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Cell-Free Hydrogel System Based on Tissue-Specific Extracellular Matrix for In Situ Adipose Tissue Regeneration Jun Sung Kim, Ji Suk Choi, and Yong Woo Cho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16783 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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ACS Applied Materials & Interfaces

Cell-Free Hydrogel System Based on TissueSpecific Extracellular Matrix for In Situ Adipose Tissue Regeneration Jun Sung Kim,# Ji Suk Choi,# and Yong Woo Cho* †

Department of Chemical Engineering, Hanyang University, Ansan, Gyeonggi-do 426-791,

Republic of Korea

KEYWORDS: in situ adipose tissue regeneration, cell-free scaffold system, adipose-derived soluble extracellular matrix, thermosensitive ECM hydrogel, host cell recruitment

ABSTRACT: Well-designed scaffolds provide appropriate niches that can effectively recruit the host cells and induce differentiation of recruited cells into desired cell types, facilitating the in situ tissue regeneration. Here we report a tissue-specific extracellular matrix (ECM) hydrogel composed of adipose-derived soluble ECM (sECM) and methylcellulose (MC) as a cell-free scaffold system for adipose tissue regeneration. The sECM-MC hydrogels showed a thermosensitive sol-gel phase transition and rapidly forming a soft hydrogel with the stiffness of 3.8 kPa at body temperature. An in vivo study showed that the sECM-MC hydrogel facilitated

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the infiltration of host cell populations, particularly adipose-derived stem cells (ASCs) and adipose tissue macrophages (ATMs) that directly contribute to the adipose tissue regeneration. Moreover, the hydrogel significantly enhanced host-derived adipogenesis and angiogenesis without exogenous cells or bioactive molecules. Our results indicate that the sECM-MC hydrogels provide mechanical and biochemical cues for host-derived adipose regeneration. Overall, the sECM-MC hydrogels are a highly promising cell-free therapeutic approach for in situ adipose tissue regeneration.

INTRODUCTION

The use of adipose-derived stem cells (ASCs) have been standard approaches in the reconstruction of adipose tissue defects, including congenital or traumatic defects, oncological resection, and degenerative diseases.1,2 Although, stem cells have clear beneficial effects in adipose tissue regeneration, stem cell-based therapy should be considered in correlation with the extensive cell expansion, immune rejection, limited survival and retention after treatment, and difficulties of controlling cell fate.3 One of the promising approaches to overcoming these limitations is cell-free systems based on tissue-specific scaffolds, which can actively participate in recruitment of body’s endogenous cells for tissue regeneration.4-8 For in situ adipose tissue regeneration, many studies have been reported adipo-inductive scaffolds designed by structural, mechanical, and biochemical parameters. However, most efforts have still concentrated on the scaffolds for delivering exogenous stem cells and/or bioactive molecules (e.g., basic fibroblast growth factor; bFGF) that enhance the recruitment, angiogenesis, and adipogenesis of stem cells.6,9-12


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Intact ECMs derived from living tissues have emerged as an ideal biomaterial for a broad range of regenerative medicine because the inherent mechanical and biochemical cues of ECM can provide an appropriate microenvironment for controlling cellular behaviors, such as cell recruitment, proliferation, and differentiation.13-16 In particular, adipose-derived ECM has comparable mechanical and biochemical properties to native adipose tissue, providing highly supportive niche for adipogenic differentiation of ASCs both in vitro and in vivo.9,17 In our previous study, we reported the thermosensitive, injectable hydrogels based on solubilized adipose ECM and methylcellulose (MC) for delivering stem cell in skin wounds.18 Thermosensitive ECM hydrogels are attractive for therapeutic applications because of easy incorporation and simple delivery of cells or agents, direct injection into defective sites even of irregular shape, and robust biological activity.19 We have shown that the soluble ECM (sECM) retains abundant ECM proteins (e.g., soluble elastin, collagen, laminin, fibronectin, and glycosaminoglycans) and 25 endogenous adipokines (e.g., hepatocyte growth factor; HGF, platelet-derived growth factor; PDGF, endothelial growth factor; EGF, insulin-like growth factors-1; IGF-1, transforming growth factor-β1; TGF- β1, and stem cell factor; SCF). Moreover, in vivo results have provided evidence that sECM-MC hydrogels have the ability to improve the engraftment of transplanted human ASCs and accelerate the full-thickness skin wound healing. In this study, we focused on the inherent host cell-recruiting and adipo-inductive abilities of sECM-MC hydrogels. The sECM-MC hydrogels were characterized in terms of sECM content by LC-MS/MS, mechanical properties by rheometry and atomic force microscopy (AFM), and in vivo injectability. The infiltration of mouse ASCs and adipose tissue macrophages (ATMs) within the hydrogels, host-derived adipogenesis and angiogenesis were also evaluated using a mouse subcutaneous injection model.


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MATERIALS AND METHODS Materials. All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN, USA). Dialysis tubing (MWCO 6,000 – 8,000) was obtained from Thermo Fisher Scientific (Rockford, IL, USA). Sircol acid/pepsin-soluble collagen, Blyscan sulfated GAG and Fastin elastin assay kits were supplied by Biocolor (Carrickfergus, Northern Ireland). QuantiMatrixTM Human laminin and fibronectin ELISA kits were supplied by KOMA BIOTECH (Seoul, Korea). 4,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Scientific (Rockford, IL, USA). Anti-mouse CD29 was purchased from BioLegend (San Diego, CA, USA). Anti-mouse CD163 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Fluorochromeconjugated goat anti-rabbit IgG was purchased from Thermo Fisher Scientific. Blood vessel staining kit (ECM590) was purchased from Merck Millipore (Billerica, MA, USA). Penicillin/streptomycin (P/S), collagenase type II, Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL, Life Technologies Inc. (Carlsbad, CA, USA). All other chemicals were reagent grade and used as received. Extraction of Soluble Extracellular Matrix (sECM) from Human Adipose Tissue. sECM was prepared from human adipose tissue as described previously.18,20 Briefly, human adipose tissue was obtained with informed consent as approved by the Institutional Review Board of the Catholic University of Korea, College of Medicine. The adipose tissue obtained by liposuction was washed several times with distilled water to remove blood components. Adipose tissue/distilled water (1:1) mixture was pulverized for 3 min and centrifuged at 16,500 ×g for 5 min. The tissue suspension was washed by 3.4 M NaCl, centrifuged at 20,000 ×g for 30 min, and


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then denatured in 4 M urea buffer containing protease inhibitor cocktail for 12 h. The mixture was centrifuged at 20,000 ×g for 60 min to remove insoluble materials. The residues were reextracted in 4 M guanidine containing protease inhibitor cocktail for 12 h and were centrifuged again. The supernatant was filtered through 40-µm mesh and dialyzed in dialysis tubing against 30 volumes of tris-buffered saline (TBS) for 24 h. The dialysate was centrifuged at 20,000 ×g for 60 min and the final soluble ECM was lyophilized. All procedures were performed at 4 ºC. The concentration of purified sECM was measured using a BCA assay. Liquid Chromatography Mass Spectrometry (LC-MS) for Protein Identification. sECM was electrophoresed on 15% SDS-PAGE gels and protein bands were excised from the stained SDS gels. The selected protein bands were analyzed by liquid chromatography-coupled electrospray ionization mass spectrometry (LC-MS/MS; Proteome Tech. Inc., Seoul, Korea) and identified by a National Center for Biotechnology Information (NCBI) BLAST search on the basis of probability. Preparation of sECM and Methylcellulose (MC) Hydrogels. sECM solution (30 wt%) was prepared by dissolving 3 g of lyophilized sECM in 10 mL of phosphate buffered saline (PBS) for 24 h at 4 °C. Methylcellulose (MC) solution (8.3 wt%) was prepared by dispersion technique. Briefly, 0.75 g of MC (viscosity of 15 cP) was thoroughly wetted in 10 mL of PBS and heated at 90 °C for 60 min. Then, the solution was allowed to equilibrate overnight at 4 °C. Subsequently, sECM and MC solution were mixed at a volume ratio of 1:4 to achieve final concentration of 6 wt% sECM and 6 wt% MC, and the mixture was homogenously stirred at 4 °C. The sECM-MC solution was kept at 4 °C until use.


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Rheological Analysis. The rheological characteristics of sECM-MC hydrogels were determined with a rheometer (Bohlin Gemini HR Nano, Malvern, UK). sECM-MC pre-gel solutions were immediately loaded onto the rheometer plate pre-cooled to 10 °C. After loading, the shear viscosity was measured at a frequency of 1 Hz. The temperature was then increased to 40 °C at a rate of 3 °C per minute to induce gelation. To measure the gelation time, G’ and G’’ were monitored as a function of time at a frequency of 1 Hz. The operation temperature was maintained at 37 °C. Viscosity Measurement. The viscosity of sECM-MC hydrogels were determined by a viscometer (DV-II Pro, Brookfield, Middleboro, MA, USA). Each sample (20 mL) was poured into a holder, kept at 4 and 37°C, and recorded in five times. Atomic Force microscopy. The stiffness of sECM-MC hydrogel was measured by a forcedistance (FD) curve-based atomic force microscopy (TT-AFM, AFM Workshop, Signal Hill, CA, USA). The hydrogels were probed at 4 random regions using a cantilever with spring constant of 0.2 N/m. The contact mechanics between an indenting probe and a hydrogel surface were converted to plots of force versus indentation depth. Young’s modulus of hydrogel was calculated by the Derjaguin-Muller-Toporov (DMT) model.21 In Vivo Studies. The lyophilized sECM and MC powders were sterilized by ethylene oxide gas. sECM-MC pre-gel solutions alone or pre-gel solutions were injected subcutaneously into the backs of male mice (6 weeks old, BALB/cAnNCrj-nu/nu, weighting 18−23 g) using a 28-gauge needle. The grafts were explanted, and fixed with 4% paraformaldehyde at 1, 2, and 3 weeks after the injections. Eight mice per each experimental group were analyzed.


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Histological, Immunofluorescence and Immunohistochemical Analysis. For histological and immunofluorescence examinations, specimens were fixed in 4% paraformaldehyde, embedded in OCT compound, and frozen at -70 °C. The frozen samples were sliced into 5-µm sections using a cryostat. The sections were washed with distilled water and 30% isopropanol to remove the OCT compound, and then stained with the hematoxylin–eosin, oil red O, and DAPI working solutions. To visualize host cell infiltration and vascularization in injected hydrogels, anti-mouse CD29 for adipose-derived stem cell, anti-mouse CD11c+ for M1 macrophage, antimouse CD163 for M2 macrophage, anti-von Willebrand Factor antibody for endothelial cell, and fluorescein-conjugated anti-mouse IgG were used. Reverse Transcription Polymerase Chain Reaction (RT-PCR). Total mRNA was isolated from tissues using an easy-BLUE reagent (Intron, Korea). PCR analysis was using ONE-STEP RT-PCR PreMix Kit (Intron, Korea) according to the manufacturer’s protocol. The cDNA was used as a template for PCR analysis with primers specific for human peroxisome proliferating activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (aP2), adiponectin, leptin, and β-actin. The expression of a housekeeping gene, β-actin was used as an internal control. PCR products were separated by electrophoresis in 1.5% agarose gels and stained with Loadings TAR (DyneBio, Korea). Statistical Analysis. Experimental data were expressed as means ± standard deviation (SD). The Student’s two-tailed t-test with SPSS 17.0 statistical software (SPSS, Chicago, IL) was used for comparison, and statistical significance was accepted at p < 0.05. RESULTS


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Protein Composition in Human Adipose Tissue-Derived sECM. The sECM was extracted from human adipose tissues as described previously.18 For identification of protein contents in sECM, the SDS-PAGE-separated proteins were visualized as discrete bands by Coomassie blue staining (Supplementary Figure S1) and the selected protein bands were analyzed by LCMS/MS. The sECM contained a complex protein composition that is representative of the original adipose tissues.18,22 A total of 113 proteins were identified in sECM with a significant Mascot score of > 40 (Table 1). A number of the identified proteins are classified as ‘extracellular matrix’ proteins, including glycoproteins (collagen I, VI, XV, XVIII, fibronectin, laminins, nidogen, and fibrillin-1) and proteoglycans (biglycan, decorin, perlecan, osteoblast specific factor 2, and osteoglycin). Adipokines, such as fatty-acid-binding protein were also identified in sECM. Supporting Information Table S1 provides detailed information on the identification of proteins.

Table 1. Protein contents in soluble ECM by LC-MS/MS. Proteins

Accession number

Biglycan

gi|179433

COL1A1 and PDGFB fusion transcript

gi|3288487

Collagen, type VI

gi|62988748

Collagen alpha 1, type VI

gi|30030


gi|13603394


gi|119591510

Collagen, type XV

gi|3893879

Collagen alpha 1, type XVIII

gi|1082300

Decorin

gi|181519

Fatty acid-binding protein, adipocyte

gi|4557579

Fibrillin

gi|227918

Fibronectin

gi|225477

EGF-like, fibronectin type III and laminin G domains

gi|39645793

Heparan sulfate proteoglycan perlecan

gi|11602963

Intermediate filament protein

gi|28317

Laminin A3

gi|509806


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Laminin, alpha 4, isoform CRA_b

gi|119568652

Laminin alpha5 chain precursor

gi|20147503

Laminin B1

gi|186837

Laminin B2

gi|1103585

Lamin A/C (LMNA) protein

gi|21619981

Lumican, extracellular matrix protein

gi|642534

Nidogen

gi|189209

Osteoblast specific factor 2

gi|393317

Osteoglycin (Mimecan isoform 2 precursor)

gi|7661704

Proline-arginine-rich end leucine-rich repeat protein (Prolargin precursor)

gi|4506041

Vimentin

gi|340219

*All proteins identified in different gel slices of 1-D SDS-PAGE gel (Fig. S1). The peptide fragmentation data from tandem mass spectrometry (LC-MS/MS) were searched against the nonredundant NCBInr database using the MASCOT search software. The list includes all significant hits (score > 40).

Characteristics of sECM-MC Hydrogels. As previously described, sECM-MC solutions were prepared by simply blending sECM (6 wt%) and MC (6 wt%) solutions at low temperature.18 The sECM-MC solution was transparent and slightly viscous at 4 °C, while the solution rapidly formed a hydrogel within 1 min at 37 °C. The sol-gel transition was monitored by measuring the storage modulus (G′) and loss modulus (G″) as functions of temperature and time. At low temperatures, the sECM-MC solution displayed a viscous liquid state in which the G″ was predominant over G′. With increasing temperature, G′ increased and eventually exceeded G″, characteristic of the transition from a sol state to a gel state (Figure 1A). Depending on the temperature, the viscosity of sECM-MC increased significantly from 752 cp to 2,000 cp (Figure 1B). The stiffness of sECM-MC hydrogel was measured using an AFM with a contact mode silicon probe. The dynamic elastic modulus of hydrogel was approximately 3.8 kPa, indicating that the sECM-MC hydrogel has stiffness value similar to adipose tissue (Figure 1C).23


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Figure 1. (A) Sol-gel transition behaviors of sECM-MC hydrogels (sECM 6 wt% and MC 6 wt%). G': storage modulus (○) and G'': loss modulus (●). (B) Viscosity and (C) AFM-based force-distance curves of sECM-MC hydrogels.

In Vivo Availability of sECM-MC Hydrogels. The sECM-MC pre-gels were injected into the dorsal subcutaneous region of nude mice through a 28-gauge needle to assess in vivo injectability, biocompatibility, and adipose tissue formation. Figure 2 displays the representative macroscopic images of the sECM-MC hydrogels at 1, 2, and 3 weeks after injection. The sECMMC pre-gel solutions formed hydrogels immediately after injection. The hydrogels adhered to surrounding tissues and were easily identified with no any signs of infection throughout the experimental period of three weeks. Two weeks after injections, the grafts had a yellowish appearance and blood vessels were observed. Overall, sECM-MC hydrogels exhibited high injectability and biocompatibility with the surrounding tissues.


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Figure 2. Macroscopic appearance of grafts in nude mice. The sECM-MC hydrogels were individually injected into the back of each nude mouse. At 1, 2 and 3 weeks after subcutaneous injection, the mice were sacrificed. The black scale bars represent 5 mm.

Cell Infiltration into the sECM-MC Hydrogels. Histological and immunofluorescence staining were used to assess the host cell infiltration into the injected hydrogels (Figure 3). The results of H&E and DAPI staining showed that a large proportion of host cells were infiltrated and homogeneously distributed within hydrogels. The number of infiltrated host cells greatly increased up to 2 weeks post-injection and gradually decreased from 2 to 3 weeks (Figure 3). A large number of the infiltrated cells into the grafts were stained with CD29, which is a specific


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marker for adipose-derived stem cell or preadipocyte24 and CD29-positive cells significantly increased at 2 weeks post-injection. Interestingly, a large number of cells were stained with antiCD163, which is a marker for M2 adipose tissue macrophage (ATM). ATMs are classified into two major subtypes (M1 macrophages and M2 macrophages) and have a pivotal role in regulating adipose tissue function. Particularly, resident M2 ATMs induce anti-inflammation, tissue repair, and constructive tissue remodeling, depending on microenvironmental stimuli.25 At 1 week, CD163+ ATMs were sparsely detected, while at 2 and 3 weeks, a high density of CD163+ ATMs were observed in the entire region of the remodeled grafts. These results suggest that ECM proteins and adipokines of sECM-MC hydrogels could provide the proper signals for ATM recruitment26 and infiltrated ATMs could result in affirmative effect on the adipose tissue regeneration.


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Figure 3. Histological and immunofluorescence evaluation of the grafts at 1, 2 and 3 weeks after injection. For assessment of host cell infiltration, sections were stained with hematoxylin-eosin and DAPI. The infiltrated host cells were stained using CD29 (green) and CD163 (green) for mouse adipose-derived stem cells/preadipocytes and adipose tissue macrophages (ATMs), respectively. Sections were counterstained with DAPI, which stained nuclei blue. The scale bars represent 50 µm (black) and 100 µm (red).

Adipose Tissue Remodeling. The in vivo adipogenesis was analyzed by oil red O staining and by RT-PCR (Figure 4). Grafts showed adipogenesis with accumulated intracellular lipid


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droplets and the oil-red O positive area in grafts was increased significantly from week 2 to 3. At 3 weeks, well-organized lobule-like structures of adipose tissue were observed (Figure 4A). The expression of mouse adipogenic genes, PPARγ, adiponectin, aP2, and leptin, was evaluated by RT-PCR (Figure 4B). Fresh mouse adipose tissue was used as a positive control. The expression of the mouse gene (aP2) for adipogenesis was clearly observed in the grafts. Other adipogenic genes, PPARγ, adiponectin, and leptin, were also expressed. Von Willebrand Factor staining showed the microvessel formation at 3 weeks (Figure 5). A large number of microvessles and a few longitudinal microvessels were observed throughout the graft. The results suggest that angiogenic factors, such as VEGF and PDGF-BB, in sECM-MC hydrogel can contribute to angiogenesis by recruiting and activating host endothelial cells.


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Figure 4. (A) Histological evaluation of grafts, stained by Oil red O, at 1, 2 and 3 weeks after injection of sECM-MC hydrogels. The black scale bars represent 200 µm. (B) RT-PCR analysis of gene expression for adipogenic differentiation in the grafts sacrificed at 3 weeks. Mouse adipose tissue was used as a positive control. Each gene was analyzed using mouse-origin primers designed for peroxisome proliferative activated receptor gamma (PPARγ), adipocyte fatty acid-binding protein (aP2), adiponectin, and leptin. β-actin was used as an internal control. MAT: mouse adipose tissue, H: sECM-MC hydrogel.

Figure 5. Distribution and density of newly formed blood microvessels were assessed using a von Willebrand Factor (light brown) antibody for mouse endothelial cells on 3 weeks. The red dotted lines indicate interface between injected hydrogel and rat tissue. The arrows denote areas


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that show the formation of newly formed blood vessels. Sections were counterstained with hematoxylin, which stains the nuclei purple. Scale bars represent 200 µm.

DISCUSSION For the in situ regeneration of adipose tissue, we focused on tissue-specific niches provided by sECM-MC hydrogel, such as mechanical stiffness and biochemical cues that mimic microenvironment of native adipose tissue. The mechanical stiffness of hydrogel play a crucial role in cellular behaviors, including cell migration, proliferation, and stem cell differentiation through mechanotransduction pathway.27,28 A variety of hydrogel systems were used to investigate cellular behaviors on the elasticity of native tissues, including brain (~ 0.2 – 1 kPa), adipose tissue (2 – 4 kPa), muscle (~ 10 kPa), and osteoids (~ 30 – 45 kPa).23,27 For instance, MSCs cultured on hydrogels that mimic the stiffness of muscle, adipose or bone showed the expression of myogenic, adipogenic or osteogenic transcription factors, respectively, in the absence of specific exogenous growth factors.27,29 The endothelial cells cultured on soft hydrogels stimulated angiogenesis and network formation compared to stiff gels.30,31 The sECMMC pre-gel solution used in this study formed a soft gel with favorable stiffness (~ 4 kPa) for promoting adipogenic differentiation of stem cells. Although the in vivo stiffness of sECM-MC hydrogel is highly dynamic due to various biological processes, such as cell infiltration and ECM degradation, it is clear that the their stiffness property influences the cell-matrix adhesion, focal adhesions, and cellular tension, resulting in change of cellular phenotype towards adipocyte of host ASCs.32-34 From a biochemical point of view, the adipose-derived sECM contains various proteins, such as collagens type I-VI, elastin, fibronectin, basement membrane proteins (Table 1 and


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Supporting Information Table S1), and bioactive molecules.22 These ECM proteins are crucial for maintaining the structural integrity of adipocytes and plays a pivotal role in adipose tissue development and expansion.35 For examples, laminins are present in the basement membrane surrounding mature adipocytes and upregulated during adipogenesis. The α4 chain of laminin is known to be involved in adipogenesis of preadipocytes and lipogenesis of adipocyte.36,37 Another major ECM protein in adipose tissue is collagen VI. Collagen type VI forms microfibrils in the interface between the basement membrane and thick bundles of collagen type I. Collagen VI regulates the adipose tissue expansion and insulin sensitivity through their interactions with multiple ECM proteins.38,39 Adipocyte fatty acid-binding protein (aP2) contained in soluble ECM involves systemic glucose and lipid metabolisms.40 In addition to ECM proteins, bioactive molecules present within the soluble ECM are considered as important cues for recruitment and activation of host ASCs, ATMs, or endothelial cells that are primarily responsible for adipose tissue remodeling.18,22,41,42 Among bioactive molecules, for example, SCF, HGF, VEGF, IGF-1, and PDGF-AB are known as stem cell-activating factors that induce the recruitment of host ASCs from the neighboring adipose tissue into reconstructed site and their adipogenesis.43-45 Several endothelial progenitor cell (EPC)-activating factors, such as VEGF, SCF, and monocyte chemoattractant protein (MCP)-1/-3, and stromal cell-derived factor-1 (SDF-1), are also involved in the recruitment of EPCs from the bone marrow or peripheral circulation and neovascularization process.46 Anti-inflammatory cytokines, such as TGF-β and interleukin-10 (IL-10), are known to regulate migration and infiltration of resident M2 ATMs, which provide critical signals that support adipogenesis in adipose tissue remodeling.47,48 Although the mechanisms of ECM-mediated tissue remodeling are not fully understood, recent studies have proved that intact ECM scaffolds release chemoattractant molecules for recruiting endogenous


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cells through their in vivo degradation process.26,49,50 Likewise, we suggest that the ECM fragments and bioactive molecules released from adipose-ECM hydrogel could contribute to a host cell recruitment and differentiation into tissue-specific cells. Overall, the mechanical and biochemical cues of sECM-MC hydrogels would act synergistically in adipose tissue remodeling. We propose that the adipose tissue-specific sECM-MC hydrogel will be useful as a cell-free therapeutic system for in situ adipose tissue regeneration.

CONCLUSIONS The sECM-MC hydrogels were used as an injectable cell-free system for adipose tissue regeneration. The sECM-MC hydrogels showed thermosensitve sol-gel phase transition and rapidly forming a soft hydrogel after subcutaneous injection in mice. In vivo study using nude mice showed that the sECM-MC hydrogels provide appropriate mechanical and biochemical niches that induce infiltration and differentiation of host cells, eventually resulting in the formation of new adipose tissue. Our findings suggest that sECM-MC hydrogel is a cell-free therapeutic system actively participating in recruitment of host endogenous cells for adipose tissue regeneration.

ASSOCIATED CONTENT Supporting Information Protein contents of soluble ECM analyzed by LC-MS/MS are available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Tel. +82 (0)31 400 5279, Fax. +82 (0)303 3475 4712 Author Contributions #

These authors contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This research was supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea) under Industrial Technology Innovation Program (Grant No.10062127). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT & Future Planning (NRF 20100027955 and NRF-2016R1C1B1006882).

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