
Consequently, the ELR-based hydrogel acts as a drug-
delivery system. Despite the fact that there are other ex-
amples in which rhBMP-2 and ELRs are combined as an
encapsulation system,
46
this approach allows a more effi-
cient production and application by taking advantage of
recombinant DNA technology.
The excellent cell adhesion found on surfaces coated with
ELR-E-RGD was similar to that obtained in other studies
using RGD-containing ELRs.
47
As such, this work demon-
strates that the inclusion of RGD sequences in the final ELR
molecule by genetic-engineering methods promotes cell
attachment and therefore provides a more ECM-mimetic
environment that is also osteoconductive. ELR-E-BMP-2-
coated substrates did not support cell adhesion due to the
absence of cell adhesion motifs in the ELR itself and in
BMP-2. With regard to cell viability, the lack of differences
between the negative control (medium only) and the media
supplemented with the recombinamers is in agreement with
previous studies in which a cell culture medium was sup-
plemented with ELRs.
48
Regarding the clinical and biochemical results of the
implant process, although initially affected by the surgery
per se, animal gait recovered rapidly to normal conditions.
The lack of change in the biochemical parameters showed
that neither the surgical procedure nor the subsequent pos-
sible matrix biodegradation had any effect on the animals,
thus showing good biocompatibility.
The images obtained in the tomographic study with 3D
reconstruction of the samples show promising results since
the signal patterns processed in this work are correlated to
bone tissue with similar characteristics to the surrounding
tissue, with complete closure of the defect being achieved in
six out of seven samples. Although a defect *1 mm in di-
ameter was still visible in the remaining animal, this was
only the case in three tomographic slices and may simply be
a consequence of a lack of time for the regeneration process
in this particular animal. However, the bone formed had the
same characteristics as the other samples, and therefore, it
can be concluded that these ELR-based matrices have a high
osteogenic potential to restitute a bone defect of 6 mm di-
ameter and 6 mm depth ad integrum in 90 days, most
probably due to fusion of the BMP-2 protein to the ELR,
which results in a BMP-2-loaded hydrogel.
The histological analyses showed that the FBI was re-
placed by dense, new lamellar bone. Although a few remnants
of the ELR were observed at 90 days postimplantation, they
were surrounded by congestive vessels and dense laminar
bone. This supports the accepted knowledge through which
new bone is only formed in the presence of blood irrigation.
49
This new bone is arranged randomly, with an irregular ar-
rangement in various different directions, thus suggesting that
the ELR-based hydrogels act as a carrier for BMP-2, with
osteoprogenitor cells colonizing these hydrogels, depositing
osteoid matrix, and mineralizing as pagetoid-like bone,
probably driven by the network arrangement of the ELR-
based hydrogels.
50
The new trabeculae obtained show a pe-
culiar shape, as if they were obtained by the confluence of
rounded isolated bone formations. The numerous layers of
prominent osteoblasts and various shapes observed, which
appear to simulate pseudostratification, could be a result of
the activity of BMP-2.
22,51,52
As observed in vivo from the microscopic results, the
ELR-based hydrogel was found to be biodegraded as bone
formation occurred since the cells involved in this phe-
nomenon were stimulated by the BMP-2 released into the
microenvironment, probably slowly enough to allow the
differentiation of stem and progenitor cells. As such, in this
situation, elastase (matrix metalloproteinase-12, MMP-12)
secretion by osteoclasts might be increased as a conse-
quence of matrix remodeling due to the formation of de
novo bone tissue, as suggested before.
53
This could lead to
degradation of the ELR-based hydrogel, which is sensitive
to MMP-12 as a result of inclusion of the Val-Pro-Val-Ala-
Pro-Gly (VPVAPG) sequence, as described previously.
54
On the contrary, ELRs without cleavable domains are not
1368 COLETTA ET AL.
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supposed to be biodegraded. In this regard, Sallach et al.
reported a long-term stability (up to 1 year) of a physically
crosslinked ELR-based hydrogel, similar to the one used in
our work, when implanted in vivo.
55
In our case, biodegra-
dation might happen simultaneously with bone regeneration,
thus resulting in a resorbable matrix that maintains bone
integrity until full regeneration. In addition, the peptides
resulting from the degradation of VPVAPG have been re-
ported to exhibit a strong cell proliferation activity that may
promote tissue repair, as described previously.
56
Further-
more, RGD sequences provide anchoring points for cells
that help them to migrate and proliferate inside the scaffold,
thereby promoting self-regeneration of the damaged tissue.
Although several approaches have been developed in the
field of tissue engineering, to the best of our knowledge this is
the first work describing the use of ELR-based hydrogels for
the successful regeneration of a bone defect in vivo.Previous
examples make use of ELRs in combination with other ma-
terials,
57,58
andmostofthemhaveonlybeentestedin vitro,
although with promising results.
59
Besides, the ELR-based
hydrogel described in this study overcomes different issues
regarding the use of biomaterials in bone tissue engineering.
For instance, BMP-2 is not only loaded inside the hydrogel,
but it is part of it. Hence, there is no need to add this oste-
ogenic factor during the preparation of the scaffold, in con-
trast to other works,
60
reducing its cost. In addition, this
acellular system has shown to be able to promote optimal
bone regeneration, while other studies report good outcomes
only in the presence of mesenchymal stromal cells.
61,62
On
the contrary, another acellular scaffold has been described,
showing its usefulness in bone regeneration.
63
However, this
system is not injectable and thus requires the use of invasive
methods for its implantation. Moreover, the adaptation of this
scaffold to the shape of the defect depends on the mold used
in its development, reducing its versatility.
In conclusion, this work shows that a mixture of the
originally designed ELRs is able to self-assemble into an
appropriate BMP-2 carrier, namely an injectable and bio-
degradable hydrogel, which allows the slow release of this
osteogenic factor, thereby stimulating progenitor and stem
cell differentiation and osteoblast proliferation. Further-
more, the resulting ELR-based hydrogels also demonstrated
an osteoconductive behavior since they provide an ECM-
like environment as a result of the inclusion of RGD se-
quences. These two bioactivities (RGD and BMP-2), together
with elastase sensitiveness, were easily included in the final
ELR molecules in a controlled manner, due to their recom-
binant nature. Endogenous cells were able to migrate and
proliferate into these hydrogels, thereby favoring bone neo-
formation at the femoral injury, as confirmed by CT, radi-
ography, and histology.
Acknowledgments
The authors are grateful for funding from the Euro-
pean Commission (NMP-2014-646075, HEALTH-F4-2011-
278557, PITN-GA-2012-317306, and MSCA-ITN-2014-
642687), the MINECO of the Spanish Government
(MAT2013-42473-R and MAT2013-41723-R), the Junta de
Castilla y Leo
´n (VA244U13 and VA313U14), and the
Centro en Red de Medicina Regenerativa y Terapia Celular
de Castilla y Leo
´n. Dante J. Coletta has been funded by the
Consejo Nacional de Investigaciones de Ciencia y Tecno-
logı
´a de la Nacio
´n (CONICET, Argentina). They also thank
Dr. Pedro Esbrit from the Jime
´nez Dı
´az Foundation.
Disclosure Statement
No competing financial interests exist.
References
1. Amini, A.R., Laurencin, C.T., and Nukavarapu, S.P. Bone
tissue engineering: recent advances and challenges. Crit
Rev Biomed Eng 40, 363, 2012.
2. Khademhosseini, A., and Langer, R. A decade of progress
in tissue engineering. Nat Protocols 11, 1775, 2016.
3. Yoseph, B.-C. Biomimetics. Boca Raton: CRC Press, 2005,
pp. 495.
4. Costa, H.S., Mansur, A.A.P., Pereira, M.M., and Mansur,
H.S Engineered hybrid scaffolds of poly(vinyl alcohol)/
bioactive glass for potential bone engineering applications:
synthesis, characterization, cytocompatibility, and degra-
dation. J Nanomater 2012, 16, 2012.
5. Santolini, E., West, R., and Giannoudis, P.V. Risk factors
for long bone fracture non-union: a stratification approach
based on the level of the existing scientific evidence. Injury
46(Suppl 8), S8, 2015.
6. Chrcanovic, B.R., Albrektsson, T., and Wennerberg, A.
Dental implants inserted in male versus female patients: a
systematic review and meta-analysis. J Oral Rehabil 42,
709, 2015.
7. Cointry, G.R., Capozza, R.F., Feldman, S., Reina, P., and
Ferretti, J.L. Estructura y biomeca
´nica o
´sea. In: Ardila, E.
ed. Osteoporosis en Iberoame
´rica, 2nd edition. Me
´xico DF
(Me
´xico): El Manual Moderno, 2012, pp. 33.
8. Compston, J. Osteoporosis: social and economic impact.
Radiol Clin North Am 48, 477, 2010.
9. Curran, D., Maravic, M., Kiefer, P., Tochon, V., and Far-
dellone, P. Epidemiology of osteoporosis-related fractures
in France: a literature review. Joint Bone Spine 77, 546,
2010.
10. Cointry, G.R., Capozza, R.F., Feldman, S., Reina, P.,
Grappiolo, I., Ferretti, S.E., Mortarino, P., Chiappe, M.A.,
and Ferretti, J.L. Los huesos son estructuras gene
´ticas,
metabo
´licas, biomeca
´nicas, o todo a la vez. Actual osteol 5,
184, 2009.
11. Melton, L.J., 3rd, Achenbach, S.J., Atkinson, E.J., Ther-
neau, T.M., and Amin, S. Long-term mortality following
fractures at different skeletal sites: a population-based co-
hort study. Osteoporos Int 24, 1689, 2013.
12. Kremenetzky, A., Kremenetzky, L., and Feldman, S.
Aplicacio
´n de aloinjerto o
´seo como cemento biolo
´gico.
Revista de la Asociacio
´n Argentina de Ortopedia y Trau-
matologı
´a71, 61, 2006.
13. Alghazali, K.M., Nima, Z.A., Hamzah, R.N., Dhar, M.S.,
Anderson, D.E., and Biris, A.S. Bone-tissue engineering:
complex tunable structural and biological responses to in-
jury, drug delivery, and cell-based therapies. Drug Metab
Rev 47, 431, 2015.
14. Zhang, S., Zhang, X., Cai, Q., Wang, B., Deng, X., and
Yang, X. Microfibrous beta-TCP/collagen scaffolds mimic
woven bone in structure and composition. Biomed Mater 5,
065005, 2010.
15. Arealis, G., and Nikolaou, V.S. Bone printing: new fron-
tiers in the treatment of bone defects. Injury 46(Suppl 8),
S20, 2015.
BONE REGENERATION MEDIATED BY ELR-BASED HYDROGELS 1369
Downloaded by 157.88.202.121 from online.liebertpub.com at 01/02/18. For personal use only.

16. Salgado, A.J., Oliveira, J.M., Martins, A., Teixeira, F.G.,
Silva, N.A., Neves, N.M., Sousa, N., and Reis, R.L.
Chapter 1—Tissue engineering and regenerative medicine:
past, present, and future. In: Stefano Geuna, I.P.P.T., and
Bruno, B., eds. International Review of Neurobiology.
Cambridge, MA: Academic Press, 2013, pp. 1.
17. Detsch, R., and Boccaccini, A.R. The role of osteoclasts in
bone tissue engineering. J Tissue Eng Regen Med 9, 1133,
2015.
18. van der Stok, J., Lozano, D., Chai, Y.C., Amin Yavari, S.,
Bastidas Coral, A.P., Verhaar, J.A.N., Go
´mez-Barrena, E.,
Schrooten, J., Jahr, H., Zadpoor, A.A., Esbrit, P., and
Weinans, H. Osteostatin-coated porous titanium can im-
prove early bone regeneration of cortical bone defects in
rats. Tissue Eng Part A 21, 1495, 2015.
19. Romagnoli, C., D’Asta, F., and Brandi, M.L. Drug delivery
using composite scaffolds in the context of bone tissue
engineering. Clin Cases Miner Bone Metab 10, 155, 2013.
20. Inoda, H., Yamamoto, G., and Hattori, T. Histological in-
vestigation of osteoinductive properties of rh-BMP2 in a rat
calvarial bone defect model. J Craniomaxillofac Surg 32,
365, 2004.
21. Chen, D., Zhao, M., and Mundy, G.R. Bone morphogenetic
proteins. Growth Factors 22, 233, 2004.
22. dos S Kotake, B.G., Salzedas, L.M., Ervolino, E., Calzzani,
R.A., Sebald, W., and Issa, J.P. Bone recuperation after
rhBMP-2 insertion in alcoholic animals-experimental
study. Curr Pharm Des 21, 3557, 2015.
23. Sie
´ssere, S., de Sousa, L.G., Issa, J.P.M., Iyomasa, M.M.,
Pitol, D.L., Barbosa, A.P.A., Semprini, M., Sebald, W.,
Bentley, M.V.B., and Regalo, S.C.H. Application of low-
level laser irradiation (LLLI) and rhBMP-2 in critical bone
defect of ovariectomized rats: histomorphometric evalua-
tion. Photomed Laser Surg 29, 453, 2011.
24. Abdala, P.M.F., Iyomasa, M.M., Sato, S., Bentley,
M.V.L.B., Pitol, D.L., Regalo, S.C.H., Sie
´ssere, S., and
Issa, J.P.M. Osteoinductivity potential of rhBMP-2 associ-
ated with two carriers in different dosages. Anat Sci Int 85,
181, 2010.
25. Issa, J.P.M., do Nascimento, C., Bentley, M.V.L.B., Del
Bel, E.A., Iyomasa, M.M., Sebald, W., and de Albu-
querque, Jr, R.F. Bone repair in rat mandible by rhBMP-2
associated with two carriers. Micron 39, 373, 2008.
26. Haidar, Z.S., Hamdy, R.C., and Tabrizian, M. Delivery of
recombinant bone morphogenetic proteins for bone regen-
eration and repair. Part A: current challenges in BMP de-
livery. Biotechnol Lett 31, 1817, 2009.
27. Schmidmaier, G., Schwabe, P., Strobel, C., and Wild-
emann, B. Carrier systems and application of growth fac-
tors in orthopaedics. Injury 39, S37, 2008.
28. Girotti, A., Orbanic, D., Iba
´n
˜ez-Fonseca, A., Gonzalez-
Obeso, C., and Rodrı
´guez-Cabello, J.C. Recombinant
technology in the development of materials and systems for
soft-tissue repair. Adv Healthc Mater 4, 2423, 2015.
29. Rodrı
´guez-Cabello, J.C., Martı
´n, L., Alonso, M., Arias,
F.J., and Testera, A.M. ‘‘Recombinamers’’ as advanced
materials for the post-oil age. Polymer 50, 5159, 2009.
30. Urry, D.W. Molecular machines: how motion and other
functions of living organisms can result from reversible
chemical changes. Angew Chem Int Ed Eng 32, 819, 1993.
31. Martin, L., Arias, F.J., Alonso, M., Garcia-Arevalo, C.,
and Rodriguez-Cabello, J.C. Rapid micropatterning by
temperature-triggered reversible gelation of a recombinant
smart elastin-like tetrablock-copolymer. Soft Matter 6,
1121, 2010.
32. Ruoslahti, E., and Pierschbacher, M.D. Arg-Gly-Asp: a
versatile cell recognition signal. Cell 44, 517, 1986.
33. Lombard, C., Arzel, L., Bouchu, D., Wallach, J., and
Saulnier, J. Human leukocyte elastase hydrolysis of pep-
tides derived from human elastin exon 24. Biochimie 88,
1915, 2006.
34. Goy, D.P., Gorosito, E., Costa, H.S., Mortarino, P., Pede-
monte, N.A., Toledo, J., Mansur, H.S., Pereira, M.M.,
Battaglino, R., and Feldman, S. Hybrid matrix grafts to
favor tissue regeneration in rabbit femur bone lesions. Open
Biomed Eng J 6, 85, 2012.
35. Coletta, D.J., Lozano, D., Rocha-Oliveira, A.A., Mortarino,
P., Bumaguin, G.E., Vitelli, E., Vena, R., Missana, L.,
Jammal, M.V., Portal-Nunez, S., Pereira, M., Esbrit, P., and
Feldman, S. Characterization of hybrid bioactive glass-
polyvinyl alcohol scaffolds containing a PTHrP-derived
pentapeptide as implants for tissue engineering applica-
tions. Open Biomed Eng J 8, 20, 2014.
36. Rodrı
´guez-Cabello, J.C., Girotti, A., Ribeiro, A., and Arias,
F.J. Synthesis of genetically engineered protein polymers
(recombinamers) as an example of advanced self-assembled
smart materials. In: Navarro, M., and Planell, J.A., eds.
Nanotechnology in Regenerative Medicine: Methods and
Protocols. Totowa, NJ: Humana Press, 2012, p. 17.
37. Orozco, L., Munar, A., Soler, R., Alberca, M., Soler, F.,
Huguet, M., Sentis, J., Sanchez, A., and Garcia-Sancho, J.
Treatment of knee osteoarthritis with autologous mesenchy-
mal stem cells: a pilot study. Transplantation 95, 1535, 2013.
38. McKay, W.F., Peckham, S.M., and Badura, J.M. A com-
prehensive clinical review of recombinant human bone
morphogenetic protein-2 (INFUSE

Bone Graft). Int Or-
thop 31, 729, 2007.
39. Feldman, S., Cointry, G.R., Leite Duarte, M.a.E., Sarrio
´, L.,
Ferretti, J.L., and Capozza, R.F. Effects of hypophysec-
tomy and recombinant human growth hormone on material
and geometric properties and the pre- and post-yield be-
havior of femurs in young rats. Bone 34, 203, 2004.
40. Meyer, D.E., and Chilkoti, A. Genetically encoded syn-
thesis of protein-based polymers with precisely specified
molecular weight and sequence by recursive directional
ligation: examples from the elastin-like polypeptide system.
Biomacromolecules 3, 357, 2002.
41. McPherson, D.T., Xu, J., and Urry, D.W. Product purifi-
cation by reversible phase transition following Escherichia
coli expression of genes encoding up to 251 repeats of the
elastomeric pentapeptide GVGVP. Protein Expr Purif 7, 51,
1996.
42. Fernandez-Colino, A., Arias, F.J., Alonso, M., and
Rodriguez-Cabello, J.C. Self-organized ECM-mimetic
model based on an amphiphilic multiblock silk-elastin-like
corecombinamer with a concomitant dual physical gelation
process. Biomacromolecules 15, 3781, 2014.
43. Christensen, T., Hassouneh, W., Trabbic-Carlson, K., and
Chilkoti, A. Predicting transition temperatures of elastin-
like polypeptide fusion proteins. Biomacromolecules 14,
1514, 2013.
44. Iba
´n
˜ez-Fonseca, A., Alonso, M., Arias, F.J., and
Rodrı
´guez-Cabello, J.C. Fo
¨rster resonance energy transfer-
paired hydrogel forming silk-elastin-like recombinamers by
recombinant conjugation of fluorescent proteins. Bioconjug
Chem 28, 828, 2017.
1370 COLETTA ET AL.
Downloaded by 157.88.202.121 from online.liebertpub.com at 01/02/18. For personal use only.

45. Gibbs, D.M.R., Black, C.R.M., Dawson, J.I., and Oreffo,
R.O.C. A review of hydrogel use in fracture healing and
bone regeneration. J Tissue Eng Regen Med 10, 187, 2016.
46. Bessa, P.C., Machado, R., Nurnberger, S., Dopler, D.,
Banerjee, A., Cunha, A.M., Rodriguez-Cabello, J.C., Redl,
H., van Griensven, M., Reis, R.L., and Casal, M. Ther-
moresponsive self-assembled elastin-based nanoparticles
for delivery of BMPs. J Control Release 142, 312, 2010.
47. de Torre, I.G., Wolf, F., Santos, M., Rongen, L., Alonso,
M., Jockenhoevel, S., Rodriguez-Cabello, J.C., and Mela,
P. Elastin-like recombinamer-covered stents: towards a
fully biocompatible and non-thrombogenic device for car-
diovascular diseases. Acta Biomater 12, 146, 2015.
48. Pina, M.J., Girotti, A., Santos, M., Rodriguez-Cabello, J.C.,
and Arias, F.J. Biocompatible ELR-based polyplexes
coated with MUC1 specific aptamers and targeted for
breast cancer gene therapy. Mol Pharm 13, 795, 2016.
49. Tomlinson, R.E., and Silva, M.J. Skeletal blood flow in
bone repair and maintenance. Bone Res 1, 311, 2013.
50. Missana, L., Nagai, N., and Kuboki, Y. Comparative his-
tological studies of bone and cartilage formations induced
by various BMP-carrier composites. J Oral Biosci 36, 9,
1994.
51. Issa, J.P.M., Defino, H.L.A., Netto, J.C., Volpon, J.B.,
Regalo, S.C.H., Iyomasa, M.M., Sie
´ssere, S., and Tiossi,
R. Evaluation of rhBMP-2 and natural latex as poten-
tial osteogenic proteins in critical size defects by histo-
morphometric methods. Anat Rec (Hoboken) 293, 794,
2010.
52. Issa, J.P.M., Do Nascimento, C., Lamano, T., Iyomasa,
M.M., Sebald, W., and De Albuquerque, R.F., Jr. Effect of
recombinant human bone morphogenetic protein-2 on bone
formation in the acute distraction osteogenesis of rat
mandibles. Clin Oral Implants Res 20, 1286, 2009.
53. Hou, P., Troen, T., Ovejero, M.C., Kirkegaard, T., Andersen,
T.L., Byrjalsen, I., Ferreras, M., Sato, T., Shapiro, S.D.,
Foged, N.T., and Delaisse
´, J.-M. Matrix metalloproteinase-
12 (MMP-12) in osteoclasts: new lesson on the involvement
of MMPs in bone resorption. Bone 34, 37, 2004.
54. Taddese, S., Weiss, A.S., Jahreis, G., Neubert, R.H.H., and
Schmelzer, C.E.H. In vitro degradation of human tropoe-
lastin by MMP-12 and the generation of matrikines from
domain 24. Matrix Biol 28, 84, 2009.
55. Sallach, R.E., Cui, W., Balderrama, F., Martinez, A.W.,
Wen, J., Haller, C.A., Taylor, J.V., Wright, E.R., Long,
R.C., Jr., and Chaikof, E.L. Long-term biostability of self-
assembling protein polymers in the absence of covalent
crosslinking. Biomaterials 31, 779, 2010.
56. Rodriguez-Cabello, J., Ribeiro, A., Reguera, J., Girotti, A.,
and Testera, A. Chapter 14—Elastin-like systems for tissue
engineering. In: Reis, R.L. (ed). Natural-Based Polymers
for Biomedical Applications. Cambridge, UK: Woodhead
Publishing, 2008, p. 374.
57. Tejeda-Montes, E., Klymov, A., Nejadnik, M.R., Alonso,
M., Rodriguez-Cabello, J.C., Walboomers, X.F., and Mata,
A. Mineralization and bone regeneration using a bioactive
elastin-like recombinamer membrane. Biomaterials 35,
8339, 2014.
58. Prieto, S., Shkilnyy, A., Rumplasch, C., Ribeiro, A., Arias,
F.J., Rodrı
´guez-Cabello, J.C., and Taubert, A. Biomimetic
calcium phosphate mineralization with multifunctional elastin-
like recombinamers. Biomacromolecules 12, 1480, 2011.
59. Vila, M., Garcı
´a, A., Girotti, A., Alonso, M., Rodrı
´guez-
Cabello, J.C., Gonza
´lez-Va
´zquez, A., Planell, J.A., Engel,
E., Buja
´n, J., Garcı
´a-Honduvilla, N., and Vallet-Regı
´,M.
3D silicon doped hydroxyapatite scaffolds decorated with
elastin-like recombinamers for bone regenerative medicine.
Acta Biomater 45, 349, 2016.
60. Perez, R.A., Kim, J.-H., Buitrago, J.O., Wall, I.B., and
Kim, H.-W. Novel therapeutic core–shell hydrogel scaf-
folds with sequential delivery of cobalt and bone morpho-
genetic protein-2 for synergistic bone regeneration. Acta
Biomater 23, 295, 2015.
61. Jo, S., Kim, S., Cho, T.H., Shin, E., Hwang, S.J., and Noh,
I. Effects of recombinant human bone morphogenic
protein-2 and human bone marrow-derived stromal cells on
in vivo bone regeneration of chitosan–poly(ethylene oxide)
hydrogel. J Biomed Mater Res A 101A, 892, 2013.
62. Ren, Z., Wang, Y., Ma, S., Duan, S., Yang, X., Gao, P.,
Zhang, X., and Cai, Q. Effective bone regeneration using
thermosensitive poly(N-isopropylacrylamide) grafted gela-
tin as injectable carrier for bone mesenchymal stem cells.
ACS Appl Mater Interfaces 7, 19006, 2015.
63. Nguyen, T.B.L., and Lee, B.-T. A combination of biphasic
calcium phosphate scaffold with hyaluronic acid-gelatin
hydrogel as a new tool for bone regeneration. Tissue Eng
Part A 20, 1993, 2014.
Address correspondence to:
Jose
´Carlos Rodrı
´guez-Cabello, PhD
BIOFORGE Lab
Universidad de Valladolid
Paseo de Bele
´n, 19
Valladolid 47011
Spain
E-mail: roca@bioforge.uva.es
Received: January 23, 2017
Accepted: March 3, 2017
Online Publication Date: August 22, 2017
BONE REGENERATION MEDIATED BY ELR-BASED HYDROGELS 1371
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