Capillary Force Seeding of Hydrogels for Adipose-Derived Stem Cell Delivery in Wounds

To optimize hydrogel cell seeding, a capillary force-based approach was developed and compared with previously established cell seeding methods. Adipose-derived mesenchymal stem cell (ASC) viability and functionality following capillary hydrogel seeding were analyzed. Capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Abstract

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a “stem cell niche” that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Introduction

Normal wound healing is a complex process involving the coordination of multiple cell and cytokine signaling pathways [1]. These mechanisms can be overwhelmed in the setting of complex injuries and/or underlying disease states, such as diabetes and vascular insufficiency, and ultimately result in the formation of a chronic, nonhealing wound. Chronic wounds affect up to 6.5 million U.S. patients and cost in excess of U.S. $25 billion annually [2]. Although a variety of treatment modalities are available, stem cell-based therapies hold particular promise in this setting because of their strong cytokine profile and potential for multilineage differentiation [3]. To optimize this therapeutic approach, biocompatible delivery systems are needed to promote cell survival and cytokine release within the harsh wound environment, with the ideal scaffold recapitulating architectural features of human skin to restore the cell-matrix interactions critical for tissue regeneration [4].

Our group previously demonstrated that a 5% soft collagen-pullulan hydrogel can be fabricated to closely resemble the three-dimensional collagen network of human dermis at a microscopic level and is biocompatible with multiple cell types [5]. Pullulan, a linear homopolysaccharide produced by the fungus Aureobasidium pullulans, was specifically chosen for hydrogel construction in conjunction with collagen, because it is biodegradable and nontoxic, making it an attractive biomaterial for tissue engineering approaches [6, 7]. Accordingly, application of unseeded hydrogels in murine excisional wounds was found to increase both the recruitment of stromal cells and formation of vascularized granulation tissue, leading to an improvement in wound closure [5]. Evaluating the capacity of hydrogels for the delivery of cell-based therapies, we have also demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSCs) could be engrafted into the hydrogel by coculture over 14 days, resulting in an increase in BM-MSC stemness factor transcription and growth factor and cytokine secretion [8]. Additionally, application of BM-MSC-seeded hydrogels to murine excisional wounds was found to augment both wound closure rates and angiogenesis when compared with wounds that were untreated or injected with BM-MSCs [8].

Although BM-MSC delivery to wounds using a hydrogel offers a promising therapeutic opportunity, a source of mesenchymal stem cells other than the bone marrow would be more practical for widespread clinical use. Adipose-derived mesenchymal stem cells (ASCs) have several potential advantages over BM-MSCs, including their ease of harvest from human lipoaspirates [9–11], as well as their ability to proliferate rapidly and secrete high levels of proangiogenic cytokines [10]. Furthermore, the number of BM-MSCs available for isolation from bone marrow drops significantly as people age, potentially requiring larger volumes of bone marrow harvest, which carries greater risk than superficial fat harvest [12].

Promising preliminary data on the use of human ASCs in vivo has demonstrated their ability to heal critical size calvarial defects [13], as well as augment vascularization of composite ischemic tissues [14]. Prior work has also shown encouraging results using ASCs embedded in various matrices to improve excisional wound closure [15–20], although the clinical translatability of these studies is limited by the prolonged matrix seeding protocols (up to 7 days) needed to produce these constructs. In the present study, we describe a capillary seeding method to rapidly engraft ASCs into a lyophilized 5% collagen-pullulan hydrogel at the point of care. Using this efficient hydrogel seeding technique and a splinted murine excisional wound model [21], we further demonstrate that both murine and human ASC-seeded hydrogels augment wound closure and angiogenesis and are well suited for clinical adaptation.

Materials and Methods

Animals

All mice were housed in the Stanford University Veterinary Service Center in accordance with NIH and institution-approved animal care guidelines. All procedures were approved by the Stanford Administrative Panel on Laboratory Animal Care. All assays were performed in triplicate unless otherwise stated.

Murine Adipose-Derived Mesenchymal Stem Cell Isolation

Wild-type and luciferase+/green fluorescent protein (GFP)+ ASCs were isolated from the inguinal fat pads of 8–12-week-old mice (C57BL/6J and FVB-Tg(CAG-luc,−GFP)L2G85Chco/J, respectively; Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). Fat pads were minced, and digested for 1 hour at 37°C using collagenase I (Roche, Indianapolis, IN, http://www.roche.com). The reaction was stopped, and the cells were spun down to obtain the stromal vascular fraction (SVF). The SVF was resuspended, strained, and plated on plastic culture dishes. The medium was changed every 48 hours until cells reached 90% confluence. The cells were used at or before passage 2 unless otherwise indicated.

Hydrogel Fabrication and Cell Seeding Optimization

A 5% collagen-pullulan hydrogel was produced as described previously [8]. Capillary force seeding was assessed against adaptations of three previously described scaffold seeding approaches (injection, centrifugal, and orbital culture) [22], with each technique described in detail below (Fig. 1A, 1B). For this and all subsequent hydrogel-based analyses, dehydrated hydrogel was cut into 6-mm circles using a punch biopsy tool and seeded with 2.5 × 10⁵ ASCs (n = 4 hydrogels per analysis). Following the respective seeding technique, hydrogels were placed in excess Dulbecco’s modified Eagle’s medium (DMEM) solution supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY, http://www.lifetech.com) and cultured for cell viability and scanning electron microscopy structural analyses. Seeding efficiency was also determined by counting residual cells in cell seeding medium for each methodology with a hemocytometer. Following this comparative analysis, capillary seeding was used for all subsequent experiments.

Figure 1.

Efficacy of a novel capillary technique for scaffold seeding. (A): A 5% collagen-pullulan hydrogel contains a porous architecture that interfaces with a droplet of suspended adipose-derived mesenchymal stem cells (ASCs) on a hydrophobic surface. Cells are actively engrafted via a combination of hydrophobic, entropic, and capillary forces, the last a function of hydrogel pore width and liquid properties of the ASC solution. (B): Capillary seeding was compared with centrifugal, injection, and orbital seeding approaches (left upper to right lower corner). (C): Approximate duration of seeding techniques. (D): Quantification of cell seeding efficiency, with capillary and orbital shaker seeding demonstrating a consistently high efficacy. (E): Quantification of seeded cell viability at 72 hours, with capillary seeding resulting in a significantly enhanced survival as compared with centrifuge and injection techniques. (F): Scanning electron micrographs focusing on hydrogel structure demonstrate that although capillary seeding conserves hydrogel microarchitecture (top micrograph, white arrows indicate intact scaffold), injection seeding damages scaffold architecture (bottom micrograph, gray arrows indicate damaged scaffold). ∗, p < .05. Scale bar = 100 μm. Abbreviations: θ, contact angle; γ, surface tension; ρ, liquid density; g, gravitational force; r, pore radius.

To achieve capillary seeding, 2.5 × 10⁵ murine ASCs (mASCs) suspended in 15 μl of DMEM solution was pipetted onto hydrophobic wax paper, and the hydrogel was immediately placed on top. Cells were absorbed actively into the pores of the scaffold by capillary, hydrophobic, and entropic forces, which became visibly saturated within 1 minute (completely hydrated with negligible medium remaining on wax paper upon lifting of the hydrogel). Centrifugal seeding was achieved by combining 2.5 × 10⁵ mASCs (diluted in 200 μl of medium) and a hydrogel in a 1.5-ml Eppendorf tube. Following saturation of the hydrogel in excess medium, the tube was subjected to three rounds of centrifugation at 3,000 rpm for 2 minutes, interrupted by vortexing for 10 seconds. Injection seeding was completed by injecting 2.5 × 10⁵ mASCs suspended in 30 μl of medium into the center of each hydrogel using a 25-gauge needle. Orbital seeding was achieved by placing each hydrogel in 100 μl of medium on a 48-well plate, followed by application of 2.5 × 10⁵ mASCs suspended in 15 μl of medium on top of each hydrogel and rocking on an orbital shaker for 1 hour at 37°C.

Scanning Electron Microscopy Analysis

High-resolution scanning electron microscopy of ASC-seeded hydrogels was completed using a Hitachi 3400N VP scanning electron microscope (Hitachi High Technologies America, Inc., Schaumburg, IL, http://hitachi-hta.com) at the Stanford Cell Sciences Imaging Facility.

In Vitro Cell Viability/Migration/Proliferation

A live-dead assay was performed to assess ASC viability following hydrogel seeding according to the manufacturer’s instructions (Live/Dead Cell Viability Assay; Life Technologies). To confirm cell migration through the hydrogel, a modified Transwell assay was performed. Briefly, ASCs were seeded by capillary force onto 6-mm hydrogels and placed in the top chamber of an 8.0-μm HTS Transwell 96-well plate (Corning Life Sciences, Tewksbury, MA, http://www.corning.com/lifesciences) with mouse platelet-derived growth factor BB as the chemoattractant. Twenty-four hours later, membranes were removed and fixed with 4% paraformaldehyde. Nuclei were stained with VectaShield Mounting Medium with 4[prime],6-diamidino-2-phenylindole (DAPI) and analyzed using fluorescence microscopy. ASC proliferation was compared between hydrogel-seeded cells and plated cells using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Vybrant MTT Cell Proliferation Assay Kit; Invitrogen, Grand Island, NY, http://www.invitrogen.com).

In Vitro Real-Time Quantitative PCR Analysis

ASCs were capillary-seeded onto scaffolds or plated into each well of a 6-well plate and incubated at 37°C in 5% CO₂ for 24–48 hours. Total RNA was harvested from hydrogel-seeded and plated ASCs as previously described [8] and converted to cDNA through reverse transcription (Superscript First-Strand Synthesis Kit; Invitrogen). Real-time quantitative polymerase chain reactions (PCRs) were performed using 2× Universal TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) and TaqMan gene expression assays for murine Pou5f1 (Oct4, Mm00658129g), Cxcl12 (stromal cell-derived factor-1/Sdf-1, Mm00445552_m1), Ccl2 (monocyte chemoattractant protein-1/Mcp-1, Mm00441242_m1), Fgf-2 (fibroblast growth factor-2, Mm00433287_m1), Igf-1 (insulin-like growth factor-1, Mm00439560_m1), Vegf-a (vascular endothelial growth factor-A, Mm01281447_m1), Eng (endoglin, Mm00468256_m1), Hgf (hepatocyte growth factor, Mm01135193_m1), and Angpt1 (angiopoietin 1, Mm00456503_m1) using a Prism 7900HT Sequence Detection System (Applied Biosystems). The levels of murine Actb (β-actin, Mm01205647_g1) were quantified in parallel as an internal control, and gene expression was normalized.

In Vitro Stemness Factor/Angiogenic Cytokine Quantification and Western Blot

Total protein was collected from murine ASCs capillary-seeded onto hydrogels or plated for 24–48 hours with RIPA buffer (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) in combination with a protease inhibitor. Angiogenic cytokine protein levels were quantified using a Mouse Angiogenesis Array Kit (R&D Systems, Minneapolis, MN, MN, http://www.rndsystems.com). Pixel density of each spot in the array was quantified and normalized to controls using ImageJ (NIH, Bethesda, MD).

For Western blot analysis, protein was separated on a 4%–12% polyacrylamide gel (Invitrogen) and then transferred to a nitrocellulose membrane (Invitrogen). Anti-Oct4 (1:800; Abcam, Cambridge, MA, http://www.abcam.com) and anti-β-actin were used as the primary antibodies. A horseradish peroxidase-conjugated secondary antibody was used (1:10,000) and detected using the ECL Plus Western Blotting Detection Kit (GE Healthcare, Waukesha, WI, http://www.gehealthcare.com).

In Vitro Flow Cytometric Analysis of Cell Stemness

Plated and hydrogel-seeded murine ASCs were analyzed via flow cytometry for expression of alkaline phosphatase using a monoclonal anti-alkaline phosphatase (ALP) antibody (Abcam; 2° fluorescein isothiocyanate-conjugated anti-Rb antibody; Life Technologies) following cell fixation and permeabilization. Mesenchymal stem cell markers were assessed via flow cytometry using the following anti-murine monoclonal antibodies: CD90-PeCy7 (eBioscience, San Diego, CA, http://www.ebioscience.com) and CD44-APC (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). All analyses were performed on an LSRII Flow Cytometer (BD Biosciences).

In Vitro Immunofluorescence

2.5 × 10⁵ murine ASCs seeded onto coverslips or onto hydrogel scaffolds for 24 hours were fixed in 4% paraformaldehyde for 1 hour and then incubated with a primary antibody against Oct4 (1:200; Abcam), followed by Alexa Fluor 594-conjugated secondary antibody (Invitrogen). Cell nuclei were stained with DAPI.

In Vivo Excisional Wound Model

Eight- to twelve-week-old male C57Bl/6 mice (Jackson Laboratory) were randomized to two treatment groups: unseeded hydrogel control or murine ASC-seeded hydrogel. As previously described [5], two 6-mm full-thickness wounds per mouse were excised from either side of the midline. Each wound was held open by donut-shaped silicone rings fastened with 6-0 nylon sutures to prevent wound contraction. For mice in the unseeded hydrogel control group, a 6-mm piece of hydrogel saturated with phosphate-buffered saline (PBS) was placed in each wound bed. For mice in the ASC-seeded hydrogel group, a 6-mm piece of hydrogel-seeded by capillary force with ASCs (suspended in saline) was placed in the wound bed. All wounds were covered with an occlusive dressing (Tegaderm; 3M, St. Paul, MN, http://www.3m.com). Digital photographs were taken on days 0, 1, 3, 5, 7, 9, 11, and 14. The wound area was measured using ImageJ software (NIH) (n = 6 wounds per group). This model was repeated in its entirety with human ASCs and 8–12-week-old nude male B6.Cg-Foxn1nu/J mice (Jackson Laboratory).

In Vivo Bioluminescence Imaging

Viability of ASCs was assessed in vivo in wild-type mice using bioluminescence imaging (n = 6 wounds per condition). Wounded mice treated with 2.5 × 10⁵ luciferase+ ASCs either seeded on hydrogels or injected circumferentially in the wound bed (four injection sites at the 12, 3, 6, and 9 o’clock positions, as previously described) [8] were anesthetized and injected with 150 mg/kg luciferin in PBS intraperitoneally. Images were obtained 10 minutes later with a cooled CCD camera using the Xenogen IVIS 200 System (Caliper Life Sciences, Mountain View, CA, http://www.perkinelmer.com). Luminescence was quantified as units of total flux in an area of interest subtracted from the background luminescence. Images were taken on days 0 and 3 and every other day thereafter until day 14.

In Vivo ASC Localization

Hydrogel-only and murine GFP+ ASC-seeded hydrogel-treated wounds were harvested on day 10 from wild-type mice (FVB/NJ; Jackson Laboratory) and immediately embedded in OCT (Sakura Finetek, Torrance, CA, http://www.sakura.com) for histologic localization of GFP+ cells and CD31 immunohistochemical stain as described below.

Human ASC Isolation

Human lipoaspirates were collected from healthy, adult female patients with approval from the Stanford Institutional Review Board and digested in a similar fashion as described for murine adipose tissue. The freshly obtained human SVF was purified via fluorescence-activated cell sorting (FACS) to obtain ASCs (defined as the CD45−/CD31−/CD34+ cell fraction) using the following mouse anti-human monoclonal antibodies: CD31-PE, CD45-PeCy7, and CD34-APC (BD Biosciences). This surface marker profile was chosen to exclude hematopoietic and endothelial cells and was used in combination with propidium iodide to eliminate dead cells. FACS was performed on a BD FACSAria (BD Biosciences), with sorted cells collected for immediate use (2.5 × 10⁵ cells per wound) without culture expansion.

Assessment of Wound Vascularity

Wound vascularity was assessed utilizing hematoxylin and eosin (H&E) histological examination and/or immunohistochemical staining for the endothelial cell marker CD31 (n = 6 wounds per condition). Briefly, wounds from the excisional model were harvested upon closure and either processed for paraffin sectioning or immediately embedded in OCT (Sakura Finetek). H&E immunohistochemical staining of 7-μm-thick paraffin sections was used to assess microvessel density. For dermal microvessel counts, luminal structures containing red blood cells were considered microvessels. For each condition, four high-powered fields at ×400 were examined for three separate wound samples by three independent blinded observers.

Immunohistochemical staining of 7-μm-thick frozen sections for CD31 was also used to quantify wound vascularity as described previously [8]. Briefly, slides were fixed in precooled acetone for 10 minutes, washed in PBS, and blocked in a humidified chamber for 2 hours. Primary antibody (1:100 Rb α CD31, Ab28364; Abcam) was incubated overnight at 4°C, followed by secondary antibody staining (1:400 AF547 Gt α Rb; Life Technologies). Cell nuclei were visualized with the nuclear stain DAPI. ImageJ (NIH) was used to binarize immunofluorescent images taken with the same gain, exposure, and excitation settings as previously described [8]. Intensity threshold values were set automatically, and quantification of CD31 staining was determined by pixel-positive area per high power field.

Wound Angiogenic Cytokine Quantification

mASC-treated and control wounds were harvested at day 5, snap frozen in liquid nitrogen, and stored at −80°C. Total protein was isolated from wounds using RIPA buffer (Sigma-Aldrich) in combination with a protease inhibitor, and levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were quantified using a mouse quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN).

Statistical Analysis

All values are expressed as means ± SEM. Statistical significance across seeding methods was determined using a one-way analysis of variance, with subsequent comparisons between individual methods completed using a Tukey post hoc analysis. Subsequent data analyses were performed using a Student’s t test. p values ≤ .05 were considered statistically significant.

Results

Efficiency of Hydrogel Seeding via Capillary Force

To determine the most effective cell-seeding methodology, a rapid, capillary force technique (Fig. 1A) was assessed against three previously described scaffold seeding approaches (injection, centrifugal, and orbital culture) [22] (Fig. 1B), with regards to seeding time and efficiency, cell survival, and maintenance of structural integrity of the hydrogel. In comparison with the other protocols, capillary force seeding possessed the most optimal combination of speed, efficiency, cell survival, and maintenance of hydrogel structure (Fig. 1C, 1F). Specifically, capillary seeding led to ASC engraftment within 1 minute (Fig. 1C) and was found to be significantly more efficient than centrifugal seeding (99.38% ± 0.38 vs. 18.22% ± 2.7, p < .01) (Fig. 1D). Capillary seeding was also associated with greater cell viability as compared with both centrifugal and injection seeding (p < .02) (Fig. 1E). Finally, scanning electron microscopy evaluation of scaffolds revealed that injection seeding substantially disrupted the hydrogel microarchitecture as compared with capillary and other seeding approaches (Fig. 1F). Given the overall superiority of the capillary seeding approach, this technique was utilized for all subsequent experiments.

ASCs Are Biocompatible With Biomimetic Pullulan-Collagen Hydrogel Scaffolds

Engrafted ASCs were further investigated for biocompatibility within the hydrogel. Scanning electron microscopy analysis of capillary seeded hydrogels demonstrated that ASCs became suspended in the three-dimensional matrix and formed cytoplasmic extensions projecting in and around scaffold micropores (Fig. 2A). A live-dead assay was next performed to determine longer-term cell viability in vitro, which remained greater than 96% over a 14-day time frame (Fig. 2B). Engrafted cells also retained their ability to migrate through the hydrogel, a crucial function for in vivo application of cells to the wound bed, as demonstrated by a Transwell migration assay (Fig. 2C). Finally, ASC proliferation/metabolic activity was determined using an MTT assay. Plated ASCs demonstrated an increase in metabolic activity over a 7-day period, whereas metabolic activity in hydrogel engrafted ASCs did not increase (p < .05) (Fig. 2D). Given the sustained cell viability observed following hydrogel engraftment in vitro, these data suggested that the hydrogel preserved ASCs in a quiescent state and created a functional niche.

Figure 2.

ASCs are biocompatible with a pullulan-collagen hydrogel. (A): Electron microscopy images reveal adipose-derived mesenchymal stem cells (ASCs) integrated into the hydrogel scaffold with cytoplasmic extensions into the surrounding three-dimensional matrix (left). Cells (white arrowheads) are found interspersed around, between, and within pores (black arrowheads) in a dynamic three-dimensional environment (right). Scale bars = 30 μm. (B): A live-dead assay demonstrates >96% cell viability in the hydrogel through day 14. Right: At 14 days, live cells appear green, and dead cells appear red. Scale bar = 100 μm. (C): A Transwell migration assay at 24 hours reveals that ASCs (GFP+ cells indicated by white arrowheads) have migrated onto a permeable membrane below the hydrogel. Scale bar = 100 μm. (D): MTT proliferation assay demonstrates a steady increase in metabolic activity among plated ASCs compared with a relatively constant metabolic activity among hydrogel-seeded ASCs. ∗, p < .05. Data are means ± one SEM.

Hydrogel Engrafted ASCs Demonstrate Augmented Wound Healing Potential In Vitro

In order to determine the effects of hydrogel engraftment on ASC wound healing potential, plated murine ASCs and hydrogel-seeded ASCs were compared for their expression of stemness-related proteins, growth factors, and cytokines related to wound healing. After 24–48 hours of being plated or seeded in hydrogels, ASC RNA was isolated, and quantitative reverse transcription-PCR was performed, revealing a significant increase in expression of the stemness-related transcription factor Oct4 in hydrogel-seeded versus plated cells (2.28 ± 0.73 vs. 0.18 ± 0.17, p = .02) (Fig. 3A). Western blotting and immunofluorescence staining confirmed an increase in Oct4 expression among hydrogel-engrafted ASCs compared with plated cells (Fig. 3B, 3C). Flow cytometric analysis for the pluripotency-related marker ALP and mesenchymal stem cell markers (CD90 and CD44) further demonstrated an enhancement of ASC stemness following hydrogel seeding (Fig. 3D).

Figure 3.

Hydrogel engraftment augments adipose-derived mesenchymal stem cell (ASC) stemness. (A): Quantitative reverse transcription reveals increases in Oct4 transcriptional levels among hydrogel-seeded ASCs compared with plated cells. (B): Immunoblot confirms the increased presence of Oct4 protein expression in hydrogel-seeded ASCs compared with plated ASCs. (C): Immunofluorescence staining similarly demonstrates that ASCs cultured within the hydrogel express increased levels of Oct4 compared with plated cells. (D): Flow cytometric analysis demonstrates increased expression of selected stemness and mesenchymal stem cell markers upon hydrogel seeding. Left: Representative histograms with gray histogram representing the negative control. Right: Quantification. ∗, p < .05. Data are means ± 1 SEM. Scale bar = 100 μm. Abbreviations: ALP, alkaline phosphatase; DAPI, 4[prime],6-diamidino-2-phenylindole; GFP, green fluorescent protein; Oct4, octamer-binding transcription factor 4.

In addition, hydrogel seeding of ASCs resulted in augmented gene expression of multiple growth factors and cytokines related to angiogenesis and wound healing when compared with standard culture techniques (Fig. 4A). Relative expression of Sdf-1 was significantly increased in hydrogel-seeded ASCs (30.48 ± 4.61 vs. 0.80 ± 0.04, p = .0002), in addition to Mcp-1 (3.44 ± 0.31 vs. 0.23 ± 0.01, p = 6.07 × 10⁻⁶), Fgf-2 (2.77 ± 0.38 vs. 1.79 ± 0.17, p = .04), Igf-1 (2.66 ± 0.06 vs. 1.08 ± 0.04, p = 2.94 × 10⁻⁹), Vegf-a (2.59 ± 0.31 vs. 0.62 ± 0.01, p = .0002), Eng (1.00 ± 0.06 vs. 0.43 ± 0.01, p = 2.49 × 10⁻⁵), Hgf (0.93 ± 0.03 vs. 0.38 ± 0.05, p = 1.03 × 10⁻⁵), and Angpt1 (0.10 ± 0.01 vs. 0.003 ± 5.79 × 10⁻⁵, p = 6.86 × 10⁻⁶). To confirm the transcriptional data, protein was isolated, and the relative levels of selected angiogenesis related proteins were quantified using a murine angiogenesis array (Fig. 4B). Significantly increased protein levels of MCP-1 (60.54 ± 4.11 vs. 40.23 ± 3.70, p = .03), SDF-1 (25.24 ± 11.15 vs. 5.65 ± 0.74, p = .04), and HGF (17.79 ± 0.04 vs. 11.72 ± 0.56, p = .004) were found in samples isolated from hydrogel-seeded ASCs compared with those plated under standard conditions. The augmentation of ASC stemness and angiogenesis-related proteins suggested that the hydrogel scaffold may be an effective cell delivery system for enhancing wound regeneration.

Figure 4.

Hydrogel engraftment augments adipose-derived mesenchymal stem cell (ASC) growth factor and cytokine expression. (A): Multiple growth factors and cytokines demonstrate increased transcriptional levels among hydrogel-seeded ASCs compared with plated cells. (B): Protein confirmation of the upregulation of selected angiogenesis related genes via angiogenic array. ∗, p < .05. Data are means ± one SEM. Abbreviation: Angpt1, angiopoietin 1; Eng, endoglin; Fgf-2, basic fibroblast growth factor; Hgf, hepatocyte growth factor; Igf-1, insulin like growth factor 1; Mcp-1, monocyte chemotactic protein 1; Sdf-1, stromal cell-derived factor 1; Vegf-a, vascular endothelial growth factor-a.

ASC-Seeded Bioscaffolds Result in Sustained Cell Delivery to Excisional Wounds

Given these promising in vitro findings, in vivo experiments were performed to determine whether a pullulan-collagen hydrogel enhanced cell viability. Murine stented excisional wounds were therefore treated with local injection or hydrogel delivery of luciferase-expressing ASCs, and bioluminescence imaging revealed a significant improvement in cell viability with hydrogel delivery of ASCs over a 14-day time period (Fig. 5A, 5B). At 1 hour following ASC treatment, bioluminescence had already decreased dramatically between hydrogel bioscaffold and local injection groups (342.31 ± 63.86 vs. 72.73 ± 29.28, p = .003). By day 9, there was no further evidence of viable cells in injection-treated mice, whereas cell viability was sustained in the hydrogel treatment group through day 11 (p < .05).

Figure 5.

Hydrogels promote sustained ASC delivery to murine wounds. (A): In vivo imaging of luciferase+ ASCs delivered to murine excisional wounds by local injection or topical bioscaffold reveals prolonged cell viability in the hydrogel treatment group. (B): Graphical representation of luciferase signal in ASC-seeded hydrogel-treated wounds compared with local ASC injection. ASC-seeded hydrogels result in a significant increase in cell viability and a sustained period of cell delivery relative to injected cells. (C): Covisualization of GFP+ ASCs with CD31 staining demonstrates the presence of hydrogel-delivered ASCs in the perivascular space (white arrowheads). ∗, p < .05. Data are means ± one SEM. Scale bar = 100 μm. Abbreviations: ASC, adipose-derived mesenchymal stem cell; D, day; DAPI, 4[prime],6-diamidino-2-phenylindole; GFP, green fluorescent protein; hr, hour; sec, second.

Having demonstrated that ASCs engraft within the wound, visualization of GFP+ ASCs in conjunction with a cell-specific marker was performed on day 10 wounds to investigate ASC localization. Using CD31 as a marker for blood vessel endothelium, GFP+ ASCs delivered into wounds via a hydrogel scaffold were found within the perivascular space (Fig. 5C), consistent with an active role in supporting wound neovascularization.

ASC-Seeded Hydrogels Improve Wound Closure and Vascularization by Increased Proangiogenic Cytokine Expression

Having established that delivery of ASCs to wounds is sustained using a pullulan-collagen hydrogel, further experiments were conducted to determine whether wound healing was improved. Wild-type mice were subjected to the stented excisional wound model, and wounds were followed for 14 days. Mice that were treated with mASC-seeded hydrogels healed significantly faster than control mice treated with PBS-soaked hydrogels (Fig. 6A), despite similar scaffold resorption kinetics in both groups. Wound area was significantly smaller in the mASC hydrogel-treated group compared with control wounds at days 9 and 11 (day 9: 26.88 mm² ± 2.56 vs. 41.79 mm² ± 4.49, p = .04; day 11: 1.38 mm² ± 0.9 vs. 18.06 mm² ± 4.85, p = .02;), and mASC hydrogel-treated wounds closed on average 3 days earlier than controls (p < .05).

Figure 6.

Murine and human adipose-derived mesenchymal stem cell (ASC)-seeded hydrogels improve cutaneous wound healing and vascularization. (A): Wound closure rates were significantly faster among mASC-seeded hydrogels at days 9 and 11, and wounds closed an average of 3 days earlier than controls. (B): CD31 staining confirmed a significant increase in microvessels among the mASC-seeded hydrogel group. DAPI was the nuclear stain. Scale bar = 100 μm. (C): Quantification of CD31 stained pixels. (D): Evaluation of angiogenic cytokine levels within the wound demonstrates significantly higher levels of VEGF and HGF with ASC treatment. (E): Representative excisional wounds demonstrate a more rapid and earlier time to wound closure among mice treated with hASC-seeded hydrogels compared with controls. (F): Wound closure rates were significantly faster following hASC-seeded hydrogel treatment at days 7, 9, and 11 and closed an average of 2.3 days earlier than controls. (G, H): CD31 staining and pixel quantification confirmed a significant increase in microvessels among the mASC-seeded hydrogel group. ∗, p < .05; # indicates significance in time to closure. Scale bars = 100 μm. All data are means ± one SEM. Abbreviations: D, day; DAPI, 4[prime],6-diamidino-2-phenylindole; hASC, human adipose-derived mesenchymal stem cell; HGF, hepatocyte growth factor; mASC, murine adipose-derived mesenchymal stem cell; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

Additionally, wounds treated with mASC-seeded hydrogels were significantly more vascular than controls (Fig. 6B, 6C; supplemental online Fig. 1). CD31 staining of tissue sections confirmed these results with evidence of increased vascularity among mASC-seeded hydrogel wounds, as compared with unseeded hydrogel controls at day 14 (20,010.37 pixels ± 3,839.92 vs. 6,113.68 ± 1,258.67, p = .003). Additionally, H&E-stained tissue sections of day 14 wounds showed a significant increase in microvessel density among the mASC-seeded hydrogel treatment group compared with unseeded hydrogel samples (7.29 ± 1.48 vs. 3.70 ± 0.42, p = .01).

To better understand any ASC cytokine contributions to the wound environment, ELISA assays were performed on day 5 mASC-treated and control wounds. Significantly higher levels of proangiogenic VEGF and HGF cytokine expression were detected in mASC-seeded hydrogel-treated wounds (113.98 pg/ml ± 3.47 vs. 68.23 pg/ml ± 8.95, p = .03 and 589.08 ± 102.33 vs. 299.53 ± 30.49, p < .01, respectively) (Fig. 6D). These data suggested that the proangiogenic profile of ASC-seeded hydrogels was maintained in vivo and translated to significantly augmented vascularization through multiple paracrine signaling pathways.

Human ASC-Seeded Hydrogels Augment Wound Closure and Vascularization in Nude Mice

Given the promising effects of murine ASCs on wound healing, fresh, unexpanded human ASCs (hASCs) were isolated via FACS from healthy, adult-derived lipoaspirates and analyzed for the presence of a similar beneficial influence. Immunocompromised mice were subjected to the splinted excisional wound model and were treated with either hASC-seeded hydrogels or PBS-soaked controls (Fig. 6E, 6F). The wound area was significantly smaller in the hASC hydrogel-treated group compared with control wounds at days 7, 9, and 11 post injury (day 7: 31.09 mm² ± 4.46 vs. 51.94 ± 7.76, p = .04; day 9: 15.34 ± 2.81 vs. 28.22 mm² ± 3.90, p = .02; and day 11: 2.04 mm² ± 1.43 vs. 15.64 mm² ± 3.78), and hASC hydrogel-treated wounds closed on average 2.3 days earlier than controls (p < .01).

Similar to the beneficial effects of hydrogel delivery of murine ASCs, wounds treated with hASC-seeded hydrogels were significantly more vascular than controls based on CD31 staining (17,230.75 pixels ± 2,681.98 vs. 7,494.82 pixels ± 1,239.38, p = .001) (Fig. 6G, 6H). These human data indicated a similar efficacy across cell sources and supported the use of fresh hASCs within the hydrogel, obviating the need for time-consuming ex vivo expansion prior to application.

Discussion

Innovative treatment options are needed to address the significant morbidity and costs associated with chronic and complex acute wounds. In the present study, we have presented a method of almost instantly seeding ASCs into a lyophilized 5% collagen-pullulan hydrogel via capillary force and demonstrated the efficacy of this cell-based therapy for wound healing applications.

Prior research on scaffold seeding methodologies has focused on increasing seeding efficacy, because a densely seeded construct is crucial for proper tissue formation [23]. Nonetheless, increasingly complex approaches can promote a high seeding density at the expense of time, with protocols often lasting up to several hours or even requiring overnight incubation [24]. To maximize both seeding time and efficiency, a rapid capillary force approach was developed (combining hydrophobic, entropic, and capillary forces to promote active, “bottom-up” cell engraftment) and compared with three previously described seeding methodologies: “top-down” seeding on an orbital shaker, seeding through centrifugation, and direct-injection seeding [22]. Of these techniques, we observed a consistently high seeding efficacy only for orbital shaker seeding and our capillary protocol, with capillary seeding having the additional advantage of being significantly faster than orbital shaking (on the order of minutes as opposed to hours). In fact, capillary seeding was the only seeding methodology that allowed for efficient, rapid cell engraftment, with preservation of cell viability and scaffold microarchitecture, making it highly translatable to the clinical setting.

Utilizing this seeding approach for all subsequent analyses, we further demonstrated the biocompatibility of ASCs within the hydrogel scaffold, with seeded cells demonstrating a sustained viability and migratory capacity in vitro. Moreover, although ASCs cultured under standard conditions demonstrated a steady increase in metabolic activity associated with cellular proliferation, ASCs seeded within hydrogel scaffolds showed minimal proliferation and maintained baseline levels of metabolic activity over 7 days. Given that there was no significant cytotoxicity observed with hydrogel culture conditions, these data suggest that the hydrogel induces ASC quiescence and thus may act as a functional niche for this stem cell population. This is consistent with prior studies demonstrating a preservation of cells in the undifferentiated state when embedded in a hyaluronic acid hydrogel, with concomitant maintenance of full differentiation capacity [25].

Although ASCs are easily accessible and implantable in a hydrogel, the retention of cell stemness remains a key variable. Similar to embryonic stem cells, human bone marrow-derived adult mesenchymal stem cells have been shown to regulate plasticity through the expression of embryonic transcription factors, such as the master transcriptional regulator Oct4 [26]. Oct4, which is expressed in developing cells of the early blastomere and associated with cell self renewal and pluripotency [27], has also been shown to be expressed in both murine and human ASCs [28, 29] but decreases with multiple passages presumably because of the disruption of the stem cell niche. Engraftment of ASCs in the hydrogel, however, resulted in increased transcriptional and protein levels of Oct4, further suggesting that the hydrogel bioscaffold provides a niche-like environment for ASCs and promotes delivery of cells with enhanced stemness characteristics to the wound. ASC upregulation of the pluripotency marker ALP [30] and the mesenchymal stem cell marker CD44 [31] following hydrogel seeding supports this conclusion.

The therapeutic potential of ASC-seeded hydrogels was also demonstrated by transcriptional analyses of plated versus hydrogel-seeded ASCs. Both plated and hydrogel-seeded ASCs expressed numerous growth factors and proangiogenic cytokines, substantiating previous findings of the wide spectrum of ASC growth factor/cytokine expression [10]. Nonetheless, we found that ASC engraftment in the hydrogel significantly augmented expression of multiple factors in vitro, including Sdf-1, Mcp-1, Fgf-2, Igf-1, Vegf-a, Eng, Hgf, and Angpt1. These factors play a role in the early inflammatory phase of wound healing, recruit progenitor cells, and facilitate angiogenic processes critical to wound repair and regeneration. Providing insight into the mechanistic underpinnings of hydrogel-associated changes in ASC gene expression, prior investigations comparing multicellular aggregates of ASCs to plated ASCs have demonstrated a similar upregulation of growth factors, with concomitant increases in wound healing potential [32]. Although this suggests that the three-dimensional environments of a cell aggregate and hydrogel scaffold are both capable of augmenting the proangiogenic and regenerative potential ASC-based therapies through recapitulation of the stem cell niche, the major translational advantage of the hydrogel to clinical applications is its ability to be seeded with freshly obtained cells without the need for ex vivo expansion.

Prolongation of ASC survival following application is another potential approach to maximize regenerative impact. Our laboratory has previously demonstrated that hydrogel seeding of BM-MSCs enhances their survival in the harsh wound environment as compared with standard cell injection [8]. We observed a similar increase in in vivo ASC survival following hydrogel seeding herein, with the combined data supporting a dual role of the hydrogel for delivery of cells to the wound environment: enhancement of proregenerative signaling and prolongation of survival.

Extrapolating this methodology to the clinical setting, the relative ease of lipoaspirate-based ASC collection and immediate hydrogel cell seeding makes our technique ideal for the rapid application of autologous cells to wounds. This approach could theoretically be accomplished in one procedure and would circumvent the immunoreactive potential of allogenic cell sources. To demonstrate the in vivo regenerative potential of ASC-seeded hydrogels, murine and human cells were separately applied to a splinted murine excisional wound model, which “humanizes” murine wounds by forcing them to close by re-epithelialization and granulation tissue formation rather than skin contraction [21]. Expanding upon the previously described beneficial effect of ASCs in nonsplinted wound models [19, 20], hydrogels seeded with both culture-expanded murine ASCs and freshly isolated human ASCs were found to significantly improve wound healing at multiple time points compared with unseeded hydrogels, as well as accelerate time to closure and increase wound vascularity. Additionally, the effect on wound closure rates was more pronounced than that reported with shorter-term ASC delivery to similarly splinted wounds using a different bioscaffold [18], highlighting the influence of both matrix composition and cell delivery time on therapeutic efficacy.

Given the enhanced vasculogenic profile of hydrogel-seeded ASCs, as well as the known paracrine effects of mesenchymal stem cells [33–35], the beneficial effects of ASC-seeded hydrogels on vascularization and wound healing observed herein were almost certainly the result of increased ASC-derived growth factors and cytokines within the wound. Nonetheless, the long-term fate of the applied ASCs within cutaneous wound is controversial, because the differentiation of locally administered ASCs into epithelial and endothelial cells within cutaneous wounds has been reported by several groups [36, 37]. Investigating the fate of hydrogel-delivered ASCs within healing wounds, we observed cells predominately in the vicinity of blood vessels, although colocalization to the endothelium was not seen. Quantification of ASC-treated and control wounds also revealed significantly greater expression of multiple hydrogel-inducible and vasculogenesis-related cytokines within the wound environment. These data support a paracrine mechanism of action for ASC support of neovascularization rather than direct differentiation, regardless of delivery technique.

Collectively, these findings demonstrate not only the regenerative potential of human ASC-seeded hydrogels following wounding, but also the clinically appealing procedural ability to go from cell collection to application in a span of hours. Although the efficacy of ASC-seeded hydrogels remains to be determined in the setting of pathological healing, such as diabetes and aging, the promising results of this study suggest this therapeutic combination would be similarly efficacious in settings where angiogenesis is impaired.

Conclusion

Our biocompatible 5% collagen-pullulan hydrogel can be rapidly seeded with ASCs via capillary force and provides a functional niche that promotes ASC stemness and growth factor/angiogenic cytokine expression. When applied to excisional wounds, both murine and human ASC-seeded hydrogels promote faster wound healing and enhance angiogenesis and regenerative cytokine expression. ASC-seeded hydrogels are highly translatable because of the ease of cell harvest and potential for immediate application.

Author Contributions

R.K.G., R.C.R., and D.D.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.S., R.K., L.J.A., J.L., M.T.C., and K.P.: collection and/or assembly of data; J.N.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; J.R.: conception and design, provision of study material, final approval of manuscript; M.T.L. and G.C.G.: conception and design, financial support, manuscript writing, final approval of manuscript.

Disclosure of Potential Conflicts of Interest

G.C.G., J.R., R.C.R., and D.D. are uncompensated patent holders.