An injectable decellularized matrix that improves mesenchymal stem

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Tissue Engineering and Regenerative Medicine

An injectable decellularized matrix that improves mesenchymal stem cell engraftment for therapeutic angiogenesis Gun-Jae Jeong, Seuk Young Song, Mikyung Kang, Seokhyeong Go, Hee Su Sohn, and Byung-Soo Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00617 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 9, 2018

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ACS Biomaterials Science & Engineering

An injectable decellularized matrix that improves mesenchymal stem cell engraftment for therapeutic angiogenesis

Gun-Jae Jeong1, Seuk Young Song1, Mikyung Kang2, Seokhyeong Go2, Hee Su Sohn1, Byung-Soo Kim1,3,*.

1

School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic

of Korea 2

Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, Republic of

Korea 3

Institute of Chemical Processes, Seoul National University, Seoul, Republic of Korea

*Author to whom the correspondence should be addressed: Byung-Soo Kim, Ph.D., E-mail: [email protected], Tel.: +82-2-880-1509, Fax: +82-2888-1604

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Stem cell therapy has great potential for the treatment of ischemic diseases, but poor engraftment of implanted stem cells limits the therapeutic efficacy. Here, we developed an approximately 80-µm injectable decellularized matrix (IDM) to increase the angiogenic efficacy of mesenchymal stem cells by improving the engraftment of the stem cells implanted in to an ischemic tissue. Adhesion of human adipose tissue-derived stem cells (hADSCs) to the IDM enhanced the cell viability and upregulated angiogenic factors in vitro under either cell adhesion-suppressive conditions or hypoxic conditions, which simulated the microenvironment of ischemic tissues. In a murine ischemic-hindlimb model, hADSCs that were attached to the IDM and subsequently injected into an ischemic region showed better grafting and angiogenic factor expression. The hADSC-IDM implantation subsequently promoted the formation of microvessels, attenuated fibrosis, and increased blood perfusion in the ischemic region, as compared to implantation of hADSCs only. The IDM may be an effective off-the-shelf material that can enhance therapeutic efficacy of stem cell therapy for ischemic diseases.

Keywords : angiogenesis, cell implantation, injectable decellularized matrix, ischemic disease, stem cell therapy

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1. Introduction Stem cell therapy has great potential for therapeutic angiogenesis against ischemic diseases. Endothelial progenitor cells derived from embryonic stem cells contribute to postnatal neovascularization by directly participating in blood vessel formation.1 Mesenchymal stem cells (MSCs) derived from bone marrow and adipose tissues mainly induce angiogenesis through paracrine secretion of angiogenic growth factors.2-3 Despite this strong potential, stem cells implanted into an ischemic region have shown low therapeutic efficacy because of their low survival rate, which is a large obstacle in clinical trials.4 In an ischemic region, implanted stem cells are immediately exposed to harsh conditions and are prone to apoptosis.5 One of the major types of implanted cell apoptosis is anoikis, which is apoptosis due to the loss of cell adhesion to an extracellular matrix (ECM).5 Ex vivo cultured stem cells are usually harvested for implantation from culture plates by proteolytic enzyme (e.g., trypsin) treatment. Because this treatment causes a loss of the ECM from the harvested cells, cell adhesion signals are downregulated in the harvested and subsequently implanted cells.6 Indeed, within a few days after cell transplantation, widespread implanted-cell death has been observed in a number of animal studies.7-8 Moreover, clinical studies on the treatment of ischemic diseases have shown that stem cell implantation has insignificant therapeutic efficiency most likely due to poor survival of the implanted cells.4, 9-11 To improve the engraftment of stem cells implanted into an ischemic tissue, several strategies have been developed. The strategies include preconditioning of stem cells,12-13 genetic modifications of stem cells,12,

14-15

combination of stem cell therapy with growth factor

delivery,16 and the use of tissue engineering scaffolds.17 Although preconditioned stem cells show higher engraftment, the risk of cell damage and differentiation during the 3



preconditioning procedure should be addressed before clinical trials.12 Genetic modification of stem cells has been set back by low transfection efficiency, the mutagenic potential of target genes, and cytotoxicity.18-19 A combination with growth factor delivery can enhance stem cell survival, but local and controlled-release system must be developed to avoid potential side effects and to preserve the growth factor activity in vivo.20 In tissue engineering, synthetic polymer scaffolds or a biologically derived ECM can serve as a cell carrier for implantation. These matrices provide implanted cells with a cell-adhesion cue for cell survival following implantation.21-23 Several studies have revealed that cell implantation with a hydrogel containing ECM components enhances therapeutic efficacy in animal models of myocardial infarction, limb ischemia, and bone fracture.24-26 Hence, here we tested a hypothesis that injection of therapeutic cells-adherent to an injectable decellularized matrix (IDM)-into an ischemic site may improve grafting of the cells and enhance the angiogenic efficacy of these cells. For fabrication of the IDM, we applied a roller cutter to confluently cultured human adiposederived stem cells (hADSCs) and treated the cell layer with a decellularization solution to obtain an IDM with particle size of approximately 80-µm (Figure 1). hADSCs were allowed to adhere to the IDM. It was investigated whether the construct of hADSCs and IDM (cellIDM) enhances the cell viability and upregulates angiogenic factors in vitro under either cell adhesion -suppressive or hypoxic conditions, which simulate the microenvironment of ischemic tissues. Next, cell-IDM was implanted into an ischemic region of mouse hindlimbs to test whether the implantation of cell-IDM improves the angiogenic efficacy of hADSCs by improving the engraftment of the implanted hADSCs (Figure 1).

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2. Materials and Methods

2.1 Cell culture hADSCs were purchased from Lonza (Walkersville, MD). hADSCs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Waltham, MA) supplemented with 10 % (v/v) of fetal bovine serum (FBS, Gibco, Waltham, MA) and antibiotics in a 5 % (v/v) CO2 incubator at 37 °C. hADSCs at passage 4-6 were used in the experiments.

2.2 Fabrication of IDM hADSCs were cultured for 2 weeks in a 150-mm dish. Then, the cell layer was washed with phosphate buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA) and 10 ml of PBS was added. The cell layer was cut using an EZ Passage Roller Cutter (Invitrogen). The roller cutter was pulled in one direction, and this movement was repeated after the dish was turned by 90˚ to make a lattice (Figure 1). Then, the cell layer was decellularized with decellularization buffer (0.5% Triton X-100 and 20mM NH4OH in PBS) as reported previously.27 After that, the IDM was carefully scratched off from the dish surface using a cell scraper (Corning, Corning, NY). IDM was collected by centrifugation at 2000 g for 20 min, washed twice with PBS to remove the decellularization solution, and treated with 100 U/ml DNase (Worthington Bio, Lakewood, NJ) overnight. The IDM was lyophilized and stored at – 20 ˚C prior to experiments.

2.3 IDM characterization 5



Cultured hADSCs, the decellularized matrix, and IDM were immunostained with an antifibronectin antibody (Abcam, Cambridge, UK). For DNA quantification, cultured hADSCs, the decellularized matrix layer, and IDM were obtained from 106 cultured cells. Total DNA samples were prepared from the cell samples by means of a DNA extraction kit (Bioneer, Dajeon, Korea). The DNA content of the samples was measured on a Nanodrop 2000 (Thermo Fisher Scientific). Next, the DNA amount in each sample made from 106 hADSCs was calculated.

2.4 A cell viability assay hADSCs were cultured on agarose (2% in PBS)-coated dishes, which prevent cell adhesion to the dishes. After cultivation for various periods, live and dead cells were detected by staining with fluorescein diacetate (FDA, Sigma, St. Louis, MO) and ethidium bromide (EB, Sigma), respectively. The dead cells stained red due to the nuclear permeability of EB. The viable cells, which are capable of converting the non-fluorescent FDA into fluorescein, stained green.

2.5 Cell attachment to the IDM To evaluate cell attachment to the IDM, this material and cells were labeled with coumarin (100 µg/ml, green, Sigma) and PKH-26 (Sigma), respectively. The labeled IDM (4 mg) and cells (106 hADSCs) were incubated in 1 ml of PBS at 37 °C and 5% CO2 for various periods. The percentage of cells attached to the IDM was determined under a fluorescence microscope (Nikon TE2000, Tokyo, Japan).

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2.6 The mouse model of hindlimb ischemia Hindlimb ischemia was induced in athymic mice (BALB/c, female, 6 weeks- old, 20–25 g, Orient, Seoul, Korea) as described elsewhere.1, 28 After anesthesia with ketamine (100 µg/g) and xylazine (10 µg/g)), the femoral artery and its branches of the mice were ligated with 6-0 black silk sutures (Ailee, Busan, Korea). Then, the external iliac artery and the upstream arteries were ligated. The femoral artery was excised from its proximal site of origin. All the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Seoul National University (approval No. SNU-160303-6).

2.7 Treatment of hindlimb ischemia Instantly after the hindlimb ischemia modeling, the mice were subdivided into three groups (n = 12 per group): no treatment (sham), hADSC injection (Cell group), and hADSC-IDM construct injection (Cell-IDM group). The sham group served as the control. The Cell group received an injection of 106 trypsinized-hADSCs resuspended in 100 µl of PBS into the gracilis muscle in the medial thigh on day 0. Mice in the Cell-IDM group received an injection of the hADSC (106 cells)-IDM (4 mg) construct resuspended in 100 µl of PBS. Needles with 26 G were used for Cell-IDM injection. We injected the cells slowly to avoid shearing damage.

2.8 Live imaging of implanted cells To evaluate the hADSC grafting and retention after the implantation, hADSCs were labeled with VivoTrack 680 (PerkinElmer, Waltham, MA) and injected into ischemic muscle (106 hADSCs in 100 µl of PBS per mouse). Luminescence intensity was monitored and quantified 7



for 14 days by means of an IVIS spectrum live imaging system (PerkinElmer, Waltham, MA).

2.9 Laser Doppler imaging analysis Laser Doppler imaging (laser Doppler perfusion imager; Moor Instruments, Devon, UK) was performed for serial noninvasive physiological evaluations of blood perfusion in mouse ischemic hindlimbs. The mice were monitored by serial scanning of the surface blood flow in hindlimbs on days 0, 7, 14, 21, and 28 after treatment. Color-coded digital images were analyzed to quantify blood flow in the ischemic hindlimbs, and the mean perfusion values were subsequently calculated.

2.10 Immunohistochemistry On days 7 and 28 after hindlimb ischemia treatment, limb muscles were collected from euthanized mice. After that, these samples were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA). After a sample freezing step, the samples were cut into 10-µm-thick slices at −21 °C. The ischemic-region slices obtained on day 28 after hindlimb ischemia treatment were subjected to immunofluorescent staining to quantify microvessels with an anti-smooth muscle α-actin (SM α-actin) antibody (Abcam, Cambridge, UK) and anti-CD31 antibody (Santa Cruz Biotechnology, Dallas, TX). The samples from day 7 after hindlimb ischemia treatment were processed by immune-fluorescent staining to assess cell grafting and viability with an anti-human nuclear antigen (HNA) antibody and anticaspase-3 antibody (Abcam). Fluorescein isothiocyanate–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used to visualize the signals. The slices were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and examined under the fluorescence microscope. 8


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2.11 Histological examination At 28 days after treatment, the thigh muscles of the ischemic limbs were excised from euthanized mice. The ischemic-limb muscle specimens were fixed in formaldehyde, dehydrated in a graded series of ethanol solutions, and embedded in paraffin. The paraffinembedded specimens were sliced into 4-µm-thick sections and stained with hematoxylin and eosin (H&E) to assess muscle degeneration and tissue inflammation. Masson’s trichrome collagen staining was also conducted to evaluate tissue fibrosis in the ischemic regions.

2.12 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) qRT-PCR analysis was conducted to quantify relative mRNA expression. Total RNA was extracted from samples (n = 4 per group) with 1 ml of the TRIzol reagent (Invitrogen) and 200 µl of chloroform. The lysed samples were centrifuged at 8000 g for 10 min at 4 °C. The RNA pellet was washed with 75% (v/v) ethanol in water and dried. After that, the samples were dissolved in RNase-free water. For qRT-PCR, the SYBR green real-time PCR master mix (Thermo Fisher Scientific) was applied. β-Actin served as the internal control.

2.13 Western blot analysis The murine ischemic-limb samples were lysed with an electric homogenizer in lysis buffer (Cell Signaling Technology, Danvers, MA). Protein concentrations were determined by a bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL). Equal amounts of protein from each sample (25 µg) were mixed with sample buffer, loaded, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a 10-15% (v/v) 9



resolving gel. Proteins separated by SDS-PAGE were transferred to an Immobilon-P membrane (Millipore, Billerica, MA) and then probed with antibodies against SM-α, CD31, HGF, VEGF, and SDF-1α (anti-CD31 from Santa Cruz Biotechnology and the others from Abcam) for 1 hr at room temperature. Next, the membranes were submerged in a solution of a horse radish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. The blots were developed using a Gel logic imaging system (Carestream Health, Rochester, NY). Bands were imaged and quantified in the Image J software (NIH, Bethesda, MD).

2.14 Statistical analysis Quantitative data were expressed as mean ± SD. OriginPro 8 software (OriginLab, Northampton, MA) served for one-way analysis of variance. “Statistically significant” means that the p value is less than 0.05.

3. Results

3.1 Formation of the cell-IDM construct The IDM as an hADSC implantation vehicle was prepared from cultured hADSCs (Figure 1) because hADSCs are expected to interact best with the ECM produced by the same cells (i.e., hADSCs). The amount of IDM obtained from 106 cultured hADSCs was approximately 2 mg. To characterize this IDM, immunostaining for fibronectin and quantification of DNA content were performed. Immunostaining and western blot analysis showed that fibronectin, a major ECM component for cell adhesion, was left over in a large amount to make the IDM (Figure 10


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2a and Figure S1) and maintain its shape after the decellularization process (Figure 2a). The DNA quantification in cultured hADSCs, the decellularized ECM, and DNase-treated decellularized ECM suggested that the IDM produced by decellularization and DNase treatment contained no DNA (Figure 2b). The size of rectangular IDM pieces was approximately 80 µm. Several hADSCs were attached to a piece of the IDM after incubation for 30 min (Figure 2c).

Figure 1. A schematic diagram of the injectable decellularized matrix (IDM) preparation and the in vivo experiment. Layers of confluently cultured cells were cut with a roller cutter, 11



followed by decellularization and DNase treatment. Prior to cell injection, a cell-IDM construct was formed by mixing the IDM and hADSCs. Injection of the cell-IDM construct was expected to enhance therapeutic efficacy of hADSCs in a mouse model of hindlimb ischemia by improving the hADSC grafting and paracrine factor secretion by hADSCs, as compared to injection of hADSCs alone .

Figure 2. IDM characterization and cell-IDM construct formation. (a) Before and after decellularization of a cultured-hADSC layer. ECM proteins were stained with an antifibronectin antibody (green). Cell nuclei were stained with DAPI (blue). Scale bars = 100 µm. (b) DNA content was determined before and after decellularization and DNase treatment of the hADSC layer composed of 106 cells and the ECM. (c) Before and after hADSC attachment to the IDM. For cell attachment to the IDM, a mixture of hADSCs and the IDM was incubated for 30 min. The IDM was stained with the anti-fibronectin antibody (green). 12


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hADSCs were stained with PKH26 (red). Scale bars = 100 µm.

3.2 Optimization of incubation time for hADSC attachment to the IDM To optimize the incubation time for hADSC attachment to the IDM, the percentage of cells attached to the IDM was determined after incubation of a mixture of fluorescently labeled hADSCs and the IDM in PBS for various periods. The mixture ratio was 106 cells to 4 mg IDM. The amount of IDM generated from 106 cultured hADSCs was approximately 2mg. Nonetheless, we used 4mg IDM per 106 hADSCs, rather than 2 mg of the IDM because the loss of the ECM during the IDM fabrication process cannot provide a sufficient amount of the ECM for cell attachment. We used PBS to incubate the cell-IDM mixture because PBS is generally employed to make a cell suspension for cell implantation in the clinic. hADSCs started to attach to the IDM quickly, which may be due to instant cell attachment to fibronectin of the IDM. The percentage of hADSCs attached to the IDM increased with incubation time until 30 min (Figure 3a). Aggregation between individual cell-IDM constructs was not observed. The cell viability stayed unchanged until 30 min and decreased at 60 min (Figure 3b). According to these results, we selected 30 min as the optimal incubation time of the hADSC-IDM mixture for the subsequent in vitro and in vivo experiments. Both the cell concentration and IDM concentration would affect the optimal incubation time of the hADSC-IDM mixture. If the IDM concentration increases without cell concentration change, the optimal incubation time would decrease because the probability of cell-IDM contact would increase. Low cell concentration would prolong the optimal incubation time. However, the incubation time of hADSC-IDM mixture should be less than 60 min because the cell viability decreased after 60 min in PBS. 13



Figure 3. Optimization of incubation time of the hADSC-IDM mixture for hADSC adhesion to the IDM. The mixture was composed of 4 mg-IDM and 106 hADSCs in PBS. (a) The percentage of cells attached to the IDM was determined at various incubation time points. The IDM and cells were labeled with coumarin (green) and PKH-26 (red), respectively; *p < 0.05 as compared to time point 0 min; n = 5 per group. (b) Viability of the cells after 14


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incubation of the hADSC-IDM mixture for various periods. Live and dead cells were labeled with FDA (green) and EB (red), respectively; *p < 0.05 as compared to time point 0 min; n = 5 per group.

3.3 In vitro prevention of hADSC anoikis by the IDM To determine whether hADSC attachment to the IDM prevents anoikis of hADSCs under either cell adhesion-suppressive or hypoxic conditions, hADSCs were cultured in agarosecoated plates, which prevent cell adhesion to culture plates, under either normoxic (20% oxygen) or hypoxic conditions (1% oxygen), which mimic the environment of an ischemic tissue. Hypoxia in an ischemic tissue promotes reactive oxygen species production,29 which prevent implanted-cell adhesion to the ECM and subsequently causes anoikis of the implanted cells.30 Thus, cell culture in agarose-coated plates under hypoxic conditions can mimic the environment of an ischemic tissue. The quantity of the IDM mixed with hADSCs was 4 mg per 106 cells in the 1x IDM group and 10 mg per 106 cells in the 2.5x IDM group. Cell culture without the IDM served as the control (the No IDM group). After 24 hr culture in agarose-coated plates under normoxic conditions, the 1x IDM group and the 2.5x IDM group showed higher cell viability than the No IDM group did (Figure 4a). There was no significant difference in cell viability between the 1x IDM group and the 2.5x IDM group. This result indicated that hADSC attachment to the IDM can prevent anoikis of hADSCs under cell adhesion-suppressive conditions. Cell culture in agarose-coated plates under hypoxic conditions for 24 hr also yielded improved cell viability in the 1x IDM group and the 2.5x IDM group (Figure 4b). This result suggested that hADSC attachment to the IDM may enhance hADSC viability after implantation into an ischemic tissue. Because cell viability 15



was not significantly different between the 1x IDM group and 2.5x IDM group, 1x IDM was selected for the subsequent experiments. Next, qRT-PCR analysis was performed to test whether the hADSC-IDM construct can prevent apoptosis of hADSCs (Figure 5). hADSCs (the Cell group) and the hADSC-IDM construct (Cell-IDM group) were cultured in agarosecoated plates under hypoxic conditions for 24 hr. Pro-apoptotic gene (BAX) expression decreased and anti-apoptotic gene (Bcl-2) expression increased in the Cell-IDM group as compared to the Cell group.

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Figure 4. Reduced anoikis (i.e., cell death due to loss of cell adhesion to the ECM) of hADSCs under the influences of the cell-IDM construct. hADSCs were cultured under (a) normoxic (20% oxygen) or (b) hypoxic (1% oxygen) conditions for 24 hr in agarose-coated plates, which prevent cell adhesion to the dishes. The 1x IDM group denotes the construct composed of 4 mg IDM and 106 hADSCs. The 2.5x IDM group represents the construct 17



composed of 10 mg IDM and 106 hADSCs. hADSCs without the IDM served as the control. Live and dead cells were labeled with fluorescein diacetate (FDA, green) and ethidium bromide (EB, red), respectively. Cell viability was determined after 24 hr culture; *p < 0.05 as compared to the control group (No IDM); n = 5 per group.

Figure 5. Reduced apoptotic signal expression and enhanced angiogenic paracrine factor expression in the cell-IDM construct 24 hr after cultivation in agarose-coated dishes under hypoxic (1% O2) conditions. Expression levels of a pro-apoptotic factor (BAX), antiapoptotic factor (Bcl-2), and angiogenic factors (VEGF and HGF) were evaluated by realtime PCR analysis; *p < 0.05 as compared to the Cell group; n = 4 per group.

3.4 In vitro upregulation of angiogenic factor expression qRT-PCR analysis was carried out to determine whether the hADSC-IDM construct can upregulate expression of angiogenic paracrine factors in hADSCs (Figure 5). hADSCs (the Cell group) and the hADSC-IDM construct (the Cell-IDM group) were cultured in agarose18


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coated plates under hypoxic conditions, which mimic the environment of an ischemic tissue, for 24 hr. mRNA expression of angiogenic factors (VEGF and HGF) increased in the CellIDM group as compared to the Cell group.

3.5 In vivo engraftment of hADSCs implanted into the ischemic tissue Live imaging analysis was performed during 14 days after cell implantation to study in vivo engraftment of hADSCs injected into the ischemic tissue. hADSCs were labeled with VivoTrack 680 prior to injection. The relative luminescence intensity was higher in the CellIDM group than in the Cell group throughout the 14-day period (Figure 6a). This result suggested that the engraftment of hADSCs implanted into the ischemic tissue was enhanced by hADSC adhesion to the IDM prior to the injection. Immunohistochemcal analysis was conducted to examine apoptosis of the implanted hADSCs on day 7 (Figure 6b). Caspase-3 and HNA double staining of the cells revealed apoptotic hADSCs. The density of HNApositive cells was higher in the Cell-IDM group than in the Cell group, implying that more hADSCs survived in the ischemic tissue because of being implanted as the hADSC-IDM construct. Besides, the density of caspase-3 and HNA double stained cells was lower in the Cell-IDM group than in the Cell group, indicating that apoptosis of hADSCs implanted into the ischemic tissue decreased when the cells were implanted as the hADSC-IDM construct.

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Figure 6. Enhanced hADSC grafting after injection of the cell-IDM construct into a hindlimb ischemic region in mice. (a) In vivo imaging of VivoTrack 680-stained hADSCs on days 0, 3, 7, and 14 after implantation. The fluorescence intensity was quantified and normalized to that of each animal on day 0; *p

An injectable decellularized matrix that improves mesenchymal stem

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