Effective Wound Healing by Antibacterial and Bioactive Calcium

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Bio-interactions and Biocompatibility

Effective Wound Healing by Antibacterial and Bioactive Calcium-Fluoride-Containing Composite Hydrogel Dressings Prepared Using In Situ Precipitation Seol-Ha Jeong, Da-Yong Shin, In-Ku Kang, Eun-Ho Song, Yun-Jeong Seong, Ji-Ung Park, and Hyoun-Ee Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00198 • Publication Date (Web): 12 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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ACS Biomaterials Science & Engineering

Effective Wound Healing by Antibacterial and Bioactive Calcium-FluorideContaining Composite Hydrogel Dressings Prepared Using In Situ Precipitation Seol-Ha Jeonga, Da-Yong Shina, In-Ku Kanga, Eun-Ho Songa, Yun-Jeong Seonga, Ji-Ung Parkb, Hyoun-Ee Kima,c,*

a Department of Materials Science and Engineering, Seoul National University, Seoul, South Korea b Department of Plastic and Reconstructive Surgery, Seoul National University College of Medicine, Seoul, South Korea c Biomedical Implant Convergence Research Center, Advanced Institutes of Convergence Technology, Suwon, South Korea * Corresponding author: Tel.: +82 2 880 8320; fax.: +82 2 884 1413. E-mail address: [email protected]

Keywords: Nanocomposite hydrogel, Calcium fluoride, In situ precipitation, Antibacterial, Wound healing

Abstract In this study, we report the development of a hyaluronic acid (HA)-based composite hydrogel containing calcium fluoride (CaF2) with good biocompatibility and antibacterial properties for multifunctional wound dressing applications. CaF2 was newly selected for incorporation within HA because it can release both Ca2+ and F− ions, which are well-known ions for affecting cell proliferation and inhibiting bacterial growth, respectively. In particular, an insitu precipitation process enables easy control over the released amount of F− ions by simply adjusting the precursor solutions (calcium chloride (CaCl2) and ammonium fluoride (NH4F)) used for the CaF2 precipitation. This study introduces a calcium fluoride (CaF2)-containing composite hydrogel with good biocompatibility and antibacterial properties for multifunctional wound-dressing applications. CaF2 particles were uniformly embedded within a HA based pure hydrogel using an in situ precipitation process. By varying the CaCl2 and NH4F concentrations used in the precipitation as well as the precipitation time, composite 1 ACS Paragon Plus Environment

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hydrogels with different ion-release profiles were obtained. By controlling the precipitation time, especially for 10 min and after 30 min, large differences in the ion-release profiles as a function of CaF2 concentration were observed. A shorter precipitation time resulted in faster release of fluoride, whereas for the 30-min and 1-h samples, sustained ion release was achieved. Colony tests and live/dead assays using Escherichia coli and Staphylococcus aureus revealed a lower density of bacteria on the CaF2 composite hydrogels than on the pure hydrogel for both strains. In addition, improved cellular responses such as cell attachment and proliferation were also observed for the CaF2 composite hydrogels than for the pure hydrogel. Furthermore, the composite hydrogels exhibited excellent wound healing efficiency, as evidenced by an in vitro cell migration assay. Finally, monitoring of the wound closure changes using a full-thickness wound in a rat model revealed the accelerated wound healing capability of the CaF2 composite hydrogels compared with that of the pure hydrogel. Based on our findings, these CaF2 composite hydrogels show great potential for application as advanced hydrogel wound dressings with antibacterial properties and accelerated woundhealing capabilities.

1. Introduction Wound healing is a global issue that poses several challenges as the aging population continues to grow. Indeed, unfavorable delayed healing conditions, including those associated with diabetes 1, ulcers 2, and persistent infections, lead to wound-healing failure accompanied by severe suffering.3 Therefore, the need to develop an effective wound dressing that enables expeditious wound healing has received considerable attention. An ideal wound dressing must prevent infection, allow gaseous exchange, and provide a hydrated environment.4 Recently, commercial wound dressing materials made of hydrogels have been widely been reported. Their advantages are owing to the presence of hydrophilic polymer chains, which can absorb a large amount of water.5 Hydrogel dressings can also 2 ACS Paragon Plus Environment

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provide moisture to wounds and properly absorb exudates from the wound. These dressings can be made from either synthetic or natural polymers. Among the dressings available in hydrogel form, hyaluronic acid (HA) has been recognized as exerting a positive effect on the wound-healing process because of its excellent biocompatibility, bioactivity, and biodegradability.6-8 However, its poor mechanical properties in the hydrated state make it difficult to achieve the necessary structural stability. Also, the demand for more advanced platforms for efficient wound therapy and the desire to achieve expeditious wound healing with several functionalities, such as bioadhesivity, bioactivity, or antibacterial activities, has resulted in great progress in the development of advanced wound dressing materials. One strategy to maximize the therapeutic effects of advanced hydrogel wound dressings is to incorporate several agents or other materials into the dressings to create composite hydrogel forms.9 For example, to achieve antibacterial effects, silver (Ag) nanoparticles10 and antibiotic-loaded inorganic carriers such as silica nanoparticles11 have been incorporated in hydrogels. Clusters of Ag atoms ranging in size from 1 to 100 nm have already been shown to be highly effective against bacteria and other eukaryotic microorganisms. In addition, smaller Ag nanoparticles up to 20 nm in size induce highly effective antibacterial effects via a reactive-oxygen-species-mediated cell death mechanism, which is induced by both these particles and the released Ag+ ions; thus, this finding makes Ag called a “nanoweapon against bacteria”.12 However, these Ag-based systems can be toxic in a cellular environment during the wound-healing process. Other systems utilizing inorganic carriers with antibiotics have also been widely developed.13 To overcome the rapid release of drugs and control the drug delivery systems, the microporous structure of silica nanoparticles has been extensively studied. Nevertheless, since silica itself is highly hydrophilic and porous, the burst release of drugs and fast dissolution of silica particles are unavoidable. To overcome these drawbacks of Ag- and silica-based systems, we investigated another antibacterial agent, fluorine ions (F−). Fluoride is a well-known inorganic material that 3 ACS Paragon Plus Environment


interferes with bacterial metabolism by inhibiting glycolytic enzymes14-15, and the effect of F− ions on antibacterial activity using various fluoride composites such as magnesium fluoride16 and calcium fluoride (CaF2)17 has been widely reported. In our work, CaF2 nanoparticles were selected for incorporation in a HA-based hydrogel to design a novel composite hydrogel system for advanced wound healing with antibacterial effects. Because Ca2+ ions are known to greatly affect fibroblast contraction and proliferation, resulting in a decrease of wound size18-19, we expected that a CaF2-embedded composite hydrogel would simultaneously achieve both biocompatibility and antibacterial property for wound healing. CaF2 in the composite hydrogel may offset the negative effects of toxicity of traditional agents, as well as its high crystallinity among fluoride composites, which may prevent the burst release of ions under physiological conditions. As there have not been any reports on the effect of CaF2 on the wound-healing process to date, this work highlights the potential for developing CaF2 composite hydrogels using the in situ precipitation process described in our previous work. This process enables easy control over the released amount of F− ions by simply adjusting the precursor solutions (calcium chloride (CaCl2) and ammonium fluoride (NH4F)) used for the CaF2 precipitation. In this study, the surface morphologies, chemical compositions, rheological properties, and F−-ion release behaviors of CaF2 composite hydrogels were characterized, and their antibacterial capabilities were evaluated using bacteria colony tests and live/dead assays. Moreover, the cellular responses were also compared with those of the pure hydrogel using cell attachment, proliferation, and migration assays. Finally, the potential wound-healing efficiency of the composite hydrogels was investigated using a full-thickness wound in a rat model and compared with that for the pure hydrogel.

2. Experimental Methods 2.1. Materials and fabrication of CaF2 composite hydrogels 4 ACS Paragon Plus Environment

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HA sodium salt (Mw: 1.8–2.5 MDa, cosmetic grade, 99.84+% purity) produced by fermentation of Streptococcus zooepidemicus was purchased from Bioland Co., Ltd. (Cheonan, Korea). All the other chemicals were purchased from Sigma–Aldrich unless otherwise specified. Triethylamine, glycidyl methacrylate, tetrabutylammonium bromide, 2hydroxy-4´-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), poly(ethylene glycol) diacrylate (PEGDA, M.W. of 700 g/mol), calcium chloride (CaCl2), and ammonium fluoride (NH4F) were used for the experiments.

For the pure hydrogel, glycidyl methacrylated HA (GMHA) was synthesized using a previously proposed protocol.20 A mixture of 1 wt% GMHA and 10 wt% PEGDA with a volume ratio of 8:2 was prepared. To crosslink the pure hydrogel, the mixture solutions were poured into the plastic mold with the diameter of 12 mm and exposed to ultraviolet (UV) light with the addition of 2.2% of Irgacure 2959 for 15 min. The pure hydrogel was then immersed in CaCl2 solution overnight and subsequently dipped in NH4F solution to induce CaF2 precipitation within the gel. The concentration of the CaCl2 solution was controlled from 0.125 M to 0.25 M and that of the NH4F solution was also adjusted to maintain a molar ratio of 1:2 with the CaCl2 solution. A schematic diagram of this process is presented in Scheme 1. The precipitation time was controlled from 10 min to 1 h. For characterization tests including SEM observation, in vitro ion release tests, and in vitro cell tests, 400 ul of the disc-shaped hydrogels were prepared with the diameter of 12 mm. For rheological property evaluation, 1 ml of the hydrogels were prepared with the diameter of 20 mm.

2.2. Characterization of CaF2 composite hydrogels The surface morphologies of the hydrogels were examined using field-emission scanning electron microscopy (FE-SEM; SUPRA 55VP, Carl Zeiss, Germany), and the phase of the precipitated CaF2 in the composite hydrogels was determined using XRD (D8-Advance, 5 ACS Paragon Plus Environment


Bruker, Germany). The results were compared with those for the pure hydrogel. The chemical structures of the hydrogels were identified using attenuated total reflectance FTIR spectroscopy (Nicolet 6700, Thermo Scientific, USA) for wavenumbers ranging from 650 to 4000 cm−1 with an average of 32 scans. Before the assessment, the hydrogels were washed with phosphate-buffered saline (PBS) and distilled water (DW) and the samples were lyophilized overnight to maintain the structure of the samples. TGA (Discovery TGA, TA Instruments, USA) was also performed to analyze the relative content of CaF2 in the composite hydrogels. The temperature was increased to 800 ºC at a heating rate of 20 ºC min−1 under N2 condition with a flow rate of 20 mL min−1; the weight change was monitored, and finally, the percentage of residue after combustion was determined. The equilibrium water content (EWC) of the hydrogels was evaluated. The swollen hydrogels in DW at 37 ºC were weighed (Ws). Then, the hydrogels were dried in a vacuum oven and then re-weighed (Wd). The EWC was calculated using the following equation: EWC (%) = (Ws – Wd)/Ws × 100. The amount of CaF2 and EWC of the hydrogels are summarized in Table 1.

2.3. Rheological behaviors of CaF2 composite hydrogels The viscoelastic properties of the hydrogels were determined using a rheometer (DHR-2, TA Instruments, USA) with parallel-plate geometry (diameter of 20 mm, gap of 3 mm). Dynamic oscillatory frequency sweep was performed at room temperature and constant strain (1%, in the linear viscoelastic range) over the frequency range of 0.1–100 rad/s. The storage moduli (G′) were measured as a function of frequency.

2.4. Fluorine ions (F−) release behaviors

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The ion-release behaviors of fluorine ions (F−) from the CaF2 composite hydrogels in PBS solution at 37°C were monitored for 10 days using a fluoride-ion electrode (Orion Star A214, Orion Research Inc., UK) connected to an ion analyzer (901, Orion Research, UK) after dilution with a total ionic strength adjustment buffer solution (TISAB).

2.5. In vitro cell tests The in vitro cellular responses were monitored using cell attachment tests and MTS assays using a mouse fibroblast cell line (L929; CCL-1, Mus musculus) obtained from ATCC®. Fibroblasts at densities of 1 × 104 cells/mL were seeded on the hydrogels for the cell attachment tests. The cells were cultured in minimum essential medium alpha (α-MEM; Welgene, Korea) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin in a humidified incubator containing 5% CO2 at 37°C for 3 days. After being fixed using 4% paraformaldehyde, the cells on the samples were stained with fluorescent phalloidin and 4',6-diamidino-2-phenylindole (DAPI) for 20 and 5 min, respectively. The cells attached on the hydrogels were visualized using confocal laser scanning microscopy (CLSM; FluoView FV1000, Olympus, Japan). Then, 3 × 104 cells/mL of cells were seeded on the hydrogels for the cell proliferation tests. After culturing for 3 and 5 days, the level of cell proliferation was measured using the MTS assay (CellTiter 96 Aqueous One Solution, Promega, USA). Human umbilical vein endothelial cells (HUVECs, ATCC, USA) were used for the in vitro cell migration assay. In brief, the cells were seeded on 4-well plates and cultured until a cell monolayer was formed. Then, a pipette tip was used to create a scratch, and the plates were gently washed with the α-MEM medium supplemented with FBS to rinse the detached cells. The pure hydrogel and CaF2 composite hydrogel were placed on each plate and incubated for 24 h. After culturing, images were obtained using a photomicroscope (ZEISS,



Axiophot, Germany) after staining with crystal violet for 20 min, and the migration ratio was calculated using the following equation: Migration ratio (%) = (M0−Mt)/M0 x 100%, Here, M0 and Mt represent the initial and healed scratch distance, respectively.

2.6. Assessment of antibacterial activity of CaF2 composite hydrogels To confirm the antibacterial activity of the CaF2 composite hydrogels, both qualitative and quantitative methods were employed using two bacterial strains, Escherichia coli (E. coli, ATCC, ATCC 8739, Rockville, MD, USA) and Staphylococcus aureus (S. aureus, ATCC, ATCC 6538, Rockville, MD, USA) in Luria–Bertani broth (LB broth; BD DifcoTM, 244620, USA). A concentration of 0.5 × 105 CFU/mL of bacteria was introduced onto the surface of the pure hydrogel and CaF2 composite hydrogel. The bacterial suspensions inoculated on each sample were incubated at 37°C for 12 h. Bacterial live/dead assays and colony tests were conducted using qualitative and quantitative methods, respectively. For the live/dead assays, the samples were carefully rinsed with new LB broth. The samples were then stained with SYTO 9 and propidium iodide at a ratio of 1:1 for 15 min. The stained bacteria were examined using CLSM (live: green, dead: red). For the colony tests, the hydrogels were rinsed with 1 mL of PBS immediately after incubation, and the rinsed samples were transferred into 3 mL of fresh PBS in a 15-mL sterilized tube. The tubes containing bacteria incubated on the samples were then vortexed for 1 min to detach all the bacteria from the surfaces of the samples. The viable bacteria in the PBS were examined by standard serial dilution and spreading on a LB agar plate. The agar plate was incubated at 37°C for 15 h, and colony images were obtained using a digital camera.

2.7. In vivo assessment


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For the in vivo animal tests, 13 male 6-week-old Sprague–Dawley rats (KOSA Bio Inc., Seongnam, Korea) were used to examine the wound-healing efficiency of the CaF2 composite hydrogels compared with that of the pure hydrogel. The rats were fed a standard diet, and the wound closure was examined using full-thickness wounds. The rats were anesthetized using an intraperitoneal injection of zoletil (30 mg/kg) and rumpun (5 mg/kg). After the anesthetic was administered, the dorsum was shaved, sterilized with diluted betadine solution, and draped in a sterile manner. For the full-thickness wound preparation, an 8-mm-diameter wound was created using a medical biopsy punch, and hydrogel dressings of identical size were placed on the wounds. Each hydrogel dressing was covered with a transparent film and compressive dressing bandages for solid fixation. This animal experiment was approved by the Institutional Animal Care and Use Committee of GENOSS (GEN-IACUC 1612-03).

2.8. Wound closure and histological observation The wound closure was examined at different times and was quantified by measuring the change of the size of the wounds. At each measurement time, the hydrogel dressings were exchanged with new ones. After 7 and 14 days of treatment, the tissues were harvested, fixed in 10% formalin overnight, and embedded in paraffin. After sectioning the paraffin block in the vertical direction of the skins, histological sections were stained according to routine hematoxylin and eosin (H&E) and Masson’s trichrome protocols. For the histological observation, microscopic images of the stained sections were obtained using a panoramic digital slide scanner (Pannoramic 250 Flash III, 3DHISTECH Ltd., Hungary).

2.9. Immunohistochemistry Immunohistochemical detection was performed using anti-CD86 antibodies (1:200, OABF00403, Aviva System Biology, USA), following the manufacturer’s instructions and the literature.21 Briefly, tissue section samples were deparaffinized and hydrated in graded 9 ACS Paragon Plus Environment


ethanol solutions and DW. Endogenous peroxidase was inactivated with 3% hydrogen peroxide for 10 min at 37°C. After quenching using endogenous peroxidase, high-temperature antigen retrieval was performed (citrate buffer 10 mM, pH 6.0). The slides were incubated with anti-CD86 antibodies for 1 h at 37°C and were developed with Dako Envision SystemHRP labeled polymer anti-mouse (K4000, DAKO, USA) in chromogen solution and counterstained with Harris hematoxylin at 37°C. A Tris buffer at pH 7.6 was used as the washing solution. Microscopic images of the stained sections were obtained using the panoramic digital slide scanner.

2.10. Statistical analysis All the experimental results are expressed as the mean ± standard deviation (SD) for n > 3. The difference between the two groups was determined using a one-way analysis of variance (ANOVA) followed by Tukey post-hoc comparison test, and p < 0.05 and p < 0.01 were considered to be statistically significant (*p < 0.05 and **p < 0.01).

3. Results

3.1. Characterization of CaF2 composite hydrogels A schematic diagram of the sequential immersion in precursor solutions for certain time interval and in situ precipitation process used to fabricate the CaF2 composite hydrogels is presented in Scheme 1.

Scheme 1. Schematic illustration of fabrication of CaF2 composite hydrogels via in situ precipitation process.


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Briefly, after immersion in two precursors, a Ca2+ ion-rich solution and a F−-ion-rich solution, in sequence, CaF2 rapidly precipitated in the hydrogels. The photo-crosslinked hydrogels formed from a mixture of HA and poly(ethylene glycol) (PEG) were transparent, whereas the CaF2 composite hydrogels were opaque because of the precipitation of ceramic particles, as shown in the scanning electron microscopy (SEM) images in the insets in Figure 1.

Figure 1. SEM images of (A) pure gel and (B) CaF2 composite gel (HA-CaF2 composite gel 0.25 M for 30 min precipitation) and high-magnification SEM images of (C) pure gel and (D) CaF2 composite gel. (E) FT-IR spectra and (F) XRD patterns of pure gel and CaF2 composite gel.

Table 1 summarizes the amounts of each reagent added to fabricate the pure gel, CaF2 composite gel-0.125 M, and CaF2 composite gel-0.25 M, and the amount of precipitated CaF2 inside the composite hydrogel. The amount of CaF2 in the composite hydrogels increased with increasing the precursors concentration from approximately 10 wt% for CaF2 composite gel0.125 M to about 18 wt% for CaF2 composite gel-0. 25 M, according to the TGA results. The EWC slightly decreased from 98.7 % to around 97 % with increasing CaF2 content, implying that the network density increased with the addition of CaF2; nevertheless, all the hydrogels exhibited high water content. 11 ACS Paragon Plus Environment


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Table 1. Calculated CaF2 content of HA-CaF2 composite hydrogels with the corresponding amount of two reactants, CaCl2 and NH4F to each CaF2 content and equilibrium water content (EWC) Sample

CaCl2 (M)

NH4F (M)

Pure gel CaF2 composite gel-0.125 M CaF2 composite gel-0.25 M

0 0.125 0.250

EWC in DW (%)

0 0.250

CaF2 content measured by TGA (wt%) 0 10.06 ± 3.02

0.500

18.11 ± 1.57

97.01 ± 0.57

98.71 ± 0.18 97.44 ± 0.29

The pure hydrogel had a porous microstructure with pore sizes of several hundred µm (Figure 1(A)). The CaF2 composite hydrogel also exhibited a highly porous structure with pore sizes similar to that of the pure hydrogel. The microstructure of the composite hydrogel consisting of interconnected pores indicates that the network structure of the hydrogel was maintained after the in situ precipitation process (Figure 1(B)). However, the nanostructures, and especially the surface morphologies of the two hydrogels greatly differed. The pure hydrogel exhibited a smooth surface matrix, as shown in Figure 1(C), whereas large numbers of spherical particles were densely deposited on the surface of the CaF2 composite hydrogels (Figure 1(D)), showing roughened surface. The size of the precipitated CaF2 nanoparticles was around 300 – 500 µm, and their homogeneous distribution was observed. Fouriertransform infrared (FTIR) spectra of the pure hydrogel and CaF2 composite hydrogel are presented in Figure 1(E). For the pure hydrogel, the broad band between 3500–3000 cm−1 corresponds to stretching vibration of the –OH group, and the peak at around 1700 cm−1 corresponds to characteristic C=O stretching. According to literature, the residue glycidyl methacrylate to the HA backbone showed the presence of the methacrylate C=C bonds (at around 900 and 1490 cm−1 ), which did not react during crosslinking. Sodium carboxylate (– COO–), and C–O–C stretching vibrations were detected at around 1400, and 1025 cm–1, respectively. For the CaF2 composite hydrogel, the broad band corresponding to the –OH group was diminished because the presence of CaF2 hindered the –OH groups of the pure 12 ACS Paragon Plus Environment

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hydrogels. Other bands were still detected in the CaF2 composite hydrogel, indicating that overall, the hydrogel forms were maintained. No significant peaks were detected in the X-ray diffraction (XRD) pattern of the pure hydrogel (Figure 1(F)); however, that of the composite hydrogels contained typical peaks of CaF2 ceramics corresponding to (1 1 1), (2 2 0), (3 1 1), and (4 0 0) planes. These broad peaks indicated the small crystallite size of the CaF2 particles resulting from the in situ precipitation process and hydrogel matrix.

3.2. Rheological properties and ion-release behaviors of CaF2 composite hydrogels Upon incorporating the CaF2 particles in the gel matrix, the water content significantly decreased and the G´ values dramatically increased compared with those of the pure hydrogel (Figure 2(A)). Regardless of their content, the CaF2 particles were characterized by a linear G´ graph with frequency-independent behavior. The degree of storage modulus at certain frequency (at around 1 Hz) slightly increased from around 800 to 1110 Pa as increasing the amount of CaF2 from 10 to 18 wt%.

Figure 2. (A) Rheological behavior of pure gel and CaF2 composite gel in frequency sweep mode. (B) Fluoride-ion release profiles of CaF2 composite gels with different CaF2 concentrations and precipitation times.

The ion-release profiles were dependent on CaF2 precipitation time, rather than the CaF2 concentration as shown in Figure 2(B). No large difference in the concentrations of the released ions were observed for different CaCl2 concentrations; however, by controlling the 13 ACS Paragon Plus Environment


precipitation time, especially for 10 min and after 30 min, large differences in the ion-release profiles as a function of CaF2 concentration were observed. A shorter precipitation time resulted in faster release of fluoride, whereas for the 30-min and 1-h samples, sustained ion release was achieved. 3.3. In vitro cell tests Notably, the CaF2 composite hydrogel exhibited excellent bioactivity compared with that of the pure hydrogel. In particular, the morphology of the fibroblasts on the CaF2 composite gel appeared more polarized and stretched with proliferation in large numbers in contrast with the few round-shaped cells on the pure HA hydrogels (Figure 3(A) & (B)).

Figure 3. CLSM images of L929 fibroblasts cultured on (A) pure gel and (B) CaF2 composite gel (HACaF2 composite gel 0.25 M for 30 min precipitation) and HUVEC cultured on (C) pure gel and (D) CaF2 composite gel after 2 days of culturing.


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The human umbilical vein endothelial cells (HUVECs) also appeared to prefer the surface of the composite hydrogel over the pure one, with increased numbers of the cells being detected (Figure 3(C) & (D)). To elucidate the wound healing efficiency of the CaF2 composite gel, an in vitro cell migration assay was conducted using both HUVECs and fibroblasts (Figure 4). The stained images showed the migration behaviors of both cells, and the quantitative results indicated that the migration ratio of the CaF2 composite gel group was significantly higher than that of the pure hydrogel group. These findings indicated that the composite hydrogel stimulated the migration of both cells compared with the pure hydrogel.



Figure 4. Migration behavior of HUVECs and L929 fibroblasts on hydrogels. (A) HUVECs and (B) L929 fibroblasts 0 and 24 h after being scratched (BF: bright field and CV: crystal-violet-stained images) and (C) migration ratio of HUVECs and L929 fibroblasts (scale bar = 1 mm, *** p

Effective Wound Healing by Antibacterial and Bioactive Calcium

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