Patent Application
20240189475
This application is based on and claims priority from Korean Patent Application No. KR10-2022-0173566, filed on Dec. 13, 2022, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
The present disclosure relates to a nanofiber scaffold including polycaprolactone (poly(ε-caprolactone; PCL), decellularized extracellular matrix (dECM) and usnic acid (UA), and a preparation method thereof.
A microbial community, biofilm formation and subsequent infection may produce various substances, including toxins, which impede wound healing and complicate a regeneration process. A scaffold for skin regeneration fabricated to avoid wound infection and eradicate infected pathogens needs to have excellent antibacterial activity.
Usnic acid (UA) is a secondary metabolite of sea moss and is a molecule with strong antibacterial activity against various pathogens. The UA also has significant potential in the fields of pharmacology and tissue engineering applications due to its unique biological and physiological activities such as antioxidant, anti-inflammatory, antiviral, fibroblast migration, growth factor release enhancement and wound healing properties. However, due to its low solubility in water, its usability is somewhat limited. To improve a delivery range, the UA is mounted to various delivery systems for utilization such as liposomes, films, and nanoparticles.
Decellularization is a technique to remove cells while maintaining important physiologically active molecules such as glycosaminoglycans, collagens, proteoglycans and growth factors and core morphological, structural, mechanical and biochemical properties. Decellularized extracellular matrix (dECM) is widely used in tissue engineering due to its biocompatibility, unique properties, and numerous biological properties. However, due to the loss of structural and mechanical properties and the increase in solubility, the dECM is not applied alone to the fabrication of nanofiber scaffolds for skin regeneration.
Nanofibers fabricated utilizing electrospinning technology have similar structural properties to natural ECM in terms of their interconnected porous structure, and thus have attracted considerable attention in the field of skin tissue engineering applications. Electrospinning may be used with a variety of synthetic polymers, including polycaprolactone (PCL), due to their unique properties such as mechanical properties, biodegradability and biocompatibility. The PCL is a widely used semi-crystalline aliphatic polyester approved for biomedical use by the U.S. Food and Drug Administration (FDA). However, due to its hydrophobicity and low response to cells, the PCL is mixed and used with various natural polymers such as collagen, gelatin, alginate, and chitosan to control biological and mechanical properties and provide structural functions suitable for tissue regeneration.
The inventors of the present disclosure prepared polycaprolactone-based nanofibers utilizing usnic acid and pig skin-derived decellularized extracellular matrix and identified their structural properties, antibacterial activity, and skin regeneration ability to complete the present disclosure.
Accordingly, an example embodiment of the present disclosure provides a nanofiber scaffold including polycaprolactone (poly(ε-caprolactone; PCL), decellularized extracellular matrix (dECM) and usnic acid (UA), and a preparation method thereof.
According to the example embodiment of the present disclosure, there is provided the nanofiber scaffold including the PCL, the dECM, and the UA.
In addition, according to another example embodiment of the present disclosure, there is provided a method of preparing a nanofiber scaffold, in which the method includes: mixing polycaprolactone, decellularized extracellular matrix, and usnic acid (phase 1); preparing a nanofiber mat by electrospinning the mixture (phase 2); cross-linking the nanofiber mat (phase 3); and freeze-drying the cross-linked nanofiber mat (phase 4).
In addition, according to yet another example embodiment of the present disclosure, there is provided a wound dressing including the nanofiber scaffold according to the example embodiment of the present disclosure.
The PCL/dECM/UA nanofibers for skin regeneration of an example embodiment of the present disclosure may be fabricated utilizing electrospinning technology, which enables commercial production. In addition, the nanofiber of an example embodiment of the present disclosure has a uniform nanofiber structure, good fluid absorption and retention, moderate release rate, high mechanical stability, thermal stability, and semi-crystalline structure, and exhibits excellent antibacterial efficacy against Candida albicans and four other fungi/bacteria, and good compatibility with human-derived fibroblasts. It was identified through an in vivo full-thickness incisional wound healing mouse model that the fabricated hydrogels significantly improved wound closure by day 21, and histomorphometric evaluation of wound site tissues revealed rapid re-epithelialization, differentiated dermis and epidermis with minimal scar tissue remaining. Accordingly, the fabricated nanofibers exhibited excellent antibacterial activity and skin regeneration efficacy, making them applicable for wound healing applications.
In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.
Hereinafter, the present disclosure will be described in more detail. However, the present disclosure may be implemented in many different forms, the present disclosure is not limited by the example embodiments described herein, and the present disclosure is defined only by the claims to be described later.
The terms used herein are presented for the description of the specific embodiments but are not intended to limit the present disclosure. In the entire specification, when a certain portion “comprises or includes” a certain component, this indicates that the other components are not excluded and may be further included unless specially described otherwise.
An example embodiment of the present disclosure relates to a nanofiber scaffold including polycaprolactone (poly(ε-caprolactone; PCL), decellularized extracellular matrix (dECM) and usnic acid (UA).
The nanofiber scaffold may be in the form of a nanofiber of the decellularized extracellular matrix and polycaprolactone containing usnic acid, but is not limited thereto.
The nanofiber scaffold may be prepared by electrospinning, but is not limited thereto.
The nanofiber scaffold may include the polycaprolactone and the decellularized extracellular matrix in a weight ratio of 8 to 2:2 to 8, for example, the polycaprolactone and the decellularized extracellular matrix in a volume ratio of 8:2, 6:4, 4:6 or 2:8, but is not limited thereto.
The nanofiber scaffold may be cross-linked, but is not limited thereto.
The nanofiber scaffold is prepared by a method including: mixing polycaprolactone, decellularized extracellular matrix, and usnic acid (phase 1); preparing a nanofiber mat by electrospinning the mixture (phase 2); cross-linking the nanofiber mat (phase 3); and freeze-drying the cross-linked nanofiber mat (phase 4), but is not limited thereto.
Hereinafter, the nanofiber scaffold of an example embodiment of the present disclosure will be described based on the preparation method of the nanofiber scaffold of an example embodiment of the present disclosure.
First, the preparation method of the nanofiber scaffold of an example embodiment of the present disclosure includes mixing polycaprolactone, decellularized extracellular matrix, and usnic acid (phase 1).
In phase 1, the polycaprolactone and the decellularized extracellular matrix may be mixed in a weight ratio of 8 to 2:2 to 8, for example, the polycaprolactone and the decellularized extracellular matrix may be mixed in a volume ratio of 8:2, 6:4, 4:6 or 2:8, but is not limited thereto.
In addition, the preparation method of the nanofiber scaffold of an example embodiment of the present disclosure includes: preparing a nanofiber mat by electrospinning the mixture (phase 2); cross-linking the nanofiber mat (phase 3); and freeze-drying the cross-linked nanofiber mat (phase 4).
In phase 2, the electrospinning may be performed using a technique commonly used in the pertinent field, but is not limited thereto.
In phase 3, the nanofiber mat may be immersed in EDC and NHS in ethanol to cross-link, but is not limited thereto.
In addition, an example embodiment of the present disclosure relates to a wound dressing including the nanofiber scaffold of an example embodiment of the present disclosure.
Pure PCL, usnic acid (UA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), fluorescein diacetate (FDA), propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), and 1, 1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Sigma-Aldrich (USA). Dulbecco's minimum Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin and trypsin (250 U/mg) were purchased from GIBCO © (Invitrogen Corporation, USA). Potato dextrose broth (PDB), tryptic soy broth (TSB), and brain heart infusion (BHI) were purchased from Difco Laboratory Inc. (Detroit, MI, USA). All other chemicals and solvents used in example embodiments of the present disclosure were of analytical grade and the water was deionized.
Preparation and Isolation of dECM
Decellularized extracellular matrix (dECM) was extracted from pig skin. First, the fat adhered to the lower side portion of the skin was physically removed and washed with distilled water. Then, the skin was finely cut and put in 0.25% of Trypsin-EDTA for 6 hours at 37° C. After incubation, debris was removed from the pig skin, washed with PBS (phosphate-buffered saline), and immersed in PBS containing 1% of Triton X-100 and 25 mM of EDTA for 24 hours. The pig skin was then washed with PBS for 24 hours at 4° C. and sterilized with 0.1% of peracetic acid in 4% of EtOH. Then, the pig skin was washed several times with distilled water, lyophilized, and dissolved in 1 M acetic acid containing pepsin for 7 days. The pH of the mixture was adjusted to 7.4 using NaOH solution under cold conditions, lyophilized again, and dissolved in HFIP for the preparation of nanofiber scaffolds.
10% (w/v) of dECM and 10% (w/v) of PCL were dissolved in HFIP for Use Gap Code preparation, and PCL and dECM were mixed in different mass ratios (100:0, 80:20, 40:60, 60:40, 20:80, and 0:100) in the presence of usnic acid (UA) (3 mg/mL). The solution was electrospun through a 22-gauge needle into a stainless steel rotating collector wrapped in aluminum foil using a syringe pump (KD Scientific Inc., Holliston, MA, USA) with a high voltage power supplier under different operating conditions (Table 1). The nanofiber mat was completely immersed in pure ethanol containing EDC (20 mM) and NHS (5 mM) for 30 minutes, washed with pure ethanol, and lyophilized.
dECM)
indicates data missing or illegible when filed
The surface morphology of the nanofiber scaffold was analyzed using SEM. The electrospun composite nanofiber scaffold was sputter-coated with a silver layer using Emi-Tech K500X and visualized by SEM (FE-SEM, Hitachi S-2700, Japan) at an accelerating voltage of 5 kV. The average fiber diameter was analyzed with image analysis software (ImageJ Wayne, Rasband). In addition, the pore size distribution of the cross-linked scaffold was evaluated with image analysis software (ImageJ Wayne, Rasband) and the porosity was studied using the liquid-phase displacement method.
Fourier-transform infrared spectrometer (FTIR) analysis was used for chemical characterization of pure UA and nanofiber dECM, PCL, PCLU, PEU 2:8, PEU 4:6, PEU 6:4, and PEU8:2 samples. All spectra were recorded at 4 cm intervals in the frequency range of 4000 to 650 cm1 at room temperature using an FTIR (JASCO (FT-4100), Tokyo, Japan) spectrometer.
Pure UA, PCL, and composite nanofiber scaffolds were characterized by X-ray diffraction analysis (Philips X'pert MPD diffractometer, Eindhoven, Netherland) in the range of diffraction angles (20) from 10 to 600 at a scanning rate of 6°/min with CuKα radiation generated at 36 kV and 200 mA.
Thermal properties of pure UA, PCL, and composite nanofiber scaffolds were measured at a constant heating rate of 10° C./min under nitrogen gas in the temperature range of 50 to 700° C. using a thermogravimetric (TGA) Pyris 1 TGA analyzer (Perkin Elmer TGA-7, Waltham, MA, USA). Melting properties were analyzed using differential scanning calorimetry (DSC) (Perkin Elmer, Diamond, Waltham, MA, USA) at a constant heating rate of 10° C./min under an argon gas flow of 50 mL/min in the temperature range from room temperature to 250° C.
The tensile strength of the prepared nanofiber scaffold was identified using a universal tensile machine (LR5K Plus, Lloyd Instruments). A rectangular strip (15 mm×5 mm×T; T is the thickness) was placed and a crosshead speed of 1 mm/min was applied at room temperature. Three independent samples from each group were used for analysis.
The nanofiber scaffold was placed horizontally on a flat surface, and a droplet of DMEM (FBS free) was placed at room temperature. It was photographed with a digital camera and the contact angle was analyzed with image analysis software (ImageJ Wayne, Rasband) using three independent photographs per group.
The prepared nanofiber scaffold was immersed in 1×PBS (pH 7.4) at 37° C. until equilibrium in an expansion state was reached. The nanofiber scaffolds were collected at regular time intervals, excess PBS and surface moisture were removed with blotting paper, and left at room temperature for 1 minute. Then, the equilibrium expansion ratio was calculated according to the following equation. In the equation below, Wd and Ws are dry weight and expanded weight, respectively.
Swelling (%)=[(Ws−Wd)/Wd]×100
The prepared nanofiber scaffold was immersed in 1×PBS (pH 7.4) at 37° C. At regular intervals, the remaining samples were taken out, washed with deionized water and lyophilized. Weight loss was calculated according to the equation below. In the equation, Wi and Wt are the initial dry weight and the weight at different times, respectively.
Degradation (%)=[(Wi−Wt)/Wi]×100
The release properties of usnic acid (UA) were identified. The prepared nanofiber scaffold was immersed in 1×PBS (pH 7.4), and the release medium was withdrawn at set time intervals and analyzed with a microplate reader (Gen 5™ ELISA BioTek, USA) at 280 nm. The released UA was calculated according to the equation below. In the equation, Wi and Wt mean the initial amount of UA in the scaffold and the amount released at a specific time, respectively.
Release (%)=[(Wi−Wt)/Wi]×100
Cell culture studies were conducted using HDF fibroblasts and HaCaT keratinocytes in DMEM supplemented with 10% of FBS, 100 units/mL of penicillin, and 0.1 mg/mL of streptomycin at 37° C. in a humidified 5% CO2 atmosphere.
One hour prior to transfer to a 48-well microplate, the nanofiber scaffold was punched using an 8 mm diameter biopsy punch and exposed to UV irradiation. Cytocompatibility, cell adhesion, and proliferation on scaffolds were studied using the MTT assay at 1 and 5 days post seeding. First, 1×104 cells were placed on an 8 mm scaffold and incubated in a humid environment at 37° C. On days 1 and 5 after seeding, cell-cultured nanofiber scaffolds were washed using 1×PBS (pH 7.4) and incubated in DMEM supplemented with MTT (1 mg/mL) for 3 hours in the same environment. The incubated solution was then aspirated and re-incubated with DMSO for 30 minutes at 37° C. The absorbance of the solution was measured at 540 nm using a microplate reader (Gen 5™ ELISA BioTek, USA).
To study cytotoxicity, cell adhesion, proliferation, and migration on nanofiber scaffolds, live/dead cell staining was performed using FDA (5 μg/mL) and PI (20 μg/mL) in medium without FBS for 15 minutes at 37° C. on day 1 and day 5 after seeding the cells onto the nanofiber scaffold. After staining the cells with FDA and PI, the solution was aspirated and washed twice with 1×PBS (pH 7.4). Green fluorescence of live cells and red fluorescence of dead cells were detected using a fluorescence microscope (Leica DMI300B Microsystems, Wetzlar, Germany).
Bacterial cultures of Pseudomonas aeruginosa (PAO1 KCTC 1637), Staphylococcus aureus (KCTC 1916), Cutibacterium acnes (KCTC 3314), and Klebsiella pneumoniae (ATCC 4352) were purchased from Korean Collection for Type Cultures (KCTC, Daejeon, Korea). Streptococcus mutans (KCCM 40105), Staphylococcus epidermidis (KCCM 40003), and fungal strain Candida albicans (KCCM 11282) were purchased from Korean Culture Center of Microorganisms (KCCM, Sudaemun Seoul, Korea). The growth medium for the culture of C. albicans is PDB supplemented with 5% of glucose, and the agar medium is PDA. P. aeruginosa, S. aureus, K. pneumoniae, and S. epidermidis used TSB as the growth medium and tryptic soy agar (TSA) as the agar medium. C. acnes was grown and cultured in BHI supplemented with 1% of glucose and BHA (brain heart infusion agar) in a CO2 incubator (NAPCO 5400; General Laboratory Supply, Pasadena, USA) in a 10% CO2 humid atmosphere. The growth temperature of all bacterial strains and C. albicans was 37° C.
The antibacterial activity of nanofiber scaffolds against bacterial and fungal pathogens was identified by solid agar labeling and disk diffusion in liquid growth media. Cultures of bacterial pathogens and C. albicans grown overnight were diluted to an equivalent OD600 value of 0.05 (same as the Mcfarland standard for bacterial inoculum), and were dispensed (100 μL) onto the labels of TSA (for bacterial culture) and PDA (for fungal cell culture) plates using a cotton swab. Spreading of the cell culture was performed by forming regular bacterial cultures on agar plates. An 8 mm diameter of nanofiber scaffold was placed on the surface of an agar plate including a bacterial or fungal cell culture and incubated at 37° C. for 24 hours. After incubation, inhibition of diameter was measured.
Two nanofiber scaffolds (8 mm diameter) were placed on a 24-well microplate containing bacterial or fungal cell cultures. Bacterial cell cultures were incubated for 24 hours and C. albicans cell cultures were incubated for 48 hours while shaking the microplate at 150 rpm at 37° C. C. acnes cell cultures in BHI supplemented with glucose (1%) on a 24-well microplate were incubated with nanofiber scaffolds in a CO2 incubator (NAPCO 5400; General Laboratory Supply, Pasadena, USA) at 37° C. and 10% CO2 humid atmosphere for 48 hours under anoxic conditions. After incubation, the cell culture (100 μL) was serially diluted (10−8 dilutions) with TSB (for bacterial culture) and PDB (C. albicans). After spreading the diluted cell culture (100 μL) on TSA and PDA agar plates, bacterial cells were grown by incubation at 37° C. for 24 hours and C. albicans cells for 48 hours. C. acnes bacterial cell cultures were spread-plated on BHA and incubated for 24 hours at 37° C. under anoxic conditions. The total number of colonies on each agar plate was counted to measure colony-forming units (CFU).
Biofilm inhibitory properties of the nanofiber scaffold against P. aeruginosa and K. pneumoniae were identified. An 8 mm diameter of nanofiber scaffold was used to identify the biofilm inhibitory activity. The biofilm inhibitory activity of the nanofiber scaffold was identified by measuring the CFU of cells adhered to the surface. The nanofiber scaffold was placed at the bottom of a 24-well microtiter plate and the diluted cell culture was dispensed (1:100 dilution in its respective growth medium). Microtiter plates were incubated for 24 hours at 37° C. under static conditions. After incubation, the free-floating suspension cell culture was discarded and the nanofiber scaffold was washed 3 times with sterile growth medium and transferred to a centrifuge tube (1.5 mL). The centrifuge tube containing the nanofiber scaffold was resuspended with 500 μL of TSB (bacteria) and PDB (C. albicans). The tube was vortexed for 5 minutes and then sonicated for 10 minutes. The cell suspension (100 μL) from the centrifuge tube was diluted up to 108 dilutions with each sterile growth medium. Diluted cell cultures (100 μL) were spread-plated on agar plates and incubated at 37° C. for 24 hours. The percent inhibition of the biofilm was calculated according to the equation below. In the following equation, Nc represents the average number of microbial colonies in the control group, and Nsi represents the average number of microbial colonies in the experimental group.
Inhibition (%)=(Nc−Nsi)/Nc×100
The in vivo wound healing properties of nanofiber scaffolds were evaluated using 8-week-old normal male ICR mice (6 mice per group) weighing 30 g and acclimatized for 7 days under a controlled environment (relative humidity: 40 to 70%, temperature: 20 to 24° C.). All mice were anesthetized in a chamber containing 20% of isoflurane in isopropanol for 20 minutes and masked throughout the surgery. After shaving the hair and disinfecting with povidone-iodine and 70% of ethanol, a full-thickness 2.25 cm2 square excisional wound was created on the skin of the back. The prepared PCL, PCLU, PCL:dECM 4:6 (PE 4:6), and PEU 4:6 nanofiber scaffolds were positioned using sutures. After surgery, the wound areas of all experimental groups were photographed on days 0, 7, 14, and 21 with a digital camera, and wound closure was measured with image analysis software (ImageJ Wayne, Rasband) and plotted as a relative percentage of initial wound size. For histomorphological evaluation, mice were sacrificed on day 21 after surgery, and wound sites and tissues therearound were collected.
The collected tissue samples were fixed for 1 day using a 10% of formalin solution, dehydrated using alcohol and xylene successively, and wax infiltrated using a tissue processor (Leica TP1020, Leica Biosystems, Nussloch, Germany). Embedded tissues (Leica EG1160, Leica Biosystems, Nussloch, Germany) were sectioned at 5 μm, mounted on glass slides, dewaxed with xylene and then dehydrated with alcohol and then water. Nuclei were stained with hematoxylin, excess hematoxylin was washed away with water and ECM and cytoplasm were counterstained with eosin. The stained tissue was dehydrated by applying grading alcohol up to 100%, washed in xylene, and placed on a cover-slip using a mounting medium.
In addition, collagen deposition was evaluated by staining the dissected tissues with Masson's trichrome staining (Masson's Trichrome stain kit; KTMRT, American MasterTech, Lodi, CA, USA). First, deparaffinized incisions were immersed in Bouin's fluid preheated at 60° C. for 60 minutes, washed in running tap water, stained with working Weigert's hematoxylin for 5 minutes, and washed with tap water. Then, the deparaffinized incisions were impregnated in Biebrich scarlet for 15 minutes, washed with tap water, incubated with phosphotungstic/phosphomolybdic acid for 10 minutes, impregnated in aniline blue for 5 minutes, washed with distilled water, transferred to 1% of acetic acid, dehydrated with absolute ethanol, and placed on a cover-slip using a mounting medium. Finally, the stained incisions were observed using an optical microscope (Leica Microsystems UK Ltd, Milton Keynes, UK) and representative photographs were taken.
All quantitative data are presented as the mean±standard deviation (SD) from three independent experiments performed using fresh reagents. Significant differences between groups were evaluated using one-way ANOVA according to Duncan's test using GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). *p<0.05 and **p<0.01 were selected as having statistical significance compared to control.
Bead-free, randomly arranged, smoothly and homogeneously distributed nanofiber structures were obtained by controlling operating conditions such as voltage, distance between drum collector and needle, and flow rate of polymer solution, regardless of polymer solution concentration. (A), (B) and (C) of
The FTIR spectra of the PCL, dECM, UA, and cross-linked composite PEU nanofiber scaffolds, and shifts in the characteristic absorption bands are shown in (A) of
(B) of
TGA ((C) of
The miscibility and homogeneity of the dECM, PCL, and UA nanofiber blends were identified by DSC analysis. Theoretically, perfectly miscible and homogeneous complexes form a single-phase transition instead of the formation of mixed phases and other phase transitions of each pure material used in the complex. The results ((D) of
The mechanical integrity of nanofiber scaffolds is essential for their application in tissue regeneration as it is important to supply basic biomechanical fitness to cells before tissue regeneration. The mechanical properties of nanofiber PCL, PCLU, and PEU scaffolds with different dECM contents were analyzed, and typical tensile stress-strain curves ((E)(a) of
Since the hydrophilic nature of composite nanofiber scaffolds is one of the most important properties for water absorption, the wettability of PCL, PCLU, and composite PEU nanofiber scaffolds was identified using water contact angle. (A) and (B) of
The swelling capacity of tissue-engineered skin substitutes is important to absorb exudates, which are main ingredients that stimulate cell migration and proliferation, while providing a moist environment at the wound site. The expansion ratios of PCL, PCLU, and all PEU composite nanofiber scaffolds are shown in (C) and (D) of
The prepared nanofiber scaffolds also need to have suitable degradation properties that allow successful tissue regeneration and replacement of biological tissue with structures. Weighted analysis ((E) of
As can be seen in (F) of
The disk diffusion assay results showed a clear zone in which all types of bacterial and fungal pathogens were inhibited in the presence of the UA-integrated scaffold, and the diameter of the inhibition zone increased as the dECM content in the nanofiber scaffold increased. Among the pathogens experimented, C. acnes and S. mutans showed larger inhibition zones on both UA-integrated PCLU and PEU scaffolds ((A) of
The antibacterial effect of the nanofiber scaffolds against pathogens was experimented in liquid broth medium and representative agar plates, showing the appearance and disappearance of bacterial and strain cell cultures ((A) of
Based on the above results, it was concluded that the increased concentration of dECM showed a higher antibacterial effect against pathogens compared to the control group. The high antibacterial effect by high content of dECM suggests that the high content of dECM in nanofiber scaffolds results in high release properties accompanied by increased swelling, surface erosion and degradation. However, the antibacterial effect of PCLU clarifies the antibacterial effect of UA despite the minimal release and fiber degradation. Previous studies have shown that UA-loaded nanofibers, UA-encapsulated nanomaterials and hydrogels show antibacterial effects similar to those of the present disclosure.
The formation of microbial biofilms on biological/non-biological surfaces, including medical products, is a major source of microbial infection and poses a serious threat to human health. A biofilm is made of a homogeneous group of microbial cells encapsulated in a self-produced extracellular polymeric material. Microbial biofilms act as barriers and protect against host immune responses, so they are one of the major obstacles to the treatment of infections and cause failure of antibacterial treatment. Biofilm formation by pathogenic microorganisms, such as those found at wound sites, is a major cause of wound infection treatment failure. In an example embodiment of the present disclosure, the inhibitory ability of the PEU nanofiber scaffold against biofilm formation of microbial pathogens related to burn wound infection, such as P. aeruginosa and K. pneumoniae (PMID: 26500905 and 18158583), was identified. It was identified through antibacterial assays that the nanofiber scaffolds had effective antibacterial and antifungal activities against C. acnes, S. aureus, S. epidermidis, and S. mutans. However, the nanofiber scaffold showed no antibacterial effect against K. pneumoniae and P. aeruginosa (
Cell viability and proliferation as a function of time on nanofiber scaffolds are indicators of the scaffold's cell compatibility and suitability for tissue engineering applications. Through the MTT assay, it was identified that the nanofiber scaffold satisfied the basic cell compatibility requirements for application in tissue engineering. Adhesion and proliferation of HDF and HaCaT cells at 1 and 5 days after culture on pure PCL, PCLU, and composite PEU nanofibers are shown in (A) and (B) of
The biocompatibility of the scaffold was further evaluated using FDA and PI staining for live/dead cell labeling. (C) of
(A) and (B) of
To identify the influence of the composite nanofiber scaffold on skin tissue regeneration, a histomorphometric test was performed on the center of the wound site on day 21 after staining the wound with hematoxylin and eosin (H&E) staining ((C) of
In addition, (D) of
From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0173566 | Dec 2022 | KR | national |
