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Pristine Gellan Gum–Collagen Interpenetrating Network Hydrogels as Mechanically Enhanced Anti-inflammatory Biologic Wound Dressings for Burn Wound Therapy

  • Jian Yao Ng
    Jian Yao Ng
    Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
    More by Jian Yao Ng
  • Xiao Zhu
    Xiao Zhu
    Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
    More by Xiao Zhu
  • Devika Mukherjee
    Devika Mukherjee
    Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
  • Chi Zhang
    Chi Zhang
    Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
    More by Chi Zhang
  • Shiqi Hong
    Shiqi Hong
    Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
    More by Shiqi Hong
  • Yogesh Kumar
    Yogesh Kumar
    Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
    More by Yogesh Kumar
  • Rajeev Gokhale
    Rajeev Gokhale
    Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
  • , and 
  • Pui Lai Rachel Ee*
    Pui Lai Rachel Ee
    Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
    NUS Graduate School for Integrative Sciences and Engineering, 21 Lower Kent Ridge Road, 119077, Singapore
    *Email: [email protected]. Phone: + 65 6516 2653; Fax: +65 6779 1554.
Cite this: ACS Appl. Bio Mater. 2021, 4, 2, 1470–1482
Publication Date (Web):February 3, 2021
https://doi.org/10.1021/acsabm.0c01363

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

Gellan gum is a biologically inert natural polymer that is increasingly favored as a material-of-choice to form biorelevant hydrogels. However, as a burn wound dressing, native gellan gum hydrogels do not drive host’s biology toward regeneration and are mechanically inadequate wound barriers. To overcome these issues, we fabricateda gellan gum–collagen full interpenetrating network (full-IPN) hydrogel that can house adipose-derived mesenchymal stem cells (ADSCs) and employ their multilineage differentiation potential and produce wound-healing paracrine factors to reduce inflammation and promote burn wound regeneration. Herein, a robust temperature-dependent simultaneous IPN (SIN) hydrogel fabrication process was demonstrated using applied rheology for the first time. Subsequently after fabrication, mechanical characterization assays showed that the IPN hydrogels were easy to handle without deforming and retained sufficient mass to effect ADSCs’ anti-inflammation property in a simulated wound environment. The IPN hydrogels’ increased stiffness proved conducive for mechanotransduced cell adhesion. Scanning electron microscopy revealed theIPN’s porous network, which enabled encapsulated ADSCs to spread and proliferate, for up to 3 weeks of culture, further shown by cells’ dynamic filopodia extension observed in 3D confocal images. Successful incorporation of ADSCs accorded the IPN hydrogels with biologic wound-dressing properties, which possess the ability to promote human dermal fibroblast migration and secrete an anti-inflammatory paracrine factor, TSG-6 protein, as demonstrated in the 2D scratch wound assay and ELISA, respectively. More importantly, upon application onto murine full thickness burn wounds, our biologic wound dressing enhanced early wound closure, reduced inflammation, and promoted complete skin regeneration. Altogether, our results highlight the successful mechanical and biological enhancement of the inert matrix of gellan gum. Through completely natural procedures, a highly applicable biologic wound dressing is introduced for cell-based full thickness burn wound therapy.

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1. Introduction

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Full thickness burns are severe perennial medical issues of unmet need. Severe burn wounds are difficult to heal primarily because of persistent chronic inflammation. (1) Perpetual release of cytokines and metalloproteinases by immune cells impedes the deposition of a new extracellular matrix (ECM), leading to scarring and incomplete skin regeneration. (2) Although the use of skin grafts has improved burn treatment outcomes, donor scarcity and surgical complications have fostered the use of biologic scaffolds as alternatives. (3) These scaffolds are often hydrogels or hydrocolloids impregnated with wound-healing factors and/or contained stem cells that intend to regulate the innate repair and inflammatory mechanisms. (4−6)
Hydrogels are a 3D cross-linked network of polymers that have the ability to imbibe large amounts of water. Their aqueous nature provides a soothing sensation on burn wounds and mimics the microenvironment where cells thrive. (7) Existing hydrogel wound products are made of biologically inert synthetic polymers. (8) However, natural polymers, which are inherently more biocompatible and biodegradable, are becoming the material-of-choice to form hydrogels for biomedical applications. (9) A recent review has specifically highlighted microbial polysaccharides as an underutilized class of hydrogel-forming natural polymers. (10) In which, gellan gum stands out as it forms hydrogels rapidly under physiological conditions. However, unmodified gellan gum is biologically inert and mechanically too weak to serve as an effective wound barrier. (11,12) Therefore, pristine gellan gum hydrogels do not stimulate burn wounds toward immunomodulation or enhanced wound healing. (13) As a result, the use of gellan gum hydrogels for a skin tissue engineering approach to full thickness burn wounds is much curtailed and underdeveloped. (12)
Adipose-derived mesenchymal stem cells (ADSCs) are multipotent stromal cells with regenerative capacity to drive host’s tissue repair at wound sites. When intradermally applied, ADSCs release paracrine wound-healing factors that increased cell migration and wound closure, as well as regulated wound inflammation. (14) In addition, ADSCs can differentiate into multiple skin cell lineages and enable complete skin regeneration of appendages such as hair follicles, sebaceous, and sweat glands. (15) To confer gellan gum hydrogels with bioactivity, we sought to encapsulate ADSCs within and transform them into biologic wound dressings.
However, gellan gum’s chemical structure is devoid of integrin-binding domains. (13) The lack of cell–matrix adhesion would trigger encapsulated ADSCs to undergo a form of programmed cell death known as “anoikis”. (16) Drawing inspiration from the native ECM, it is practicable to incorporate a secondary integrin-presenting protein polymer network, such as collagen, to confer gellan gum hydrogels with cell adhesivity. (17) As compared to the other ECM proteins, collagen is able to form a hydrogel network under physiological conditions, which facilitates ADSC encapsulation. The formation of a secondary interlaced network can further enhance gellan gum hydrogel's mechanical strength. (18) In spite of the recent emerging interest in enhancing biopolymer hydrogel functionality through interpenetrating networks (IPNs), (19) the lack of mechanism-driven gelation methodology has prevented a conclusive report of a gellan gum-collagen IPN cell-laden hydrogel wound dressing. This is because gellan gum and collagen have opposing cooling and heating gelation mechanisms. (20,21) Upon mixing, the precursor blend may fall below gellan gum’s concentration-dependent transition temperature of coil–helix (Tc–h). (22) After which, gellan gum polymers anneal and the precursor solution may become too viscous for cell resuspension. Fortunately, common rheological principles govern these temperature-dependent gelations. A de novo formulation approach using applied rheology could circumnavigate the aforementioned formulation barriers.
Here, we describe a facile method of synthesis to mechanically and biologically enhance the properties of chemically pristine gellan gum, putting together an ADSC-laden IPN hydrogel tailored for the treatment of full thickness burn wounds. In comparison to existing strategies, our approach to transform gellan gum into a biologic scaffold is devoid of synthetic chemistry and eliminates the hazards associated with reported chemical cross-linking agents. (23) Successful IPN fabrication was demonstrated with systematic changes to the IPN hydrogel’s rheological properties along the fabrication process. Then, a comprehensive set of characterization studies was performed on the resulting IPN hydrogels. Physical properties such as ease of handling, porosity, equilibrium wound fluid uptake, degradation, and rheology of the IPN hydrogels were examined for consideration of adequacy. The effect of incorporating collagen on ADSC’s growth and spread was extensively studied, to prove that the resulting IPN hydrogel is a cell-conducive biologic scaffold. Upon successful ADSC encapsulation, a myriad of further in vitro and in vivo burn wound model evaluations was conducted to conclude the transformation of an inert gellan gum polymer into an efficacious biologic wound dressing.

2. Materials and Methods

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2.1. Materials

Low-acyl gellan gum (Gelzan CM, G1910), magnesium chloride (MgCl2, 208337), sodium hydroxide (NaOH, S8045), sodium bicarbonate (NaHCO3, S5761), potassium chloride (KCl, P9541), low-glucose (D5523) and high-glucose (D1152) Dulbecco’s Modified Eagle’s medium (DMEM), Accutase solution (A6964), penicillin/streptomycin (P4333), paraformaldehyde (PFA, P6148), tumor necrosis factor alpha (TNF-α), and 10% neutral buffered formalin (NBF) were purchased from Sigma-Aldrich (St Louis, MO, USA). Sodium chloride (NaCl, S34900) was obtained from Unichem Ltd (Mumbai, Maharashtra, India). All salts were received in anhydrous form. 0.22 μm polyethersulfone (PES) filters were acquired from sartorius stedim (Göttingen, Germany). Type-1 rat tail collagen (SC-136157) was procured from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Fetal bovine serum (FBS) was obtained from Hyclone (South Logan, UT, USA). Ultrapure grade phosphate buffered saline (10× PBS) was procured from Vivantis Technologies Sdn Bhd (Subang Jaya, Selangor Darul Ehsan, Malaysia). CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) kit was purchased from Promega (Madison, WI, USA). Triton-X was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Phalloidin-iFluor 488 Reagent (ab176753) was acquired from Abcam (Cambridge, UK). TNF α-stimulated gene 6 (TSG-6) enzyme-linked immunosorbent assay (ELISA) kit was obtained from RayBiotech (Peachtree Corners, GA, USA). Tegaderm was purchased from 3M (Maplewood, MN, USA). Human Dermal Fibroblast (HDF), bone marrow-derived Mesenchymal Stem Cells (hMSC, PT-2501), and Adipose-derived Stem Cells (ADSC, PT-5006) were purchased from Lonza Bioscience (Basel, Switzerland). All other materials obtained are of research grade and used as received.

2.2. Gellan Gum–Collagen IPN Hydrogel Synthesis

Aqueous solutions of gellan gum and MgCl2 (cross-linker) were separately prepared by dissolving their respective powders in deionized water. All gellan gum solutions were progressively heated to 90 °C and kept at this temperature for 30 min until a completely homogeneous solution was obtained. After which, the gellan gum solutions were cooled to 37 °C and maintained at this temperature until further use. Type-1 collagen solution was neutralized according to the manufacturer’s protocol. The neutralized collagen was always prepared sterile and kept stable on ice until further use.
The optimized final concentrations of gellan gum, collagen, and MgCl2 are 0.4% (w/v), 1 mg/mL, and 0.02% (w/v) respectively. To prepare 60 μL of the IPN hydrogel, 18 μL of 3.333 mg/mL collagen was pipetted and mixed well with 31.5 μL of 0.762% (w/v) gellan gum. This mixture was then heated at 37 °C for 30 min for collagen fibrillogenesis to occur. After which and at room temperature, 10.5 μL of 0.114% (w/v) MgCl2 was added to cross-link the gellan gum polymers. Synthesis of IPN hydrogels composed of different final concentrations of the components followed this volume ratio.

2.3. Physical Characterization of Hydrogels

2.3.1. Qualitative Inversion Test

Successful gelation was qualitatively demonstrated using inversion tests. 400 μL of hydrogels were separately prepared in 2 mL Eppendorf tubes (n = 3). The Eppendorf tubes were inverted 5 s after each step of synthesis. The gelation criterion is defined at which the clear solution did not flow upon inversion of the Eppendorf tubes.
To illustrate that IPN hydrogelscould be handled without deformation, 600 μL of IPN hydrogel was cast from the bore of a 3 mL syringe (Terumo, Tokyo, Japan). After MgCl2 was added, the IPN hydrogel was left at room temperature for 5 min before being extruded onto a dark flat surface.

2.3.2. Scanning Electron Microscopy

The microstructure of the IPN hydrogels was analyzed using scanning electron microscopy (SEM). Lyophilized hydrogel samples (n = 3) (Alpha 1-2 LD+, Christ Martin, Germany) were first frozen in liquid nitrogen before being separated at their cross sections. Then, the dissected hydrogels were quickly mounted onto pin stubs with their cross sections facing the electron emission source. The samples were subsequently sputter coated with gold (JFC-1100, JEOL, Japan) and examined with a scanning electron microscope (JSM 6510, JEOL, Japan). The pore size was calculated with reference to an in-built scale bar.

2.3.3. Equilibrium SWF Uptake and In Vitro Hydrogel Degradation Via Gravimetric Method

Simulated wound fluid (SWF) was prepared according to the previously described method. (24) To determine equilibrium SWF uptake, 600 μL of hydrogels were blot-dried, weighed (Wi), and separately transferred into 15 mL Falcon tubes (n = 3). The hydrogels were immersed and incubated in 2 mL of SWF at 37 °C under constant agitation of 60 rpm. At referred time points, the supernatant was decanted, and the remaining hydrogels were blot-dried and reweighed (Wu). Percentage mass increase of the wet hydrogels due to SWF uptake was calculated according to eq 1. Fresh 2 mL of SWF was replenished after each time point.
(1)
After the hydrogels have reached their equilibrium swelling capacity, they were examined further for degradation in SWF, 1× PBS, and culture media for up to a total of 28 days. Similarly, at referred time points, the supernatant was decanted, and the remaining hydrogels were blot-dried and reweighed (Wd). Percentage mass remaining of the wet hydrogels was calculated according to eq 2. 2 mL of fresh SWF. 1× PBS or culture media was replenished after each time point.
(2)

2.4. Rheometry of IPN Hydrogels

Rheological characterization of the hydrogels was performed with an oscillatory rheometer (MCR 302, Antor Paar, Austria), using plate–plate geometry (diameter = 8 mm, working gap = 1 mm). 100 μL of freshly prepared hydrogels were quickly transferred onto the base plate of the rheometer prior to each testing. Tests were performed using three different samples per hydrogel composition (n = 3). After lowering the upper cone plate to a gap of 1 mm with the base plate, the hydrogel sample was allowed to stabilize for 1 min. All measurements were conducted at 25 °C.
Amplitude sweeps were always first conducted to determine the limit of the linear viscoelastic region (LVR). Storage (G′) and loss (G″) moduli were obtained in the range of 0.1 to 100% shear strain at a constant frequency of 1 Hz. The linearity limit was determined using a straight ruler on the graph plotted. The shear-dependent behavior and inner structure of hydrogels were studied via frequency sweeps. G′ and G″ moduli measurements were completed from minimum to maximum frequency between 0.1 and 100 rad/s. Shear strain was set to a constant value, below the predetermined LVR prior to the initiation of each measurement.
To understand the temperature- and time-dependent gelation behavior of IPN hydrogels, constant dynamic mechanical rheometry was carried out. Both temperature and time sweeps were conducted at a constant 1% shear strain and a constant frequency of 1 Hz. For temperature sweeps, measurements at −0.5 °C–1 from 45 to 15 °C were conducted on gellan gum-collagen precursor samples, immediately after the cross-linker was added. While for time sweeps, hydrogel samples were not allowed for equilibration and G′/G″ measurements were taken every 5 s up to 2 min.

2.5. In Vitro Evaluation of ADSC-Laden IPN Hydrogels as Biologic Wound Dressings

2.5.1. In Vitro Expansion of HDF, hMSC, and ADSC

HDF, hMSC, and ADSC received from Lonza Bioscience were thawed and cultured on Nunc T-75 cell culture flasks (Roskilde, Denmark), according to the manufacturer’s protocol. All cells were cultured and expanded under basal conditions, using 90% (v/v) high-glucose (HDF) or low-glucose (stem cells) DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin. HDF was used between passages 3 and 20. The stem cells were used between passages 3 and 5. All cells were harvested between 80 and 90% confluency.

2.5.2. Preparation of Biologic Wound Dressings

ADSCs were harvested using the Accutase solution according to the manufacturer’s protocols and resuspended in the precursor solution before being cast into the wells of a 48-well plate. Cell density was kept constant at 1000 cells/μL of the hydrogel. For cell culture, 600 μL of culture media was added to every 60 μL of ADSC-laden hydrogels. All cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. The culture media were replaced every 2–3 days (Mon, Wed and Fri).

2.5.3. Cell Viability and Proliferation of Cells Cultured in IPN Hydrogels Via Metabolic Activity Assay

To determine the cell viability of encapsulated cells, (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfofenyl)-2H-tetrazolium) (MTS) assays were conducted according to the manufacturer’s protocol. At referred time points, 10 μL of MTS was incubated with cells per 100 μL of culture media for 3 h. Measurement of total metabolic activity was performed on a multiwell microplate reader (Hidex Sense, Turku, Finland) at OD490. Cell-free hydrogels treated in the same way were used as ‘‘blanks”. Cells encapsulated in pure 1 mg/mL type-1 collagen hydrogels were used for comparison purpose.

2.5.4. Morphological Assessment of Encapsulated ADSCs Via CLSM

Cell adhesion and migration of encapsulated ADSCs were analyzed with confocal laser scanning microscopy (CLSM). The cells’ F-actin cytoskeleton was stained with Phalloidin-iFluor 488 reagent according to the manufacturer’s protocol. Briefly, 500 μL of each reagent required for the staining process was used for each well of the 4-chamber glass slide (Thermo fisher, MA, USA) containing 200 μL of ADSC-laden IPN hydrogels (n = 2). Confocal z-stack images were captured with Zeiss LSM710 (Carl Zeiss AG, Oberkochen, Germany) using a 20×/0.8 objective, with a 488 nm argon laser as the excitation source. The images were digitally stacked using Imaris 9.1.3 (Belfast, UK) to produce 3D overviews of encapsulated cells in the hydrogels.

2.5.5. Determination of the Effect of Secretome of Biologic Wound Dressing on Cell Migration (Wound Closure) Via 2D Scratch Assay

To assess if ADSC-laden IPN hydrogels could secrete chemotactic wound-healing factors to promote wound closure, in vitro 2D scratch assays were performed. First, ADSC-conditioned media were prepared by incubating 600 μL of ADSC-laden IPN hydrogels in 2 mL of serum-free culture media for 72 h in a 6-well plate. A total of 10 mL ADSC-conditioned media was collected. Gel-conditioned media were prepared in the same manner using cell-free IPN hydrogels. After the media were collected, they were centrifuged at 1500 rpm for 10 min, and the supernatant was stored at −80 °C until further use. Nonconditioned culture media supplemented with 10% (v/v) FBS (normal media) or without FBS were used as positive and negative controls, respectively. For this experiment, all culture media contain 10 ng/mL of TNF-α to induce the secretion of TSG-6 protein from encapsulated ADSCs.
The scratch assays were conducted using HDF and the CytoSelect 24-well Wound-Healing Assay Kit (Cell Biolabs Inc., CA, USA), according to the manufacturer’s protocol. After the wound gaps were formed, the HDF was exposed to 500 μL of ADSC-conditioned, gel-conditioned, normal, or serum-free media (n = 3). The differential rates of wound closure were monitored with a light microscope (Olympus CKX41, Japan). Blue-dotted lines were digitally drawn to demarcate the edges of cell migration at referred time points. Perpendicular lengths between the two blue-dotted lines denote the extend of wound gaps. The migration rate was determined according to eq 3. Wound gaps at other different time-points (Lc) were first subtracted from the initial wound gap (Li) and the resulting values were divided by Li for the calculation of % wound closure.
(3)

2.6. In Vivo Evaluation of ADSC-Laden IPN Hydrogels in the Murine Burn Wound Model

The NUS Institutional Animal Care and Use Committee (IACUC) approved all in vivo procedures. Forty-two 6–8 weeks old C57BL/6NTac female mice (InVivos Pte Ltd, Singapore) were randomly divided into four groups; comprising the ADSC-laden IPN hydrogels, cell-free IPN hydrogels, cell-free IPN hydrogels soaked in ADSC-conditioned media (ADSC-conditioned media hydrogels), and untreated (controls). Six samples were used per group. Three samples were followed throughout for wound closure and skin regeneration assessments. The other three samples were examined for percentage of polymorphonuclear (PMN) leucocytes and the number of macrophage infiltration after 7 days of treatment.
Inhalational induction anesthesia was achieved with 5% isoflurane (Attane, JD Medical, USA) and maintained at 1.5% isoflurane. Hairs on the lower right posterior dorsum area of mice were cleanly removed using Veet hair removal cream (Reckitt Benckiser Group, UK). Full thickness burn injuries were generated as previously described, (25) with minor modifications (Figures S1, S2 & Table S1). Preoperative subcutaneous (SC) Buprenorphine (0.1 mg/Kg BW) was given 30 min prior to burn wound creation. Next, a stainless-steel bar (96.2 g) was heated in a 100 °C water bath for 15 min before its template hot surface of 6 mm × 5 mm was placed on the shaven posterior-dorsum of each mouse for 30 s. Postoperative SC buprenorphine was given 8-hourly for 48 h. After which, the burn wounds were excised by removing the eschar, the wounds were then treated with 60 μL of hydrogels. All wounds, including controls, were covered with a secondary Tegaderm wound dressing. Analgesic SC buprenorphine was given 12-hourly until further assessment by a veterinary surgeon. Mice were housed individually in environmentally enriched cages throughout the experiment. They were euthanized by CO2 inhalation followed by cervical dislocation at the referred time points.

2.6.1. Histological Procedure

Skin explants were immediately harvested on days 7 and 21 after euthanasia. They were fixed overnight in 10% NBF at room temperature before being embedded in paraffin tissue blocks. One histological section was obtained per skin explant sample. Representative sections were stained with routine protocols for H&E and Masson’s trichrome. The photomicrographs of each sections were taken with an Olympus DP71 camera mounted on a BX51 microscope.

2.6.2. IHC Analysis

Skin explant samples were stained with the primary antibody of rabbit antihuman F4/80 (Abcam, Cambridge UK) using the Bond Refine Detection Kit (DS9800, Leica Biosystems, Wetzlar, Germany), according to the manufacturer’s protocol. Briefly, the primary antibody was added at 1:100 dilution for 15 min. The sections were then developed using Bond-mixed DAB chromogen for 7 min, and subsequently counterstained with hematoxylin for 5 min.

2.6.3. Image Analysis

2.6.3.1. Wound Closure Assessment
The percentage of wound closure was calculated using ImageJ by analyzing the planimetric digital images taken on days 0, 7, 14, and 21 post-treatment for all four groups of animals (n = 3 per time point), according to eq 4. The wound areas at other different time-points (Ac) were first subtracted from the wound area on day 0 (Ai), and the resulting values were divided by Ai for the calculation of % wound closure. Wounds were considered completely closed when the wound area was equal to zero.
(4)
2.6.3.2. Quantification of Percentage PMN Leukocytes and the Number of F4/80+ Macrophages Infiltration after 7 days of Treatment
The lobed nuclei of PMN leukocytes were stained dark red with H&E. Percentage area of PMN leukocyte infiltration was determined using ImageJ against the lighter background (n = 3). The number of F4/80+ macrophages was counted using a manual cell counter (n = 3). For both quantifications, we analyzed a random selection of three high power fields (100×) per dermal region under the site of injury, representing a total of nine fields per mouse.

2.6.3.3. Skin Regeneration and Dermal Differentiation

Skin appendage regeneration was assessed after 21 days of treatment on Masson’s Trichrome-stained histological samples (n = 3). Skin regeneration was determined based on the presence of a mature epithelial structure with collagenous hair follicles and sebaceous glands. To distinguish these cells from the surrounding connective tissues, their nuclei and cytoplasm were stained dark red and red, respectively. The degree of dermal differentiation was graded according to the criteria: grade 1, thin, dense, and monotonous fibrosis; 2, thicker but still dense and monotonous fibrosis; 3, two layers but not completely discrete; and 4, two discrete layers with superficial fibrosis and loose alveolar tissue within the deep layer.

2.7. Statistical Analysis

Statistical analysis was performed using GraphPad Prism v7.0.3. Data are presented as mean ± standard deviation (SD). One-way ANOVA was carried out between different experimental groups. Subsequent individual student t-tests were performed to determine statistically significant differences (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001).

3. Results and Discussion

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3.1. Synthesis and Characterization of IPN Hydrogels

In this study, an ADSC-laden IPN hydrogel based on gellan gum and collagen was successfully formulated (Figure 1). The sequential fabrication process was systematically illustrated using the qualitative inversion tests (Figure 2A). ADSCs were first suspended in the precursor solution of neutralized type-1 collagen and low-acyl gellan gum. Heat-induced polymerization of collagen transformed the clear, free-flowing precursor solution into a solid mass capable of holding its own weight. This indicated that a semi-IPN hydrogel of cross-linked collagen network interspersed with uncross-linked gellan gum polymers was formed. Next, gellan gum chains were cross-linked with the addition of MgCl2, and the same solid mass of a full-IPN hydrogel retained its structural integrity.

Figure 1

Figure 1. Assembly of ADSC-laden gellan gum–collagen IPN hydrogel from low-acyl gellan gum and type-1 rat-tail collagen polymers. Schematic representation of fabrication strategy through the sequential collagen fibrillogenesis and gellan gum cross-linking. Solutions of ice-cold neutralized type-1 collagen and preheated low-acyl gellan gum were mixed to form a precursor solution. This solution was kept sterile and at 37 °C until further use. If cell encapsulation was necessary, the precursor solution was used to resuspend ADSCs. Next, this precursor solution was cast into any container of desired dimensions before being heated at 37 °C for 30 min for collagen fibrillogenesis to form the semi-IPN. The gellan gum polymers were then cross-linked by the addition of the magnesium chloride solution.

Figure 2

Figure 2. Physical characterization of IPN hydrogels. (A) (from left to right) 0.4% gellan gum, 0.4% gellan gum and 1 mg/mL collagen precursor solution, semi-IPN hydrogel formed after heating precursor solution at 37 °C for 30 min, and full-IPN hydrogel formed after cross-linking semi-IPN with 0.02% MgCl2. (B) Gross image of the IPN hydrogel conforming to a cylindrical shape (scale bar: 1 cm). SEM images at (C) 50× and (D) 150× zoom (scale bar: 100 μm). (E) SWF uptake and (F) degradation profiles of the IPN hydrogels. The mean (n = 3) and SD are shown.

In order to illustrate the IPN hydrogels’ robustness in being handled, we extruded a cylindrical specimen of about 1.2 cm in diameter from the bore of a 3 mL syringe. The IPN hydrogel specimen could be deposited onto a flat surface without breaking and deforming (Figure 2B). Furthermore, the precursor solution was able to conform to the shape of their container before cross-linking. This suggested that our IPN hydrogel possess the ability to contour undulating wound surfaces, providing a more encompassing wound barrier function.

3.1.1. Porous Microstructure of IPN Hydrogels

To reveal their internal morphology, the cross sections of IPN hydrogels were immediately examined by SEM after they were formed. Internal microstructure of the IPN hydrogels appears to be highly fibrous and porous, the pores have a longest diameter of ∼150 μm (Figure 2C,D). We rationalized that an adequate porosity of the IPN hydrogels is necessary to enable cell motility, as well as exchange of nutrients and waste materials with the external environment. Moreover, the IPN hydrogels’ fibrous structure is representative of the native skin ECM. (26)

3.1.2. Equilibrium SWF Uptake and Hydrogel Degradation in SWF

The in vitro SWF uptake profile of IPN hydrogels was evaluated for over 1 day in a simulated wound environment. Although a denser network may reduce an IPN hydrogels’ swelling capacity, our variant still managed to absorb up to 157.9 ± 7.46% of its original wet weight in the presence of SWF, after ∼120 min of incubation (Figure 2E). Given that the capacity to swell is an important criterion for hydrogel wound-dressing materials to protect wounds from maceration, our IPN hydrogels could serve to effectively manage burn wound exudate.
For physically cross-linked hydrogels, gradual disruption of cross-linkage through the ion-exchange mechanism will result in the seepage of polymers and decrease in gel mass. Hence, reduction in swollen weights of the IPN hydrogels was used as a measure of degradation. The in vitro degradation profile of IPN hydrogels was evaluated for 21 days after they had reached their equilibrium swelling capacity. Our results showed that 80% of the IPN hydrogels remained after 12 days of incubation with SWF (Figure 2F). Hence, the structural integrity of the IPN hydrogels is likely to retain on wound beds for at least a week. This is important because the recruitment of cells involved in curtailing inflammation peaks between five to seven days after an injury. (27) Besides, prior studies have also shown that the inflammatory stage of wound healing should not last beyond four to six days. (28,29) As a result, the rate of our IPN hydrogel degradation will be appropriate for residing ADSCs to exert its full anti-inflammatory effects.

3.2. Rheological Properties of IPN Hydrogels

Cellular behaviors such as cell adhesion, migration, and differentiation are highly dependent on mechanical cues from cell-hydrogel mechanosensing interactions. (30,31) To understand how the stiffness of our IPN hydrogels affects ADSCs, we examined their rheological properties extensively. First, a linearity limit of 1.1% shear strain, which thereafter resulted in a drastic hydrogel deformation, was obtained from the amplitude sweep measurements (Figure 3A). Subsequent rheological measurements of the IPN hydrogels were performed with 1% shear strain, within the LVR. Next, we performed frequency sweeps (Figure 3B) to show that IPN hydrogel’s storage moduli (G′) were higher than loss moduli (G″) throughout the range of frequencies of shear, demonstrating its stable gel-like property.

Figure 3

Figure 3. Rheological properties of IPN hydrogels. Storage modulus (G′) and loss modulus (G″) of IPN hydrogels were determined in (A) amplitude, (B) frequency, (C) temperature, and (D) time sweeps. Functions of G′ and G″ show constant plateau values within the LVR of <1.1% shear strain in amplitude sweeps. Hydrogels display stable gel-like properties when G′ remains > G″ in frequency sweeps over the frequency range of 0.1–100 rad/s. For temperature-dependent functions of G′ and G″, sol/gel transition is achieved at the crossover point G′ = G″. Damping factor: tan(δ) = G″/G′, this value should be ≤0.1 when gellan gum polymers anneal at Tc–h to form gel-like materials. When the damping factor is ≤0.1, G′ is at least 1-log greater than G″. Displayed graphics are representative of each condition. The mean (n = 3) and SD are shown.

The incorporation of a secondary collagen network strengthened the overall hydrogel mechanical properties. A systematic increase of the storage modulus from 621.1 ± 83.4 Pa of pure gellan gum hydrogels, to 1303.9 ± 144.6 Pa of semi-IPN, and to 2353.9 ± 110.2 Pa of full IPN was observed (Tables S2, S3). Gel yield was also increased from 85.9 ± 5.2% of pure gellan gum hydrogels to 95.8 ± 1.52% of IPN hydrogels (Table S4). The final storage modulus of the IPN hydrogels is about ∼3× and ∼16× higher than pure gellan gum and type-1 collagen hydrogels’ storage moduli, respectively. David et al. attributed this phenomenon to the entanglement enhancement effect whereby the two interlaced yet separate polymeric networks are tightened because of topological constraints. (32) Evidently, such a mechanical strengthening effect is more pronounced in full-IPNs as compared to semi-IPNs whereby the secondary polymer is not cross-linked.
In terms of cell adhesion and cell fate, the resulting stiffness of the IPN hydrogels was also a good fit for ADSCs. Although type-1 collagen is known to induce osteogenesis even under basal conditions, (33) previous studies suggested that the key precipitator in stem cell differentiation for hydrogel-based 3D culture is the bulk matrix stiffness. (34,35) Osteogenesis was only observed when the complex modulus of hydrogels is above 37 kPa. In contrast, the IPN hydrogel cannot be too soft as previous studies have suggested that only hydrogels with a storage modulus of >1600 Pa supported mechanotransduced cell adhesion. (36) With a storage modulus of 2353.9 ± 110.2 Pa, the optimal stiffness of our IPN hydrogels was obtained after an extensive polymer concentration optimization process [gellan gum: 0.3–0.5% (w/v); collagen: 1–2 mg/mL; and MgCl2: 0.02–0.03% (w/v)].
Apart from stiffness, the concentration of collagen was also adjusted from the commonly reported 2 to 1 mg/mL to prevent phase separation with gellan gum and facilitate its complete bulk gel interpenetration (Table S3). (21,37) In contrast to multiphasic gellan gum hydrogels, (19) uniform interpenetration is critical for single-phase IPN hydrogels to achieve homogenous properties. In addition, the cross-linker (Mg2+) was specifically chosen for its anti-inflammatory properties, (38) and its concentration was carefully adjusted for enhanced ADSC survival (Figure S3). Overall, we navigated the complex interplay of the IPN’s components and developed a hydrogel that is conducive for ADSC encapsulation as well as biologic wound healing.
More importantly, the physical mechanical strengthening effect of an IPN has allowed us to employ a low concentration of 0.4% (w/v) gellan gum. To confer sufficient mechanical strength, unmodified gellan gum hydrogels have been commonly formulated at final concentrations of >0.7% (w/v), and cross-linked using ≥0.3% (w/v) of a divalent cation, which has a high Tc–h (>40 °C). (23) When exposed to a colder temperature of 37 °C, the temperature maintained for cells, gellan gum polymers anneal and form a viscous gel-like material that is difficult to handle for cell suspension. To gain a deeper understanding of how using a lower concentration of gellan gum has lowered its Tc–h and thereby allowed us to fabricate the IPN hydrogel, we performed decreasing temperature sweeps on the precursor solution. As shown in Figure 3C, the crossover of G′ & G″ occurred at a Tc–h of ∼36 °C, which is ∼1.5 °C lower than that of the IPN precursor formed with 0.5% (w/v) gellan gum (Figure S4). Furthermore, this implied that the precursor solution formed with the optimized 0.4% (w/v) gellan gum and 0.2% (w/v) MgCl2 remained liquid like at 37 °C for adequate cell resuspension and rapidly turned viscous to maintain even cell distribution upon aspiration onto the colder cell culture plate surface.
In another aspect, time sweep measurements were conducted to determine the duration required for gellan gum polymers interspersed within the IPN networks to achieve gelation. Upon addition of MgCl2, a low damping factor of ∼0.1 associated with higher storage than loss moduli was observed from the beginning of the measurements (Figure 3D). This indicates that gelation was achieved within 5 s of adding the cross-linker. Given together with the fact that temperature on the human skin surface is 34 °C, (39) it implies that our ADSC-suspended precursor solution could first be heated at 37 °C for collagen fibrillogenesis, before being aspirated onto a patient’s burn wound for rapid MgCl2 cross-linking. The quick gelling time on skin surface temperature may also allow our biologic wound dressing to be delivered in situ via an injectable system. (40)

3.3. In Vitro Evaluation of ADSC-Laden IPN Hydrogels

3.3.1. IPN Hydrogels Facilitated Cell Growth and Adhesion for Encapsulated ADSCs

Metabolic activity of the encapsulated cells, determined via the colorimetric MTS assay, was used as an indirect measure of cell viability. ADSCs encapsulated within our IPN hydrogels gradually proliferated over the full duration of incubation (Figure 4A). Their cell viability was also slightly higher as compared to ADSCs encapsulated within the gold-standard pure type-1 collagen hydrogels. Next, to ascertain the cell-adhesive microenvironment of our IPN hydrogels, we observed changes in the cytoskeleton of encapsulated ADSCs over 21 days of culture using CLSM. After 3 days of culture, encapsulated ADSCs began to spread and form cellular protrusions (filopodia) into the IPN hydrogel matrix (Figure 4B). From day 7 onwards, the cellular protrusions appeared more elongated while progressive interconnected networks with the neighboring cells were also observed. Visually, cell density also increased in accordance with the cell viability data. In clear disparity, cells remained in their rounded morphology in the absence of collagen (pure gellan gum hydrogels), their viability rapidly reduced after just 7 days of incubation (Figure S5A–C).

Figure 4

Figure 4. Validation of the cell-conducive environment of IPN hydrogels. (A) Cell viability of ADSCs encapsulated within type-1 collagen and IPN hydrogels. Cell viability was quantified on referred time points using the MTS assay. ADSC proliferation is shown by the increasing percentage of viable cells in comparison to that determined on day 0 of culture. The mean (n = 3) and SD are shown. (B) Change in the morphology of ADSCs encapsulated within IPN hydrogels. F-actin (cytoskeleton) of cells was stained green with the Phalloidin-iFluor 488 reagent and z-stack images were captured with CLSM using Zeiss LSM 710 to analyze the entirety of the cell-laden hydrogels at referred time points over a culture duration of 21 days. Cell adhesion to, and cell spread within, IPN hydrogels could be observed in all three dimensions (scale bar: 100 μm).

It is known that collagen is not degraded by simple hydrolysis but by proteolysis. (41) Although we did not examine our hydrogel’s degradation profile in the presence of collagenase and cells, a recent study has shown that MSCs residing in hydrogels with a stiffness of ∼2400 Pa have negligible cell-induced degradation. (42) Furthermore, polymerized collagen fibers in the IPN hydrogel facilitated ADSC growth and adhesion throughout the 21 days of incubation (Figure 4B). This result is a significant improvement of gellan gum-based biomaterials for cell-based wound-healing purposes. In a previous study by Cerqueira et al., (43) an ADSC-laden semi-IPN hydrogel construct based on spongy-like gellan gum and hyaluronic acid was designed for excisional skin wound regeneration. (43) However, unlike collagen fibrils, (21,44) hyaluronic acid polymers do not undergo gelation under physiological conditions and lack cell adhesive properties. (45) As a result, the free hyaluronic acid polymers have negligible retention, ∼50% of the hydrogels’ mass was lost in vitro after 21 days in the presence of hyaluronidase while cell adhesion was only shown for up to 2 days of culture. In contrast, our collagen-infused IPN hydrogels provided a dynamic, cell-conducive environment for encapsulated ADSCs to adhere, spread, and proliferate for up to 21 days of cell culture. Together and conclusively, our results suggested the successful incorporation of ADSCs into the IPN hydrogels, enabling the formulation of an effective biologic wound dressing.

3.3.2. Secretome of Biologic Wound Dressings Improved Cell Migration of HDF (Wound Closure)

HDFs are cells that migrate over wound beds to restore skin integrity after an injury. This process was simulated in our 2D scratch assays (Figure 5A). In response to growth and migratory factors found in FBS (normal media), HDF migrated and covered up to 82.4 ± 4.45% of the wound gap after 48 h of incubation (Figure 5B). Whereas, the lack of serum supplementation (serum-free media) prevented the migration of HDF significantly, achieving a percentage wound closure of only 18.9 ± 6.81%. In contrast, the ADSC-conditioned media appeared to provide chemotactic stimuli to HDF, promoting HDF migration up to as far as 71.5 ± 3.6% of the wound gap. The cell-free IPN hydrogels did not release any substrates that could promote cell growth or migration. Percentage wound closure for HDF exposed to gel-conditioned media only managed 22.1 ± 10.8% after 48 h of incubation.

Figure 5

Figure 5. Effect of ADSC-conditioned media on the migration rate of HDF. (A) Representative images of migration of HDF over a period of 48 h into a “wounded” area created by the presence of a mechanical insert producing a 0.9 mm cell-free distance. The blue-dotted lines denote the edges of cell migration and the distances between them were used to calculate (B) percentage wound closure (migration) rates of HDF when they were exposed to different kinds of media (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001) (n.s. = not significant). The mean (n = 3) and SD are shown.

3.4. In Vivo Evaluation of the Safety and Efficacy of Biologic Wound Dressing

Key health parameters of the mice such as change in the body weight, signs of dehydration and general alertness were examined at least thrice a week for all mice. All mice survived the procedure and were taken into statistical considerations.

3.4.1. Biologic Wound Dressings Promoted Murine Full Thickness Burn Wound Closure

Wound size areas of each mice from each group were followed at referred time points to determine if the hydrogel treatment exerted any effect on wound closure (Figure 6A). ADSC-laden hydrogels (biologic wound dressing) had a significant effect on early wound closure, wounds treated exhibited significantly smaller wound size areas against the controls at days 3 and 7 (P < 0.0001) (Figure 6B). All wounds progressively close over the duration of experiment, reaching complete wound closure by day 21 of treatment. Although wounds exposed to ADSC-conditioned media hydrogels achieved significant faster wound closure than controls by day 3, the effect was significantly lesser than ADSC-laden hydrogels (P < 0.0001). Moreover, this effect could not be sustained beyond 3 days of therapy.

Figure 6

Figure 6. Effect of hydrogel treatment on the wound closure rate of full-thickness burn wounds. (A) Representative macroscopic images of the wounds were taken at referred time points over a duration of 21 days (scale bar: centimeter ruler). Using ImageJ, wound areas of the images were analyzed and expressed against that of day 0’s to determine (B) percentage wound closure for up to 21 days of treatment (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001). The mean (n = 3) and SD are shown.

Hence, the results from both the in vitro 2D scratch assay and the in vivo study have demonstrated that the ADSC-laden IPN hydrogels enhanced cell migration and promoted early wound closure. These effects could be attributed to the wide variety of chemotactic factors secreted by ADSCs that promote the movement of various types of cells to wound beds for granulation to take place. They include interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), and Chemokine (C–C motif) ligand 5 (CCL5). (46) On the other hand, in the absence of ADSCs, soluble extracts from cell-free IPN hydrogels (gel-conditioned) exerted a minimal effect on HDF cell migration. While both cell-free and ADSC-conditioned media IPN hydrogels induced significantly less early murine wound closure than ADSC-laden IPN hydrogels.
Nonetheless, it is important to note that type-1 collagen is the main connective tissue component in skin ECM. Therefore, we postulate that, by exposing the skin cells to their native biochemical environment, the type-1 collagen-containing IPN hydrogels were able to promote their migration and recruitment on burn wounds. Besides, it was previously illustrated that hydrogels capable of promoting local cell–cell interaction could potentiate the release of paracrine wound-healing factors by mesenchymal stem cells. (47) Hence, in contrast to the gellan gum-hyaluronic acid and gellan gum-gelatin variants, (43,48) early wound closure profiles were observed with all types of our hydrogels, including cell-free and ADSC-conditioned media hydrogels, when applied on the in vivo murine burn wound model.

3.4.2. Immunomodulation of Biologic Wound Dressings Via Reduction in Percentage of PMN Leukocytes and Macrophage Infiltration at Murine Full Thickness Burn Wounds

PMN leukocytes are the predominant cells recruited to sites of cutaneous injury during the acute inflammatory phase of wound healing, which typically spans 1–3 days. (49) Persistent residing of PMN leukocytes and/or macrophages beyond 7 days of treatment may indicate chronic nonhealing inflammatory states. Therefore, we could indirectly compare the treatment groups’ differential immunosuppressive effects by quantifying the amount of PMN leukocytes and/or macrophages wound infiltration on day 7 of therapy. Our results showed that significant lower amounts of PMN leukocytes (Figure 7A–D) and macrophages (Figure 7F–I) were accumulated at the wound dermis layer after 7 days of treatment with all types of hydrogels. However, ADSC-laden hydrogels promoted the most significant reduction in inflammatory cell recruitment as compared to the other hydrogel treatment groups (P < 0.0005) (Figure 7E,J). This result could be attributed to the secretion of TSG-6 protein by encapsulated ADSCs, which has been demonstrated to be a powerful anti-inflammatory factor. (50)

Figure 7

Figure 7. Effect of hydrogel treatment on wound inflammation. H&E and bright-field immunolabeling for Immunohistochemistry (IHC) sections of the wound dermal region that were (A,F) untreated, treated with (B,G) cell-free hydrogels, (C,H) ADSC-conditioned media hydrogels, or (D,I) ADSC-laden hydrogels. (Scale bar: 100 μm). The lobed nuclei of PMN leukocytes (indicated by arrows) were stained dark red, while F4/80+ macrophages were stained dark brown (indicated by arrows). Using ImageJ and manual cell counter, (E) percentage areas of dark red infiltrates and (J) number of dark brown cells were analyzed, respectively (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001). The mean (n = 3) and SD are shown.

In a previous study, TSG-6 was shown to behave as a MAPK inhibitor by suppressing the P38 and JNK signaling pathways. (51) It was further revealed that attenuation of these pathways led to reduced levels of inflammatory cytokines and inflammatory cells infiltration at wound site after severe burns. Using ELISA, we showed that ADSC encapsulated within our IPN hydrogels could secrete TSG-6 upon stimulation with a wound cytokine, 10 ng/mL of TNF-α for 72 h. Our results showed that 2.03 ± 0.379 ng/mL of TSG-6 was secreted (Figure S6). This is in agreement with a previous study whereby MSCs’ transcription of TSG-6 was found to be upregulated by about 20-fold in the presence of TNF-α, a wound cytokine. (14) More importantly, this biochemical effect of TSG-6 protein was translated into tissue-level application by our ADSC-laden IPN hydrogels. This explains the significant reduction in inflammatory PMN leukocyte recruitment to burn wound sites when biologic wound dressings were applied, which is in agreement with previous studies that wounds treated with ADSCs have reduced numbers of inflammatory cells. (52)
Cell-free and ADSC-conditioned media hydrogels were also able to reduce PMN leucocyte recruitment. Previous study has demonstrated that polymerized type-1 collagen has the propensity to downregulate inflammation. (53) However, as with wound closure, these effects, even when supplanted with ADSC-conditioned media, were less prominent than ADSC-laden hydrogels. This is likely because, unlike the in vitro environment where there is no disruption to the supply of wound healing factors, proteolysis and drainage of exudates occur continuously on tissue-level wound beds. (54) Therefore, the presence of ADSCs is likely necessary for continual secretion, and hence paracrine activity, so that significant wound-healing effects could be derived in the physiological environment.

3.4.3. Biologic Wound Dressings Promoted Complete Skin Regeneration and Dermal Differentiation of Murine Full Thickness Burn Wounds

The presence of collagenous structures denotes an active dermal regeneration process, as epithelial maturation involves the infiltration of collagenous skin appendages. We examined Masson trichrome-stained sections after 21 days of treatment and observed that ADSC-laden hydrogels promoted skin regeneration; treated wounds displayed a maturing dermal layer with the presence of hair follicles and sebaceous glands (Figure 8A–D). To a lesser extent, skin regeneration could also be observed for wounds treated with cell-free and ADSC-conditioned media hydrogels. The results from histology correlated with less scarring of the wounds. As observed macroscopically in Figure 6A. However, the improvement in terms of degree of dermal differentiation for treated wounds was not statistically significant, as compared to controls (Figure 8E).

Figure 8

Figure 8. Effect of hydrogel treatment on promoting skin regeneration. Masson’s trichrome staining for sections of wounds that are (A) untreated, treated with (B) cell-free hydrogels, (C) ADSC-conditioned media hydrogels, or (D) ADSC-laden hydrogels to indicate distinct collagen structures (indicated by arrows) in the dermis layer by day 21. (Scale bar: 100 μm). (E) Degree of dermal differentiation of wounds after 21 days of treatment, obtained after a graded qualitative analysis. Nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison tests were conducted (P < 0.05). The mean (n = 3) and SD are shown.

Full thickness burns are characterized by the complete loss of skin appendages leading to irreversible scarring. (55) Despite being the current gold standard of treatment, skin grafts are deficient in enabling complete skin regeneration. (56) Biologic wound dressings, particularly stem cell-based ones, are designed to fulfil this huge medical gap, and have been their idiosyncrasy of research focus. Developing hydrogels that possess this capability have become quintessential research objectives. In this study, we demonstrated epithelial repair and mature epithelial morphology with hair follicles and sebaceous glands after 21 days of hydrogel application. Given that ADSCs can migrate to wound sites and differentiate into skin cells, (57) it explains why our results suggested that this effect was exhibited more by the ADSC-laden hydrogels. Excessive inflammation in the early stages of healing has been identified as a causative factor in the formation of scarring and fibrosis. (58) Therefore, as cell-free and ADSC-conditioned media hydrogels were able to slightly reduce inflammation, slight skin regeneration could also be observed.
To our knowledge, this is the first time unmodified gellan gum, without the need for chemical or pharmaceutical modifications, has been proposed as a biologic wound dressing for third-degree burns. Taken together, the results in this study ratify the significance of understanding the mechanistic gelation of gellan gum and collage and serve to instruct the development of other compelling IPN hydrogel for wound dressings. In addition, our biologic wound dressing adds to a rapidly growing body of synthetic cell-based constructs as viable alternatives to the current gold standard therapy of applying autologous skin grafts for full thickness burn wounds.

4. Conclusions

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Altogether, we have shown that by understanding the temperature-dependent gelation requirements of gellan gum and collagen through rheology, mechanically and biologically enhanced IPN hydrogels between the two natural polymers could be fabricated without chemical modifications. The IPN hydrogels recapitulated a cell-conducive environment for ADSCs, allowing the stem cells to be delivered to third-degree burn wound beds. As a result, we have successfully transformed pristine gellan gum into a biologic wound dressing, capable of improving early wound closure, reducing inflammation, and promoting complete skin regeneration for third-degree burn wounds. Apart from autologous skin grafts, burn specialists could now be armed with an additional option of our biologic wound dressing to effectively manage severe burns.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.0c01363.

  • In vivo full-thickness burn wound creation procedure; pathologist’s evaluation of the degree of burn wound created for the in vivo full-thickness burn wound model; Tc–h of the IPN precursor solution formed with 0.5% (w/v) gellan gum; optimization of the gelation protocol of IPN hydrogels based on rheology and cytotoxicity; gel yield of IPN and pure gellan gum hydrogels; cellular environment of pure gellan gum hydrogels; and ELISA assay for human TSG-6 (PDF)

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Author Information

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  • Corresponding Author
    • Pui Lai Rachel Ee - Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, SingaporeNUS Graduate School for Integrative Sciences and Engineering, 21 Lower Kent Ridge Road, 119077, SingaporeOrcidhttp://orcid.org/0000-0002-7277-6233 Email: [email protected]
  • Authors
    • Jian Yao Ng - Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
    • Xiao Zhu - Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
    • Devika Mukherjee - Department of Pharmacy, Faculty of Science, National University of Singapore, 5 Science Drive 2, 117545, Singapore
    • Chi Zhang - Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
    • Shiqi Hong - Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
    • Yogesh Kumar - Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
    • Rajeev Gokhale - Roquette Singapore Innovation Center, Helios, 11 Biopolis Way, #05-06, 138667, Singapore
  • Notes
    The authors declare the following competing financial interest(s): Authors Chi Zhang, Shiqi Hong, Yogesh Kumar, and Rajeev Gokhale were employed by the company Roquette Asia Pacific Pte Ltd, Singapore. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

    Authors C. Z., S. H., Y. K., and R. G. were employed by the company Roquette Asia Pacific Pte Ltd, Singapore. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

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The authors would like to acknowledge research funding and facilities provided by the National University of Singapore and Ministry of Education Academic Research Fund (R148000287114) and Roquette Asia Pacific Pte Ltd (R148000251592) awarded to P.L.R. Ee and NUS- President's Graduate Scholarship to J.Y. Ng. We thank Lee Shu Ying and her colleagues at the confocal unit of Yong Loo Lin School of Medicine (YLLSOM) for their assistance in confocal imaging. We also acknowledge Dr Ong Chee Bing and his team at Advanced Molecular Pathology Laboratory (AMPL)–Institute of Molecular and Cell Biology (IMCB) A*Star for their assistance with histological staining and imaging.

Abbreviations

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ADSC

adipose-derived stem cell

BW

body weight

CCL5

chemokine ligand 5

CLSM

confocal laser scanning microscopy

DMEM

Dulbecco’s Modified Eagle Medium

ECM

extracellular matrix

ELISA

enzyme-linked immunosorbent assay

FBS

fetal bovine serum

G′

storage modulus

G”

loss modulus

H&E

hematoxylin and eosin

HDF

human dermal fibroblast

hMSC

human mesenchymal stem cell

IACUC

institutional animal care and use committee

IHC

immunohistochemistry

IL-6

interleukin-6

IL-8

interleukin-8

IPN

interpenetrating network

LVR

linear viscoelastic region

MCP-1

monocyte chemoattractant protein-1

MTS

(3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfofenyl)-2H-tetrazolium)

NBF

neutral buffered formalin

OD

optical density

PBS

phosphate-buffered saline

PES

polyethersulfone

PMN

polymorphonuclear

SC

subcutaneous

SD

standard deviation

SEM

scanning electron microscopy

SWF

simulated wound fluid

Tc-h

transition temperature of coil-helix

TNF-α

tumor necrosis factor-alpha

TSG-6

TNF α-stimulated gene 6.

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  • Abstract

    Figure 1

    Figure 1. Assembly of ADSC-laden gellan gum–collagen IPN hydrogel from low-acyl gellan gum and type-1 rat-tail collagen polymers. Schematic representation of fabrication strategy through the sequential collagen fibrillogenesis and gellan gum cross-linking. Solutions of ice-cold neutralized type-1 collagen and preheated low-acyl gellan gum were mixed to form a precursor solution. This solution was kept sterile and at 37 °C until further use. If cell encapsulation was necessary, the precursor solution was used to resuspend ADSCs. Next, this precursor solution was cast into any container of desired dimensions before being heated at 37 °C for 30 min for collagen fibrillogenesis to form the semi-IPN. The gellan gum polymers were then cross-linked by the addition of the magnesium chloride solution.

    Figure 2

    Figure 2. Physical characterization of IPN hydrogels. (A) (from left to right) 0.4% gellan gum, 0.4% gellan gum and 1 mg/mL collagen precursor solution, semi-IPN hydrogel formed after heating precursor solution at 37 °C for 30 min, and full-IPN hydrogel formed after cross-linking semi-IPN with 0.02% MgCl2. (B) Gross image of the IPN hydrogel conforming to a cylindrical shape (scale bar: 1 cm). SEM images at (C) 50× and (D) 150× zoom (scale bar: 100 μm). (E) SWF uptake and (F) degradation profiles of the IPN hydrogels. The mean (n = 3) and SD are shown.

    Figure 3

    Figure 3. Rheological properties of IPN hydrogels. Storage modulus (G′) and loss modulus (G″) of IPN hydrogels were determined in (A) amplitude, (B) frequency, (C) temperature, and (D) time sweeps. Functions of G′ and G″ show constant plateau values within the LVR of <1.1% shear strain in amplitude sweeps. Hydrogels display stable gel-like properties when G′ remains > G″ in frequency sweeps over the frequency range of 0.1–100 rad/s. For temperature-dependent functions of G′ and G″, sol/gel transition is achieved at the crossover point G′ = G″. Damping factor: tan(δ) = G″/G′, this value should be ≤0.1 when gellan gum polymers anneal at Tc–h to form gel-like materials. When the damping factor is ≤0.1, G′ is at least 1-log greater than G″. Displayed graphics are representative of each condition. The mean (n = 3) and SD are shown.

    Figure 4

    Figure 4. Validation of the cell-conducive environment of IPN hydrogels. (A) Cell viability of ADSCs encapsulated within type-1 collagen and IPN hydrogels. Cell viability was quantified on referred time points using the MTS assay. ADSC proliferation is shown by the increasing percentage of viable cells in comparison to that determined on day 0 of culture. The mean (n = 3) and SD are shown. (B) Change in the morphology of ADSCs encapsulated within IPN hydrogels. F-actin (cytoskeleton) of cells was stained green with the Phalloidin-iFluor 488 reagent and z-stack images were captured with CLSM using Zeiss LSM 710 to analyze the entirety of the cell-laden hydrogels at referred time points over a culture duration of 21 days. Cell adhesion to, and cell spread within, IPN hydrogels could be observed in all three dimensions (scale bar: 100 μm).

    Figure 5

    Figure 5. Effect of ADSC-conditioned media on the migration rate of HDF. (A) Representative images of migration of HDF over a period of 48 h into a “wounded” area created by the presence of a mechanical insert producing a 0.9 mm cell-free distance. The blue-dotted lines denote the edges of cell migration and the distances between them were used to calculate (B) percentage wound closure (migration) rates of HDF when they were exposed to different kinds of media (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001) (n.s. = not significant). The mean (n = 3) and SD are shown.

    Figure 6

    Figure 6. Effect of hydrogel treatment on the wound closure rate of full-thickness burn wounds. (A) Representative macroscopic images of the wounds were taken at referred time points over a duration of 21 days (scale bar: centimeter ruler). Using ImageJ, wound areas of the images were analyzed and expressed against that of day 0’s to determine (B) percentage wound closure for up to 21 days of treatment (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001). The mean (n = 3) and SD are shown.

    Figure 7

    Figure 7. Effect of hydrogel treatment on wound inflammation. H&E and bright-field immunolabeling for Immunohistochemistry (IHC) sections of the wound dermal region that were (A,F) untreated, treated with (B,G) cell-free hydrogels, (C,H) ADSC-conditioned media hydrogels, or (D,I) ADSC-laden hydrogels. (Scale bar: 100 μm). The lobed nuclei of PMN leukocytes (indicated by arrows) were stained dark red, while F4/80+ macrophages were stained dark brown (indicated by arrows). Using ImageJ and manual cell counter, (E) percentage areas of dark red infiltrates and (J) number of dark brown cells were analyzed, respectively (*p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001). The mean (n = 3) and SD are shown.

    Figure 8

    Figure 8. Effect of hydrogel treatment on promoting skin regeneration. Masson’s trichrome staining for sections of wounds that are (A) untreated, treated with (B) cell-free hydrogels, (C) ADSC-conditioned media hydrogels, or (D) ADSC-laden hydrogels to indicate distinct collagen structures (indicated by arrows) in the dermis layer by day 21. (Scale bar: 100 μm). (E) Degree of dermal differentiation of wounds after 21 days of treatment, obtained after a graded qualitative analysis. Nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison tests were conducted (P < 0.05). The mean (n = 3) and SD are shown.

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    • In vivo full-thickness burn wound creation procedure; pathologist’s evaluation of the degree of burn wound created for the in vivo full-thickness burn wound model; Tc–h of the IPN precursor solution formed with 0.5% (w/v) gellan gum; optimization of the gelation protocol of IPN hydrogels based on rheology and cytotoxicity; gel yield of IPN and pure gellan gum hydrogels; cellular environment of pure gellan gum hydrogels; and ELISA assay for human TSG-6 (PDF)


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