A Low Molecular Weight Hyaluronic Acid Derivative Accelerates Excisional Wound Healing by Modulating Pro-Inflammation, Promoting Epithelialization and Neovascularization, and Remodeling Collagen

Recent knowledge of the cellular and molecular mechanisms underlying cutaneous wound healing has advanced the development of medical products. However, patients still suffer from the failure of current treatments, due to the complexity of healing process and thus novel therapeutic approaches are urgently needed. Previously, our laboratories produced a range of low molecular weight hyaluronic acid (LMW-HA) fragments, where a proportion of the glucosamine moieties were chemically N-acyl substituted. Specifically, N-butyrylation results in anti-inflammatory properties in a macrophage system, and we demonstrate the importance of N-acyl substituents in modulating the inflammatory response of LMW-HA. We have set up an inter-institutional collaborative program to examine the biomedical applications of the N-butyrylated LMW-HA (BHA). In this study, the potentials of BHA for dermal healing are assessed in vitro and in vivo. Consequently, BHA significantly promotes dermal healing relative to a commercial wound care product. By contrast, the “parent” partially de-acetylated LMW-HA (DHA) and the re-acetylated DHA (AHA) significantly delays wound closure, demonstrating the specificity of this N-acylation of LMW-HA in wound healing. Mechanistic studies reveal that the BHA-mediated therapeutic effect is achieved by targeting three phases of wound healing (i.e., inflammation, proliferation and maturation), demonstrating the significant potential of BHA for clinical translation in cutaneous wound healing.


Preparation and Characterization of LMW-HA Derivatives
The low molecular weight hyaluronic acid (LMW-HA) derivatives were synthesized and characterized as described previously [1]. The LMW-HA solutions were prepared in sterilized ultrapure water, and the endotoxin level in solutions was measured using an Endotoxin Assay kit (GenScript, Piscataway, NJ, USA) according to the manufacturer's instructions.
In addition, a gel formulation was prepared for in vivo studies. Briefly, 30 g of alginate, 450 g of glycerin, 2 g of ethylparaben, and 0.5 g of calcium gluconate were prepared in 1 L of sterilized ultrapure water to produce the Blank-Gel. In addition, LMW-HA solutions prepared as described above were added into the Blank-Gel to form the LMW-HA-Gel.

Wound Healing Efficacy
Four excisional full-thickness wounds (~1.8 cm 2 ) per animal were made with disinfected surgical scissors deep into the dermis, without damaging the subdermal vasculature on the dorsal surface (Day 0) ( Figure S10). On the same day, the wounds were treated daily with or without 0.2 mL of LMW-HA-Gel ([c] = 0.05, 0.1, 0.25, 0.5 and 1 mg/mL), and the wound diameter was measured at Day 3, 7, 10 and 14 ( Figure S3). The wound closure rate (6 wounds per group) was calculated as (1-Sn/S0) × 100%, where Sn = the wound surface area at a predetermined day, thus, S0 = the wound surface area at Day 0.
In addition, four excisional full-thickness wounds (~1.8 cm 2 ) per animal were made as described above. From Day 0, the wounds were treated daily with 0.2 mL of Blank-Gel (negative control group), carboxymethyl chitosan (CMC) ([c] of CMC = 5 mg/mL in CHITIN ® , a commercial wound care product purchased from Shijiazhuang Yishengtang Medical Supplies Ltd., Shijiazhuang, China) (positive control group), and LMW-HA-Gel ([c] of LMW-HA = 0.25 mg/mL) (Figure 1b), and the wound diameter was measured at Days 3, 7, 10 and 14. The wound closure rate (6 wounds per group) was calculated as described above.

In Vitro Studies
The Human Umbilical Vein Endothelial Cells (HUVEC) cell line was purchased from the American Type Culture Collection (ATCC, USA). Cells were maintained in RPMI-1640 medium (Corning, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and a Penicillin-Streptomycin Nystatin solution (Biological Industries, Israel) at 37 °C under a 5% CO2 atmosphere.
Cell proliferation was examined using a Matrigel-based (a liquid laminin/collagen gel) Endothelial Cell Tube Formation Assay [2]. Briefly, 200 μL of Matrigel (Corning) per well were added into 24-well plates. When the Matrigel solidified, HUVEC were seeded at a density of 3× 10 5 cells per well for 24 h. Cells were then treated with 0.05 mg/mL de-acetylated LMW-HA (DHA), AHA and N-butyrylated LMW-HA (BHA) in fresh growth medium for 48 h. Subsequently, these cells were observed using a microscope (Olympus BX53, Tokyo, Japan).
Cell migration was studied using the scratch assay [3]. Briefly, HUVEC were seeded in 6-well plates at a density of 4 × 10 5 cells per well to reach 100% confluence. The scratches were made by pipette tips through the monolayer in the middle of the plate. Cells were then treated with serumfree medium containing LMW-HA derivatives at 0.05 mg/mL. After 48 h, images were obtained using a microscope (Olympus BX53).

In Vivo Studies
Four excisional full-thickness wounds (~1.8 cm 2 ) per animal were made as described above, in which one wound was used as the untreated control, and three others were daily treated with 0.2 mL of Blank-Gel, CMC ([c] of CMC = 5 mg/mL), and LMW-HA-Gel ([c] of LMW-HA = 0.25 mg/mL) from Day 0. The whole wound specimen, without the surrounding healthy tissues, was collected for the following experiments (6 wounds were used on each time point per group in one experiment).
Determination of mRNA expression via reverse transcription polymerase chain reaction (RT-PCR) was performed as follows: The wounds within TriZol Up reagent (TransGen Biotech, Beijing, China) were homogenated using a tissue grinder (Scientz, Ningbo, Zhejiang, China). The homogenates were centrifuged at 10,000 g for 15 min at 4 °C to remove the insoluble debris, and the supernatant was collected for a reverse transcription polymerase chain reaction (RT-PCR). Firststrand cDNA was obtained from total RNA samples using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China). Quantitative real-time RT-PCR was carried out using the StepOnePlus™ Real-Time PCR System (Thermo Scientific). RT-PCR was performed under the following conditions: An initial denaturation step at 94 °C for 30 s, followed by 45 cycles of 5 s at 94 °C, annealing for 30 s at 60 °C. The primers used were listed in Table S1. The quantitative level of each target mRNA was measured as a fluorescent signal, corrected according to the signal for β-actin RNA.
In the H&E staining assay, the inflammatory cell infiltration, fibroblast proliferation and blood vessel formation were observed under a microscope (Olympus BX53). To quantify the inflammatory cell infiltration, the integrated optical density (IOD) and the number (n) of positive cells in slides were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., USA). The quantitative level in the inflammatory cell infiltration with the treatment of Blank-Gel, CMC and BHA-Gel was determined as the IOD/n corrected according to the untreated control group. To quantify the fibroblast proliferation, three areas of the epidermis layer in one slide were randomly selected to measure the mean of the epidermal thickness using Image-Pro Plus 6.0 software. The quantitative level in fibroblast proliferation with the treatment of Blank-Gel, CMC and BHA-Gel was determined as the mean of the epidermal thickness corrected according to untreated control group. In addition, the development of blood vessels was quantified based on the mean of new blood vessels in slides.
In Masson's trichrome staining assay, collagen depositions were observed under a microscope (Olympus BX53). As described above, the quantitative level in the collagen deposition with the treatment of Blank-Gel, CMC and BHA-Gel was measured as the IOD/n, corrected according to the untreated control group.

Figure S5.
Healing rate (%) treated AHA-Gel (AHA = 0.25 mg/mL) on Days 3, 5, 7, 10 and 14 when compared with the wound area on Day 0 (n = 6). ## p < 0.01 relative to untreated control group; && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to CMC. Figure S6. The protein level of TNF-α in wounds from rats (n = 6) treated with DHA-Gel was determined by ELISA, and was shown as the fold change to those of rats in the untreated control group. ## p < 0.01 relative to untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC. Figure S7. The protein level of TNF-α in wounds from rats (n = 6) treated with AHA-Gel was determined by ELISA and was shown as the fold change to those of rats in the untreated control group. ## p < 0.01 relative to untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC. Figure S8. The proliferation of Human Umbilical Vein Endothelial Cells (HUVEC) was assessed with AHA and DHA using the Endothelial Cell Tube Formation Assay (100x, Bar = 50 μm).