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


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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.
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 magnification, the ELR hydrogel shows mineralized amorphous regions surrounded by osteoblast-like cells (black arrows). Microhemorrhage (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
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Bone Regeneration Mediated by a Bioactive
and Biodegradable Extracellular Matrix-Like Hydrogel
Based on Elastin-Like Recombinamers
Dante J. Coletta, MD,
** Arturo Iba´n˜ ez-Fonseca, MSc,
** Liliana R. Missana, PhD,
** Marı´a V. Jammal, PhD,
Ezequiel J. Vitelli, MD,
Mariangeles Aimone, BSc,
Facundo Zabalza, BSc,
Joa˜o P. Mardegan Issa, PhD,
Matilde Alonso, PhD,
Jose´ Carlos Rodrı´guez-Cabello, PhD,
and Sara Feldman, PhD
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
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.
addition, changes of the age pyramid toward an older popu-
lation have led to an increasing number of bone fractures.
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
LABOATEM, Osteoarticular Biology, Tissue Engineering and Emerging Therapies Laboratory, School of Medicine, National Rosario
University, Rosario, Argentina.
BIOFORGE Lab, University of Valladolid, CIBER-BBN, Valladolid, Spain.
Experimental Pathology and Tissue Engineering Laboratory, Dental School, National Tucuma
´n University, Tucuma
´n, Argentina.
Tissues Laboratory, Proimi-Biotechnology-Conicet, Tucuma
´n, Argentina.
˜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.
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.
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.
They should also stimulate an optimal cell
response and be liable to be replaced by de novo formed
tissue, acting as a provisional substitute.
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
For instance, the osteoinductive bone
morphogenetic protein-2 (BMP-2)
has been shown to
enhance the formation of bone tissue in situations that lead
to bone degradation, such as alcohol dependence
and os-
teometabolic diseases.
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.
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-
As an example, elastin-like recombinamers
(ELRs), thus named due to their polymeric and recombinant
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
), 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
while above that T
(e.g., physiologic temperature), the
ELR self-assembles hydrophobically, undergoing a phase
In this study, two different ELRs have been de-
veloped, based on a previously described hydrogel-forming
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
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
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
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.
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.
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
. 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
-PTS system (Charles River Laboratories). This
process allowed the production of two different ELRs, both of
which were derived from a previously synthesized block
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
(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 37C, collected at var-
ious time points (10, 20, 30, 45, 60, 90, and 120 min), and
then stored frozen at –20C 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,
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-
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 4C. The wells were washed twice with 1·
phosphate-buffered saline (PBS; Gibco), blocked with 1%
bovine serum albumin for 2 h at 37C, 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 4C 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
M) as in INFUSE
Graft (5.77 $10
M; Medtronic, USA).
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.
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.
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
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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.
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
calculated by DSC for
the ELRs dissolved in PBS (pH 7.4) was found to be 15.8C
and 15.3C 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 37C (Supplementary Fig. S5). In
addition, hydrogels were formed above the 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
TOF mass spectrometry for
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-
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
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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.
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
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
Downloaded by from at 01/02/18. For personal use only.
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.
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
Initially, it was shown that the 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
is similar to that described previously for the non-
bioactive ELR, which was found to be 13.0C,
although an
increase of 2.8C and 2.3C 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.
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.
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,
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.
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.
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.
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.
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.
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.
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.
On the contrary, ELRs without cleavable domains are not
Downloaded by from at 01/02/18. For personal use only.
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.
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.
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-
andmostofthemhaveonlybeentestedin vitro,
although with promising results.
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,
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.
the contrary, another acellular scaffold has been described,
showing its usefulness in bone regeneration.
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.
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-
´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.
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Address correspondence to:
´Carlos Rodrı
´guez-Cabello, PhD
Universidad de Valladolid
Paseo de Bele
´n, 19
Valladolid 47011
Received: January 23, 2017
Accepted: March 3, 2017
Online Publication Date: August 22, 2017
Downloaded by from at 01/02/18. For personal use only.
... Elastin-like recombinamers elastase-sensitive domain BMP-2, RGD Crosslinking / Bone regeneration [65] MMP7-sensitive peptide, maleimide-modified hyaluronic acid, RGD SDF-1α and BMP-2 Crosslinking 5 min Bone regeneration [66] Poly(ethyleneglycol) diacrylate (PEGDA), cathepsin-K-sensitive peptide GGGMGPSGWGGK (GPSG) / Crosslinking 1 h Selective degradation [67] Polyethylene glycol ID-SW3 Crosslinking 10 min Cell differentiation [68] PEG norbornene, thiolated chondroitin sulfates, GRGDS, MMP7-sensitive peptide hMSCs Crosslinking 8 min Cartilage regeneration [69] pH Carboxymethyl chitosan, amorphous calcium phosphate BMP-9 ...
... BMPs can effectively induce the osteogenic differentiation of mesenchymal stem cells (MSCs) [104]. Coletta et al. [65] developed two different bioactive materials, elastin-like recombinamers (ELRs) containing BMP-2 or a cell adhesion peptide motif (Arg-Gly-Asp: RGD), to form a BMP-2 loaded extracellular-matrix-like hydrogel. The material degrades via an enzymatic reaction through the addition of an elastase-sensitive domain into the ELR, so the scaffold should not be removed after bone healing. ...
... Chitosan is obtained via deacetylation of chitin and is composed of b-(1/4)-2amido-2-deoxy-D-glucan (glucosamine) and b-(1/4)-2-acetamido-2-deoxy-D-glucan (acetyl glucosamine) units [108]. Chitosan can be dissolved in acidic solutions with a pH less than or equal to 6.5, leading to the protonation of its amine groups [65,67]. The sol-gel transition occurs when β-glycerophosphate is added to neutralize the positive charge during the preparation process so that chitosan can exist as a soluble form in the neutral solution. ...
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Bone and cartilage regeneration is an area of tremendous interest and need in health care. Tissue engineering is a potential strategy for repairing and regenerating bone and cartilage defects. Hydrogels are among the most attractive biomaterials in bone and cartilage tissue engineering, mainly due to their moderate biocompatibility, hydrophilicity, and 3D network structure. Stimuli-responsive hydrogels have been a hot topic in recent decades. They can respond to external or internal stimulation and are used in the controlled delivery of drugs and tissue engineering. This review summarizes current progress in the use of stimuli-responsive hydrogels in bone and cartilage regeneration. The challenges, disadvantages, and future applications of stimuli-responsive hydrogels are briefly described.
... Thus, we can obtain proangiogenic (biodegradable and bioactive) hydrogels with these ELRs, which are also injectable due to the control of the gelation time upon mixing both ELR-azide and ELR-cyclooctyne solutions. Hydrogels based on ELRs are being increasingly used because of their biocompatibility (Ibáñez-Fonseca et al., 2018), and they have already proven their ability to promote tissue regeneration (e.g., osteochondral, bone, skin, skeletal muscle tissue, and myocardium) (Coletta et al., 2017;Pescador et al., 2017;Ibáñez-Fonseca et al., 2020;Contessotto et al., 2021;Stojic et al., 2021). Moreover, Staubli et al. demonstrated that the use of the aforementioned ELR-based hydrogels promotes cell adhesion, in vivo angiogenesis, and integration into the host tissue (Staubli et al., 2017). ...
... In addition, these MSCs showed even more elongated and flat morphology (Cipriani et al., 2019). Other works describe an increased number of protrusions in MSC, demonstrating their adhesion to the biomaterial in vitro and how the hydrogel was present after 28 days in vivo (Staubli et al., 2017;Coletta et al., 2017). It seems that the REVD tetrapeptide has a key role in this adhesion (Girotti et al., 2020). ...
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Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)–based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti–human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti–CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.
... However, their degradation products may lead to the formation of an acidic local microenvironment around the area of degradation which may cause damage to the surrounding cells [11]. Currently, special efforts and progress is being made towards the synthesis of enzyme-sensitive biomaterials, containing recognition sites for proteases such as matrix metalloproteinases (MMPs) [12][13][14] and serine proteases such as urokinase [15] plasmin [16], elastase [17] or trypsin [18], which could be used for tissue engineering as well as for drug delivery purposes. For example, trypsin-recognized hydrogels could be useful for encapsulating drugs for delivery into the small intestine [18] while the design of scaffolds with MMP-cleavable sequences could be relevant for the development of anti-cancer drug delivery platforms, as matrix metalloproteinases are highly overexpressed in tumor tissues [19,20]. ...
... Moreover, the importance of MMP-mediated degradation during capillary morphogenesis justifies the presence of MMP-degradable peptide sequences in synthetic hydrogels used to promote and study vasculogenesis [16]. Additionally, the presence of protease-sensitive domains in hydrogels used for tissue engineering applications may confer scaffold biodegradability, thus, avoiding the need for scaffold removal after tissue regeneration [17]. ...
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One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH2), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine.
... It has excellent potential to promote bone defect repair when loaded into hydrogel scaffolds as an active factor. It is beneficial to repair bone defects [127]. ...
Full-text available
Large bone defects due to trauma, infections, and tumors are difficult to heal spontaneously by the body’s repair mechanisms and have become a major hindrance to people’s daily lives and economic development. However, autologous and allogeneic bone grafts, with their lack of donors, more invasive surgery, immune rejection, and potential viral transmission, hinder the development of bone repair. Hydrogel tissue bioengineered scaffolds have gained widespread attention in the field of bone repair due to their good biocompatibility and three-dimensional network structure that facilitates cell adhesion and proliferation. In addition, loading natural products with nanoparticles and incorporating them into hydrogel tissue bioengineered scaffolds is one of the most effective strategies to promote bone repair due to the good bioactivity and limitations of natural products. Therefore, this paper presents a brief review of the application of hydrogels with different gel-forming properties, hydrogels with different matrices, and nanoparticle-loaded natural products loaded and incorporated into hydrogels for bone defect repair in recent years.
Bone grafting, as the current gold-standard for large scaled bone damage of various causes has faced challenges from both the source and appliance. Emerging new tissue engineering substi-tutes are demonstrating more options and possibilities, with their improved biocompatibility, accessibility, and customizable function. Amongst them, injectable gels (IGs) are a class of gel material displaying astonishing non-invasive property and surgical viability. While possessing responsiveness towards specific stimuli, they change their physical form in vivo, thus serve as wonderful biomaterials and drug delivery systems. In this review, we illustrated the mechanics of stimuli-responsive IGs developed during the pass decade. Two branches of crosslinked gels - co-valent and non-covalent crosslinked IGs and their composition and customization were intro-duced. In conclusion, we summarized the present trend in bone tissue engineering research, and made an outlook for future. We hope that this comprehensive review could provide a proper reference for the development of new IGs. This article is protected by copyright. All rights reserved.
The involvement of the extracellular matrix (ECM) in tumor progression has motivated the development of biomaterials mimicking the tumor ECM to develop more predictive cancer models. Particularly, polypeptides based on elastin could be an interesting approach to mimic the ECM due to their tunable properties. Here, we demonstrated that elastin-like recombinamer (ELR) hydrogels can be suitable biomaterials to develop breast cancer models. This hydrogel was formed by two ELR polypeptides, one containing sequences biodegradable by matrix metalloproteinase and cyclooctyne and the other carrying arginylglycylaspartic acid and azide groups to allow cell adhesion, biodegradability, and suitable stiffness through "click-chemistry" cross-linking. Our findings show that breast cancer or nontumorigenic breast cells showed high viability and cell proliferation for up to 7 days. MCF7 and MCF10A formed spheroids whereas MDA-MB-231 formed cell networks, with the expression of ECM and high drug resistance in all cases, evidencing that ELR hydrogels are a promising biomaterial for breast cancer modeling.
Elastin‐like peptides (ELPs) are thermoresponsive biopolymers inspired by the characteristic repetitive sequences of natural elastin. As ELPs exhibit temperature‐dependent reversible self‐assembly, they are expected to be biocompatible thermoresponsive materials for drug delivery carriers. One of the most widely studied ELPs in this field is the repetitive pentapeptide, (VPGXG)n. We previously reported that phenylalanine‐containing ELP (Fn) analogs, in which the former Val residue of the repetitive sequence (VPGVG)n is replaced by Phe, show coacervation with a short chain length (n = 5). Owing to their short sequences, Fn analogs are easily modified in amino acid sequences via simple chemical synthesis, and are useful for investigating the relationship between peptide sequences and temperature responsiveness. In this study, we developed Fn analogs by replacing Phe residue(s) with other amino acids or introducing another amino acid at the N‐terminus. The temperature responsiveness of the Fn analogs changed drastically with the substitution of a single Phe residue, suggesting that aromatic amino acids play an important role in their self‐assembly. In addition, the self‐assembling ability of Fn was enhanced by increasing the bulkiness of the N‐terminal amino acids. Therefore, the N‐terminal residue was considered to be important for hydrophobicity‐induced intermolecular interactions between the peptides during coacervation.
Elastin‐like recombinamers (ELRs) are proteinaceous biopolymers obtained by recombinant technology and which sequence is inspired by natural elastin. Genetic engineering allows total control over amino acid sequence and design of ELR structures in a versatile way that can include bioactive, structural, or functional domains. ELRs can be used as precursors of bioactive and biocompatible hydrogels generated by either physical or chemical interchain cross‐linking. This chapter describes ELR‐based hydrogels, their principal features, and applications in biomedical field. We focus on the use of these hydrogels as advanced scaffolds mimicking extracellular matrix in tissue engineering, reviewing the most interesting and recent examples of musculoskeletal, cardiovascular, skin, and neural tissue regeneration. Advanced drug delivery devices based on ELRs hydrogels are also reviewed concerning their application in disease therapies, such as type 2 diabetes, ischemia, or glaucoma, and focusing mainly on cancer therapy.
Bone formation and repair represent a clinical challenge. In this work, we designed and synthesized strontium Astragalus polysaccharide (APS-Sr), a novel polysaccharide compound that should have therapeutic effects on both anti-inflammation and promoting bone formation. Using material characterization techniques, including SEM, FITR, XRD, etc., we verified the successful synthesis of this compound. Moreover, we examined the potential of this compound for promoting bone repair and inhibiting inflammatory response by cell proliferation assay, ALP and Alizarin Red staining experiments and RT-qPCR. The biological experiment results showed that APS-Sr can effectively inhibit inflammatory factors, promote osteogenic differentiation and up-regulate the bone growth factors. It is therefore believed that APS-Sr should be a promising polysaccharide compound in bone-related biomedical applications.
Biomaterials are indispensable for tissue engineering, which plays a pivotal role in the skeletal tissue repair. However, biomaterials currently used such as animal extracts and chemically synthesized polymers display unsatisfactory bioactivity and safety. In recent years, modular protein engineering-based (MPE) biomaterials composed of polypeptides produced by molecular cloning and protein synthesis have greatly developed due to their lower batch-to-batch variation, avoidance of possible pathogens and, most importantly, sequence-tunable property. In this review, we first briefly describe the properties of different MPE biomaterials classified by the structural domains of polypeptides, and techniques to engineer the polypeptide sequence and synthesize MPE biomaterials at will. Then, we focus on the application of bio-designed MPE biomaterials in skeletal tissue engineering. Different structural domains of polypeptides are used individually or covalently fused with different bioactive motifs to generate a variety of MPE biomaterials. The sequence (protein modules) of MPE biomaterials would determine and guide their cytocompatibility, their effects on cell fate and ECM formation, the mechanical properties and functions during the in vivo skeletal tissue repair. Moreover, we propose several bio-design strategies and potential directions to develop MPE biomaterials for better performing skeletal tissue engineering and to achieve fast skeletal tissue regeneration. Combinations of material science and protein engineering would provide solutions to the obstacles in regenerative medicine. This article provides a board review of skeletal tissue engineering in a polypeptide sequence-guided way by using MPE biomaterials.
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Statement of significance: Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage.
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Bone loss and failure of proper bone healing continues to be a significant medical condition in need of solutions that can be implemented successfully both in human and veterinary medicine. This is particularly true when large segmental defects are present, the bone has failed to return to normal form or function, or the healing process is extremely prolonged. Given the inherent complexity of bone tissue - its unique structural, mechanical, and compositional properties, as well as its ability to support various cells - it is difficult to find ideal candidate materials that could be used as the foundation for tissue regeneration from technological platforms. Recently, important developments have been made in the implementation of complex structures built both at the macro- and the nano-level that have been shown to positively impact bone formation and to have the ability to deliver active biological molecules (drugs, growth factors, proteins, cells) for controlled tissue regeneration and the prevention of infection. These materials are diverse, ranging from polymers to ceramics and various composites. This review presents developments in this area with a focus on the role of scaffold structure and chemistry on the biologic processes that influence bone physiology and regeneration.
In the last decade, recombinant structural proteins have become very promising in addressing different issues like the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, like hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e. the green Aequorea coerulescens EGFP and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G’ = 60-4000 Pa), by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-AcEGFP, hence enabling Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods and a FRET efficiency of 0.06-0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.
Tremendous progress has been achieved in the field of tissue engineering in the past decade. Several major challenges laid down 10 years ago, have been studied, including renewable cell sources, biomaterials with tunable properties, mitigation of host responses, and vascularization. Here we review advancements in these areas and envision directions of further development.
The search for new and biocompatible materials with high potential for improvement is a challenge in gene delivery applications. A cell type specific vector made of elastin-like recombinamer (ELR) and aptamers has been specifically designed for the intracellular delivery of therapeutic material for breast cancer therapy. A lysine enriched ELR was constructed and complexed with plasmid DNA to give positively charged and stable polyplexes. Physical characterization of these polyplexes showed a particle size of around 140 nm and a zeta potential of approximately +40 mV. The incorporation of MUC1-specific aptamers into the polyplexes resulted in a slight decrease in zeta potential but increased cell transfection specificity for MCF-7 breast cancer cells with respect to a MUC1-negative tumour line. After showing the transfection ability of this aptamer- ELR made vector facilitated mainly by macropinocytosis uptake, we demonstrated its application for suicide gene therapy using a plasmid containing the gene of the toxin PAP-S. The strategy developed in this work about using ELR as polymeric vector and aptamers as supplier of specificity to deliver therapeutic material into MUC1-positive breast cancer cells shows promising potential and continues paving the way for ELRs in the biomedical field.
Bone defects can be congenital or acquired resulting from trauma, infection, neoplasm and failed arthroplasty. The osseous reconstruction of these defects is challenging. Unfortunately, none of the current techniques for the repair of bone defects has proven to be fully satisfactory. Bone tissue engineering (BTE) is the field of regenerative medicine (RM) that focuses on alternative treatment options for bone defects that will ideally address all the issues of the traditional techniques in treating large bone defects. However, current techniques of BTE is laborious and have their own shortcomings. More recently, 2D and 3D bone printing has been introduced to overcome most of the limitations of bone grafts and BTE. So far, results are extremely promising, setting new frontiers in the management of bone defects.
Non-union continues to be the most devastating complication after fracture fixation. Its treatment can be prolonged and often unpredictable. The burden to the patient, surgeon and health care system can be immense. Strategies to prevent it and or identify early its development are desirable in order to improve the clinical course of the affected patients and their outcomes. We undertook a systematic review of the literature in order to identify the most common and important risk factors based on the hierarchy of level of evidence. Accordingly, a stratification scale was formed which highlighted 10 risk factors including; an open method of fracture reduction, open fracture, presence of post-surgical fracture gap, smoking, infection, wedge or comminuted types of fracture, high degree of initial fracture displacement, lack of adequate mechanical stability provided by the implant used, fracture location in the poor zone of vascularity of the affected bone, and the presence of the fracture in the tibia. Clinicians should take in to account these findings when managing patients with long bone fractures, particularly the femur and tibia in order to minimise the risk of non-union.
In this study, thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAm) was grafted onto gelatin via atom transfer radical polymerization (ATRP). The chemical structure of PNIPAAm-grafted gelatin (Gel-PNIPAAm) was confirmed by XPS, ATR-IR and 1H NMR characterizations. Gel-PNIPAAm aqueous solution exhibited sol-to-gel transformation at physiological temperature, and was studied as injectable hydrogel for bone defect regeneration in a cranial model. The hydrogel was biocompatible and demonstrated the ability to enhance bone regeneration in comparison with the untreated group (control). With the incor-poration of rat bone mesenchymal stem cells (BMSCs) into the hydrogel, the bone regeneration rate was further significantly enhanced. As indicated by micro-CT, histological (H&E and Masson) and immunohistochemical (osteocalcin and osteopontin) staining, at 12 weeks post-implantation, newly formed woven bone tissue was clearly detected in the hydrogel/BMSCs treated group, showing indistinguishable boundary with surrounding host bone tissues. The results suggested that the thermo-sensitive Gel-PNIPAAm hydrogel was an excellent injectable delivery vehicle of BMSCs for in vivo bone defect regeneration.
The field of biomedicine is constantly investing significant research efforts in order to gain a more in-depth understanding of the mechanisms that govern the function of body compartments and to develop creative solutions for the repair and regeneration of damaged tissues. The main overall goal is to develop relatively simple systems that are able to mimic naturally occurring constructs and can therefore be used in regenerative medicine. Recombinant technology, which is widely used to obtain new tailored synthetic genes that express polymeric protein-based structures, now offers a broad range of advantages for that purpose by permitting the tuning of biological and mechanical properties depending on the intended application while simultaneously ensuring adequate biocompatibility and biodegradability of the scaffold formed by the polymers. This Progress Report is focused on recombinant protein-based materials that resemble naturally occurring proteins of interest for use in soft tissue repair. An overview of recombinant biomaterials derived from elastin, silk, collagen and resilin is given, along with a description of their characteristics and suggested applications. Current endeavors in this field are continuously providing more-improved materials in comparison with conventional ones. As such, a great effort is being made to put these materials through clinical trials in order to favor their future use. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.