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Back to Journal »International Journal of Nanomedicine» Volume 13

Authors Dhand C, Balakrishnan Y, Ong ST, Dwivedi N, Venugopal JR, Harini S, Leung CM, Low KZW, Loh XJ, Beuerman RW, Ramakrishna S, Verma NK, Lakshminarayanan R 

Published on August 3, 2018, Volume 2018: 13 pages 4473-4492

DOI https://doi.org/10.2147/IJN.S159770

Single anonymous peer review

Editor approved for publication: Dr. Thomas J Webster

Chetna Dhand,1,2,* Yamini Balakrishnan,3,* Seow Theng Ong,4,* Neeraj Dwivedi,5 Jayarama R Venugopal,6 Sriram Harini,1 Chak Ming Leung,3 Kenny Zhi Wei Low,7 Xian Jun Loh,7 Roger W Beuerman,1,2 Seeram Ramakrishna,8 Navin Kumar Verma,1,4 Rajamani Lakshminarayanan1,2 1Anti-Infective Research Group, Singapore Eye Research Institute, The Academia, Discovery Tower, Singapore; 2Ophthalmology and visual science academic clinical projects, Duke-National University of Singapore School of Medicine, Singapore; 3 Department of Bioengineering, National University of Singapore, Singapore; 4 Dermatology and Dermatology and Dermatology, Nanyang Technological University Lee Kong Chian School of Medicine, Singapore; 5 Department of Electrical and Computer Engineering, National University of Singapore, Singapore; 6 Faculty of Industrial Science and Technology, Pahang University, Malaysia, Kambang, Malaysia; 7 Department of Mechanical Engineering, Faculty of Engineering, Nanofiber and Nanotechnology Center, National University of Singapore, Singapore; 8 Department of Soft Materials, Institute of Materials Research and Engineering, A*STAR ( Singapore Institute of Science, Technology and Research) *These authors made the same contribution to this work. Introduction: In order to find a cross-linking agent with multi-functional properties, this work investigated its usefulness as a quaternary ammonium organosilane (QOS) as an electrospinning agent. Potential cross-linking agent for collagen nanofibers. We assume that quaternary ammonium ions improve electrospinning properties by reducing surface tension and imparting antibacterial properties, while siloxanes formed after alkali hydrolysis can crosslink materials and methods: by adding different concentrations of QOS (0.1%-10% w/w ) QOS collagen nanofibers are electrospun and cross-linked in situ after contact with ammonium carbonate. QOS cross-linked scaffolds have characterized their biological properties and are based on the compatibility, cell adhesion and metabolic activity of their bioco primary human dermal fibroblasts and human fetal osteoblasts. Results and discussion: Studies have shown that: 1) QOS cross-linking increases the flexibility of originally rigid collagen nanofibers and improves thermal stability; 2) QOS cross-linked mat shows strong antibacterial activity and 3) the biological phase of the composite mat The capacity depends on the content of QOS in the stock solution-at low QOS concentrations (0.1% w/w), the pad promotes the proliferation and growth of mammalian cells, while at higher QOS concentrations, cytotoxic effects are observed. Conclusion: This study shows that QOS cross-linked pads have anti-infective properties and provide a niche for cell growth and proliferation, thereby providing a useful method, which is important for hard and soft tissue engineering and regenerative medicine. Keywords: anti-infective wound dressing, cytocompatible nanofiber, electrospinning, cost-effective crosslinking agent, tissue regeneration, antibacterial

Bacterial infections and soft tissue injuries are often encountered after deep skin injuries or severe open fractures, which can lead to significant morbidity, mortality, and economic loss. 1-3 The treatment of superficial infections usually includes systemic antibiotics for 10-14 days, while the treatment time for fracture fixation device infections is extended to 6 weeks. Antibiotic-eluted biodegradable polymer stents have been shown to have the potential to deliver drugs at the site of infection. In the past few years, electrospinning of biodegradable and biocompatible polymers has been extensively studied for possible biomedical applications, such as tissue engineering and regenerative medicine. 4-7

Recent studies have demonstrated the prospect of electrospinning antibiotic-loaded electrospun nanofibers directly onto implants, fixation devices, or deproteinized cancellous bones, which can maintain antibacterial activity for a long time. 8-10 In vivo studies have confirmed that the implants or fixtures coated with electrospun nanofibers loaded with antibiotics can prevent osteolysis and improve osseointegration compared with naked implants. Although nanofibers loaded with antibiotics can effectively prevent and eradicate microbial colonization in infected parts, such systems also carry considerable risks. Excessive release of antibiotics in the early stage can cause allergies and even allergic reactions. Over time, their gradual consumption from the application site will increase the risk of antibiotic-resistant strains. 11

The biomedical applications of electrospun nanofibers are rapidly developing in two directions. One is to produce complex nanostructures, such as core-shell, Janus nanofibers, or a combination of them, through a one-pot electrospinning process. 12-15 The second is to combine/integrate external molecules (metal ions, drugs, cross-linking agents, etc.) into nanofiber molecular assembly to enhance its functional performance to achieve precise applications. The work reported in this article is a typical example of the second method and a major advancement of our previous research, which combines the beneficial properties of polycatecholamine crosslinking and mineralization. 16,17 Looking for a crosslinking agent with both durability and antibacterial properties for the stent, we are looking for a quaternary ammonium organosilane (designated as QOS) that has broad-spectrum antibacterial properties and is widely used in various medical and industrial applications And the structure of octadecyl dimethyl (3-trimethoxysilyl propyl)-ammonium chloride is encouraged. The alkyl quaternary ammonium group in 18-20 QOS imparts antibacterial and surface active properties, while the acid or alkali hydrolyzable siloxane group undergoes condensation polymerization to form a stable Si-O-Si network. The purpose of this work is to study the biocompatibility and antibacterial properties of QOS cross-linked electrospun nanofibers, and to explore their potential applications in soft and hard tissue engineering.

Bovine skin collagen type I (Col) was obtained from Cosmo Bio Collaborative Ltd. (Tokyo, Japan). Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride or QOS solution (in 42% methanol), 1,1,1,3,3,3-hexafluoro- 2-propanol (HFP), DMEM, nutritional blend F-12, antibiotics, glutaraldehyde, alizarin red-S, cetylpyridinium chloride (CPC), hexamethyldisilazane (HMDS), Hoechst, p-nitrophenyl phosphate, triton X-100, DMSO (99+%), ammonium carbonate and sodium chloride were from Sigma-Aldrich Co. (St. Louis, Missouri, USA). CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) dye, propidium iodide and Alexa Fluor 647 phalloidin were from Molecular Probe® (Thermo Fisher Scientific, Waltham, MA, USA). Human fetal osteoblasts (hFObs) and human dermal fibroblasts (hDFs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum (FBS) and trypsin-EDTA for cell culture were purchased from Thermo Fisher Scientific. Mueller Hinton Broth (MHB) was purchased from Acumedia (Michigan, Michigan, USA). All the above-mentioned chemicals are analytically pure and can be used without further purification. In order to study the antibacterial properties of QOS cross-linked mats, the following Gram-positive microbial stains were used: ATCC strain, Staphylococcus aureus (SA) 29213, Staphylococcus epidermidis (SE) 12228, MRSA 700699, clinical isolate, MRSA 21595 (From the wound).

Electrospinning of organosilane loaded collagen scaffold

To prepare the original collagen pad, dissolve the collagen in HFP to form an 8% solution and stir for 12 hours. In order to design a collagen/quaternary ammonium silane (Coll_QOS) composite scaffold, the solution is prepared by dissolving 8% collagen containing different concentrations of silane (0.1%-10% w/w of collagen) in HFP: methanol (9:1) of. The coating solution was then transferred to a polypropylene plastic syringe with a 27 G stainless steel blunt needle. Pure collagen nanofibers are electrospun from a high-voltage source (Gamma High Voltage Research, Inc., FL, USA) with an applied voltage of 13 kV, and the distance between the needle and the collector is 13 cm. However, for the Coll_QOS pad, the nanofibers were obtained at a voltage/distance setting of 15 kV/5 cm. Collect fibers at a constant flow rate of 1 mL h-1. Collect nanofibers on a flat aluminum foil for mechanical, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) research. Nanofibers are collected on a cover glass (CS, Ø15 mm) for contact angle measurement and cell culture experiments, and collected on a gold-plated copper grid for transmission electron microscopy (TEM) analysis. All electrospinning experiments are carried out at room temperature with a humidity of 55%. The collected electrospun mats were dried in a vacuum dryer for 24 hours to remove any residual HFP and stored in a drying cabinet to avoid contamination. The QOS-loaded mat was then placed in a sealed desiccator containing 5 grams of (NH4)2CO3 powder for 48 hours to induce the polymerization of the incorporated organosilane and the crosslinking of the nanofibers. The pads are marked as follows: as-spun collagen pad-ES_Coll; as-spun collagen pad with n% QOS-Coll_n%QOS, where n = 0.1, 0.5, 1, 5 and 10; (NH4)2CO3 treated with n% QOS As-spun collagen cushion-Coll_n%QOS_XL.

Morphological analysis of electrospun fibers using SEM and TEM

Field emission scanning electron microscope (FE-SEM) (FEI-QUANTA 200F, Netherlands) is equipped with an energy dispersive X-ray spectrometer (EDXS) accessory. After sputter coating, SEM and EDXS analysis are performed under 15kV acceleration voltage. Pad with platinum (JEOL JSC) -1200 Fine Coating Machine, Japan). FE-SEM was used for the morphological analysis of nanofiber scaffolds to reveal: 1) the influence of integrating different concentrations of organosilane in the collagen scaffold; 2) the influence of ammonium carbonate treatment on the morphology of the Coll_n%QOS pad and 3) the incorporation of Organosilane and cross-linking treatments stimulate cell adhesion/diffusion and cell morphology to the collagen scaffold. An EDXS study was performed to scan the distribution of silane within the collagen scaffold and to detect calcium mineralization induced by osteoblasts. ImageJ image analysis software (National Institutes of Health, Bethesda, Maryland, USA) was used to evaluate the average fiber diameter of various scaffolds. Randomly select at least 50 nanofibers from their respective SEM images (3-4 micrographs focusing on different areas) to estimate their diameter. Use JEOL JEM-3010 instrument for TEM research. Since the morphology of nanofibers may vary with targets with different conductivity, we analyzed and compared the SEM and TEM images of all supports on the same collection substrate-SEM is aluminum foil, and TEM is gold-plated copper grid.

The electrospun nanofiber scaffold was mechanically tested using a benchtop tensile testing machine (Instron 5345, Norwood, MA, USA) under ambient conditions using a load cell with a capacity of 10 N and a constant strain rate of 1 mm min-1. For testing, the samples were cut into rectangular strips with a size of 1 × 3 cm2, and the thickness of each sample was measured using a micrometer. Different mechanical parameters, including Young's modulus, failure stress (strength), toughness (area under the curve) and failure strain are calculated from the obtained stress-strain diagram.

The SDT 2960 thermogravimetric analyzer (TA Instruments, New Castle, DE, USA) (TGA) was used to conduct thermogravimetric studies on various stents at a heating rate of 20°C min-1 between 25°C and 900°C. A dynamic nitrogen atmosphere with a flow rate of 70 mL min-1. The dynamic normalization algorithm given by the instrument is used to determine the thermal parameters describing the various stages of temperature-induced weight loss.

Water contact angle (WCA) research

VCA Optima surface analysis system (AST Products, Billerica, MA, USA) was used to study the dynamic contact angle of different collagen pads at ambient temperature. Drop deionized water (1 μL) carefully on the surface of the mat, and take pictures continuously for 2 minutes at 5 second intervals.

The XPS study was done using Kratos AXIS UltraDLD (Kratos Analytical Ltd., Wharfside, Manchester, UK) in ultra-high vacuum (~10-9 Torr) using a monochromatic Al-Kα X-ray source (1,486.71 eV). By recording its high-resolution element spectra, in-depth analysis of different nanofiber mats in different chemical states has been carried out. The high-resolution spectrum uses different Gauss-Lorentz components for deconvolution during the analysis, and the Shirley mode is used to subtract the background.

hFOb cells were cultured in DMEM/F12 medium (1:1) supplemented with 10% FBS and antibiotic cocktail, while hDF cells were cultured in DMEM supplemented with 10% FBS and antibiotic cocktail in 75 cm2 cell culture flasks. The cells were cultured in a humidified CO2 incubator at 37°C and supplemented with fresh medium every 3 days. For cell seeding, the nanofiber scaffold prepared on 15 mm CS was first sterilized by ultraviolet light for 1 hour, and then placed in a 24-well plate with a sterilized stainless steel ring to prevent the scaffold from lifting. In order to remove residual solvent, the scaffold was washed three times with PBS (pH 7) for 15 minutes each time, and then soaked in the culture medium overnight. In order to seed the cells on different scaffolds, trypsin-EDTA was used to collect the cells and re-seeded after counting the cells with trypan blue using a hemocytometer. hFOb and hDF cells were seeded on ES_Coll, Coll_0.1%QOS_XL, Coll_0.5%QOS_XL and Coll_1%QOS_XL scaffolds at a density of 1 × 104 and 0.8 × 104 cell wells-1, respectively. The cells were seeded on CS and ES_Coll as controls.

Cell adhesion, proliferation and differentiation detection

CellTracker Green CMFDA is a cell penetrating dye, which is easily destroyed by intracellular esterases in living cells to produce green fluorescent calcein. After the cells were cultured for 6 days, the complete medium was removed from the sample and the cells were treated with 25 μM C​​MFDA in serum-free medium at 37°C. After 2 hours of treatment, the CMFDA medium was replaced with complete medium and incubated overnight. Before confocal microscopy, cells were stained with Hoescht and propidium iodide (to detect dead cells) for 30 minutes. Then, a Plan-Apochromat ×40/1.3 oil immersion objective lens with Zeiss LSM800 Airyscan confocal microscope was used to obtain confocal z-stack images through 405, 488 and 561 nm laser excitation.

In order to check the morphology of cells grown on various scaffolds on day 6, the cells were fixed with 4% (v/v) formaldehyde and stained with Alexa Fluor 647-Phalloidin (to display cells) and Hoechst (to display nuclei). A Zeiss LSM800 Airyscan Plan-Apochromat ×40/1.3 oil immersion objective lens was used to obtain confocal images with z-stack through 405 and 640 nm laser excitation. All confocal images were prepared using Zen 2.1 lite imaging software (Carl Zeiss Meditec AG, Jena, Germany).

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS, inner salt; CellTiter 96® AQueous One Assay) is used to evaluate the cell proliferation of hFOb and hDF on various scaffolds. Draw the cell number and absorbance calibration curve of the two cell lines to calculate the MTS measured absorbance reading as the cell number. The basis of MTS detection is the reaction of metabolically active living cells with MTS tetrazolium salt to generate formazan dye, which can be quantified by the absorbance value at 490 nm. For this assay, the scaffold for cell growth was first washed with PBS and incubated with 20% MTS solution in serum-free medium for 3 hours. Thereafter, transfer the solution in 100 μL aliquots to a 96-well culture plate, and use a spectrophotometer plate reader (FLUOstar OPTIMA; BMG Lab Technologies, Germany) to record the absorbance value at 490 nm.

ALP is a homodimeric protease responsible for nucleating minerals by providing free PO43− ions through the decomposition of organic phosphates, and its expression is related to cell differentiation. The enzyme-linked immunosorbent assay (Sigma Life Science, St Louis, MO, USA) using alkaline phosphate yellow liquid replacement system was used to evaluate the ALP activity of hFOb cells seeded on various collagen scaffolds. ALP catalyzes the hydrolysis of colorless p-nitrophenyl phosphate (PNPP) into yellow products, p-nitrophenol and phosphate. On days 3, 6, and 9 (ps) after inoculation, the complete medium was removed and the scaffold was washed three times with PBS. Then incubate the scaffold with 400 μL PNPP solution for 30 minutes. After that, 200 μL of 2 M NaOH solution was added to stop the reaction. Then transfer the subsequent yellow solution to a 96-well plate and evaluate the absorbance at 405 nm in a Tecan plate reader.

Alizarin Red-S (ARS) staining assay

ARS is a dye that can be selectively combined with calcium salts and used in calcium mineral histochemistry. Therefore, ARS can be used to qualitatively and quantitatively assess the degree of mineralization on different stents. First, hFOb cells grown on various nanofiber scaffolds were washed three times with PBS, and then fixed with 70% ethanol for 1 hour. After fixation, the cells were washed three times with deionized water, and then stained with 40 mM ARS reagent at room temperature for 20 minutes. Later, the stent was washed several times with deionized (DI) water and observed under an optical microscope. For quantitative evaluation, the stain was eluted with 10% CPC for 60 minutes, and the absorbance of the solution was recorded at 540 nm on a Tecan microplate reader.

FE-SEM cell morphology analysis

The cell morphology of hFOb and hDF cultured in vitro was analyzed by FE-SEM (FEI-QUANTA 200F) at 6 days ps. The cell-seeded scaffold was washed with PBS to remove non-adherent cells, and fixed with 3% glutaraldehyde at room temperature. The cell scaffold is then dehydrated using a series of graded alcohol solutions, and finally dried overnight in HMDS. The dried cell structure was then sputter coated with platinum and observed under FE-SEM at an accelerating voltage of 10 KV.

Antibacterial properties of QOS cross-linked collagen cushion

Quaternary ammonium compounds have been used as a class of effective preservatives. With this in mind, we evaluated the antibacterial properties of QOS cross-linked collagen nanofibers using the micro broth dilution method in accordance with the protocol of the Clinical and Laboratory Standards Association. For the microbroth growth inhibition assay, a nanofiber mat containing 0.1%, 0.5%, and 1% QOS and weighing 10.2±0.3 mg was used in 1 ml of MHB containing bacterial culture at 105 colony forming units (CFU) at 37 Incubate at °C for 24 hours ml-1. Bacterial cultures without any pads served as positive growth controls. After the 24-hour incubation period, prepare 1 log (10-fold) serial dilutions of the bacterial suspension (with or without pads) in PBS, and then pour 100 μL of each dilution onto the MHA and place it at 37°C Incubate for 24 hours. Count the CFU and estimate the reduction factor (Rf) using the following equation:

Where Nc is the number of living cells (CFU) in the positive growth control, and Nd is the number of living cells (CFU) in the silane-containing nanofiber mat.

The influence of QOS on the diameter of electrospun collagen nanofibers

SEM and TEM analysis revealed the morphological changes of electrospun collagen nanofibers with different concentrations of QOS in the stock solution before and after exposure to (NH4)2CO3. The electrospun collagen nanofibers showed a smooth bead-free morphology with an average diameter (φ) of 252 ± 58 nm (Figure 1A). As the QOS concentration in the coating solution increases, smooth fibers appear, and the average diameter gradually decreases to 196±42 nm for Coll_0.1%QOS to 155±67 nm for Coll_0.5%QOS and 141±35 for Coll_1%QOS nm (Figure 1B-D), probably due to the increase in conductivity of the doping solution and the decrease in surface tension. 21-23 As the QOS concentration increases to 5% and 10%, the average diameter increases to 205 ± 35 nm. Coll_5%QOS is 315±42 nm, and Col_10%QOS is 10% (Figure S1A and B). In the case of as-spun Coll_10% QOS, the number of "welded connections" increased, indicating that partial cross-linking or interaction between collagen chains and QOS may contribute to the increase in average diameter.

Figure 1 FE-SEM display of (A) ES_Coll, (B) Coll_0.1%QOS, (C) Coll_0.5%QOS, (D) Coll_1%QOS, (E) Coll_0.1%QOS_XL, (F) Coll_0 Micro photos. 5%QOS_XL and (G) Coll_1%QOS_XL. The inset shows the TEM image of the equivalent sample, (H) shows the average fiber diameter of various collagen scaffolds, (I) EDXS spectrum and (J) energy dispersive X-ray mapping recorded by Coll_1%QOS_XL. The scale bars of SEM, TEM and EDXS mapping are 1, 0.2 and 3 μm, respectively. Abbreviations: FE-SEM, field emission scanning electron microscope; QOS, quaternary ammonium organosilane; TEM, transmission electron microscope; EDXS, energy dispersive X-ray spectrometer.

Based on the premise that ammonia conditions may cause trimethoxysilane to hydrolyze and initiate silicate polycondensation, the as-spun mats containing different concentrations of QOS were exposed to (NH4)2CO3. The SEM image of the scaffold after exposure to (NH4)2CO3 showed all the characteristics of cross-linked nanofibers, namely, extensive inter-fiber interactions, fiber-fiber bonding, and a significant increase in nanofiber connections (Figure 1E-G; Figure S1C and D). The SEM study showed a significant increase in the average diameter of the nanofibers (Figure 1H), indicating the formation of an organosilicate coating on the collagen nanofibers. These results confirm the observation of Pirzada et al. that the increase in the average diameter of electrospun polyvinyl alcohol (PVA) nanofibers after the addition of tetraethyl orthosilicate is related to the increase in the degree of hydrolysis and the increase in aging time. 23 TEM images (insets in Figure 1E-G) revealed that after exposure of (NH4)2CO3, a smooth coating and nanofiber welds were formed at the connection points of the collagen pad loaded with QOS. The combination of elemental (Si) mapping and EDXS showed the presence and uniform distribution of QOS on the entire nanofiber collagen scaffold (Figure 1I and J). In summary, electron microscopy studies have shown that QOS forms a smooth coating and crosslinking of electrospun collagen fibers.

To confirm the QOS functionalization and cross-linking of collagen nanofibers, the FT-IR spectra of ES_Coll, Coll_1%QOS and Coll_1%QOS_XL pads are compared in Figure 2A. The original ES_Coll spectrum shows that the broad peak between 3,700 and 3,100 cm-1 is caused by the overlap of H-connected functional units, such as OH stretching for adsorbing water molecules or NH stretching in the protein backbone (amide A). The peaks at 1,243 and 1,539 cm-1 are attributed to NH bending and CN stretching (amide III and amide II peaks). The peaks at 1,454, 1,639, and 2,948 cm-1 indicate aliphatic CH2 bending, C=O stretching (amide I), and aliphatic CH2 stretching, respectively. 24,25 The QOS functionalization of collagen nanofibers showed two small peaks at 2,924.5 and 2,853.6 cm-1 due to the long chains of methylene and methyl groups in QOS, which were further strengthened after (NH4)CO3 exposure. 26 It is worth noting that a new small peak is observed at 1,237.2 cm-1, which is assigned to the carbon-siloxane (CO-Si) bond. In the Coll_1%QOS_XL spectrum, the -OH group of collagen and QOS can be supported. Effective reaction between hydrolysis of trimethoxysilyl (-Si(CH3O)3) groups.

Figure 2 (A) FTIR spectrum, (B) XPS general scan and (C) high-resolution C 1s, N 1s, O 1s and Si 2p spectra of samples ES_Coll, Coll_1%QOS and Coll_1%QOS_XL. Abbreviations: XPS, X-ray photoelectron spectroscopy; QOS, quaternary ammonium organosilane; FTIR, Fourier transform infrared.

In order to better understand the surface chemistry, XPS analysis was performed to thoroughly analyze the chemical bond interaction between collagen and QOS before and after cross-linking treatment. The wide-range XPS scans of all studied samples showed three peaks corresponding to C 1s, N 1s, and O 1s, while Coll_1%QOS and Coll_1%QOS_XL showed additional peaks at 101.85 eV due to the Si 2p component, Consistent with their expected composition (Figure 2B). The high-resolution C 1s, N 1s, O 1s and Si 2p spectra further confirmed the existence of multiple bond components in all samples and revealed the changes caused by the addition of QOS after crosslinking (Figure 2C). Perform comprehensive chemical bonding analysis through the deconvoluted C 1s, N 1s, O 1s and Si 2p high-resolution spectra of ES_Coll, Coll_1%QOS and Coll_1%QOS_XL (Figure 3). The curve fitting of C1s shows the existence of four peaks with binding energies of 284.5, 285.65, 286.45, and 287.45 eV, which are designated as C–C/C–H, C–N/C–OR, C–OR, and C = O/HN(C=O) bonding is in ES_Coll respectively, while C–C/C–H, C–N/C–O–Si/C–OR, C–OR/C–O–Si and C = O/HN(C=O) bonding, respectively in the samples Coll_1%QOS and Coll_1%QOS_XL (Figure 3A). 24 The CO-Si peaks of the CO-Si spectra in the samples Coll_1%QOS and Coll_1%QOS_XL correspond to the silane peaks. Similarly, the deconvolution of the N 1s spectra of all samples shows that there is a main peak at 399.4 eV, which belongs to For the nitrogen in the free amino group, a sub-peak at 400.9 eV is attributed to the nitrogen in the amide group (Figure 3B). The spectrum of Coll_1%QOS_XL N1s connected by 25 Cross shows an additional peak at 402 eV, which is attributed to hydrogen-bonded amines or quaternary ammonium cations. Two chemical states were observed in the high-resolution O 1s XPS spectra of different mats. The high binding energy signal at 532.3 eV (O1) corresponds to the Si-O-Si/Si-OC/OC bond, while the low binding energy signal at 530.9 eV (O2) corresponds to the C=O bond (Figure 3C). 24 Compared with the equivalent uncrosslinked Coll_1%QOS pad, the O1/O2 peak area ratio of the QOS crosslinked pad is slightly enhanced (Table 1). We deconvolved the Si 2p spectrum and designated it as the silane peak at 101.85 eV (Figure 3D). 26 It is worth noting that we did not observe any peaks related to SiO (Si metal) or SiO2. The overall XPS analysis confirmed the successful incorporation of silane into the collagen matrix and produced new bonding arrangements in terms of C-Si-O, Si-O-Si and Si-OC bonding.

Figure 3 Deconvolution of high resolution (A) C 1s, (B) N 1s, (C) O 1s and (D) Si 2p spectra of samples ES_Coll, Coll_1%QOS and Coll_1%QOS_XL. Abbreviation: QOS, quaternary ammonium organosilane.

Table 1 Different sample deconvoluted C 1s, N 1s, O 1s and Si 2p spectra under various chemical bonding curve analysis quantitative area QOS, quaternary ammonium organosilane.

Based on the combined analysis and these observations in the previously reported literature, we propose here a reasonable mechanism for QOS-mediated crosslinking of collagen nanofibers (Figure 4). 25,26 The decomposition of ammonium carbonate produces ammonia conditions and increases pH >8.5. The alkaline conditions then promote the hydrolysis of the -Si(CH3O)3 groups and initiate the condensation polymerization of the silanol groups and the crosslinking of the -OH groups present in the collagen nanofibers.

Figure 4 Proposed cross-linking mechanism of electrospun collagen by QOS alkaline hydrolysis. Abbreviation: QOS, quaternary ammonium organosilane.

Mechanical, thermal and wetting properties of collagen pads with QOS

In order to determine the influence of QOS cross-linking, we studied the mechanical properties of the original collagen pad and collagen-QOS pad (Figure 5A-C) and analyzed the tensile strength (σ), elongation at break (εb), Young’s Modulus (E') and toughness (Jlc). The various mechanical parameters estimated from the stress-strain curve are shown in Table 2. The original ES_Coll shows brittleness-like behavior, with σ and εb values ​​of 4.9 ± 0.5 MPa and 6.0% ± 1.3%, respectively. 9 Due to low elongation, the mat exhibits a high Young's modulus of 156.7 ± 29 MPa and a toughness of 0.22 ± 0.07 MJ m-3. The stress-strain curve of the cushion containing QOS shows a significant increase in elastic properties, while a decrease in mechanical strength and stiffness (Figure 5B). As the QOS content in the coating solution increases, significant increases in εb and Jlc are observed, indicating a transition from brittleness to plasticity-like transition (Figure 5C). The εb value has increased from 6.0% ± 1.3% of ES_Coll to 64.77% ± 8.15% of Coll_1%QOS_XL. Similarly, QOS crosslinking improves the toughness of nanofibers. The E'values ​​of Coll_0.1%QOS_XL, Coll_0.5%QOS_XL and Coll_1%QOS_XL are 0.33 ± 0.04, 0.41 ± 1.78 and 0.46 ± 0.17 MJ m-3 ( Figure 5C). Similar to our observations, Liu et al. reported that the crosslinking of electrospun poly(acrylic acid) (PAA)/PVA polymer with silicate sol due to the formation of a silicate network makes the fiber mat ductile. 27 Therefore, the QOS cross-linking of electrospun collagen resulted in a significant increase in the flexibility and toughness of the fiber mat compared to the original collage.

Figure 5 (A) Stress-strain curves of various collagen scaffolds; the figure shows the QOS cross-linked pair (B) peak stress (σ) and Young's modulus (E') and (C) elongation at break (εb) And the work of failure (Jlc) of collagen nanofibers. Significance value: **p<0.01; *** p<0.001; ****p<0.0001 and not significant (ns), through Student's t test or one-way analysis of variance p>0.05. (D) TGA and (E) DTA curves of different collagen nanofiber mats. (F) The photograph shows the contact angle formed by the fixed water droplets on the original and cross-linked mats. Abbreviation: QOS, quaternary ammonium organosilane.

Table 2 Mechanical properties of different concentrations of quaternary silane cross-linked electrospun collagen pad Note: **p<0.01; *** p<0.001; ****p<0.0001 and nsp>0.05, through Student t test or one-way analysis of variance .

The thermal degradation behavior of collagen cross-linked with QOS was studied by thermogravimetric analysis and differential thermogravimetric analysis (Figure 5D and E). Pure collagen shows weight loss that can be described in three different stages: 28 First, the loss of water molecules due to physical adsorption (25°C–200°C); second, weight loss refers to the thermal degradation of collagen (200°C). °C-400°C), the third is due to the carbonization of residual organic components (above 400°C). TGA studies have shown that the initial temperature of collagen decomposition (Ti) after QOS cross-linking has increased, although with the increase of silane content, no regular trend is observed in the Ti value (Table 3). Compared with the ES_Coll pad, the collagen degradation starting temperature (1Tmax) of the QOS cross-linked pad is moderately increased, and the residual weight (Wres%) is significantly increased. All these observations indicate that there is a significant interaction between collagen and silica network, which helps stabilize collagen nanofibers and improve their thermal stability. These observations confirm previous reports that silicate crosslinking improves thermal stability and reduces the weight loss of electrospun PVA or PVA/PAA stents. 23,27

Table 3 Abbreviations of thermal stability of various electrospun collagen pads: Ti, starting temperature of collagen decomposition; 1Tmax, collagen degradation temperature; 2Tmax, carbonization temperature of residual organic components; Wres, remaining weight.

After applying water droplets to the fiber surface, the change in wettability of the mat was evaluated by measuring WCA (θstatic). The results showed that the wettability of the pad did not change with the increase of QOS content (Figure 5F), indicating that silicate crosslinking did not change the water wettability of the collagen pad.

Biological characteristics of QOS cross-linked collagen scaffold

In order to explore the biological applicability of collagen QOS cross-linking, we first studied the biocompatibility of hDF cell scaffolds by MTS assay after 3-9 days. ps29,30 In order to convert the MTS absorbance value to the number of cells, a calibration method was used ( Figure S2). Cells seeded on CS and ES_Coll served as controls. The results showed that the metabolic activity of hDF inoculated on Coll_0.1%QOS_XL pad was higher than that of ES_Coll or ES, confirming the biocompatibility of organosilane crosslinking (Figure 6A). Compared with CS or ES_Coll pads, the number of cells in Coll_0.1%QOS_XL pads after 3 days increased by twice the number of days ps (p<0.0001). The SEM image showed that the morphology of the cells seeded on Coll_0.1%QOS_XL was extended, and the cells completely covered the surface of the mat 6 days after ps, indicating that the cross-linked mat provided a compatible surface for hDF adhesion and diffusion, and when seeded on ES_Col (Figure 6B). Consistent with these observations, CMFDA staining of cells seeded on Coll_0.1%QOS_XL pads confirmed that most spindle-shaped cells are feasible (Figure 6B inset). The cells seeded on Coll_0.1%QOS_XL and ES_Coll also showed strong actin stress filaments, indicating strong adhesion of hDF and higher contraction behavior (Figure 7). In addition, compared with hDF seeded on CS or ES_Coll pads, the cells seeded on Coll_0.1%QOS_XL pads showed an expanded polyhedral shape with frequent cell-to-cell connections and a good spreading phenotype (Figure 7), confirming Results from SEM and CMFDA images. However, compared with the Coll_0.1%QOS_XL pad, the cells seeded on the collagen pad with higher QOS content showed weaker metabolic activity, cell shrinkage and attachment loss, indicating the toxic effect of the cross-linking agent at high concentrations ( Figures 6 and 7). According to reports, glutaraldehyde, genipin, hexamethylene diisocyanate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) have similar crosslinking agent concentration dependence Sexual toxicity, which is attributed to the presence of unreacted/partially reacted crosslinkers. 31 –34

Figure 6 Evaluation of the biocompatibility of QOS cross-linked collagen pad to primary hDF. (A) MTS assay shows the metabolic activity of hDF inoculated on various electrospun mats. The metabolic activity is converted into the total number of cells by the calibration method. The average cell number is reported as the mean ± SD (n=3). Statistical significance only means that ES_Coll and Coll_0.1%QOS_XL (****p<0.0001) at 9 days ps (B) SEM images show that the morphology of hDF seeded on various scaffolds after 3 and 6 days is more ps High coverage rate compared with other scaffolds, hDF seeded on Coll_0.1%QOS_XL was observed. Scale bar = 10 μm. The inset is a confocal image of hDF (6 days ps) after CMFDA staining. Scale bar = 20 μm. The cytotoxicity of the scaffold containing higher concentration of QOS in the coating solution is consistent with the results of MTS. Abbreviations: CMFDA, 5-chloromethyl fluorescein diacetate; QOS, quaternary ammonium organosilane; hDF, human dermal fibroblasts; MTS, 3-(4,5-dimethylthiazol-2-yl)- 5-(3-Carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazole; SEM, scanning electron microscope; ps, later stage.

Figure 7 Confocal immunofluorescence images showing the cytoskeleton organization of hDF seeded on various substrates. Scale bar = 20 μm. Compared with CS and ES_Coll, cells seeded on Coll_0.1%QOS_XL were observed to have increased cell spread and polyhedral shape. Scale bar = 20 μm. Abbreviations: hDFs, human dermal fibroblasts; QOS, quaternary ammonium organosilanes; CS, coverslips.

Next, we investigated whether the QOS cross-linked collagen pad can provide a biocompatible surface for hFOb cells through cell proliferation (MTS) and cell differentiation (ALP) analysis. As shown in Figure 8A, compared with TCP or ES_Coll pads, when cells were seeded on Coll_0.1%QOS_XL, the cell proliferation rate was significantly increased. ALP activity is a marker of early osteoblast differentiation and the orientation of stem cells to osteoblast phenotype. 35-37 The ALP activity of various scaffolds was measured at 3, 6, and 9 days after ps, and normalized by the number of cells, as shown in Figure 8B. The results showed a significant difference in ALP activity/cell number between ES_Coll and Coll_0.1%QOS_XL (p<0.0001). However, the cells seeded on TCP have higher ALP activity/cell number than ES_Coll (p<0.001) and Coll_0.1%QOS_XL. This may be due to the enhanced cell density of Coll_0.1%QOS_XL and ES_Coll compared to TCP. As observed in the cell proliferation test, for the cells seeded on the Coll_0.5%QOS_XL and Coll_1%QOS_XL pads, less or negligible ALP activity can be detected, indicating that the cross-linking agent has cells at elevated concentrations Toxicity (data not shown).

Figure 8 (A) The metabolic activity of osteoblasts seeded on the original and cross-linked collagen pads was evaluated by MTS assay. The average ± SD from five independent measurements is reported. (B) Intracellular ALP activity of hFOb cells determined by pNPP assay on various scaffolds after 3, 6, and 9 days. The ALP results are standardized and reported by the number of cells (mmol p-nitrophenol/h/number of cells in hFob cells). These values ​​represent the mean ± SD from three independent repeated measurements. ****p<0.0001 is determined by Student's t-test or one-way analysis of variance. (C) Quantification of mineral deposits in hFOb cells by Alizarin Red-S staining. (D) Optical microscopy image shows the degree of mineralization of hFOb cells at 6 days ps. ps scale = 50 μm. Abbreviations: ALP, alkaline phosphatase; hFOb, human fetal osteoblasts; ps, after seeding; TCP, tissue culture plate.

After osteogenic differentiation, hFOb cells begin to secrete mineral matrix and enter the mineralization stage. 8,38 Here, we used ARS detection to qualitatively and quantitatively determine mineral deposits (Figure 8C and D). Since the ability to deposit minerals is a sign of mature osteoblast activity, stronger ARS staining indicates enhanced mineralization. The results of ARS staining show that, compared with TCP or ES_Coll, the mineralization of Col_0.1%QOS_XL is enhanced, which is consistent with the optical image (Figure 8D).

We next performed FE-SEM, CMFDA, and actin staining on the cells to visualize the morphology of hFOb seeded on various scaffolds at 6 days ps. The SEM images show the extended morphology accompanying mineralization on the ES_Coll and Coll_0.1%QOS_XL scaffolds, while the mineral deposits are negligible. Supplementary ARS results are observed on the CS surface (Figure 9A-C). The EDXS of sedimentary minerals confirmed the presence of peaks corresponding to calcium and phosphate ions, indicating the formation of calcium phosphate crystals (Figure 9D). For the Coll_0.5%QOS_XL and Coll_1%QOS_XL scaffolds, the cells shrink and detach from the surface (Figure 9E and F). The confocal image shows the intact living cell morphology (CMFDA staining) and the cytoskeletal structure firmly adhered to the surface of CS, ES_Coll Coll_0.1% QOS_XL (actin staining) (Figure 10), further confirming the results from the MTS and SEM studies The results obtained. In an earlier study, Torres-Giner et al. observed that compared to mats cross-linked with transglutaminase and attributed to higher cross-linking, inoculation with EDC/N-hydroxysuccinimide (NHS) The initial proliferation rate of osteoblasts on cross-linked electrospun collagen is higher than the density achieved with EDC/NHS. 39 Therefore, the presence of cross-linking and organosilicates is likely to promote cell proliferation at lower concentrations, and the increase in the amount of quaternary ammonium ions may damage the cell plasma membrane, thereby destroying cell viability.

Figure 9 SEM image shows the morphology of hFOb on various stents at 6 days ps (A) CS, (B) ES_Coll, (C) Coll_0.1% QOS_XL, (D) EDXS in the box area of ​​“c” shows the presence of phosphoric acid Calcium, (E) Coll_0.5%QOS_XL and (F) Coll_1%QOS_XL. Scale bar = 10 μm. Abbreviations: CS, cover glass; SEM, scanning electron microscope; hFOb, human fetal osteoblasts; QOS, quaternary ammonium organosilane; EDXS, energy dispersive X-ray spectrometer; ps, late stage.

Figure 10 Confocal images showing the morphology of hFOb cells seeded on various scaffolds 6 days after ps. Please note that the cells seeded on the Coll_0.1%QOS_XL scaffold have considerable cell proliferation and increased abundance of stress filaments. Scale bar = 20 μm. Abbreviations: hFOb, human fetal osteoblasts; QOS, quaternary ammonium organosilane; ps, after seeding; CS, cover glass; CMFDA, 5-chloromethylfluorescein diacetate.

Antibacterial evaluation of QOS cross-linked collagen scaffold

QOS has broad-spectrum antibacterial properties and represents an effective category of disinfectants, which produce rapid sterilization properties by destroying microbial cell membranes. 19,40,41 The antibacterial properties of different QOS cross-linked mats were studied to understand whether QOS retains its antibacterial potential in the collagen field. The antibacterial activity of the QOS cross-linked collagen pad is expressed as a reduction factor (inhibition percentage) relative to the positive control, and is listed in Table 4. Interestingly, all QOS cross-linked mats showed significantly reduced viability (>99.9%) of Gram-positive bacterial strains, including Staphylococcus aureus/MRSA, which is the main cause of osteomyelitis, thus confirming the cross-linking The antibacterial properties of QOS are retained.

Table 4 Antibacterial potential of QOS cross-linked collagen cushion in reducing factor. Abbreviation: QOS, quaternary ammonium organosilane.

In conclusion, the cross-linked collagen pad containing 0.1% QOS (Coll_0.1%QOS_XL) pad proved to be an ideal scaffold, with high mechanical toughness, good thermal stability, antibacterial properties and they provide a favorable environment for mammalian cells The potential for growth, proliferation and differentiation.

In this work, we report a simple cross-linking strategy for electrospun collagen nanofibers using multifunctional QOS, and study the thermal, mechanical, and wettability characteristics of the cross-linked scaffold. QOS crosslinking increases the ductility of other rigid collagen nanofibers and increases the occurrence of thermal degradation. By evaluating the cell adhesion, metabolic activity and morphology of cells seeded on various scaffolds, the biocompatibility of these cross-linked scaffolds was established for dermal fibroblasts and osteoblasts. We have observed that increasing the concentration of the cross-linking agent is cytotoxic to dermal fibroblasts and osteoblasts, thereby limiting the concentration range of the cross-linking agent. However, mats cross-linked with low concentrations of QOS showed effective bactericidal activity, indicating the potential of this scaffold to avoid microbial colonization while promoting the proliferation and growth of mammalian cells. The cost-effectiveness and multifunctional characteristics of crosslinking agents have broad prospects in the production of high-performance stents. These advantages provide a wide range of opportunities for the use of such stents in many biomedical applications, where the infection of commensal pathogens needs to be controlled. The application of this stent has the potential to develop anti-infective 3D stents for soft tissue and hard tissue engineering, durable antibacterial wound dressings/bandages and implant coatings for wound management.

The author is grateful to the National Research Foundation of Singapore for its translational and clinical research flagship project (NMRC/TCR/008-SERI/2013), which is managed by the National Medical Research Council of the Ministry of Health of Singapore. This work was supported by the cooperative basic research grant from the National Medical Research Council of Singapore (NMRC/CBRG/0048/2013) and the SNEC Ophthalmology Technology Incubator Program grant granted to RL (project number R1181/83/2014). This research was supported by the National Medical Research Council of the Ministry of Health of Singapore, and its central funding program-Ophthalmology Research Core Platform Technology Optimization (INCEPTOR)-NMRC/CG/M010/2017_SERI. NKV recognizes the financial support of Lee Kong Chian School of Medicine, Nanyang Technological University Entrepreneurship Grants (L0412130 and L0412290) and AcRF-Tier I Grants (2015-T1-001-082) from the Ministry of Education of Singapore.

The authors report no conflicts of interest in this work.

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Figure S1 FE-SEM micrographs of (A) Coll_5%QOS, (B) Coll_10%QOS, (C) Coll_5%QOS_XL and (D) Coll_10%QOS_XL. Note: Scale bar = 1 μm. Yellow arrows indicate areas with adhesion/bonding between fibers. Abbreviations: SEM, scanning electron microscope; QOS, quaternary ammonium organosilane.

Figure S2 uses MTS to determine the standard (cell number and absorbance) calibration curves drawn for (A) hDF and (B) hFOb. Abbreviations: hFOb, human fetal osteoblasts; hDF, human dermal fibroblasts; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H tetrazolium.

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