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A stromal cell-derived factor-1 releasing matrix enhances the progenitor cell response and blood vessel growth in ischaemic skeletal muscle

  • Pathologists Bio-Medical Labs

Abstract and Figures

Although many regenerative cell therapies are being developed to replace or regenerate ischaemic muscle, the lack of vasculature and poor persistence of the therapeutic cells represent major limiting factors to successful tissue restoration. In response to ischaemia, stromal cell-derived factor-1 (SDF-1) is up-regulated by the affected tissue to stimulate stem cell-mediated regenerative responses. Therefore, we encapsulated SDF-1 into alginate microspheres and further incorporated these into an injectable collagen-based matrix in order to improve local delivery. Microsphere-matrix impregnation reduced the time for matrix thermogelation, and also increased the viscosity reached. This double-incorporation prolonged the release of SDF-1, which maintained adhesive and migratory bioactivity, attributed to chemotaxis in response to SDF-1. In vivo, treatment of ischaemic hindlimb muscle with microsphere-matrix led to increased mobilisation of bone marrow-derived progenitor cells, and also improved recruitment of angiogenic cells expressing the SDF-1 receptor (CXCR4) from bone marrow and local tissues. Both matrix and SDF-1-releasing matrix were successful at restoring perfusion, but SDF-1 treatment appeared to play an earlier role, as evidenced by arterioles that are phenotypically older and by increased angiogenic cytokine production, stimulating the generation of a qualitative microenvironment for a rapid and therefore more efficient regeneration. These results support the release of implanted SDF-1 as a promising method for enhancing progenitor cell responses and restoring perfusion to ischaemic tissues via neovascularisation.
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D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
European Cells and Materials Vol. 22 2011 (pages 109-123) ISSN 1473-2262
Although many regenerative cell therapies are being
developed to replace or regenerate ischaemic muscle, the
lack of vasculature and poor persistence of the therapeutic
cells represent major limiting factors to successful tissue
restoration. In response to ischaemia, stromal cell-
derived factor-1 (SDF-1) is up-regulated by the affected
tissue to stimulate stem cell-mediated regenerative
responses. Therefore, we encapsulated SDF-1 into
alginate microspheres and further incorporated these into
an injectable collagen-based matrix in order to improve
local delivery. Microsphere-matrix impregnation reduced
the time for matrix thermogelation, and also increased the
viscosity reached. This double-incorporation prolonged
the release of SDF-1, which maintained adhesive and
migratory bioactivity, attributed to chemotaxis in response
to SDF-1. In vivo, treatment of ischaemic hindlimb muscle
with microsphere-matrix led to increased mobilisation of
bone marrow-derived progenitor cells, and also improved
recruitment of angiogenic cells expressing the SDF-1
receptor (CXCR4) from bone marrow and local tissues.
Both matrix and SDF-1-releasing matrix were successful
at restoring perfusion, but SDF-1 treatment appeared to
play an earlier role, as evidenced by arterioles that are
phenotypically older and by increased angiogenic cytokine
production, stimulating the generation of a qualitative
microenvironment for a rapid and therefore more ef cient
regeneration. These results support the release of implanted
SDF-1 as a promising method for enhancing progenitor cell
responses and restoring perfusion to ischaemic tissues via
Keywords: Cytokines, hydrogels, injectables,
neovascularisation, regenerative medicine, vascular biology
*Address for correspondence:
E.J. Suuronen
Division of Cardiac Surgery, University of Ottawa Heart
40 Ruskin Street, Ottawa K1Y4W7, Canada
Telephone Number: 1-613-798-5555 x19087
FAX Number: 1-613-671-5367;
E-mail address:
Myopathies, such as ischaemic heart disease and
peripheral arterial disease (PAD) are signi cant killers in
developed nations. In particular, PAD af icts an estimated
27 million people in Europe and North America (Belch et
al., 2003). This disease manifests when the ow of blood
to extremities is acutely or chronically reduced (Selvin
and Erlinger, 2004; Shamoun et al., 2008). Attributed to
the lack of blood ow, symptoms typically include: pain,
cramping, weakness and a poor ability to heal. To restore
perfusion, current treatments include stent or vascular
transplants with surgical intervention, or amputation;
however, these options are invasive, carry risks, and
amputation signi cantly reduces quality of life. Therefore,
non-invasive options for restoring perfusion would be
invaluable for PAD patients. Recent years have seen the
generation of many novel approaches for developing
tissue substitutes to replace or regenerate ischaemic
muscle; however, the supply of suf cient and appropriate
vasculature represents a major limiting parameter for a
successful therapeutic approach (Ko et al., 2007; Kaully
et al., 2009).
To augment reparative processes, regenerative
therapies using bone marrow-, blood-, or tissue-derived
progenitor cells have emerged; however, the results of
these treatments are controversial and there is growing
evidence to suggest that non-embryonic transplanted cells
do not successfully integrate with host tissue (Murry et
al., 2006; Suuronen et al., 2007). It is now believed that
functional improvement results from neovascularisation
and the restoration of blood ow to ischaemic muscle,
and that this phenomenon is initiated, maintained, and
enhanced by paracrine factors and secondary recruitment
of progenitor cells (Cho et al., 2007; Formigli et al., 2007).
After ischaemic injury, an endogenous response
to recruit the endogenous progenitor cells is initiated
(Hofmann et al., 2005). Regarded as critical to this process
is the up-regulation and release of the cytokine stromal
cell-derived factor-1 (SDF-1) from ischaemic muscle
(Askari et al., 2003) and the subsequent recruitment of
circulating progenitor cells (CPCs), mobilised from distal
tissues, such as bone marrow, into ischaemic tissue (De
Falco et al., 2004). However, the stem cell recruitment
response is short-lived and tissue accumulation is low
(Wojakowski et al., 2004; Fazel et al., 2006). Recruited
CPCs are observed to be pro-angiogenic (Park et al.,
D. Kuraitis1,2,3, P. Zhang1, Y. Zhang1,2, D.T. Padavan1, K. McEwan1,4, T. Sofrenovic1,2, D. McKee1, J. Zhang5,
M. Grif th2,6, X. Cao4, A .Musarò3, M. Ruel1,2 and E.J. Suuronen1,2,*
1Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Canada
2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada
3DAHFMO-Unit of Histology and Medical Embryology, IIM, University of Rome La Sapienza, Rome, Italy
4Faculty of Engineering, University of Ottawa, Ottawa, Canada
5Department of Chemical and Biochemical Engineering, University of Western Ontario, London (Ont.), Canada
6Integrative Regenerative Medicine Centre, Cell Biology Building, Linköping University, Linköping, Sweden
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
2004; Suuronen et al., 2009) and are thought to augment
functional recovery by promoting neovascularisation.
This study aims to use the SDF-1 signalling mechanism
in an effort to amplify the endogenous response to
ischaemia and the recruitment of vasculogenic progenitor
cells. Herein, we report on the encapsulation of SDF-1 into
microspheres, which are added to an injectable collagen-
based matrix that thermogels at physiological temperature.
We provide evidence that treatment with an SDF-1
releasing collagen matrix improves the vasculogenic
response of ischaemic muscle, mediated by the recruitment
of progenitor cells.
All reagents were obtained from Sigma-Aldrich (Oakville,
Canada), unless otherwise indicated.
Generation of microspheres
Through a physical cross-linking reaction, blank (no
peptide added) alginate microspheres were created with
1.25 % sodium alginate (w/v). The solution was added to
a syringe and forced through a J1 Encapsulation Device
(Nisco, Zürich, Switzerland) at 1 mL/min, using a syringe
pump, and 9.8 L/min N2 gas. Microspheres were allowed
to fall into a 2 % CaCl2(w/v) cross-linking bath, and kept
stirring for 20 min before washing with phosphate-buffered
saline (PBS), and snap-freezing in liquid nitrogen. SDF-
1 loaded microspheres were created by adding 200 ng
SDF-1 per g of alginate solution during sodium alginate
solubilisation. Microspheres with both human (Cedarlane
Laboratories, Hornby, Canada) and murine SDF-1
(Biovision, San Francisco, CA, USA) were generated,
and stored at -80 ºC.
Microsphere morphology
Microspheres were thawed and suspended in PBS. Images
were taken using an inverted phase contrast microscope
(Olympus 1x81F), and microsphere size was assessed
using Image-Pro Plus. Microsphere ultrastructure was
observed using a Philips/FEI XL-30 scanning electron
microscope (Hillsboro, OR, USA); WD = 7.0 mm and
keV = 1.2. Microspheres were xed in 3 % glutaraldehyde
for 2 h, washed and subsequently dehydrated in various
ethanol dilutions (30 %, 50 %, 70 %, 80 %, 90 %, 95 %,
99 %) for 5 min each before being critically point-dried.
Specimens were mounted on stubs and coated with Pd/Au
using a Hummer Sputter Coater (Ladd Research, Williston,
Matrix preparation
As described previously (Suuronen et al., 2009), a
collagen matrix was created on ice, using a cross-
linking mixture containing a 1:1 molar ratio of
hydrochloride and N-hydroxysuccinimide (EDC/NHS;
13 mM) in 2(-N-morpholino) ethanesulfonic acid (MES)
buffer, a solution of 1 % porcine type I atelocollagen
(w/v; Nippon Ham, Tskuba, Japan) and 40 % chondroitin
sulphate-C (CSC) (w/v; Wako Chemicals, Osaka, Japan).
The cross-linked collagen solution was diluted with PBS
before adjusting the pH to 7.2 ±0.2 using 1 N NaOH
or HCl. The nal concentration of collagen and CSC
were 0.59 % (w/v) and 2.4 % (w/v), respectively. The
proportion of microspheres in the matrix was 21 % (w/v),
and when included, the SDF-1 concentration was 40 ng/
mL. SDF-1 microsphere-matrix was prepared using the
same procedure, except 400 mg of microspheres was added
in the PBS dilution step. For in vitro use, matrices were
thermogelled for 20 min at 37 °C.
Reagent sterilisation
All liquid reagents (PBS, MES, CSC, alginate) were sterile
ltered (0.45 m). Collagen (1 %) was prepared with sterile
water and the resulting solution was sterilised by exposure
to ultraviolet (UV) light for a period of 15 min.
The rheological properties of the collagen matrix were
measured using a Brookfield R/S Plus Rheometer as
previously described (Deng et al., 2010). Samples of
collagen matrix or microsphere-collagen matrix (1.5 mL)
were subjected to a constant shear rate of 5 s-1 for 20-30
min using a C50-2 spindle (spindle gap of 4 m, according
to the spindle speci cations), and the temperature was
maintained at 37 °C. Rheo3000 v1.2 software was used to
monitor viscosity (Pa·s) and time to gelation (s). The time
at which maximum viscosity was reached was considered
to be the material’s time to gelation. In addition to time
viscosity profiles, elastic storage (G’) and loss (G”)
modulus as a function of temperature at a frequency of 1
Hz was also measured.
SDF-1 release
Microspheres containing human SDF-1 were used to
assess release kinetics. Microspheres alone or embedded
in a collagen matrix were added to a 20 mL ask with 5
mL of PBS. At various time points, samples were taken
and immediately frozen at -80 ºC and fresh PBS was
added to replace the removed aliquot volume. SDF-1
content in the supernatant was assessed using an ELISA
kit (R&D Systems, Minneapolis, MN, USA), according
to the manufacturer’s protocol. Data is reported as a ratio
of concentration at time t, relative to the maximal release.
Release kinetics
To analyse SDF-1 release from microspheres (+/- matrix
embedding), various release kinetic models were used
to describe the observed release kinetics. Correlation
coef cients were determined for data t to zero-order
release (Hadjiioannou et al., 1993), rst-order release
(Bourne et al., 2002), Higuchi (Higuchi, 1963), Hixson-
Crowell (Hixson and Crowell, 1931) and Korsmeyer-
Peppas (Korsmeyer et al., 1983) models.
Cell culture
Procedures for the isolation of human circulating progenitor
cells (CPCs) were approved by the Human Research
Ethics Board of the University of Ottawa Heart Institute.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
After obtaining informed consent, total peripheral blood
mononuclear cells (PBMCs) were isolated from the blood
of healthy human volunteers by Histopaque 1077 density-
gradient centrifugation, as previously described (Ruel et
al., 2005). Cells were cultured on bronectin-coated plates
in endothelial basal medium (EBM-2; Clonetics, Guelph,
Canada) containing 5 % FBS (v/v), VEGF, R3-IGF-1, and
hEGF supplements. After 4 d in culture, supernatant and
non-adherent cells were removed, and adherent populations
were considered to be circulating progenitor cells (CPCs).
Adhesion assay
CPCs (2×104) were resuspended in 1 mL of medium and
seeded in 12-well dishes with bronectin-coated coverslips,
containing medium with 50 mg of blank microspheres or
medium with 50 mg of human SDF-1 loaded microspheres.
After 1 h at 37 ºC, medium was aspirated and adherent
cells were fixed with 4 % paraformaldehyde (PFA).
Coverslips were washed with PBS and mounted on slides
with 4’,6-diamidino-2-phenylindol-(DAPI)-containing
mounting medium (Vector Laboratories, Burlington,
Canada). Six random elds-of-view were imaged using
a Zeiss Z1 uorescent microscope and DAPI+ cells were
Migration assay
CPCs (2×104) in VEGF-free medium were added to the top
chamber of a Transwell tissue culture well (Corning, New
York, NY, USA). The lower chamber contained VEGF-free
medium with 50 mg of blank microspheres or VEGF-free
medium with 50 mg of human SDF-1 loaded microspheres.
The bottom of each well contained a bronectin-coated
coverslip. After 24 h of incubation, cells were xed, DAPI-
stained, visualised, and counted.
Chemokinesis versus chemotaxis assay
To investigate the mode of action through which SDF-1
induces CPC migration, assays, based on a previously
described protocol for the evaluation of chemokinesis
versus chemotaxis (Misiak-Tloczek and Brzezinska-
Blaszczyk, 2009), were carried out. Three treatment
conditions (represented as the presence of SDF-1 in the
upper well/lower well) were tested: SDF-1/0 (SDF-1 only
in the upper well); SDF-1/SDF-1 (SDF-1 in both upper
and lower wells); 0/SDF-1 (SDF-1 in the lower well only).
SDF-1 was used at a concentration of 9 ng/mL, as this is
the approximate amount released from microspheres after
24 h. Quanti cation was performed in the same manner as
described for the migration assay above.
Animal model
All procedures were performed with the approval of
the University of Ottawa Animal Care Committee, in
compliance with the National Institute of Health’s Guide
for the Care and Use of Laboratory Animals. Bone marrow
transplantation was performed as previously described
(Whitman et al., 2004). Brie y, female C57BL/6J mice
(8-9 weeks old, Jackson Laboratories, Bar Harbor, ME,
USA) were irradiated with a total of 900 rads from a
caesium source, delivered in 2 equal doses, 3 h apart. Donor
bone marrow cells (7×106) from green uorescent protein
(GFP) transgenic mice (C57BL/6-Tg(CAG-EGFP)10sb/J,
Jackson Laboratories) were injected into the tail vein of
irradiated recipient mice. Six weeks after transplantation,
proximal femoral arteries in left hindlimbs were ligated as
described (Limbourg et al., 2009), using 4.0 silk thread,
under 2 % iso urane. Limbs subsequently received 80 L
injection of: 1) PBS (n = 9); 2) collagen matrix (n = 8); or
3) collagen matrix containing murine SDF-1 microspheres
(n = 8). Treatments were delivered by 3 equivolumetric
injections into the adductor muscle downstream of the
ligation site, using a 27-gauge needle.
Blood perfusion of both hindlimbs was measured
before and after ligation, and at days 4, 7 and 14 post-
operatively using a multi bre needle probe (8 separate
collecting bres), and a laser Doppler blood ow monitor
(Moor Instruments, Axminster, UK).
Blood samples (~100 L) were procured from the
right saphenous veins on days 0 (pre-operative baseline),
1, 4, 7 and 14 post-operatively. PBMCs were isolated
using density-gradient centrifugation and immediately
characterised using ow cytometry as described below.
Flow cytometry
Cells were labelled with antibodies against the following
antigens: c-kit (Southern Biotech, Birmingham, AL, USA),
CXCR4 (BD Biosciences, Mississauga, Canada), and k-1
(eBioscience, San Diego, CA, USA), and analysed with
a FACSAria ow cytometer (BD Biosciences). Isotype-
matched immunoglobulin antibodies were used as controls.
Animals were sacri ced at day 14. Hindlimb muscle
tissue was collected and xed overnight in PFA before
paraffinisation. All samples were analysed in cross-
section. Samples were de-paraf nised and hydrated with
sequential washes in toluene and decreasing concentrations
of ethanol. Antigen retrieval was performed using
boiling citrate buffer. All staining was performed in PBS
containing 10 % normal horse serum (Vector Laboratories).
The following antibodies were used: anti--smooth
muscle actin (SMA; pre-diluted, Abcam, Cambridge, MA,
USA), anti-GFP (1:100; Abcam), and anti-CXCR4 (1:50;
Abcam). For all tissue sections, mounting medium with
DAPI (Vector Laboratories) was used to visualise nuclei.
All measurements and cell counts were determined from
6 random microscopic elds-of-view and averaged from
2 blinded observers.
Cytokine Antibody Arrays
Relative cytokine levels in hindlimb lysates and serum
from sacri ced animals (n = 5 per treatment group) were
analysed using RayBio Mouse Cytokine Antibody Array
Kits (Raybiotech, Norcross, GA, USA), according to the
manufacturer’s protocol.
Statistical Analysis
Unless otherwise stated, values are expressed as means
± standard error. Statistical analyses were performed
using SPSS (IBM, Somers, NY, USA). Comparisons of
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
continuous data between groups were performed with a
one-way analysis of variance and comparisons between
individual groups were performed with a two-tailed
Student’s t-test. For in vitro CPC analysis, results were
paired by donor, and subjected to a paired t-test. Probability
values of p < 0.05 were considered statistically signi cant.
Generation of SDF-1 microspheres
Microspheres had a mean diameter of 38.5 m (±14.0
m (SD); Fig. 1A). When hydrated, microspheres had a
smooth, spherical morphology (Fig. 1B), and a rougher
surface upon dehydration as visualised by scanning
electron microscopy (SEM) (Fig. 1C).
Microsphere-matrix impregnation
The addition of microspheres to the collagen matrix
reduced the time to gelation and caused the matrix to
solidify at a greater viscosity, as early as 400 s after
application (p = 0.05; Fig. 2A, 2B), which was maintained
over time. Maximum viscosity reached was greater for
the matrix containing microspheres (1.42 Pa·s), compared
to matrix alone (1.18 Pa·s; p = 0.003). Microspheres in
solution released their SDF-1 content within 1 d, but
matrix impregnation prolonged the maximal release up to
approximately 10 d (Fig. 2C). Analysis of SDF-1 release
kinetics shows that release from microspheres best ts
a rst order model, indicated by the greatest correlation
coef cient, but after impregnation in a matrix, release
follows the Higuchi model (Table 1, Fig. 2D).
Bioactivity of SDF-1 microspheres
Blank microspheres did not confer any difference in
adhesion potential with cultured CPCs, but the addition
of SDF-1 loaded microspheres supported an increase in
adhesive CPCs by 2.3-fold after 1 h of exposure (p = 0.04;
Fig. 3A-C). When CPCs were given a chemotactic stimulus
of blank or SDF-1 loaded microspheres, 3.2-fold more cells
migrated towards the SDF-1-releasing microspheres than
the blank ones after 24 h (p = 0.004; Fig. 3D-F).
Fig. 1. Characterisation of SDF-1-containing alginate microspheres. Average size of microspheres was 38.5 m
(A). Microspheres displayed a rounded morphology in saline solution (B; 400x), and a lattice structure after drying
and imaging using scanning electron microscopy (C; 379x). Scale bars = 50 m.
Order First
Order Hixson-
Crowell Higuchi Korsmeyer-
Free MS 0.93 0.95 0.94 0.84 0.92
Embedded MS 0.66 0.75 0.78 0.94 0.59
Table 1. Correlation coef cients (R2) for SDF-1 release from microspheres alone or
embedded in a collagen matrix, based on classical drug release models.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
Fig. 2. Matrix-microsphere effects. After microsphere (MS) impregnation into collagen matrix, rheological properties
of the matrix were altered: time to gelation was reduced by 16 %, and viscosity increased by 22 % (A; n = 10).
Differences in gelation were rst noted at 400 s. B shows representative modulus versus time curves, which illustrate
G’ (storage) and G” (loss) values for the different hydrogels. The burst-release effect of SDF-1 was reduced upon
incorporation into matrix; the maximal release was delayed from approximately 1 to 10 d (C; n = 3). Analysis of
release kinetics demonstrates that when microspheres are embedded in a matrix, SDF-1 release ts the Higuchi
model (D; R2 = 0.94). *p < 0.05 versus matrix without microspheres at the same time point.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
Fig. 3. Effects of blank (no SDF-1) and SDF-1 loaded microspheres on cultured primary CPCs. In the presence of
SDF-1 loaded microspheres (MS), over 2-fold more CPCs were adherent to bronectin within 1 hour of exposure
(A; n = 4); B, C are representative images of adherent CPCs in the presence of blank or SDF-1 loaded microspheres,
respectively. More CPCs migrated towards SDF-1 microspheres (D; n = 4); E, F are representative images of blank
and SDF-1 loaded microsphere-induced migration of CPCs, respectively. The effect of SDF-1 on CPC migration
was mainly chemotactic, rather than chemokinetic, as evidenced by the greater migratory effect of CPCs towards
SDF-1 when it is presented as a gradient (G). Scale bars = 100 m. *p < 0.05; **p < 0.05 vs. all others.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
SDF-1 mediated-migration of CPCs is mainly
Compared to the lack of a chemotactic stimulus (absence
of SDF-1 in the lower well), chemokinesis, stimulated
by equivalent amounts of SDF-1 in the upper and lower
chambers, increased CPC movement to the lower chamber
by 13 % (p = 0.04; Fig. 3G). The chemotactic stimulus
with SDF-1 only in the lower chamber induced the greatest
migration of CPCs, by 177 % (p = 0.006; Fig. 3G).
In vivo mobilisation of CPCs by SDF-1 treatment
Based on the in vitro chemotactic properties of SDF1
microsphere matrix, we de ned whether the construct is
able to enhance the recruitment of CPC in an in vivo system.
GFP+ (bone marrow-derived) CPCs in the peripheral
blood were analysed over time, and compared to baseline.
Interestingly, the SDF-1 microsphere matrix treatment
signi cantly increased circulating CXCR4+ cells (by 45
%; p = 0.02), k-1+ cells (by 105 %; p = 0.001), and c-kit+
cells (by 18 %; p = 0.04) at early time point post injury
(Fig. 4A) and was able to maintain elevated numbers of
circulating k-1+ cells (by 149 %; p = 0.002) and c-kit+
cells (by 48 %; p = 0.01) for an extended period (7-14 d)
post operation (Fig. 4B). Notably, the collagen matrix alone
increased circulating k+ cells (by 20 %; p = 0.009) at the
earlier evaluation and c-kit+ cells at both early (by 48 %; p
= 0.01) and late time point (by 29 %; p = 0.02; Fig. 4A, B).
Restoration of perfusion by matrix treatments
Indeed, both matrix treatments (with or without SDF-1
microspheres) were able to restore perfusion to baseline
levels by one week post-treatment, compared to PBS
controls (Fig. 4C; p < 0.04), which was maintained at two
weeks post-treatment (p < 0.05).
Arteriole density of ischaemic hindlimbs
Arteriole density in ischaemic hindlimbs was not different
between collagen and SDF-1 collagen matrices (p = 0.9);
Fig. 4. Functional effects of collagen & SDF-1 microsphere matrix treatments in vivo. At early time points after
treatment (A; days 1-4), animals receiving SDF-1 microsphere-matrix (SDF-1) had greater numbers of circulating
CPCs. This was also observed at later time points (B; days 7-14). By 7 d post-op, hindlimb perfusion was restored
to baseline levels with matrix treatments (C; n = 8-9 per group). *p < 0.05 versus PBS; **p < 0.05 versus PBS &
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
however, both matrix and SDF-1 microsphere matrix
treatments increased arteriole density compared to PBS
by 67 % (p = 0.01) & 69 % (p = 0.02), respectively (Fig.
5A-E). Mean arteriole size was greater in the SDF-1
microsphere matrix treatment group compared to PBS-
treated mice (p < 0.05; Fig. 5B-E). There was also a trend
for greater arteriole cross-sectional area with SDF-1
microsphere matrix treatment compared to collagen matrix
(p = 0.09), and for collagen matrix treatment compared to
PBS (p = 0.08; Fig. 5B).
Recruitment of bone marrow-derived cells to treated
SDF-1 microsphere matrix and collagen matrix treatments
recruited 17.7- and 4.2-fold more GFP+ cells to treated
hindlimbs, compared to PBS (p = 0.0007 and 0.02),
respectively (Fig. 6A-D). There was a trend for SDF-1
microsphere matrix treatment to recruit more GFP+ cells
than collagen matrix alone (p = 0.06; Fig. 6A).
Engraftment of CXCR4+ cells in ischaemic hindlimbs
Overall, SDF-1 microsphere matrix treatment recruited
4.3- and 1.8-fold more CXCR4+ cells to treated hindlimbs
compared to PBS and collagen (p = 0.0004 and 0.05; Fig.
7A-D). Separating the analysis of CXCR4+ cells into those
recruited from the marrow versus those recruited locally,
SDF-1 microsphere matrix treatment recruited 4.9-fold
more CXCR4+GFP+ (bone marrow-derived) and 4.5-fold
more CXCR4+GFP- (local) cells, compared to PBS and
collagen (p = 0.008 and 0.02; Fig. 7A). Collagen matrix
alone also recruited 2.8-fold more CXCR4+GFP+ cells
compared to PBS (p = 0.03; Fig. 7A).
Cytokine pro les
In the hindlimb (Fig. 8A), interleukin-1 (IL-1; p = 0.04)
and monocyte inhibitory protein-3 (MIP-3; p = 0.04)
were reduced, and vascular cell adhesion molecule-1
(VCAM-1; p = 0.03) and insulin-like growth factor-1
(IGF-1; p = 0.03) were increased with SDF-1 microsphere
Fig. 5. Assessment of vasculature in treated hindlimbs. There was a trend for increased arteriole counts with matrix
treatments (A). Larger arterioles were produced in animals receiving collagen or SDF-1 microsphere-matrix (SDF-1)
treatments, with the SDF-1 microsphere-matrix treatment leading to the largest arterioles (B). C, D, E are representative
images of arterioles (indicated by SMA+, red), and counter stained with DAPI (blue). Scale bars = 50 m. n = 6-8
per group. **p < 0.05; *0.05 < p <0.10.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
matrix treatment. In the serum (Fig. 8B): L-selectin was
increased with SDF-1 microsphere matrix treatment
(p = 0.01) and reduced with collagen alone (p = 0.01);
basic broblast growth factor (bFGF) was increased with
collagen alone (p < 0.001) and with SDF-1 microsphere
matrix treatment (p = 0.02); and VCAM-1 was reduced
with SDF-1 microsphere matrix treatment compared to
collagen matrix (p = 0.007) and PBS (p < 0.001). For both
serum and hindlimb, no signi cant differences between any
treatments were observed for the following in ammatory
cytokines: GM-CSF, TNF-, IL-1 and IL-6 (all p > 0.4).
In this study, we demonstrated that the release of SDF-1
from alginate microspheres can be effectively prolonged
by incorporation of the microspheres into a thermogelling
collagen matrix. Released SDF-1 was bioactive, supported
rapid adhesion and migration of CPCs, and also stimulated
the mobilisation of CPCs from bone marrow when
applied to ischaemic muscle. Both matrix alone and
SDF-1-releasing matrix restored perfusion and improved
arteriole density by 2 weeks, but the SDF-1 treatment best
supported the growth of arterioles. SDF-1 microsphere
matrix treatment also recruited more bone marrow-derived
cells and local CXCR4+ cells to the ischaemic muscle, and
altered levels of local angiogenic factors.
Incorporation of alginate microspheres into the collagen
matrix increased the hydrogel’s viscosity and reduced
the time to gelation. This is thought to be mediated by
the cross-linking reaction, whereby functional groups on
collagen (–NH2) and alginate (–COOH) are covalently
bound by EDC/NHS (Mitra et al., 2011), increasing
the total number of cross-links within the material and
enhancing its strength, as has been previously reported (Liu
et al., 2008). An earlier onset of gelation is advantageous
Fig. 6. Recruited bone marrow-derived cells. More GFP+ cells were engrafted in ischaemic hindlimbs treated with
collagen or SDF-1 microsphere-matrix (SDF-1), with a trend for more GFP+ cells in the SDF-1 microsphere-matrix
treatment, compared to matrix alone (A). Representative images of GFP+ cells recruited to PBS- (B), collagen
matrix- (C), or SDF-1 microsphere-matrix- (D) treated hindlimbs. Scale bars = 100 m; n = 6-8 per group. *p
0.02 vs. PBS, p = 0.06 vs. collagen.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
in a clinical setting, allowing for a reduction in the time
required for stable material integration within host tissue.
Additionally, a positive correlation between hydrogel
viscosity and resistance to degradation has been noted
(Yang et al., 2010), suggesting a better persistence in vivo
of microsphere-containing hydrogels.
During the period of release, SDF-1 kinetics from
matrix-embedded microspheres t the Higuchi model of
release whereby the initial drug concentration >> drug
solubility in the matrix, swelling is negligible, perfect sink
conditions are maintained and edge effects are negligible,
suggesting that diffusion is the primary mechanism of
SDF-1 release (Higuchi, 1963). In contrast, microspheres
alone initially demonstrated a burst release followed by a
rst order release of SDF-1. Although a burst-release effect
was observed with microspheres alone, the embedding of
microspheres in matrix was able to prolong the SDF-1
release by 10-fold. This observation can be explained
by the properties of the microspheres and the collagen-
hydrogel system. Hydrogels have an innate ability to retain
small peptides (Cadee et al., 2002; Ruvinov et al., 2010),
and incorporation of peptide-containing microspheres
into hydrogels has been shown previously to extend the
release pro le of the peptide (Kempen et al., 2008). SDF-
1 bioactivity was maintained during the microsphere
generation and cross-linking procedure; upon its release,
SDF-1 augmented rapid adhesion of CPCs, as well as
inducing chemotaxis of CPCs, rather than chemokinesis,
indicating its ability to induce CPC homing towards a
chemotactic gradient rather than the induction of random
In the ischaemic hindlimb study, marrow-mobilised
cells expressing the SDF-1 receptor CXCR4 were
increased in circulation of SDF-1 microsphere matrix-
treated animals at early time points, but this effect was later
lost. This can be explained by previous studies showing
the eventual down-regulation of CXCR4 following
cytokine-induced mobilisation from the bone marrow
(Kim et al., 2006). More speci cally, CXCR4 expression
is dose-dependent on SDF-1 levels; doses of 1 g in a
mouse model have shown CXCR4 levels equivalent to
controls that did not receive SDF-1 (Kimura and Tabata,
2010). Interestingly, higher doses of SDF-1 have the same
effect, suggesting a potential negative feedback loop for the
SDF-1/CXCR4 axis. Notably, SDF-1 microsphere matrix
treatment also stimulated an early mobilisation of k-1+
and c-kit+ cells from the bone marrow into circulation,
and maintained this effect up to 2 weeks post-treatment.
Fig. 7. Localised CXCR4+ cells. SDF-1 microsphere-matrix (SDF-1) treatment recruited more CXCR4+ cells,
from both bone marrow (GFP+) and locally (GFP-). (A) Representative images of GFP+ (green), CXCR4+ (red), or
GFP+CXCR4+ cells (yellow) in PBS- (B), collagen matrix- (C), or SDF-1 microsphere-matrix- (D) treated hindlimbs.
Scale bars = 200 m; n = 6-8 per group. *p < 0.05 vs. PBS, **p < 0.05 vs. all.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
CXCR4 is SDF-1’s exclusive receptor and CXCR4+ CPCs
are reduced over time, mechanisms other than SDF-1
release are needed to explain the increase in mobilised
CPCs expressing k-1 and c-kit. We have previously shown
that systemic transplantation of CPCs can induce a potent
response from the host’s CPCs (Suuronen et al., 2009).
Additionally, this effect has been documented in humans,
and CPC persistence in the circulation has been observed
up to 1 year after cell transplantation (Turan et al., 2010).
Therefore, it is likely that the initiation of the SDF-1/
CXCR4 mobilisation and recruitment response by SDF-1
microsphere matrix treatment activates other endogenous
progenitor cell mechanisms.
Ischaemia was induced in all mice (average of 55
% of normal perfusion). By one week post-treatment,
animals that received matrix treatment (with or without
SDF-1 microspheres) had hindlimb perfusion restored
to baseline levels, which is similar to previous reports of
matrix treatment for hindlimb ischaemia (Suuronen et al.,
2009). It has been previously demonstrated that an increase
in perfusion is attributable to increased local vasculature
(Kim et al., 2010; Suuronen et al., 2010), an observation
that was seen in the current study; matrix treatments
increased arteriole density in ischaemic hindlimbs. It was
hypothesised that SDF-1 microsphere matrix treatment
would confer superior restoration of perfusion, but
instead, the matrix-only treatment was equally effective.
Kimura and Tabata (2010) also attempted to enhance
neovascularisation using an SDF-1-releasing hydrogel,
and observed increased capillary density with SDF-1
treatment at 4 d post-treatment, which was abrogated by 10
d. Another SDF-1 release study did not show a difference
in vascular density between SDF-1 and controls at 9 d post-
treatment (Rabbany et al., 2010). It may be that the addition
of SDF-1 accelerates the regenerative response to collagen
matrix treatment. This is supported by the observation
that SDF-1 treatment increased the cross-sectional size of
arterioles. Cross-sectional area of new vasculature has been
shown to increase over a period of 4 weeks (Ruvinov et al.,
2011), demonstrating that vessel area is indicative of vessel
maturity. Therefore, it is plausible that SDF-1 treatment
had an earlier effect on neovascularisation, allowing for
more rapid growth/maturation of arterioles, compared to
both matrix and PBS treatments.
Fig. 8. Cytokine pro les. Observed differences in cytokines in hindlimb lysates (A) and serum (B), two weeks after
treatment (n = 5). *p < 0.05.
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
The SDF-1 microsphere matrix treatment recruited the
most GFP+ marrow-mobilised cells in our animal model.
Other SDF-1-release studies have also shown recruitment
of stem cells, positive for c-kit (Zhang et al. 2007; Thevenot
et al., 2010) expression. In particular, it was expected
that SDF-1 treatment would increase the recruitment and
engraftment of CXCR4+ cells. Both the matrix and SDF-1
microsphere matrix treatments increased homing of bone
marrow-derived CXCR4+ cells to the treated hindlimbs
(SDF-1 microsphere matrix treatment had the most
pronounced effect), but the SDF-1 microsphere matrix
treatment also demonstrated an improved recruitment
of CXCR4+ cells of non-bone marrow origin. Recently,
it was shown that local pools of progenitor cells co-
expressing CXCR4 and endothelial markers reside in
tissues (Sandstedt et al., 2010), suggesting a potential role
in neovascularisation. In our experiment, SDF-1 treatment
may be recruiting a similar population, as evidenced by
CXCR4+GFP- staining. Microvascular endothelial cells
have also been documented to express CXCR4 (Takagi
et al., 2009), suggesting another CXCR4-expressing
population that is possibly activated in treated hindlimbs.
Regardless, CXCR4+ fractions have been observed to
be superior to whole CXCR4- fractions with respect to
potential for invasion, neovascularisation, and restoration
of perfusion (Seeger et al., 2009).
Compared to controls, in ammatory cytokines (IL-1
and MIP-3) were reduced in hindlimbs treated with SDF-1
microsphere matrix, a result similar to a study by Thevenot
et al. (2010). The latter examined cytokine responses to
SDF-1 delivery in a synthetic scaffold; however, our results
also suggest that the SDF-1 microsphere matrix supports
a favourable environment for angiogenic activity through
local factors, as evidenced by increased IGF-1 and VCAM-
1 in hindlimb lysates. IGF-1 is a stimulator of angiogenesis
(Su et al., 2003) and is cytoprotective (Li et al., 1997), and
elevated IGF-1 is ideal for recovery. Pelosi et al. (2007)
have shown that persistent IGF-1 expression accelerates
the regenerative response and restores architecture and
structure soon after skeletal muscle injury. VCAM-1 is
a cell adhesion molecule expressed by endothelial cells.
When VCAM-1 is solubilised and detected in serum,
it is used as an indicator of dysfunctional endothelium
(Balciunas et al., 2009). Our SDF-1 microsphere matrix
treatment had signi cantly less circulating VCAM-1,
further suggesting a vasculo-protective role of this therapy.
Furthermore, both matrix treatments increased systemic
bFGF, which is a potent angiogenic cytokine (Hosseinkhani
et al., 2006).
In this study, we demonstrated that SDF-1 can be
successfully encapsulated in an alginate microsphere
system and further incorporated into an injectable matrix
for non-invasive delivery. The treatment of ischaemic
mouse hindlimbs with SDF-1-releasing matrix enhanced
progenitor cell mobilisation and recruitment. Compared to
collagen-only treatment, the addition of SDF-1 appears to
confer an earlier effect on neovascularisation, as suggested
by greater arteriole maturity. These ndings suggest that
application of an SDF-1-releasing matrix constitutes a
suitable therapy for prevalent myopathies with reduced
perfusion, with the potential to augment progenitor cell
mobilisation and homing, as well as its ability to rapidly
support neovascularisation.
DK was supported by a Canadian Institutes of Health
Research Canadian Graduate Scholarship; PZ is a recipient
of the Lawrence Soloway Research Fellowship Award;
YZ was supported by a Heart & Stroke Foundation of
Ontario Doctoral Research Award; DTP was supported by
a University of Ottawa Cardiology Research Endowment
Fellowship; KM was supported by an Ontario Graduate
Scholarship; DK and TS were supported by Heart & Stroke
Foundation of Ontario Master’s Studentships; JZ was
supported by a Natural Sciences and Engineering Research
Council of Canada postdoctoral fellowship. The authors
would like to thank Suzanne Crowe for her ow cytometry
expertise and Ann Fook Yang for assistance with SEM.
This work was supported by grant-in-aid T6793 from
the Heart and Stroke Foundation of Ontario (to EJS), by
grant MOP-77536 from the Canadian Institutes of Health
Research (to MR and EJS), by a Natural Sciences and
Engineering Research Council of Canada Research Tool
and Instruments Grant (to XC), by a Canadian Stem Cell
Network grant (to MG), and by Fondazione Roma and
7FP-Myoage (to AM). Funding sources did not have a role
in experimental design, data management, or manuscript
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Discussion with Reviewers
Reviewer II: Could the authors foresee applications for
their injectable hydrogel loaded with SDF1 microspheres
in damaged bone tissue with compromised vascularity?
Authors: Vascularisation is believed to play an important
role in bone wound healing (Giannoudis et al., 2008,
additional reference), yet defective neovascularisation
in some patients leads to an avascular fracture site. A
recent study reported that inhibiting angiogenesis in a
rat tibia osteotomy model resulted in non-union of the
fracture over the early healing stages, demonstrating the
importance of the vasculature for proper bone wound
repair (Fassbender et al., 2011, additional reference).
Notably, the local application of SDF-1 was able to increase
neo-blood vessel maturation leading to accelerated bone
regeneration, characterised by increased callus formation
in a distraction osteogenesis model (Fujio et al., 2011,
additional reference). Therefore, we can envision that a
strategy such as the system reported here may be applicable
in bone trauma, whereby blood vessel growth is stimulated
at the early stages after bone injury, possibly allowing for
more effective wound healing to occur, and without delay.
Reviewer II: In the discussion, the authors suggest that
the matrix is important to support the reperfusion and
that SDF-1 supports early neovascularisation. Would it
be possible that a burst release of SDF-1 from a hydrogel
matrix would be equivalent or even better than the material
solution proposed with a relatively long release pro le?
Authors: After the induction of limb ischaemia, the
natural in vivo response is that of a burst-release of SDF-
1 (De Falco et al., 2004, text reference). This is also
suggested by the observation that patients with acute
limb ischaemia have greater levels of SDF-1 than those
with chronic ischaemia (van Weel et al., 2007, additional
reference). However, the data so far suggests that a
prolonged release and presence of SDF-1 is superior to
burst release in improving angiogenesis. For example,
this has been demonstrated for subcutaneous implantation
(Kimura and Tabata, 2010, text reference) and for the
repair of ischaemic cardiac tissue (Segers et al., 2007,
additional reference; Sundararaman et al., 2011, additional
D Kuraitis et al. Controlled SDF-1 release for treating ischaemia
reference). However, in the present study, we observed
an increase in vessel size, but no difference in vascular
density between the two matrix treatments (+/- SDF-1
release), suggesting that the SDF-1 effect was early and
accelerated the regeneration response conferred by the
injected collagen matrix. Therefore, it is conceivable that
a burst release of SDF-1 from microspheres to augment the
body’s natural SDF-1 release response may be as good as,
or better than, prolonged release for treatment strategies
involving therapeutic matrix injections. However, we also
cannot exclude the possibility that the prolonged presence
of SDF-1 may have a role in other effects, such as the
vessel maturation process, or the prevention of apoptosis,
which have been reported previously by others (Reddy et
al., 2008, additional reference; Ho et al., 2010, additional
reference), but were not examined in the present study.
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... Moreover, SDF-1α is a potent chemokine upregulated during the early stages of wound healing. It is crucial for the homing of endothelial progenitor cells to ischemic sites and promote local angiogenesis [21][22][23][24]. Therefore, in this study, we seek to investigate if the SDF-1α GAS could be used to enhance the provasculogenic maturation of cocultures of endothelial cells and adipose-derived stem/stromal cells (ADSCs). ...
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Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1α GAS) could be used to enhance functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1α GAS with a gene-free scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the VE-cadherin. On the 14th day, the coculture on the SDF-1α GAS showed enhanced maturation than its gene-free equivalent through the elevation of provasculogenic genes (SDF-1α—7.4-fold, CXCR4—1.5-fold, eNOS—1.5-fold). Furthermore, we also found that the coculture on SDF-1α GAS secretes bioactive factors that significantly (p < 0.01) enhanced human Schwann cells’ clustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1α GAS could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between endothelial cells and ADSCs.
... Further, they were incorporated into an injectable collagen-based matrix for the treatment of ischemic hindlimb muscle. This local delivery increased the migration of progenitor cells, and also improved the recruitment of angiogenic cells (Kuraitis et al. 2011). ...
Soft tissues connect, support, or surround other structures and organs of the body, including skeletal muscles, tendon vessels, and nerves supplying these components. Also, organs such as the heart, brain, liver, and kidney are considered as soft tissues. Acute and chronic injury may cause transient or permanent damage to organs and soft tissues. If the damage is severe, the natural physiological repair and restoration mechanisms are not possible. The repair or regeneration using tissue engineered (TE) scaffolds has been considered as a clinical solution. TE approach involves the replacement of damaged parts using grafts made from natural or synthetic or composite polymers. Choosing the polymer with appropriate biological, physicochemical, and mechanical properties is the key to make a successful TE scaffold, and it is still a challenging task. Moreover, the fabrication technique and choice of cells or growth factors for encapsulation to develop the graft also play a crucial role. Therefore, in this chapter, we have highlighted the grafts developed for engineering soft tissues such as blood vessels, skin, cartilage, intervertebral disc, tendon, and skeletal muscle. We have restricted our focus on electrospun scaffolds, and injectable hydrogels prepared using polymers include collagen (Col), chitosan (CS), hyaluronic acid (HA) alginate (Alg), poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic-lactic acid) (PLGA), and their composites. This chapter will help the readers to understand the choice of materials and fabrication techniques for developing successful TE scaffolds for soft tissue engineering applications.
... With clinical application as the goal, synthetically-produced recombinant human collagen was used to circumvent immunogenic reactions that can occur with animal-derived collagen in susceptible patients due to their non-human protein composition, 44 and pathogen transmission risks. Furthermore, our collagen-based biomaterials made for the cornea have been modified for use in other systems, [45][46][47] as similar conditions such as skin ulcers in legs of diabetics, are enormous problems in LMICs. 48 While confirmation in a larger number of patients is needed, we nevertheless demonstrate that implantation with cell-free RHCIII-MPC implants is a safe, reliable option for treating patients at high risk of donor allograft rejection; providing pain relief, and regenerating tissue and nerves. ...
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New scaffold materials composed of biodegradable components are of great interest in regenerative medicine. These materials should be: stable, nontoxic, and biodegrade slowly and steadily, allowing the stable release of biodegradable and biologically active substances. We analyzed peptide-polysaccharide conjugates derived from peptides containing RGD motif (H-RGDS-OH (1), H-GRGDS-NH2 (2), and cyclo(RGDfC) (3)) and polysaccharides as scaffolds to select the most appropriate biomaterials for application in regenerative medicine. Based on the results of MTT and Ki-67 assays, we can state that the conjugates containing calcium alginate and the ternary nonwoven material were the most supportive of muscle tissue regeneration. Scanning electron microscopy imaging and light microscopy studies with hematoxylin–eosin staining showed that C2C12 cells were able to interact with the tested peptide–polysaccharide conjugates. The release factor (Q) varied depending on both the peptide and the structure of the polysaccharide matrix. LDH, Alamarblue®, Ki-67, and cell cycle assays indicated that peptides 1 and 2 were characterized by the best biological properties. Conjugates containing chitosan and the ternary polysaccharide nonwoven with peptide 1 exhibited very high antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae. Overall, the results of the study suggested that polysaccharide conjugates with peptides 1 and 2 can be potentially used in regenerative medicine.
C-X-C motif chemokine 12 (CXCL12), also known as stromal cell-derived factor-1 (SDF-1), is produced by the bone marrow microenvironment. This chemokine binds and activates its cognate receptors C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine receptor type 7 (CXCR7) to widely regulate cell proliferation, survival, differentiation, as well as homing and mobilization of hematopoietic stem cells (HSCs) in specialized niches within the bone marrow. Given this key role in hematopoiesis, it comes as no surprise that any aberrancies in CXCL12/CXCR4 or CXCL12/CXCR7 pathways might lead to excessive proliferation of HSCs, an event that leads to the development of leukemia. So far, numerous therapeutic interventions have been developed to harness CXCL12/CXCR4 and CXCL12/CXCR7 axes in leukemic cells. Plerixafor, BKT140, LY2510924, PF-06747143, ulocuplumab, and NOX-A12 are among the most well-known CXCR4 and CXCL12 modulators that their therapeutic efficacies have been evaluated in different pre-clinical and clinical studies of hematologic malignancies. To have an overview of the importance of CXCL12/CXCR4 and CXCL12/CXCR7 axes in the pathogenesis of leukemia and to gather information about the latest advances as well as challenges in targeting these axes in clinical settings, the present review has begun with a discussion about how aberrant expression of CXCL12/CXCR4 and CXCL12/CXCR7 pathways might regulate leukemogenesis and ended by outlining the key news of preclinical and clinical investigations in leukemia treatment.
Skeletal muscles normally have a remarkable ability to repair themselves; however, large muscle injuries and several myopathies diminish this ability leading to permanent loss of function. No clinical therapy yet exists that reliably restores muscle integrity and function following severe injury. Consequently, numerous tissue engineering techniques, both acellular and with cells, are being investigated to enhance muscle regeneration. Biomaterials are an essential part of these techniques as they can present physical and biochemical signals that augment the repair process. Successful tissue engineering strategies require regenerative biomaterials that either actively promote endogenous muscle repair or create an environment supportive of regeneration. This review will discuss several acellular biomaterial strategies for skeletal muscle regeneration with a focus on those under investigation in vivo. This includes materials that release bioactive molecules, biomimetic materials and immunomodulatory materials.
Critical limb ischemia (CLI) is the most severe clinical manifestation of peripheral arterial disease (PAD), resulting in the total or partial loss of limb function. Although the conventional treatment strategy of CLI (e.g., medical treatment and surgery) can improve blood perfusion and restore limb function, many patients are unsuitable for these strategies and they still face the threats of amputation or death. Therapeutic angiogenesis, as a potential solution for these problems, attempts to manipulate blood vessel growth in vivo for augment perfusion without the help of extra pharmaceutics and surgery. With the rise of interdisciplinary research, regenerative medicine strategies provide new possibilities for treating many clinical diseases. Hydrogel, as an excellent biocompatibility material, is an ideal candidate for delivering bioactive molecules and cells for therapeutic angiogenesis. Besides, hydrogel could precisely deliver, control release, and keep the bioactivity of cargos, making hydrogel-based therapeutic angiogenesis a new strategy for CLI therapy. In this review, we comprehensively discuss the approaches of hydrogel-based strategy for CLI treatment as well as their challenges, and future directions.
Cytokine signaling is critical to a range of biological processes including cell development, tissue repair, aging, and immunity. In addition to acting as key signal mediators of the immune system, cytokines can also serve as potent immunotherapies with more than 20 recombinant products currently Food and Drug Administration (FDA)‐approved to treat conditions including hepatitis, multiple sclerosis, arthritis, and various cancers. Yet despite their biological importance and clinical utility, cytokine immunotherapies suffer from intrinsic challenges that limit their therapeutic potential including poor circulation, systemic toxicity, and low tissue‐ or cell‐specificity. In the past decade in particular, methods have been devised to engineer cytokines in order to overcome such challenges and here, the myriad strategies are reviewed that may be employed in order to improve the therapeutic potential of cytokine and chemokine immunotherapies with applications in cancer and autoimmune disease therapy, as well as tissue engineering and regenerative medicine. For clarity, these strategies are collected and presented as they vary across size scales, ranging from single amino acid substitutions, to larger protein‐polymer conjugates, nano/micrometer‐scale particles, and macroscale implants. Together, this work aims to provide readers with a timely view of the field of cytokine engineering with an emphasis on early‐stage therapeutic approaches. Cytokines are master regulators of the human immune system, and dysregulation of cytokine signaling networks is a hallmark of cancer, autoimmunity, and a variety of other diseases. In recent years, methods have been developed to engineer potent, safe, and specific cytokine immunotherapies that are reviewed here with an emphasis on early‐stage and material‐based therapeutic approaches.
Introduction and hypothesisCurrent treatment modalities for anal sphincter injuries are ineffective for many patients, prompting research into restorative and regenerative therapies. Although cellular therapy with stem cells and progenitor cells show promise in animal models with short-term improvement, there are additional regenerative approaches that can augment or replace cellular therapies for anal sphincter injuries. The purpose of this article is to review the current knowledge of cellular therapies for anal sphincter injuries and discusses the use of other regenerative therapies including cytokine therapy with CXCL12.MethodsA literature search was performed to search for articles on cellular therapy and cytokine therapy for anal sphincter injuries and anal incontinence.ResultsThe article search identified 337 articles from which 33 articles were included. An additional 12 referenced articles were included as well as 23 articles providing background information. Cellular therapy has shown positive results for treating anal sphincter injuries and anal incontinence in vitro and in one clinical trial. However, cellular therapy has disadvantages such as the source and processing of stem cells and progenitor cells. CXCL12 does not have such issues while showing promising in vitro results for treating anal sphincter injuries. Additionally, electrical stimulation and extracorporeal shock wave therapy are potential regenerative medicine adjuncts for anal sphincter injuries. A vision for future research and clinical applications of regenerative medicine for anal sphincter deficiencies is provided.Conclusion There are viable regenerative medicine therapies for anal sphincter injuries beyond cellular therapy. CXCL12 shows promise as a focus of therapeutic research in this field.
Prolonged healing is a severe problem for elderly and diabetic patients. Impaired angiogenesis, stem cell differentiation, and migration have been shown to delay wound healing. The chemokine stromal cell-derived factor-1 (SDF-1) plays an essential role in recruiting cells to wound sites and is suggested to be a candidate for tissue engineering. In this study, chitosan (CHI) scaffolds were crosslinked with nontoxic genipin (Gp) and further heparinized for SDF-1 immobilization. Then, the structures were evaluated for their physicochemical properties (porosity, swelling ratio, and water vapor transmission rate (WVTR)). The interaction between SDF-1 and heparin could sustain SDF-1 release, which has been shown to enhance human umbilical vein endothelial cell (HUVEC) 2D/3D migration. The investigation of the wound-healing activity of the SDF-1-loaded CHI scaffolds revealed a better wound recovery rate in vivo in healthy and streptozotocin-induced diabetic Sprague-Dawley (SD) rats. The histological analysis illustrated that the local of SDF-1 treatment scaffold at the wound site enhanced neovascularization. The wounds treated with SDF-1 scaffolds also exhibited higher vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) expression in Western blot assays. Based on the wound-healing activity and beneficial characteristics, the SDF-1-loaded CHI scaffold demonstrates potential as a material for treating skin wounds.
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In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, (3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and (4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivities of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (Implant 3, p < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than that in the gelatin and microsphere/gelatin hydrogels (p < 0.001), however, there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
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Long bone and in particular tibia fractures frequently fail to heal. A disturbed revascularisation is supposed to be a major cause for impaired bone healing or the development of non-unions. We aim to establish an animal model, which reliably mimics the clinical situation. Human microvascular endothelial cells (HMEC-1) and primary human osteoblast like cells (POBs) were cultured with different angiogenesis-inhibitors (Fumagillin, SU5416, Artesunate and 3,5,4'-Trimethoxystilbene) released out of poly(D,L-Lactide) (PDLLA) coated k-wires and cell activity was determined. Discs containing PDLLA or PDLLA + Fumagillin/Artesunate were placed at the chorionallantoic membrane of hen eggs and the effect on vessel formation and egg vitality was observed. Tibia osteotomy was performed in rats and stabilised with K-wires coated with PDLLA + Fumagillin or with PDLLA only (control group). The healing was compared at different time points to the PDLLA control. Fumagillin and Artesunate inhibited the activity of HMEC-1 with minor effect on POBs. Artesunate caused embryonic death, whereas Fumagillin had no effects on egg vitality, but reduced the blood vessels. In the animal study all rats showed an impaired healing with reduced biomechanical stability. The Fumagillin treated tibiae had a significantly decreased callus size at day 42 and 84, less blood vessels in the early callus, a reduced histological callus size at day 10, 28 and 84, as well as an altered callus composition. This study presents a less vascularised, atrophic, tibia non-union and can be used in further investigations to analyse the pathology of atrophic non-union and to test new interventions.
Endothelium forms an inner layer of vascular wall. It plays an important role in inflammatory process, regulation of vascular tone, and synthesis of thromboregulatory substances. Leukocyte and endothelium interactions during inflammation are regulated by different families of adhesion molecules. Increased levels of soluble forms of adhesion molecules have been detected in the circulating blood in conditions such as autoimmune diseases, transplant rejection, ischemia-reperfusion injury in addition to neutrophil-and endothelial membrane-bound forms reflecting the level of endothelial dysfunction. It is known that endothelial dysfunction is a risk factor for ischemic events such as stroke, myocardial infarction, unstable angina pectoris, ventricle fibrillation, necessity of revascularisation procedures, and death from cardiovascular reasons. Clinical studies showed that cardiac surgery has an impact on vascular endothelial function as well. The amount of endotheliumderived soluble forms of vascular-1 and intercellular-1 adhesion molecules increases after cardiopulmonary bypass suggesting endothelial dysfunction. However, further investigations are needed to be done to support the evidence that endothelial dysfunction proceeding heart surgery is one of the reasons of tissue ischemia-reperfusion injury.
Stem cells are the building blocks through which tissues are developed and maintained. We and many other groups predict that stem cells will prove tremendously useful in clinical medicine. Possible uses include systems for high- throughput drug screens, as in vitro models of disease and, eventually, in treating diseases associated with cell defi- ciency. As one of the least regenerative organs in the body, the heart stands to benefit greatly from addition of new parenchymal cells. Cardiovascular researchers have risen to this challenge and, as a result, cardiac repair is arguably the most advanced program in the emerging field of regenera- tive medicine. Progress in this field has been rapid, from humble beginnings with committed skeletal (1-3) or cardiac muscle cells (4-6), moving to multipotent adult stem cells (7-9) and, most recently, to embryonic stem cells (10-13). In this brief commentary, we review some important recent developments in stem cell-based tissue repair. (Read- ers wishing additional basic and clinical information are directed to several recent in-depth reviews (14-16) on the field.) Like other areas involving stem cell-based regeneration, the field of cardiac repair has its share of controversies. These will also be touched upon, with an aim of separating experi- mental observation, on which there is much agreement, from interpretation, which varies widely at the moment.
The degradation behavior of hydrogel based on poly(aspartic acid) (PASP) which crosslinked by 1, 6-hexamethylene diamine (HD) was studied in α-chymotrypsin solution. The degradation behaviors of PASP under different crosslinking levels were evaluated as a function of biodegradation time by monitoring the changes of crosslinking density, molecular weight, rheological behavior, interior morphology, mass loss, and swelling ratio. It was found that the degradation behaviors relied heavily on the crosslinking density which calculated from the Flory-Rehner equation. A pseudo first-kinetic equation was proposed to model the mass loss behavior and the degradation rate constant was calculated under different crosslinking levels. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010
Theoretically expected rates of release of solid drugs incorporated into solid matrices have been derived for several model systems. Mathematical relations have been obtained for cases (a) where the drug particles are dispersed in a homogeneous, uniform matrix which acts as the diffusional medium and (b) where the drug particles are incorporated in an essentially granular matrix and released by the leaching action of the penetrating solvent. Release from both planar surface and a sphere is considered. The unidimensional release rates are shown to follow our earlier equation derived for release from ointment bases. Release rates from spherical pellets by both model mechanisms are shown not to follow first-order relationships. The analyses suggest that for the latter system the time required to release 50 per cent of the drug would normally be expected to be approximately 10 per cent of that required to dissolve the last trace of the solid drug phase in the center of the pellet.
Porous hydrophilic discs were prepared from two grades of poly(vinyl alcohol) of varying degree of hydrolysis. The influence of the molecular size of the tracer used (potassium chloride, phenylpropanolamine hydrochloride and bovine serum albumin), that of the addition of a second water-soluble polymer poly(N-vinyl-2-pyrrolidone) and poly(ethylene glycol)) and the effect of the tracer/excipient ratio on the release profile were examined. Finally the role of the dynamic swelling and the dissolution of the polymer matrix on the release mechanism are discussed.
Distraction osteogenesis (DO) is a unique therapy that induces skeletal tissue regeneration without stem/progenitor cell transplantation. Although the self-regeneration property of DO provides many clinical benefits, the long treatment period required is a major drawback. A high-speed DO mouse model (H-DO), in which the distraction was done two times faster than in control DO (C-DO) mice, failed to generate new bone callus in the DO gap. We found that this was caused by the unsuccessful recruitment of bone marrow endothelial cells (BM-ECs)/endothelial progenitor cells (EPCs) into the gap. We then tested the ability of a local application of stromal cell-derived factor-1 (SDF-1), a major chemo-attractant for BM-ECs/EPCs, to accelerate the bone regeneration in H-DO. Our data showed that, in H-DO, SDF-1 induced callus formation in the gap through the recruitment of BM-ECs/EPCs, the maturation of neo-blood vessels, and increased blood flow. These results indicate that the active recruitment of endogenous BM-ECs/EPCs may provide a substantial clinical benefit for shortening the treatment period of DO.