Article pubs.acs.org/Biomac
Injectable and Thermosensitive Soluble Extracellular Matrix and Methylcellulose Hydrogels for Stem Cell Delivery in Skin Wounds Eun Ji Kim, Ji Suk Choi, Jun Sung Kim, Young Chan Choi, and Yong Woo Cho* Department of Chemical Engineering, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea S Supporting Information *
ABSTRACT: Extracellular matrix (ECM) provides structural support and biochemical cues for tissue development and regeneration. Here we report a thermosensitive hydrogel composed of soluble ECM (sECM) and methylcellulose (MC) for injectable stem cell delivery. The sECM was prepared by denaturing solid ECM extracted from human adipose tissue and then blended with a MC solution. At low temperatures, the sECM-MC solution displayed a viscous solution state in which the loss modulus (G″) was predominant over the storage modulus (G′). With increasing temperature, G′ increased dramatically and eventually exceeded G″ around 34 °C, characteristic of the transition from a liquid-like state to an elastic gel-like state. After a single injection of the stem cellembedded hydrogel in full thickness cutaneous wound, the wound healed rapidly through re-epithelialization and neovascularization with minimum scar formation. The overall results suggest that in-situ-forming sECM-MC hydrogels are a promising injectable vehicle for stem cell delivery and tissue regeneration.
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INTRODUCTION
D ultrastructure of the tissue, mechanical integrity, protein composition, and biologic activity.9,23−25 In this study, we report on the development of in-situforming thermosensitive hydrogels based on human adiposederived soluble ECM (sECM) and methylcellulose (MC) for delivering stem cells in skin wounds. Human adipose tissue has been used as a rich source of ECM because it can be harvested in large quantities with minimal morbidity and contains abundant ECM proteins and soluble growth factors that are important for tissue development and regeneration.26,27 The human adipose-derived ECMs were solubilized using urea and guanidine buffer and then the extracted sECM was blended with MC to improve mechanical properties of the thermosensitive hydrogels. The sECM-MC hydrogels were characterized in terms of sECM content, rheological properties, and ability to embed stem cells. The stem cell delivery and woundhealing efficacy of the hydrogels were also evaluated using a full-thickness wound model.
In-situ-forming hydrogels that undergo sol−gel transitions in response to temperature are attractive as injectable systems for tissue therapies because of easy incorporation of cells or therapeutic agents, simplicity of implantation by injection, longterm retention of delivered cells, and improvement of cell viability by providing a three-dimensional (3-D) microenvironment for delivered cells.1−4 Although many synthetic and natural biomaterials have been used for developing in-situforming hydrogels that mimic the chemical and physical cues of the native extracellular matrix (ECM), the currently developed biomaterials are unsatisfactory in that they do not reflect the intricate functions of natural ECM in living tissues.5−7 Recently, intact ECMs derived from living tissues have emerged as a major biomaterial in regenerative medicine.8−15 The composition and spatial structure of ECMs play key roles in guiding cell migration, stimulating cell proliferation and differentiation, and modulating cellular responses.16 For this reason, many researchers have designed different processes to isolate intact ECMs from tissues and have fabricated various 3D tissue engineering scaffolds for use in regenerative medicine, particularly for patients requiring soft tissue regeneration.11,17−22 More recently, pH- or temperature-sensitive hydrogels based on ECM proteins extracted from adipose, dermis, liver, myocardium, and urinary bladder tissues have been developed as injectable scaffolds for cell delivery and tissue regeneration. The ECM hydrogels are promising in providing an artificial cell niche for controlling cellular behavior and tissue regeneration because of the well-preserved original 3© XXXX American Chemical Society
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MATERIALS AND METHODS
Materials. All chemical reagents were purchased from SigmaAldrich (St. Louis, MO, U.S.A.). Protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN, U.S.A.). Dialysis tubing (molecular weight cutoff (MWCO) 6000−8000) was obtained from Fisher Scientific (Pittsburgh, PA, U.S.A.). Amicon Ultra-15 centrifugal filter devices were supplied by Millipore (Billerica, MA, U.S.A.). Bicinchoninic acid (BCA) assay kits were purchased from Pierce (Rockford, IL, U.S.A.). Sircol acid/pepsin-soluble Received: May 6, 2015
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DOI: 10.1021/acs.biomac.5b01566 Biomacromolecules XXXX, XXX, XXX−XXX
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at 90 °C for 60 min. Then, the solution was allowed to equilibrate overnight at 4 °C. Subsequently, sECM and MC solutions were mixed at a volume ratio of 1:4 to the final concentrations of 6 wt % sECM and 6 wt % MC, and the mixture was homogeneously stirred at 4 °C. The sECM-MC solution was kept at 4 °C until use. Scanning Electron Microscopy. The microstructure of sECMMC hydrogels was observed using scanning electron microscopy (SEM; VEGA-II SBH, TESCAN, Brno, Czech Republic). The sECMMC hydrogels were frozen using liquid nitrogen, and freeze-dried. Then, these hydrogels were fixed to metal stubs and coated with platinum by sputtering at an accelerating voltage of 15 kV. Rheological Analysis. The rheological properties of the sECMMC hydrogels were investigated with a rheometer (Bohlin Gemini HR Nano, Malvern, U.K.) operating with 40 mm parallel plate geometry. The sECM-MC pregel solutions were immediately loaded onto a rheometer plate that was precooled to 10 °C. After loading, the shear viscosity was measured at a frequency of 1 Hz. The temperature was then increased to 45 °C at a rate of 3 °C per minute to induce gelation. Preparation of hASCs. The human adipose tissue-derived stem cells (hASCs) were isolated according to a previously described protocol.18 Briefly, the adipose tissue was washed with PBS containing 5% penicillin/streptomycin (P/S). After removing red blood cells, the adipose tissue was digested in PBS supplemented with 0.01 wt % collagenase type II for 1 h at 37 °C. The digested tissue was filtered through a 100 μm mesh to remove aggregated tissue and debris. The filtered suspension was centrifuged at 200 ×g for 7 min, and the resulting stromal vascular fraction (SVF) pellet was washed several times in PBS. SVF cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% P/S at 37 °C under 5% CO2. In Vitro Cell Study. The lyophilized sECM and MC powders were sterilized by ethylene oxide gas. A 50 μL aliquot of sECM-MC hydrogel containing hASCs (105 cells/mL) was placed into each well of 96-well plates and incubated at 37 °C under 5% CO2 for 3 days. Rat tail type I collagen hydrogel (3 mg/mL, Corning, NY, U.S.A.) was used as a control. The cell viability was analyzed using the Premix WST-1 Cell Proliferation Assay System and Live/Dead Viability/ Cytotoxicity kit. The Premix WST-1 reagent was added to each well, and the plates were incubated for 3 h at 37 °C. The absorbance was measured at 440 nm using a microplate reader (PowerWave XS, BioTek Instruments, Winooski, VT). The hydrogels containing hASCs were stained for 1 h with the combined Live/Dead reagent at 37 °C and observed using a fluorescence microscope (IX81; Olympus, Tokyo, Japan). In Vivo Wound Healing Study. The stem cell delivery and wound-healing efficacy of the sECM-MC hydrogels were evaluated using a full-thickness cutaneous wound model. All experiments were performed with approval from the Institutional Review Board of Hanyang University. The rat dorsal area (female, Sprague−Dawley, weighting 80−120 g, HanaBio, Hwaseong-si, Korea) was completely depilated under general anesthesia, and a full-thickness circle wound (about 169 mm2 in area) was created in the upper back area of each rat. The lyophilized sECM and MC powders were sterilized by ethylene oxide gas. A 200 μL aliquot of the sECM-MC pregel solutions with or without hASCs (105 cells/mL) were injected once into the wound bed using a syringe. The wound was then wrapped with a plastic mold for protection. At appropriate times, the rats from each group were sacrificed, and the skins, including the wounds were harvested for histological examination. For macroscopic evaluation of wound healing, the wound area was examined at 1, 2, and 3 weeks after treatment. The cutaneous wounds of the five animals were photographed, and the unhealed wound area was measured based on the image produced by the Imaging Analyzer (Bio-Rad Laboratories, Hercules, CA, U.S.A.). For histological and immunofluorescence examinations, specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced at a 6−10 μm thickness using a microtome. The sections were deparaffinized, dehydrated through a series of graded ethanol, and stained with Gomori’s trichrome. The hASCs embedded in the injected hydrogel were confirmed via immunofluorescence staining for human stem cell
collagen, Blyscan sulfated GAG, and Fastin elastin assay kits were supplied by Biocolor (Carrickfergus, Northern Ireland). QuantiMatrix human laminin and fibronectin ELISA kits were supplied by KOMA BIOTECH (Seoul, Korea). Premix WST-1 Cell Proliferation Assay System were purchased from Takara (Shiga, Japan). Live/Dead Viability/Cytotoxicity kits were obtained from Molecular Probes (Eugene, OR, U.S.A.). 4,6-Diamidino-2-phenylindole (DAPI, Thermo Scientific, Rockford, IL, U.S.A.) was used to identify nuclear components. Fluorochrome-conjugated anti-human CD29 and antihuman CD44 were purchased from BioLegend (San Diego, CA, U.S.A.). Mouse anti-rat PECAM-1 and fluorescein-conjugated bovine anti-mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Cell culture media, serum, and antibiotics were purchased from Life Technologies (Carlsbad, CA, U.S.A.). All other chemicals were reagent grade and used as received. Extraction of sECM from Human Adipose Tissue. Solid ECM was prepared from human adipose tissue, as described previously.15 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 was washed several times with distilled water to remove blood components. Distilled water was added to the adipose tissue and the tissue/water (1:1) mixture was homogenized for 3 min using a commercial blender. The tissue suspension was centrifuged at 16500 ×g for 5 min at 4 °C to remove oil components. The viscous suspension was washed with 3.4 M NaCl and centrifuged at 20000 ×g for 30 min at 4 °C. For sECM preparation, solid ECM was suspended in 4 M urea buffer containing protease inhibitor cocktail for 12 h at 4 °C. The mixture was centrifuged at 20000 ×g for 60 min at 4 °C to remove insoluble materials. The residues were re-extracted in 4 M guanidine containing protease inhibitor cocktail for 12 h at 4 °C, and were centrifuged. The supernatant was filtered through a 40 μm mesh and dialyzed extensively in dialysis tubing against 30 volumes of Tris-buffered saline (TBS) for 24 h at 4 °C. The dialysate was centrifuged at 20000 ×g for 60 min at 4 °C, and was filtered through a 40 μm mesh. The supernatant was concentrated using a 3000 MWCO Amicon Ultra-15 centrifugal filter device, and the final sECM was lyophilized. The concentration of purified sECM was measured using a BCA assay. sECM Protein Content. Protein content in the sECM was quantified using Sircol acid/pepsin-soluble collagen, Blyscan sulfated glycosaminoglycan (GAG), and Fastin elastin assay kits according to the manufacturer’s protocols. For quantification of acid/pepsin-soluble collagen, sECM was incubated with 1 mL Sircol dye reagent for 30 min at room temperature. For sulfated GAG, sECM was mixed with 1 mL Blyscan dye, and the precipitate was collected via centrifugation. For elastin, sECM was mixed with 1 mL Fastin dye. Collagen type I (rat tail), chondroitin 4-sulfate (bovine trachea), and α-elastin (bovine neck) were used as standards for the biochemical assays. Relative absorbance was measured in 96-well plates using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, U.S.A.). Laminin and fibronectin in sECM were quantified via enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (KOMA BIOTECH, Inc., Seoul, Korea). Optical density was measured at 560 nm using a microplate spectrophotometer (PowerWave XS, BioTek Instruments, Winooski, VT, U.S.A.). Growth Factor Antibody Array. Bioactive molecules in the sECM were analyzed using a growth factor antibody array kit (RayBiotech Inc., Norcross, GA, U.S.A.) according to the manufacturer’s protocol. The array glass chip containing 41 different human cytokine antibodies was blocked and incubated with diluted sECM. The glass chip was washed and subsequently treated in biotinconjugated antibodies. After incubation with fluorescent dyeconjugated streptavidin, cytokine signals were detected using a laser scanner (Axon Instruments, Union, CA, U.S.A.) using the Cy3 channel. Signal intensities were quantified with GenePix Pro software. Preparation of sECM-MC Hydrogels. The 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. MC solution (7.5 wt %) was prepared by a dispersion technique. Briefly, 0.75 g of MC (viscosity of 15 cP) was thoroughly wetted in 10 mL PBS and heated B
DOI: 10.1021/acs.biomac.5b01566 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules markers. The sections were incubated with a 10 wt % BSA blocking agent for 30 min to inhibit nonspecific binding of IgG. Sections were incubated with fluorochrome-conjugated anti-human CD29 and antihuman CD44. To visualize vascularization in injected hydrogels, mouse anti-rat PECAM-1 and fluorescein-conjugated bovine antimouse IgG were used. All sections were counterstained with 1 μg/mL DAPI for 1 min and observed with a fluorescence microscope (Olympus, Tokyo, Japan). Statistical Analysis. Experimental data are expressed as the mean ± standard deviation (SD). The Student’s two-tailed t-test with SPSS 17.0 statistical software (SPSS, Chicago, IL, U.S.A.) was used for comparison, and statistical significance was accepted at p < 0.05.
Table 1. Profiling of Growth Factors in sECM Derived from Human Adipose Tissue Using Growth Factor Antibody Assaysa growth factor HGF TGF-β1 TGF-β3 EGF EGFR HB-EGF
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RESULTS AND DISCUSSION Characterization of Human Adipose Tissue-Derived sECM. For preparing sECM, solid ECMs isolated from human adipose tissue were solubilized using urea and guanidine buffer.28 The average final yield was approximately 45 ± 0.8 mg/g of wet solid ECM. The ECM is a key component of the cell niche and provides structural and biochemical cues that are required for tissue development and regeneration.29 Therefore, preservation of the proteins and growth factors during the extraction of sECM from tissue is highly desirable for retaining the biological and functional effects of the ECM. The sECM contained large amounts of soluble elastin (16.463 ± 1.440 mg/mL), acid/pepsin-soluble collagen (4.572 ± 0.136 mg/ mL), laminin (1.038 ± 0.064 mg/mL), and small amounts of fibronectin (0.144 ± 0.054 mg/mL) and sulfated GAG (0.021 ± 0.004 mg/mL). Endogenous growth factors in the sECM were detected using different human growth factor antibody arrays. Among the 41 growth factors, 25 growth factors were significantly detected in the prepared sECM (Table 1). Notably, hepatocyte growth factor (HGF), platelet-derived growth factor-BB (PDGF-BB), endothelial growth factor (EGF), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), which are all involved in the regulation of wound healing and angiogenesis, were highly detected in the sECM. Rheological Analysis of sECM-MC Hydrogels. Thermosensitive sECM-MC solutions were successfully prepared by blending sECM and MC solutions at a final concentration of 6 wt %, respectively. The sECM-MC solution was freely flowing and slightly viscous at 4 °C, while the solution became opaque and rapidly formed a stable and highly porous hydrogel within 3 min at 37 °C (Figure 1A and Supporting Information, Figure S1). The sol−gel transition was monitored by measuring the storage modulus (G′) and loss modulus (G″) as a function of temperature (Figure 1B−E). At low temperatures, the sECMMC solution displayed a viscous solution state in which the G″ was predominant over G′. With increasing temperature, G′ increased dramatically and eventually exceeded G″, characteristic of the transition from a liquid-like state to an elastic gel-like state. In particular, the sECM-MC solutions showed different gelation temperatures and gel strengths, depending on the concentration of sECM. The gelation temperature (crossover point of G′ and G″) decreased to 44.1, 43.0, 38.0, and 33.9 °C with increasing sECM concentrations at 0, 1, 3, and 6 wt %, respectively. Among these hydrogels, the sECM-MC hydrogel containing 6 wt % sECM revealed the highest G′ value with increasing temperature, showing a typical elastic gel-like behavior. The G′ value of the 6 wt % sECM-MC hydrogel also increased with an increase in frequency from 0.01 to 10 Hz at 37 °C after incubation for 10 min at 37 °C, and the G′ was substantially larger than the G″ over the entire tested frequency
PDGFAA, AB, BB PDGF Rβ VEGF VEGF R2, R3 IGF1 IGF1R IGFBP2 IGF2 CSF1 CSF1R CSF3 SCFR PLGF AREG NT-3
functions Regulation of cell growth, cell motility, and morphogenesis Control of cell proliferation, differentiation, adhesion, and migration Control of cell differentiation and embryogenesis Stimulation of cell growth, proliferation, and differentiation Receptor for EGF Mediation of cell adhesion, migration, and cell cycle progression; Predominant growth factor for epithelialization of skin wound Regulation of cell growth, division, and angiogenesis Receptor for PDGF Regulation of angiogenesis and lymphangiogenesis Receptor for VEGF Promotion of cell proliferation, the inhibition of cell apoptosis, and regulation of neural development Receptor for IGF1 A carrier protein for IGF-1 Organ development and function in fetal stage Stimulation of neutrophil survival, proliferation, differentiation, and function Receptor for CSF1 Stimulation of neutrophil survival, proliferation, differentiation, and function Receptor for SCF A key molecule in angiogenesis and vasculogenesis during embryogenesis Regulation of mitogen of astrocytes, Schwann cells, fibroblast and T-cells Encouragement of neuron growth and differentiation
a
HGF, hepatocyte growth factor; TGF-β, transforming growth factorβ; EGF, epidermal growth factor; EGF-R, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; PDGF-AA, AB, BB, plateletderived growth factor subunit A, B; PDGF-Rβ, platelet-derived growth factor-receptor β-polypeptide; VEGF, vascular endothelial growth factor; VEGF R2, R3, VEGF receptor; IGF 1, insulin-like growth factor 1; IGF1R, IGF-1 receptor; IGFBP2, IGF binding proteins-2; IGF2, insulin-like growth factor 2; CSF1, colony stimulating factor 1; CSF1R, CSF 1 receptor; CSF3, colony stimulating factor 3; PLGF, placenta growth factor; AREG, amphiregulin; NT, neurotrophin-3.
range, suggesting that the hydrogel becomes more stable with an increase in physical cross-links (Supporting Information, Figures S1 and S2).30 Physical cross-linking has been proposed as a valuable cross-linking methodology for in-situ-forming injectable hydrogels because it is capable of producing a hydrogel without the need for chemical modification or the addition of cross-linking agents.31 MC is a typical example that undergoes a sol−gel transition via physical cross-linking. MC forms a hydrogel by the dominant hydrophobic interaction between chain segments containing methoxy substitutions as the temperature increases.32 However, MC itself is not sufficient for use as an in-situ-fast gelling system due to high gelation temperatures (40−60 °C). The gelation temperature of MC can be lowered by the addition of salting-out salts such as NaCl, NaBr, KF, (NH4)2SO4, and Na2CO3, which bind water molecules tightly.33 In the presence of salting-out salts, hydrophobic segments in the MC chains are more easily excluded from the water-solvation shells, thereby promoting the hydrophobic interactions of MC chains as the temperature C
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Figure 1. (A) Sol−gel transition behavior of sECM-MC hydrogel (sECM 6 wt % and MC 6 wt %). (B−D) G′ and G″ of sECM-MC hydrogels with different sECM concentrations as a function of temperature: (B) 0 wt %, (C) 1 wt %, (D) 3 wt %, and (E) 6 wt % with MC 6 wt %; G′, storage modulus (●); G″, loss modulus (○).
increases.34 The anionic groups of sECM proteins seem to facilitate the hydrophobic interactions of MC chains, similar to the action of salting-out salts, resulting in a lower gelation temperature.35 The concentration of the final ECM is an important determinant of its rheological properties; the gelation temperature decreased with increasing sECM concentration. The appropriate gelation temperature for in-situ-gelling and fast sol−gel phase transition of the sECM-MC hydrogel is beneficial for embedding cells within the hydrogel and the delivery system as a whole compared with other intact ECM hydrogels. Cytocompatibility of the sECM-MC Hydrogel. To assess the cytocompatibility of the sECM-MC hydrogels, hASCs were embedded in the hydrogel (sECM 6 wt % and MC 6 wt %) and incubated for 3 days. The hASCs embedded in collagen hydrogels were used as controls. The proliferation of hASCs in the hydrogels was analyzed using WST-1 assay after 1 and 3 days, which is based on the reduction of a stable tetrazolium salt to a soluble violet formazan product by viable cells (Figure 2A). The live/dead assay was used to determine the distribution of live and dead cells after 3 days in the hydrogels (Figure 2B−D). Calcein AM, which fluoresces upon the reaction of intracellular esterase, stains live cells (green), while ethidium homodimer-1, which binds to the DNA of dead membrane compromised cells, stains dead cells (red). On day 3, the majority of cells expressed green fluorescence, and dead
cells were rarely detected. The hASCs proliferated with spindlelike morphology on the 2-D control cultures, as expected, while the cells embedded in the hydrogels showed spherical morphology over the culture period. It is a noted fact that 3D hydrogels can maintain spherical cell morphology by providing in vivo-like conditions, thereby having a significant influence on cellular functions.36,37 Our results suggest that the sECM-MC hydrogels provided a cell niche that maintained cell morphology and survival and did not induce significant cytotoxicity. In Vivo hASC Delivery Efficacy. The main issues with transplanted stem cells is that they may display limited survival, low retention rate, or immune rejection in injured tissues, reducing their therapeutic efficacy in vivo.38,39 One of the promising approaches to overcoming these problems is to use injectable hydrogels. The hydrogel can provide a favorable stem cell niche that promotes stem cell retention and survival and also act as a barrier to protect the stem cells against attack by the host immune response.40,41 To study the ability of sECMMC hydrogels to deliver stem cells, the sECM-MC solution containing hASCs was subcutaneously injected into fullthickness wounds of rats through a 23-gauge needle (Figure 3A,B and Supporting Information, Figure S3). The injected hydrogel rapidly formed a transparent hydrogel at the wound site. On day 1 after injection, the cell distribution was evaluated D
DOI: 10.1021/acs.biomac.5b01566 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 2. Proliferation of hASCs embedded in sECM-MC hydrogel and collagen hydrogel for 3 days (A). Data were normalized by the absorbance of hASC embedded in collagen hydrogel on day 1. Fluorescence micrographs of hASCs cultured on a tissue culture plate (B), embedded in sECMMC hydrogel (C), and collagen hydrogel (D) on day 3. Scale bars represent 200 μm.
healing process.42 Many groups have reported that the stem cell fate could be determined by biochemical cues, such as soluble factors and ECM molecules.43 We postulated that the sECMMC hydrogels containing inherent soluble factors and proteins could provide the proper stem cell niche for cell retention, survival, and differentiation when compared with other hydrogels (e.g., synthetic polymers, recombinant protein polymers, hyaluronic acid, alginate, and agarose).44−47 The in vivo results suggest that sECM-MC hydrogels will be valuable not only in enhancing stem cell engraftment, but also in promoting the recruitment of host cells that can be guided to regenerate damaged tissues. Effect of hASC-Embedded sECM-MC Hydrogels on Wound Healing. Photographs of wounds were taken to observe changes in wound size, and histological staining was used to assess the wound healing processes on days 7, 14, and 21 (Figure 4 and 5). There was no sign of inflammation or infection in both the hydrogel and hASC-embedded hydrogel groups. Wound sizes were reduced over a period of time in all groups. On day 7, the hASC-embedded hydrogel group wound size (37.6 ± 5.9%) was significantly reduced compared with the control (65.9 ± 4.4%) and hydrogel group (57.4 ± 4.5%). The wounds treated with hASC-embedded hydrogels were covered with a continuous epidermis, and the regenerated dermis was much thicker than that of the hydrogel only. A large number of epidermal appendages were observed in the regenerated skin treated with the hASC-embedded hydrogel (Supporting Information, Figure S6). On day 14, the control group wounds were partially covered with epidermis, while those of the hydrogel and hASC-embedded hydrogel groups were fully covered with a continuous epidermis. In the wounds treated with hASC-embedded hydrogels, the microvessel density was much higher than those of the control and hydrogel only (Figure 6). However, no significant differences were observed in all groups on day 21.
Figure 3. sECM-MC solution containing hASCs was subcutaneously injected into a full-thickness wound of a rat through a 23-gauge needle (A,B). The hASCs were stained using CD29 (red) and CD44 (green) for human stem cells on day 1 after injection. Sections were counterstained with DAPI, which stains nuclei blue (C,D). H, hydrogel; I, interface between hydrogel and rat tissue; and T, rat tissue. Scale bars represent 200 μm.
by immunofluorescence staining. A large number of stem cells were stained with anti-CD29 and anti-CD44, which are specific markers for human mesenchymal stem cells. The stem cells were homogeneously distributed and integrated well within the hydrogel (Figure 3C,D). Interestingly, the results of DAPI staining showed a large proportion of host cells infiltrated within the injected hydrogels. Although some types of infiltrated host cells are involved in inflammatory responses (e.g., macrophages), the recruitment of various host cells into the hydrogel may result in affirmative effects on the wound E
DOI: 10.1021/acs.biomac.5b01566 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 6. Distribution and density of newly formed blood microvessels were assessed using a CD31 (green) antibody for rat endothelial cells on days 14 (A,C,E) and 21 (B,D,F). Arrows denote areas that show the formation of newly formed blood vessels. Sections were counterstained with DAPI, which stains the nuclei blue. Scale bars represent 200 μm.
combination of chemical, biological and mechanical processes in vivo.48−50 Märtson et al.48 reported the degradation of cellulose sponges in the rat connective tissue. Miyamoto et al.49 also reported that cellulose with low crystallinity was more easily absorbed than that with high crystallinity. MC is a watersoluble cellulose derivative with low crystallinity and slowly degraded by biological, chemical, and physical processes in vivo.50 Niche signals from degraded sECM-MC hydrogels, such as collagen, laminin, fibronectin, elastin, and GAG, are known to regulate cell−cell or cell−matrix interactions and induce secretion of soluble signals from stem cells, stimulating differentiation of stem cells as well as modulating various cellular responses.51 In addition to providing niche signals, ECM components are integral to each phase of the wound healing process from hemostasis to remodeling by interacting with cells and growth factors in a dynamic.52 For example, ECM components (e.g., collagen type I, III, IV, laminin,
Figure 4. Progression in healing of full-thickness cutaneous wounds injected with hASCs-embedded sECM-MC hydrogel, sECM-MC hydrogel alone, and control groups on days 0, 7, 14, and 21. (A) Wounds were photographed on days 0, 7, 14, and 21. (B) The wound area is expressed as a percentage of the initial wound area on day 0. Data are shown as the mean ± standard deviation (n = 3), with significance at *p < 0.05.
It is notable that the wounds covered with hASC-embedded hydrogel healed rapidly through re-epithelialization with minimal scar formation. We suggest that ECM fragments released via degradation of the sECM-MC hydrogel could play a significant role by releasing the raw materials necessary for the generation of new matrix in wounds. Several studies have shown that cellulose or cellulose derivatives are degraded by a
Figure 5. Histological evaluation of wound sections injected with hASC-embedded sECM-MC hydrogel, sECM-MC hydrogel alone, and control groups (n = 5) after dermal excision on days 7, 14, and 21. Black arrows indicate the wound edges. Insets are magnified images of the indicated rectangles and represent the regeneration of the outer layer of the skin. Muscle, keratin, and cytoplasm, red; collagen, green; and nuclei, black. Scale bars represent 200 μm. F
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fibronectin, and GAGs) and various growth factors [e.g., PDGF, TGF, EGF, fibroblast growth factor (FGF), keratinocyte growth factor (KGF), IGF-1, and HGF] recruit host stem cells, macrophages, fibroblasts, and endothelial cells into the wound site, modulating epithelialization, collagen accumulation, and angiogenesis.29,53,54 Paracrine signals secreted from hASCs (bFGF, TGF-β, VEGF)55−57 and endogenous growth factors (HGF, PDGF-BB, EGF, IGF, and VEGF) in the sECM-MC hydrogel seemed to have a synergic effect on improvement of the quality of wound healing by orchestrating events during granulation tissue formation, re-epithelialization, and angiogenesis. However, abnormal ECM production by fibroblasts and excessive contractile force mediated by myofibroblasts during the proliferation phase can result in scarring, which can lead to significant functional and cosmetic morbidity.52,58 During the proliferation and remodeling phases, some growth factors are known to play an important role in scar formation. HGF controls the equilibrium between synthesis and degradation of ECMs by stimulating the secretion of matrix metalloproteinase (MMP) in fibroblasts.59 bFGF is also known to reduce wound contraction by inhibiting the phenotypic change of fibroblasts to myofibroblasts, which is likely to be involved in contraction.60 As mentioned above, sECM-MC hydrogels degraded within the wound and the degraded ECM fragments can be used to reconstruct granulation tissue instead of fibroblasts, resulting in the inhibition of excessive ECM production. In addition, soluble signals (e.g., bFGF) secreted by embedded hASCs and endogenous growth factors (e.g., HGF) released from the sECM could contribute to a reduction in wound contraction by inhibiting the phenotypic change of fibroblasts to myofibroblasts. Based on these results, the sECMMC hydrogel is a promising injectable system that provides the therapeutic niche necessary to improve the quality of wound healing in addition to delivering therapeutic cells to the wound site.
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel.: +82 (0)31 400 5279. Fax: +82 (0)303 3475 4712. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Seoul R&BD Program (Grant No. SS110011), the Basic Research Program (Grant No. 2012008294), and the Bio & Medical Technology Development Program (Grant No. 2011-0019774) through the National Research Foundation of Korea (NRF) funded by the Korean government (MEST).
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REFERENCES
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CONCLUSIONS Injectable, thermosensitive sECM-MC hydrogels were successfully prepared by a simple blending of human adipose-derived sECM and MC solutions at low temperatures. The sECM-MC hydrogels showed a thermosensitive sol−gel phase transition and rapidly formed a hydrogel at body temperature. In vivo study using a full-thickness wound model showed that the sECM-MC hydrogels provide not only a cell niche that improves engraftment and survival of stem cells delivered to the wound, but also biological cues that accelerate wound regeneration. Our findings suggest that in-situ-forming sECMMC hydrogels are a promising injectable vehicle for stem cell delivery and soft tissue engineering.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01566. Microstructure, dynamic frequency sweep and storage and loss modulus plots of sECM-MC hydrogels with different sECM concentrations and additional in vivo images (PDF). G
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Biomacromolecules (24) Young, D. A.; Ibrahim, D. O.; Hu, D.; Christman, K. L. Acta Biomater. 2011, 7, 1040−1049. (25) Freytes, D. O.; Martin, J.; Velankar, S. S.; Lee, A. S.; Badylak, S. F. Biomaterials 2008, 29, 1630−1637. (26) Choi, J. S.; Choi, Y. C.; Kim, J. D.; Kim, E. J.; Lee, H. Y.; Kwon, I. C.; Cho, Y. W. Macromol. Res. 2014, 22, 932−947. (27) Trayhurn, P. Acta Physiol. Scand. 2005, 184, 285−293. (28) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 5142−5147. (29) Hubmacher, D.; Apte, S. S. Curr. Opin. Rheumatol. 2013, 25, 65−70. (30) Jeong, K. J.; Panitch, A. Biomacromolecules 2009, 10, 1090− 1099. (31) Yu, L.; Ding, J. Chem. Soc. Rev. 2008, 37, 1473−1481. (32) Li, L.; Thangamathesvaran, P. M.; Yue, C. Y.; Tam, K. C.; Hu, X.; Lam, Y. C. Langmuir 2001, 17, 8062−8068. (33) Cho, Y. W.; An, S. W.; Song, S. C. Macromol. Chem. Phys. 2006, 207, 412−418. (34) Xu, Y.; Wang, C.; Tam, K. C.; Li, L. Langmuir 2004, 20, 646− 652. (35) Gupta, D.; Tator, C. H.; Shoichet, M. S. Biomaterials 2006, 27, 2370−2379. (36) Meng, X.; Leslie, P.; Zhang, Y.; Dong, J. SpringerPlus 2014, 3, 80−87. (37) Baker, B. M.; Chen, C. S. J. Cell Sci. 2012, 125, 3015−3024. (38) Wei, X.; Yang, X.; Han, Z. P.; Qu, F. F.; Shao, L.; Shi, Y. F. Acta Pharmacol. Sin. 2013, 34, 747−754. (39) Phinney, D. G.; Prockop, D. J. Stem Cells 2007, 25, 2896−2902. (40) Seliktar, D. Science 2012, 336, 1124−1128. (41) Sart, S.; Ma, T.; Li, Y. BioRes. Open Access 2014, 3, 137−149. (42) Ko, I. K.; Lee, S. J.; Atala, A.; Yoo, J. J. Exp. Mol. Med. 2013, 45, e57. (43) Choi, Y. C.; Choi, J. S.; Woo, C. H.; Cho, Y. W. J. Controlled Release 2014, 193, 42−50. (44) Cai, L.; Dewi, R. E.; Heilshorn, S. C. Adv. Funct. Mater. 2015, 25, 1344−1351. (45) Parisi-Amon, A.; Mulyasasmita, W.; Chung, C.; Heilshorn, S. C. Adv. Healthcare Mater. 2013, 2, 428−432. (46) Kim, Y.-M.; Oh, S. H.; Choi, J.-S.; Lee, S.; Ra, J. C.; Lee, J. H.; Lim, J.-Y. Laryngoscope 2014, 124, E64−E72. (47) Mayfield, A. E.; Tilokee, E. L.; Latham, N.; McNeill, B.; Lam, B.K.; Ruel, M.; Suuronen, E. J.; Courtman, D. W.; Stewart, D. J.; Davis, D. R. Biomaterials 2014, 35, 133−142. (48) Märtson, M.; Viljanto, J.; Hurme, T.; Laippala, P.; Saukko, P. Biomaterials 1999, 20, 1989−1995. (49) Miyamoto, T.; Takahashi, S.-i.; Ito, H.; Inagaki, H.; Noishiki, Y. J. Biomed. Mater. Res. 1989, 23, 125−133. (50) Chandra, R.; Rustgi, R. Prog. Polym. Sci. 1998, 23, 1273−1335. (51) Li, L.; Xie, T. Annu. Rev. Cell Dev. Biol. 2005, 21, 605−631. (52) Olczyk, P.; Mencner, Ł.; Komosinska-Vassev, K. BioMed Res. Int. 2014, 2014, 747584. (53) Schultz, G. S.; Wysocki, A. Wound Repair Regen. 2009, 17, 153− 162. (54) Kim, S. H.; Turnbull, J.; Guimond, S. J. Endocrinol. 2011, 209, 139−151. (55) Uysal, C. A.; Tobita, M.; Hyakusoku, H.; Mizuno, H. Adv. Wound Care 2014, 3, 405−413. (56) Lau, K.; Paus, R.; Tiede, S.; Day, P.; Bayat, A. Exp. Dermatol. 2009, 18, 921−933. (57) Cerqueira, M. T.; Pirraco, R. P.; Santos, T. C.; Rodrigues, D. B.; Frias, A. M.; Martins, A. R.; Reis, R. L.; Marques, A. P. Biomacromolecules 2013, 14, 3997−4008. (58) Gurtner, G. C.; Werner, S.; Barrandon, Y.; Longaker, M. T. Nature 2008, 453, 314−321. (59) Jackson, W. M.; Nesti, L. J.; Tuan, R. S. Stem Cell Res. Ther. 2012, 3, 20−28. (60) Hinz, B. J. Invest. Dermatol. 2007, 127, 526−537.
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DOI: 10.1021/acs.biomac.5b01566 Biomacromolecules XXXX, XXX, XXX−XXX
Injectable and Thermosensitive Soluble Extracellular Matrix and Methylcellulose Hydrogels for Stem Cell Delivery in Skin Wounds Eun Ji Kim, Ji Suk Choi, Jun Sung Kim, Young Chan Choi, and Yong Woo Cho* Department of Chemical Engineering, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea S Supporting Information *
ABSTRACT: Extracellular matrix (ECM) provides structural support and biochemical cues for tissue development and regeneration. Here we report a thermosensitive hydrogel composed of soluble ECM (sECM) and methylcellulose (MC) for injectable stem cell delivery. The sECM was prepared by denaturing solid ECM extracted from human adipose tissue and then blended with a MC solution. At low temperatures, the sECM-MC solution displayed a viscous solution state in which the loss modulus (G″) was predominant over the storage modulus (G′). With increasing temperature, G′ increased dramatically and eventually exceeded G″ around 34 °C, characteristic of the transition from a liquid-like state to an elastic gel-like state. After a single injection of the stem cellembedded hydrogel in full thickness cutaneous wound, the wound healed rapidly through re-epithelialization and neovascularization with minimum scar formation. The overall results suggest that in-situ-forming sECM-MC hydrogels are a promising injectable vehicle for stem cell delivery and tissue regeneration.
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INTRODUCTION
D ultrastructure of the tissue, mechanical integrity, protein composition, and biologic activity.9,23−25 In this study, we report on the development of in-situforming thermosensitive hydrogels based on human adiposederived soluble ECM (sECM) and methylcellulose (MC) for delivering stem cells in skin wounds. Human adipose tissue has been used as a rich source of ECM because it can be harvested in large quantities with minimal morbidity and contains abundant ECM proteins and soluble growth factors that are important for tissue development and regeneration.26,27 The human adipose-derived ECMs were solubilized using urea and guanidine buffer and then the extracted sECM was blended with MC to improve mechanical properties of the thermosensitive hydrogels. The sECM-MC hydrogels were characterized in terms of sECM content, rheological properties, and ability to embed stem cells. The stem cell delivery and woundhealing efficacy of the hydrogels were also evaluated using a full-thickness wound model.
In-situ-forming hydrogels that undergo sol−gel transitions in response to temperature are attractive as injectable systems for tissue therapies because of easy incorporation of cells or therapeutic agents, simplicity of implantation by injection, longterm retention of delivered cells, and improvement of cell viability by providing a three-dimensional (3-D) microenvironment for delivered cells.1−4 Although many synthetic and natural biomaterials have been used for developing in-situforming hydrogels that mimic the chemical and physical cues of the native extracellular matrix (ECM), the currently developed biomaterials are unsatisfactory in that they do not reflect the intricate functions of natural ECM in living tissues.5−7 Recently, intact ECMs derived from living tissues have emerged as a major biomaterial in regenerative medicine.8−15 The composition and spatial structure of ECMs play key roles in guiding cell migration, stimulating cell proliferation and differentiation, and modulating cellular responses.16 For this reason, many researchers have designed different processes to isolate intact ECMs from tissues and have fabricated various 3D tissue engineering scaffolds for use in regenerative medicine, particularly for patients requiring soft tissue regeneration.11,17−22 More recently, pH- or temperature-sensitive hydrogels based on ECM proteins extracted from adipose, dermis, liver, myocardium, and urinary bladder tissues have been developed as injectable scaffolds for cell delivery and tissue regeneration. The ECM hydrogels are promising in providing an artificial cell niche for controlling cellular behavior and tissue regeneration because of the well-preserved original 3© XXXX American Chemical Society
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MATERIALS AND METHODS
Materials. All chemical reagents were purchased from SigmaAldrich (St. Louis, MO, U.S.A.). Protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN, U.S.A.). Dialysis tubing (molecular weight cutoff (MWCO) 6000−8000) was obtained from Fisher Scientific (Pittsburgh, PA, U.S.A.). Amicon Ultra-15 centrifugal filter devices were supplied by Millipore (Billerica, MA, U.S.A.). Bicinchoninic acid (BCA) assay kits were purchased from Pierce (Rockford, IL, U.S.A.). Sircol acid/pepsin-soluble Received: May 6, 2015
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DOI: 10.1021/acs.biomac.5b01566 Biomacromolecules XXXX, XXX, XXX−XXX
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at 90 °C for 60 min. Then, the solution was allowed to equilibrate overnight at 4 °C. Subsequently, sECM and MC solutions were mixed at a volume ratio of 1:4 to the final concentrations of 6 wt % sECM and 6 wt % MC, and the mixture was homogeneously stirred at 4 °C. The sECM-MC solution was kept at 4 °C until use. Scanning Electron Microscopy. The microstructure of sECMMC hydrogels was observed using scanning electron microscopy (SEM; VEGA-II SBH, TESCAN, Brno, Czech Republic). The sECMMC hydrogels were frozen using liquid nitrogen, and freeze-dried. Then, these hydrogels were fixed to metal stubs and coated with platinum by sputtering at an accelerating voltage of 15 kV. Rheological Analysis. The rheological properties of the sECMMC hydrogels were investigated with a rheometer (Bohlin Gemini HR Nano, Malvern, U.K.) operating with 40 mm parallel plate geometry. The sECM-MC pregel solutions were immediately loaded onto a rheometer plate that was precooled to 10 °C. After loading, the shear viscosity was measured at a frequency of 1 Hz. The temperature was then increased to 45 °C at a rate of 3 °C per minute to induce gelation. Preparation of hASCs. The human adipose tissue-derived stem cells (hASCs) were isolated according to a previously described protocol.18 Briefly, the adipose tissue was washed with PBS containing 5% penicillin/streptomycin (P/S). After removing red blood cells, the adipose tissue was digested in PBS supplemented with 0.01 wt % collagenase type II for 1 h at 37 °C. The digested tissue was filtered through a 100 μm mesh to remove aggregated tissue and debris. The filtered suspension was centrifuged at 200 ×g for 7 min, and the resulting stromal vascular fraction (SVF) pellet was washed several times in PBS. SVF cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% P/S at 37 °C under 5% CO2. In Vitro Cell Study. The lyophilized sECM and MC powders were sterilized by ethylene oxide gas. A 50 μL aliquot of sECM-MC hydrogel containing hASCs (105 cells/mL) was placed into each well of 96-well plates and incubated at 37 °C under 5% CO2 for 3 days. Rat tail type I collagen hydrogel (3 mg/mL, Corning, NY, U.S.A.) was used as a control. The cell viability was analyzed using the Premix WST-1 Cell Proliferation Assay System and Live/Dead Viability/ Cytotoxicity kit. The Premix WST-1 reagent was added to each well, and the plates were incubated for 3 h at 37 °C. The absorbance was measured at 440 nm using a microplate reader (PowerWave XS, BioTek Instruments, Winooski, VT). The hydrogels containing hASCs were stained for 1 h with the combined Live/Dead reagent at 37 °C and observed using a fluorescence microscope (IX81; Olympus, Tokyo, Japan). In Vivo Wound Healing Study. The stem cell delivery and wound-healing efficacy of the sECM-MC hydrogels were evaluated using a full-thickness cutaneous wound model. All experiments were performed with approval from the Institutional Review Board of Hanyang University. The rat dorsal area (female, Sprague−Dawley, weighting 80−120 g, HanaBio, Hwaseong-si, Korea) was completely depilated under general anesthesia, and a full-thickness circle wound (about 169 mm2 in area) was created in the upper back area of each rat. The lyophilized sECM and MC powders were sterilized by ethylene oxide gas. A 200 μL aliquot of the sECM-MC pregel solutions with or without hASCs (105 cells/mL) were injected once into the wound bed using a syringe. The wound was then wrapped with a plastic mold for protection. At appropriate times, the rats from each group were sacrificed, and the skins, including the wounds were harvested for histological examination. For macroscopic evaluation of wound healing, the wound area was examined at 1, 2, and 3 weeks after treatment. The cutaneous wounds of the five animals were photographed, and the unhealed wound area was measured based on the image produced by the Imaging Analyzer (Bio-Rad Laboratories, Hercules, CA, U.S.A.). For histological and immunofluorescence examinations, specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced at a 6−10 μm thickness using a microtome. The sections were deparaffinized, dehydrated through a series of graded ethanol, and stained with Gomori’s trichrome. The hASCs embedded in the injected hydrogel were confirmed via immunofluorescence staining for human stem cell
collagen, Blyscan sulfated GAG, and Fastin elastin assay kits were supplied by Biocolor (Carrickfergus, Northern Ireland). QuantiMatrix human laminin and fibronectin ELISA kits were supplied by KOMA BIOTECH (Seoul, Korea). Premix WST-1 Cell Proliferation Assay System were purchased from Takara (Shiga, Japan). Live/Dead Viability/Cytotoxicity kits were obtained from Molecular Probes (Eugene, OR, U.S.A.). 4,6-Diamidino-2-phenylindole (DAPI, Thermo Scientific, Rockford, IL, U.S.A.) was used to identify nuclear components. Fluorochrome-conjugated anti-human CD29 and antihuman CD44 were purchased from BioLegend (San Diego, CA, U.S.A.). Mouse anti-rat PECAM-1 and fluorescein-conjugated bovine anti-mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Cell culture media, serum, and antibiotics were purchased from Life Technologies (Carlsbad, CA, U.S.A.). All other chemicals were reagent grade and used as received. Extraction of sECM from Human Adipose Tissue. Solid ECM was prepared from human adipose tissue, as described previously.15 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 was washed several times with distilled water to remove blood components. Distilled water was added to the adipose tissue and the tissue/water (1:1) mixture was homogenized for 3 min using a commercial blender. The tissue suspension was centrifuged at 16500 ×g for 5 min at 4 °C to remove oil components. The viscous suspension was washed with 3.4 M NaCl and centrifuged at 20000 ×g for 30 min at 4 °C. For sECM preparation, solid ECM was suspended in 4 M urea buffer containing protease inhibitor cocktail for 12 h at 4 °C. The mixture was centrifuged at 20000 ×g for 60 min at 4 °C to remove insoluble materials. The residues were re-extracted in 4 M guanidine containing protease inhibitor cocktail for 12 h at 4 °C, and were centrifuged. The supernatant was filtered through a 40 μm mesh and dialyzed extensively in dialysis tubing against 30 volumes of Tris-buffered saline (TBS) for 24 h at 4 °C. The dialysate was centrifuged at 20000 ×g for 60 min at 4 °C, and was filtered through a 40 μm mesh. The supernatant was concentrated using a 3000 MWCO Amicon Ultra-15 centrifugal filter device, and the final sECM was lyophilized. The concentration of purified sECM was measured using a BCA assay. sECM Protein Content. Protein content in the sECM was quantified using Sircol acid/pepsin-soluble collagen, Blyscan sulfated glycosaminoglycan (GAG), and Fastin elastin assay kits according to the manufacturer’s protocols. For quantification of acid/pepsin-soluble collagen, sECM was incubated with 1 mL Sircol dye reagent for 30 min at room temperature. For sulfated GAG, sECM was mixed with 1 mL Blyscan dye, and the precipitate was collected via centrifugation. For elastin, sECM was mixed with 1 mL Fastin dye. Collagen type I (rat tail), chondroitin 4-sulfate (bovine trachea), and α-elastin (bovine neck) were used as standards for the biochemical assays. Relative absorbance was measured in 96-well plates using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, U.S.A.). Laminin and fibronectin in sECM were quantified via enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (KOMA BIOTECH, Inc., Seoul, Korea). Optical density was measured at 560 nm using a microplate spectrophotometer (PowerWave XS, BioTek Instruments, Winooski, VT, U.S.A.). Growth Factor Antibody Array. Bioactive molecules in the sECM were analyzed using a growth factor antibody array kit (RayBiotech Inc., Norcross, GA, U.S.A.) according to the manufacturer’s protocol. The array glass chip containing 41 different human cytokine antibodies was blocked and incubated with diluted sECM. The glass chip was washed and subsequently treated in biotinconjugated antibodies. After incubation with fluorescent dyeconjugated streptavidin, cytokine signals were detected using a laser scanner (Axon Instruments, Union, CA, U.S.A.) using the Cy3 channel. Signal intensities were quantified with GenePix Pro software. Preparation of sECM-MC Hydrogels. The 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. MC solution (7.5 wt %) was prepared by a dispersion technique. Briefly, 0.75 g of MC (viscosity of 15 cP) was thoroughly wetted in 10 mL PBS and heated B
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Biomacromolecules markers. The sections were incubated with a 10 wt % BSA blocking agent for 30 min to inhibit nonspecific binding of IgG. Sections were incubated with fluorochrome-conjugated anti-human CD29 and antihuman CD44. To visualize vascularization in injected hydrogels, mouse anti-rat PECAM-1 and fluorescein-conjugated bovine antimouse IgG were used. All sections were counterstained with 1 μg/mL DAPI for 1 min and observed with a fluorescence microscope (Olympus, Tokyo, Japan). Statistical Analysis. Experimental data are expressed as the mean ± standard deviation (SD). The Student’s two-tailed t-test with SPSS 17.0 statistical software (SPSS, Chicago, IL, U.S.A.) was used for comparison, and statistical significance was accepted at p < 0.05.
Table 1. Profiling of Growth Factors in sECM Derived from Human Adipose Tissue Using Growth Factor Antibody Assaysa growth factor HGF TGF-β1 TGF-β3 EGF EGFR HB-EGF
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RESULTS AND DISCUSSION Characterization of Human Adipose Tissue-Derived sECM. For preparing sECM, solid ECMs isolated from human adipose tissue were solubilized using urea and guanidine buffer.28 The average final yield was approximately 45 ± 0.8 mg/g of wet solid ECM. The ECM is a key component of the cell niche and provides structural and biochemical cues that are required for tissue development and regeneration.29 Therefore, preservation of the proteins and growth factors during the extraction of sECM from tissue is highly desirable for retaining the biological and functional effects of the ECM. The sECM contained large amounts of soluble elastin (16.463 ± 1.440 mg/mL), acid/pepsin-soluble collagen (4.572 ± 0.136 mg/ mL), laminin (1.038 ± 0.064 mg/mL), and small amounts of fibronectin (0.144 ± 0.054 mg/mL) and sulfated GAG (0.021 ± 0.004 mg/mL). Endogenous growth factors in the sECM were detected using different human growth factor antibody arrays. Among the 41 growth factors, 25 growth factors were significantly detected in the prepared sECM (Table 1). Notably, hepatocyte growth factor (HGF), platelet-derived growth factor-BB (PDGF-BB), endothelial growth factor (EGF), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), which are all involved in the regulation of wound healing and angiogenesis, were highly detected in the sECM. Rheological Analysis of sECM-MC Hydrogels. Thermosensitive sECM-MC solutions were successfully prepared by blending sECM and MC solutions at a final concentration of 6 wt %, respectively. The sECM-MC solution was freely flowing and slightly viscous at 4 °C, while the solution became opaque and rapidly formed a stable and highly porous hydrogel within 3 min at 37 °C (Figure 1A and Supporting Information, Figure S1). The sol−gel transition was monitored by measuring the storage modulus (G′) and loss modulus (G″) as a function of temperature (Figure 1B−E). At low temperatures, the sECMMC solution displayed a viscous solution state in which the G″ was predominant over G′. With increasing temperature, G′ increased dramatically and eventually exceeded G″, characteristic of the transition from a liquid-like state to an elastic gel-like state. In particular, the sECM-MC solutions showed different gelation temperatures and gel strengths, depending on the concentration of sECM. The gelation temperature (crossover point of G′ and G″) decreased to 44.1, 43.0, 38.0, and 33.9 °C with increasing sECM concentrations at 0, 1, 3, and 6 wt %, respectively. Among these hydrogels, the sECM-MC hydrogel containing 6 wt % sECM revealed the highest G′ value with increasing temperature, showing a typical elastic gel-like behavior. The G′ value of the 6 wt % sECM-MC hydrogel also increased with an increase in frequency from 0.01 to 10 Hz at 37 °C after incubation for 10 min at 37 °C, and the G′ was substantially larger than the G″ over the entire tested frequency
PDGFAA, AB, BB PDGF Rβ VEGF VEGF R2, R3 IGF1 IGF1R IGFBP2 IGF2 CSF1 CSF1R CSF3 SCFR PLGF AREG NT-3
functions Regulation of cell growth, cell motility, and morphogenesis Control of cell proliferation, differentiation, adhesion, and migration Control of cell differentiation and embryogenesis Stimulation of cell growth, proliferation, and differentiation Receptor for EGF Mediation of cell adhesion, migration, and cell cycle progression; Predominant growth factor for epithelialization of skin wound Regulation of cell growth, division, and angiogenesis Receptor for PDGF Regulation of angiogenesis and lymphangiogenesis Receptor for VEGF Promotion of cell proliferation, the inhibition of cell apoptosis, and regulation of neural development Receptor for IGF1 A carrier protein for IGF-1 Organ development and function in fetal stage Stimulation of neutrophil survival, proliferation, differentiation, and function Receptor for CSF1 Stimulation of neutrophil survival, proliferation, differentiation, and function Receptor for SCF A key molecule in angiogenesis and vasculogenesis during embryogenesis Regulation of mitogen of astrocytes, Schwann cells, fibroblast and T-cells Encouragement of neuron growth and differentiation
a
HGF, hepatocyte growth factor; TGF-β, transforming growth factorβ; EGF, epidermal growth factor; EGF-R, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; PDGF-AA, AB, BB, plateletderived growth factor subunit A, B; PDGF-Rβ, platelet-derived growth factor-receptor β-polypeptide; VEGF, vascular endothelial growth factor; VEGF R2, R3, VEGF receptor; IGF 1, insulin-like growth factor 1; IGF1R, IGF-1 receptor; IGFBP2, IGF binding proteins-2; IGF2, insulin-like growth factor 2; CSF1, colony stimulating factor 1; CSF1R, CSF 1 receptor; CSF3, colony stimulating factor 3; PLGF, placenta growth factor; AREG, amphiregulin; NT, neurotrophin-3.
range, suggesting that the hydrogel becomes more stable with an increase in physical cross-links (Supporting Information, Figures S1 and S2).30 Physical cross-linking has been proposed as a valuable cross-linking methodology for in-situ-forming injectable hydrogels because it is capable of producing a hydrogel without the need for chemical modification or the addition of cross-linking agents.31 MC is a typical example that undergoes a sol−gel transition via physical cross-linking. MC forms a hydrogel by the dominant hydrophobic interaction between chain segments containing methoxy substitutions as the temperature increases.32 However, MC itself is not sufficient for use as an in-situ-fast gelling system due to high gelation temperatures (40−60 °C). The gelation temperature of MC can be lowered by the addition of salting-out salts such as NaCl, NaBr, KF, (NH4)2SO4, and Na2CO3, which bind water molecules tightly.33 In the presence of salting-out salts, hydrophobic segments in the MC chains are more easily excluded from the water-solvation shells, thereby promoting the hydrophobic interactions of MC chains as the temperature C
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Figure 1. (A) Sol−gel transition behavior of sECM-MC hydrogel (sECM 6 wt % and MC 6 wt %). (B−D) G′ and G″ of sECM-MC hydrogels with different sECM concentrations as a function of temperature: (B) 0 wt %, (C) 1 wt %, (D) 3 wt %, and (E) 6 wt % with MC 6 wt %; G′, storage modulus (●); G″, loss modulus (○).
increases.34 The anionic groups of sECM proteins seem to facilitate the hydrophobic interactions of MC chains, similar to the action of salting-out salts, resulting in a lower gelation temperature.35 The concentration of the final ECM is an important determinant of its rheological properties; the gelation temperature decreased with increasing sECM concentration. The appropriate gelation temperature for in-situ-gelling and fast sol−gel phase transition of the sECM-MC hydrogel is beneficial for embedding cells within the hydrogel and the delivery system as a whole compared with other intact ECM hydrogels. Cytocompatibility of the sECM-MC Hydrogel. To assess the cytocompatibility of the sECM-MC hydrogels, hASCs were embedded in the hydrogel (sECM 6 wt % and MC 6 wt %) and incubated for 3 days. The hASCs embedded in collagen hydrogels were used as controls. The proliferation of hASCs in the hydrogels was analyzed using WST-1 assay after 1 and 3 days, which is based on the reduction of a stable tetrazolium salt to a soluble violet formazan product by viable cells (Figure 2A). The live/dead assay was used to determine the distribution of live and dead cells after 3 days in the hydrogels (Figure 2B−D). Calcein AM, which fluoresces upon the reaction of intracellular esterase, stains live cells (green), while ethidium homodimer-1, which binds to the DNA of dead membrane compromised cells, stains dead cells (red). On day 3, the majority of cells expressed green fluorescence, and dead
cells were rarely detected. The hASCs proliferated with spindlelike morphology on the 2-D control cultures, as expected, while the cells embedded in the hydrogels showed spherical morphology over the culture period. It is a noted fact that 3D hydrogels can maintain spherical cell morphology by providing in vivo-like conditions, thereby having a significant influence on cellular functions.36,37 Our results suggest that the sECM-MC hydrogels provided a cell niche that maintained cell morphology and survival and did not induce significant cytotoxicity. In Vivo hASC Delivery Efficacy. The main issues with transplanted stem cells is that they may display limited survival, low retention rate, or immune rejection in injured tissues, reducing their therapeutic efficacy in vivo.38,39 One of the promising approaches to overcoming these problems is to use injectable hydrogels. The hydrogel can provide a favorable stem cell niche that promotes stem cell retention and survival and also act as a barrier to protect the stem cells against attack by the host immune response.40,41 To study the ability of sECMMC hydrogels to deliver stem cells, the sECM-MC solution containing hASCs was subcutaneously injected into fullthickness wounds of rats through a 23-gauge needle (Figure 3A,B and Supporting Information, Figure S3). The injected hydrogel rapidly formed a transparent hydrogel at the wound site. On day 1 after injection, the cell distribution was evaluated D
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Figure 2. Proliferation of hASCs embedded in sECM-MC hydrogel and collagen hydrogel for 3 days (A). Data were normalized by the absorbance of hASC embedded in collagen hydrogel on day 1. Fluorescence micrographs of hASCs cultured on a tissue culture plate (B), embedded in sECMMC hydrogel (C), and collagen hydrogel (D) on day 3. Scale bars represent 200 μm.
healing process.42 Many groups have reported that the stem cell fate could be determined by biochemical cues, such as soluble factors and ECM molecules.43 We postulated that the sECMMC hydrogels containing inherent soluble factors and proteins could provide the proper stem cell niche for cell retention, survival, and differentiation when compared with other hydrogels (e.g., synthetic polymers, recombinant protein polymers, hyaluronic acid, alginate, and agarose).44−47 The in vivo results suggest that sECM-MC hydrogels will be valuable not only in enhancing stem cell engraftment, but also in promoting the recruitment of host cells that can be guided to regenerate damaged tissues. Effect of hASC-Embedded sECM-MC Hydrogels on Wound Healing. Photographs of wounds were taken to observe changes in wound size, and histological staining was used to assess the wound healing processes on days 7, 14, and 21 (Figure 4 and 5). There was no sign of inflammation or infection in both the hydrogel and hASC-embedded hydrogel groups. Wound sizes were reduced over a period of time in all groups. On day 7, the hASC-embedded hydrogel group wound size (37.6 ± 5.9%) was significantly reduced compared with the control (65.9 ± 4.4%) and hydrogel group (57.4 ± 4.5%). The wounds treated with hASC-embedded hydrogels were covered with a continuous epidermis, and the regenerated dermis was much thicker than that of the hydrogel only. A large number of epidermal appendages were observed in the regenerated skin treated with the hASC-embedded hydrogel (Supporting Information, Figure S6). On day 14, the control group wounds were partially covered with epidermis, while those of the hydrogel and hASC-embedded hydrogel groups were fully covered with a continuous epidermis. In the wounds treated with hASC-embedded hydrogels, the microvessel density was much higher than those of the control and hydrogel only (Figure 6). However, no significant differences were observed in all groups on day 21.
Figure 3. sECM-MC solution containing hASCs was subcutaneously injected into a full-thickness wound of a rat through a 23-gauge needle (A,B). The hASCs were stained using CD29 (red) and CD44 (green) for human stem cells on day 1 after injection. Sections were counterstained with DAPI, which stains nuclei blue (C,D). H, hydrogel; I, interface between hydrogel and rat tissue; and T, rat tissue. Scale bars represent 200 μm.
by immunofluorescence staining. A large number of stem cells were stained with anti-CD29 and anti-CD44, which are specific markers for human mesenchymal stem cells. The stem cells were homogeneously distributed and integrated well within the hydrogel (Figure 3C,D). Interestingly, the results of DAPI staining showed a large proportion of host cells infiltrated within the injected hydrogels. Although some types of infiltrated host cells are involved in inflammatory responses (e.g., macrophages), the recruitment of various host cells into the hydrogel may result in affirmative effects on the wound E
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Figure 6. Distribution and density of newly formed blood microvessels were assessed using a CD31 (green) antibody for rat endothelial cells on days 14 (A,C,E) and 21 (B,D,F). Arrows denote areas that show the formation of newly formed blood vessels. Sections were counterstained with DAPI, which stains the nuclei blue. Scale bars represent 200 μm.
combination of chemical, biological and mechanical processes in vivo.48−50 Märtson et al.48 reported the degradation of cellulose sponges in the rat connective tissue. Miyamoto et al.49 also reported that cellulose with low crystallinity was more easily absorbed than that with high crystallinity. MC is a watersoluble cellulose derivative with low crystallinity and slowly degraded by biological, chemical, and physical processes in vivo.50 Niche signals from degraded sECM-MC hydrogels, such as collagen, laminin, fibronectin, elastin, and GAG, are known to regulate cell−cell or cell−matrix interactions and induce secretion of soluble signals from stem cells, stimulating differentiation of stem cells as well as modulating various cellular responses.51 In addition to providing niche signals, ECM components are integral to each phase of the wound healing process from hemostasis to remodeling by interacting with cells and growth factors in a dynamic.52 For example, ECM components (e.g., collagen type I, III, IV, laminin,
Figure 4. Progression in healing of full-thickness cutaneous wounds injected with hASCs-embedded sECM-MC hydrogel, sECM-MC hydrogel alone, and control groups on days 0, 7, 14, and 21. (A) Wounds were photographed on days 0, 7, 14, and 21. (B) The wound area is expressed as a percentage of the initial wound area on day 0. Data are shown as the mean ± standard deviation (n = 3), with significance at *p < 0.05.
It is notable that the wounds covered with hASC-embedded hydrogel healed rapidly through re-epithelialization with minimal scar formation. We suggest that ECM fragments released via degradation of the sECM-MC hydrogel could play a significant role by releasing the raw materials necessary for the generation of new matrix in wounds. Several studies have shown that cellulose or cellulose derivatives are degraded by a
Figure 5. Histological evaluation of wound sections injected with hASC-embedded sECM-MC hydrogel, sECM-MC hydrogel alone, and control groups (n = 5) after dermal excision on days 7, 14, and 21. Black arrows indicate the wound edges. Insets are magnified images of the indicated rectangles and represent the regeneration of the outer layer of the skin. Muscle, keratin, and cytoplasm, red; collagen, green; and nuclei, black. Scale bars represent 200 μm. F
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fibronectin, and GAGs) and various growth factors [e.g., PDGF, TGF, EGF, fibroblast growth factor (FGF), keratinocyte growth factor (KGF), IGF-1, and HGF] recruit host stem cells, macrophages, fibroblasts, and endothelial cells into the wound site, modulating epithelialization, collagen accumulation, and angiogenesis.29,53,54 Paracrine signals secreted from hASCs (bFGF, TGF-β, VEGF)55−57 and endogenous growth factors (HGF, PDGF-BB, EGF, IGF, and VEGF) in the sECM-MC hydrogel seemed to have a synergic effect on improvement of the quality of wound healing by orchestrating events during granulation tissue formation, re-epithelialization, and angiogenesis. However, abnormal ECM production by fibroblasts and excessive contractile force mediated by myofibroblasts during the proliferation phase can result in scarring, which can lead to significant functional and cosmetic morbidity.52,58 During the proliferation and remodeling phases, some growth factors are known to play an important role in scar formation. HGF controls the equilibrium between synthesis and degradation of ECMs by stimulating the secretion of matrix metalloproteinase (MMP) in fibroblasts.59 bFGF is also known to reduce wound contraction by inhibiting the phenotypic change of fibroblasts to myofibroblasts, which is likely to be involved in contraction.60 As mentioned above, sECM-MC hydrogels degraded within the wound and the degraded ECM fragments can be used to reconstruct granulation tissue instead of fibroblasts, resulting in the inhibition of excessive ECM production. In addition, soluble signals (e.g., bFGF) secreted by embedded hASCs and endogenous growth factors (e.g., HGF) released from the sECM could contribute to a reduction in wound contraction by inhibiting the phenotypic change of fibroblasts to myofibroblasts. Based on these results, the sECMMC hydrogel is a promising injectable system that provides the therapeutic niche necessary to improve the quality of wound healing in addition to delivering therapeutic cells to the wound site.
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel.: +82 (0)31 400 5279. Fax: +82 (0)303 3475 4712. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Seoul R&BD Program (Grant No. SS110011), the Basic Research Program (Grant No. 2012008294), and the Bio & Medical Technology Development Program (Grant No. 2011-0019774) through the National Research Foundation of Korea (NRF) funded by the Korean government (MEST).
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CONCLUSIONS Injectable, thermosensitive sECM-MC hydrogels were successfully prepared by a simple blending of human adipose-derived sECM and MC solutions at low temperatures. The sECM-MC hydrogels showed a thermosensitive sol−gel phase transition and rapidly formed a hydrogel at body temperature. In vivo study using a full-thickness wound model showed that the sECM-MC hydrogels provide not only a cell niche that improves engraftment and survival of stem cells delivered to the wound, but also biological cues that accelerate wound regeneration. Our findings suggest that in-situ-forming sECMMC hydrogels are a promising injectable vehicle for stem cell delivery and soft tissue engineering.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01566. Microstructure, dynamic frequency sweep and storage and loss modulus plots of sECM-MC hydrogels with different sECM concentrations and additional in vivo images (PDF). G
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