WO2024143784A1 - Nanofibrous scaffold for skin regeneration, comprising polydeoxyribonucleotide derived from patiria pectinifera - Google Patents

Abstract

The present invention relates to a nanofibrous scaffold and a preparation method therefor, the nanofibrous scaffold comprising: poly(ε-caprolactone) (PCL); gelatin; and polydeoxyribonucleotide (PDRN) derived from Patiria pectinifera. The nanofibrous scaffold of the present invention has a uniform nanofiber structure, excellent fluid absorption and retention, an adequate release speed, high mechanical stability, thermal stability, and thus is suitable for a dressing for healing wounds.

Description

Nanofiber scaffold for skin regeneration containing starfish-derived polydeoxyribonucleotide

The present invention relates to a nanofiber scaffold containing polycaprolactone (poly(ε-caprolactone; PCL); gelatin; and polydeoxyribonucleotide (PDRN) derived from starfish ( Patiria pectinifera ); and a method for manufacturing the same. will be.

Dressing is a method used to improve the healing rate by covering the wound surface, which is a skin defect caused by burns, wounds, bedsores, and trauma. In 1962, Winter reported that the rate of epithelium formation in porcine wounds decreased when the wet environment was dry. Since the announcement that it is more than twice as fast as the environment, wet dressings have been developed and released one after another, and various wound treatment methods are being developed.

Wet dressings are designed to seal the wound surface and maintain a moist state. By developing and combining various hydrophilic and hydrophobic polymers, they are available in the form of film, sheet, non-woven fabric, sponge, and foam ( It is rapidly developing into various forms such as foam, rope, pellet, and powder.

The material used for such wound healing is a product that can absorb and contain a large amount of exudate discharged from the wound surface. It must have the form of a three-dimensional network structure made by cross-linking hydrophilic polymers with covalent or non-covalent bonds. Due to its hydrophilic nature, it absorbs a large amount of moisture and swells in aqueous solutions and in aqueous environments, but must have the property of not dissolving due to the cross-linked structure.

Therefore, various methods have been proposed depending on the composition or manufacturing method, but in the case of physical crosslinking by non-covalent bonds, deterioration of physical properties is likely to occur, and in the case of chemical crosslinking, it is known that there is skin toxicity due to the crosslinking agent, and ultrasonic or radiation crosslinking is known to cause skin toxicity. There are methods, but the realistic situation is that there are limitations in equipment and high costs.

Nanofibers produced using electrospinning technology are receiving considerable attention in skin tissue engineering applications because they have structural characteristics similar to extracellular matrix (ECM) in terms of their interconnected porous structure. Electrospinning can be used with a variety of synthetic polymers, including polycaprolactone (PCL), due to their unique properties such as mechanical properties, biodegradability, and biocompatibility. PCL is a widely used semi-crystalline aliphatic polyester that has been approved for biomedical use by the U.S. Food and Drug Administration (FDA). However, due to its hydrophobicity and low response to cells, it is mixed 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.

Meanwhile, polydeoxyribonucleotides (PDRN), an active mixture with a molecular weight ranging from 50 to 1,500 kDa, are mainly extracted and purified from sperm cells of rainbow trout ( Oncorhynchus mykiss ) or salmon ( Oncorhynchus keta ). PDRN is a type of DNA-derived medicine currently approved by the Food and Drug Administration and is widely used for tissue repair and wound treatment. PDRN exhibits multiple efficacies including pro-angiogenic, anti-apoptotic, anti-inflammatory and tissue repair activities. However, despite these biological effects related to tissue repair, only O. mykiss and O. keta are used as extraction raw materials for PDRN.

Accordingly, the inventors of the present invention isolated polydeoxyribonucleotide (PDRN), which has excellent skin regeneration efficacy as a DNA polymer, from starfish ( Patiria pectinifera ), and studied the structural properties and skin regeneration of nanofibers manufactured using it. The ability was confirmed and the present invention was completed.

Accordingly, the purpose of the present invention is to provide a nanofiber scaffold containing polydeoxyribonucleotide (PDRN) derived from starfish ( Patiria pectinifera ) and a method for manufacturing the same.

The present invention provides a nanofiber scaffold containing polycaprolactone (poly(ε-caprolactone; PCL); gelatin; and polydeoxyribonucleotide (PDRN) derived from starfish ( Patiria pectinifera ).

In addition, the present invention includes the steps of isolating polydeoxyribonucleotide (PDRN) from starfish ( Patiria pectinifera ) (step 1); Mixing the polydeoxyribonucleotide with polycaprolactone and gelatin (step 2); and electrospinning the mixture to produce a nanofiber mat (step 3).

Additionally, the present invention provides a wound dressing comprising the nanofiber scaffold according to the present invention.

The present invention secures polydeoxyribonucleotide (PDRN) with excellent skin regeneration efficacy from the starfish ( Patiria pectinifera ), which is an unutilized resource and a pirate organism, and applies it to a nanofiber scaffold for skin regeneration, thereby protecting unused marine life. It provided new possibilities for utilizing resources in tissue engineering and as a medical device for skin tissue regeneration using Byeolbulgasari PDRN.

Figure 1 shows the results of confirming the molecular weight distribution of PDRN derived from P. pectinifera using agarose gel electrophoresis. Lane A: DNA marker; Lane B: PDRN from P. pectinifera (20 μg).

Figure 2 shows the proliferation effect of PDRN derived from P. pectinifera in HDF. ^* p < 0.10 and ^** p < 0.05 were considered statistically significant differences compared to the untreated group.

Figure 3 shows the results of FDA/PI staining showing the proliferation effect by PDRN derived from P. pectinifera in HDF.

Figure 4 is a graph showing the results of the MTT assay confirming the cytotoxicity of P. pectinifera- derived PDRN against HaCaT. ^* p < 0.10 and ^** p < 0.05 were considered statistically significant differences compared to the untreated group.

Figure 5 shows the results of FDA/PI staining confirming the cytotoxicity of P. pectinifera- derived PDRN against HaCaT.

Figure 6 is a graph showing the results of Picro-Sirius red staining confirming the effect of collagen production on the HDF of PDRN derived from P. pectinifera . ^* p < 0.05 and ^** p < 0.10 were considered statistically significant differences compared to the untreated group.

Figures 7a and 7b show the effect of P. pectinifera -derived PDRN on cell migration for 24 hours: (A) representative images of cell migration assay results and (B) graph of wound healing rate. ^a p < 0.05 was considered to be a statistically significant difference compared to the initial scratch wound area in each group, and ^b p <0.05 was considered to be a statistically significant difference compared to the untreated group.

Figure 8 shows the effect on the expression and (B) mechanism of action of several proteins (type I collagen, type III collagen, α-SMA) related to skin regeneration in the HDF of PDRN extracted from P. pectinifera (A).

Figure 9 is a schematic diagram showing the manufacturing process of nanofibers using electrospinning.

Figure 10 shows SEM micrographs and the corresponding nanofiber diameter distributions (percentage and frequency of distribution): (a) P10G0, (b) P8G2, (c) P6G4, (d) P4G6, (e) P2G8, and (f) P0G10 nanofiber.

Figure 11 shows the mechanical properties of PCL/Gel nanofibers: (A) typical stress-strain curve, (B) tensile strength, (C) strain at maximum load, and (D) strain at maximum extension. ^# p < 0.05 was considered to be a statistically significant difference compared to P10G0 nanofiber.

Figure 12 is a graph showing (A) a representative image and (B) a dynamic water contact angle of the water contact angle of the manufactured nanofibers (n > 3). *p < 0.05 was considered to be a statistically significant difference compared to the P0G10 nanofiber group at the same time.

Figure 14 shows SEM micrographs and the corresponding nanofiber diameter distribution (percentage and frequency of distribution): (a) P, (b) PG, and (c) PGP nanofibers.

Figure 15 shows the mechanical properties of nanofibers: (A) typical stress-strain curve, (B) tensile strength, (C) strain at maximum load, and (D) strain at maximum extension. ^# p < 0.05 was considered to be a statistically significant difference compared to P nanofiber.

Figure 16 shows the PDRN release profile of PGP nanofibers in 1X PBS (pH 7.4) at 37°C. Data are presented as mean ± standard deviation (n = 4).

Figure 17 shows the FTIR spectra and peak intensities of pure PCL, gelatin, PDRN, and prepared nanofibers and their positions according to the mixing ratio. (a) pure PCL (black), (b) pure gelatin (yellow), (c) PDRN (brown), (d) P (gray), (e) PG (blue), and (f) PGP (green). ) Nanofiber.

Figure 18 is a graph showing the results of thermogravimetric analysis of pure materials and manufactured nanofibers.

Figure 19 shows differential scanning calorimetry (DSC) curves of pure materials and manufactured nanofibers.

Figure 20 shows the X-ray diffraction profiles of pure material and prepared nanofibers.

Figure 21 is a graph showing (A) a representative image and (B) a dynamic water contact angle of the water contact angle of the manufactured nanofibers (n > 3). ^# p < 0.05 was considered to be a statistically significant difference compared to the P nanofiber group at the same time.

Figure 22 shows the swelling properties of nanofibers prepared at 37°C for 96 hours in PBS (pH 7.4). Data are presented as mean ± standard deviation (n = 4).

Figure 23 shows the results of the MTT assay confirming the direct cytotoxicity and proliferation of (A) HDF and (B) HaCaT cultured on the manufactured nanofibers. ^* p < 0.05 was considered a statistically significant difference compared to the untreated group.

Figure 24 confirms the effect of the manufactured nanofibers on HDF cell viability through indirect cytotoxicity analysis using FDA/PI staining.

Figure 25 confirms the effect of the manufactured nanofibers on HaCaT cell viability through indirect cytotoxicity analysis using FDA/PI staining.

Figure 26 confirms the effect of the nanofibers prepared through indirect cytotoxicity analysis using the MTT assay on the cell viability of (A) HDF and (B) HaCaT. ^* p < 0.05 was considered a statistically significant difference compared to the untreated group.

Figure 27 is a schematic diagram showing the experimental schedule for inducing a circular full-thickness incision wound in an ICR mouse and applying a dressing of the manufactured nanofiber.

Figure 28 is a schematic diagram showing the experimental schedule for inducing a rectangular full-thickness incision wound in an ICR mouse and applying a dressing of the manufactured nanofiber.

Figure 29 shows the effect of fabricated nanofibers on a circular full-thickness wound model compared to the untreated group at 21 days after surgery: (A) representative images of wound closure kinetics and (B) average over time after surgery. Wound closure. Error bars represent standard deviation (n≥3).

Figure 30 shows the effect of fabricated nanofibers on a rectangular full-thickness wound model compared to the untreated group at 28 days after surgery: (A) representative images of wound closure kinetics and (B) average over time after surgery. Wound closure. Error bars represent standard deviation (n≥3).

Figures 31A and 31B show wound closure of nanofibers in a circular full-thickness wound model at 21 days. (A) H&E, Masson's trichrome, and Picro-Sirius Red staining results of mouse skin tissue 21 days after wounding. Scale bar 600 μm at 2X and 100 μm at 10X. (B) Relative wound length and (C) wound thickness of the circular full-thickness wound model at day 21. Error bars represent standard deviation (n≥3). ^* p < 0.05 was considered a statistically significant difference compared to the untreated (blank) group.

Figures 32A and 32B show wound closure of nanofibers in a rectangular full-thickness wound model at 28 days. (A) H&E, Masson's trichrome, and Picro-Sirius Red staining results of mouse skin tissue 28 days after wounding. Scale bar 1000 μm at 1X and 200 μm at 5X. (B) Relative wound length and (C) wound thickness of the rectangular full-thickness wound model at day 28. Error bars represent standard deviation (n≥3). ^* p < 0.05 was considered a statistically significant difference compared to the untreated (blank) group.

Figure 33 shows the effect of PGP nanofibers on the expression of several proteins (type I collagen, type III collagen, α-SMA) related to skin regeneration in (A) circular and (B) rectangular full-thickness wound models. β-actin was used as a housekeeping gene for quantification of Western blot analysis results.

Hereinafter, the present invention will be described in more detail. However, the present invention may be implemented in various different forms, and the present invention is not limited to the embodiments described herein, and the present invention is only defined by the claims to be described later.

In addition, the terms used in the present invention are only used to describe specific embodiments and are not intended to limit the present invention. In the entire specification of the present invention, 'including' a certain element means that other elements may be further included rather than excluding other elements, unless specifically stated to the contrary.

The present invention relates to a nanofiber scaffold containing polycaprolactone (poly(ε-caprolactone; PCL); gelatin; and polydeoxyribonucleotide (PDRN) derived from starfish ( Patiria pectinifera ); and a method for manufacturing the same. will be.

The nanofiber scaffold may be manufactured by electrospinning, but is not limited thereto.

The nanofiber scaffold may include the polycaprolactone and gelatin in a weight ratio of 8 to 2:2 to 8, for example, the polycaprolactone and gelatin in a weight ratio of 8:2, 6:4, 4:6. Alternatively, it may be included in a weight ratio of 2:8, but is not limited thereto.

The nanofiber scaffold may include the polycaprolactone, gelatin, and polydeoxyribonucleotide in a weight ratio of 4 to 8:2 to 6:0.005 to 0.1, for example, in a weight ratio of 6:4:0.02. It may include, but is not limited to this.

The gelatin can be used without limitation as long as it is gelatin used for wound dressing in the field, but is not limited thereto.

The nanofiber scaffold includes the steps of isolating polydeoxyribonucleotide (PDRN) from starfish ( Patiria pectinifera ) (step 1); Mixing the polydeoxyribonucleotide with polycaprolactone and gelatin (step 2); and electrospinning the mixture to produce a nanofiber mat (step 3), but is not limited thereto.

Hereinafter, the nanofiber scaffold of the present invention will be described based on the manufacturing method of the nanofiber scaffold of the present invention.

First, the method for manufacturing the nanofiber scaffold of the present invention includes the step (step 1) of isolating polydeoxyribonucleotide (PDRN) from starfish ( Patiria pectinifera ).

Next, the present invention includes mixing polydeoxyribonucleotide with polycaprolactone and gelatin (step 2).

In step 2, the polycaprolactone and gelatin may be mixed at a weight ratio of 8 to 2:2 to 8, for example, the polycaprolactone and gelatin may be mixed at a weight ratio of 8:2, 6:4, 4:6 or 2. : Can be mixed at a weight ratio of 8, but is not limited thereto.

Additionally, in step 2, the polycaprolactone, gelatin, and polydeoxyribonucleotide may be mixed at a weight ratio of 4 to 8:2 to 6:0.005 to 0.1, for example, 6:4:0.02. It can be done, but is not limited to this.

Next, the method for producing a nanofiber scaffold of the present invention includes the step (step 3) of producing a nanofiber mat by electrospinning the mixture.

In step 3, electrospinning may be performed using techniques commonly used in the field, but is not limited thereto.

Additionally, the present invention relates to a wound dressing comprising the nanofiber scaffold of the present invention.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

<Example>

matter

Starfish ( P. pectinifera ) was provided by Dongsam Fishery Village (Yeongdo-gu, Busan, South Korea). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Grand Island, NY, USA). Purchased from . 3-(4,5-Dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), fluorescein diacetate (FDA), PCL, and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit monoclonal antibodies against anti-COL type I (ab90395) and Collagen III (ab7778) and anti-α-smooth muscle actin (α-SMA) mouse monoclonal (ab7817) antibodies were purchased from Abcam (Cambridge, UK). Phosphorylated (p)-c-Jun N-terminal kinases (p-JNK; #9251), and p-p38 (#9211) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778), extracellular signal-regulated kinase (ERK) 1/2 (sc-514302), p-ERK 1/2 (sc-7383), and JNK (sc-7383) -7345), rabbit polyclonal antibody against p38 (sc-7149), and goat IgG donkey polyclonal secondary antibody (sc-2020) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat polyclonal secondary antibodies against rabbit IgG (G21234) and mouse IgG (G21040) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Grand Island, NY, USA). Other general analytical grade chemicals and reagents used in the present invention are commercially available.

Example 1

P. pectinifera Preparation of PDRN extract from

PDRN (Polydeoxyribonucleotide) was extracted from P. pectinifera (starfish) using the AccuPrep Genomic DNA extraction kit (Bioneer, Daejeon, Korea). First, P. pectinifera was washed three times with tap water to remove epiphytes, salt, and sand attached to the surface, and then carefully washed again with fresh water. P. pectinifera frozen at -20°C was freeze-dried and then homogenized to powder. Crushed P. pectinifera (1 g) was extracted in 4 ml TL buffer containing Proteinase K (2 mg/ml) and RNase A (2 mg/ml) at 60°C for 2 hours. Then, the extract was added to 4 ml of GB buffer and 8 ml of absolute ethanol and mixed with a pipette, and then the extract was transferred to a binding column tube and centrifuged at 8,000 rpm for 1 minute. W1 and W2 buffers were added to the binding column tube and centrifuged at 8,000 rpm for 1 minute each. Then, ethanol was completely removed from the binding column tube by centrifugation (13,000 rpm, 1 min). For elution of PDRN, EA buffer was added to the binding column tube and centrifuged at 8,000 rpm for 1 minute. Finally, the PDRN eluate was lyophilized and stored at -20 °C until use.

P. pectinifera Characterization of origin PDRN

The purity of PDRN was calculated based on the ratio of absorbance at 260 nm and 280 nm, and the DNA content was confirmed using absorbance at 260 nm.

Protein content was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, IL, USA). First, 20 μl of each extract in 180 μl of working reagent solution was incubated at 37 °C for 30 min. Absorbance was measured using a microplate reader (PowerWave XS2, BioTek Instruments, Inc., Winooski, VT, USA) at 562 nm. A standard curve of bovine serum albumin was prepared and protein content was calculated. Carbohydrate content was measured according to the information described in Dubois et al. 100 μl of each extract was mixed with 100 μl of 5% phenol and 500 μl of H ₂ SO ₄ and reacted at room temperature for 20 minutes. Absorbance was measured using a microplate reader at 490 nm. A glucose standard curve was prepared and the sugar content was calculated. Polyphenol content was measured according to protocols published in previous literature. 250 μl of 7.5% Na ₂ CO ₃ was added to 100 μl of each extract and reacted at room temperature for 5 minutes. Then, 300 μl of 1N Folin-Ciocalteu reagent was added and incubated for 30 minutes in dark conditions. After incubation, absorbance was measured using a microplate reader at 765 nm. A gallic acid standard curve was prepared to calculate the polyphenol content.

Agarose powder was dissolved in Tris-acetate-EDTA (TAE) buffer and heated. The heated agarose solution was poured into a mold and cooled to room temperature to prepare a transparent gel. The 2% agarose gel was then slowly transferred into an electrophoresis chamber submerged in TAE buffer with the loading well of the gel proximal to the cathode side. The agarose gel was electrophoresed for 1 hour by applying a voltage of 100 V away from light. The gel was visualized after staining with ethidium bromide using the UNOK-8000 Gel Manager System (Biotechnology, Seoul, Korea), and mRNA expression was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

cell culture

Human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were obtained from the American Type of Culture Collection (ATCC, Manassas, VA). Cells were grown in DMEM supplemented with 10% heat-inactivated FBS. Cells were incubated at 37° C. in a humid atmosphere with 5% CO ₂ . Cells were cultured until they reached a density of 70% to 80%.

Cytotoxicity of PDRN

HDF and HaCaT were each inoculated at ^a density of 1 The next day, the cells were treated with various concentrations of PDRN, and then the cells were incubated for an additional 24 hours. MTT solution (1 mg/ml in PBS) was added to the culture medium at 20 μl/100 μl of each well. After incubation for 2 hours, the supernatant was removed and the formazan crystals were dissolved in DMSO. Then, the absorbance was measured at 540 nm using a PowerWave XS2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Data are expressed as percentage of viability of visualized cells ± standard deviation of three replicate experiments.

Live/dead cell staining

To observe live/dead cells and cell proliferation of HDF and HaCaT cells treated with samples or eluates for 1 and 3 days after inoculation, HDFs and HaCaT were incubated with FDA (10 μg/ml) in serum-free medium for 15 min at 37 °C. and PI (20 μg/ml). The stained cells were then washed with PBS to remove untreated FDA and PI and residues. FDA appears as green fluorescence in live cells and PI appears as red in dead cells, and was quantitatively analyzed using a fluorescence microscope (Leica DMI3000B).

In vitro cell migration analysis

HDF was inoculated into 2 ml of culture medium in a 6-well plate at ^a density of 4 Cells were incubated for 24 hours to attach at a density of approximately 90%. After cells were attached to the plate, a wound line was created with a 2 mm wide plastic pipette tip, and unattached cells were washed with PBS. Then, cells were treated with PDRN at different concentrations and allowed to migrate. Pictures were taken at a magnification of 50 was carried out.

Collagen production efficacy analysis

Collagen production was assessed by Picro-Sirius red staining. HDFs were incubated with a staining solution made of Sirius red (0.1%, Sigma) dissolved in a saturated aqueous solution of picric acid (1.3% in water, Sigma) for 2 hours, washed three times with PBS, and then dehydrated. The stained crystals were dissolved in 0.1 M NaOH, and the absorbance was measured using a PowerWave XS2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Data were expressed as a percentage of the average collagen production rate ± standard deviation of visualized cells in three replicate experiments.

Western blot analysis

To evaluate the cell proliferation effect of PDRN on HDFs, HDFs were grown in 100 cm ² dishes at a density of approximately 2 × 10 ⁶ cells. Cells were treated with PDRN at various concentrations and incubated for 24 hours to check the collagen expression level, and incubated for 30 minutes to analyze the degree of activation of Smad2/3 and mitogen-activated protein kinase (MAPK) pathways.

For in vivo analysis, mice were sacrificed, skin tissue was removed, and tissue samples were stored in a nitrogen gas tank until further analysis. Frozen tissue samples were homogenized with a Tissue Lyser (SpeedMill PLUS, Jena, Germany) in lysis buffer. Specimens were destroyed using steel beads at 30 cycles/second for 5 minutes.

Samples were lysed in lysis buffer (20mM Tris-HCl at pH 7.4, 5mM EDTA, 10mM Na ₄ P ₂ O ₇ , 100mM NaF, 2mM Na ₃ VO ₄ , 1% NP-40, 10 mg/ml aprotinin, After dissolving in 10 mg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride) for 30 minutes, the residue was removed by centrifugation at 13,000 rpm for 15 minutes at 4°C. The protein concentration of cell lysates was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc., Grand Island, NY, USA). Then, 30 μg of protein was separated by electrophoresis using 10% SDS-PAGE and transferred to Amersham Protran Premium 0.45-μm nitrocellulose blotting membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membrane was blocked with 5% skim milk powder in TBS-T (25mM Tris-HCl at pH 7.4, 137mM NaCl, 2.65mM KCl, 0.05% Tween-20) for 2h at room temperature and then incubated with primary antigen (1: 1,000 dilution) and incubated for 24 hours. After washing with TBS-T, the membrane was incubated with secondary antibody (1:5,000 dilution) for 2 hours at room temperature. Bands were visualized using the CAS-400SM Davinch-Chemi image™ system (Young Hwa Scientific Co. LTD., Seoul, Korea), and protein expression was analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). was quantified. In addition, Western blot was repeated three times for in vitro and in vivo analysis.

<Result>

P. pectinifera Characterization of PDRN from

The purity and DNA concentration of PDRN extracted from P. pectinifera were 1.24 ± 0.01 and 176.67 ± 5.20 μg/ml, respectively. The chemical composition of PDRN is shown in Table 1, and it contains a slightly higher content of protein. These results are similar to the general composition of P. pectinifera, which consists of large amounts of water, proteins and lipids, and small amounts of carbohydrates. In addition, it was confirmed through agarose electrophoresis that PDRN derived from P. pectinifera had a small base pair range (<400 bp) (Figure 1).

To confirm the effect of HFIP used in nanofiber production on the stability of PDRN extracted from P. pectinifera , the purity and DNA concentration of PDRN in two solvents (DW and HFIP) were compared based on absorbance values. Regardless of the type of solvent, the purity and DNA concentration of PDRN extracted from P. pectinifera were maintained (Table 2). These results indicate that PDRN extracted from P. pectinifera is not affected by HFIP, and HFIP was selected and used as a solvent for producing nanofibers through electrospinning technology.

[Table 1]

[Table 2]

In vitro cytotoxic and proliferative activity

In vitro cytotoxicity and proliferative activity of PDRN derived from P. pectinifera were evaluated using MTT assay and FDA/PI staining. For the MTT assay, HDF and HaCaT cells were treated with increasing concentrations of PDRN for 1, 3, and 5 days, and for FDA/PI staining, they were treated with increasing concentrations of PDRN for 1 and 3 days. In HDF cells, PDRN (5-200 μg/ml) significantly increased cell proliferation after 3 days of treatment, with treatment at 50 μg/ml PDRN showing the highest proliferation (Figures 2 and 3). Additionally, PDRN induced HDF proliferation at concentrations of 5, 10, and 50 μg/ml. On the other hand, PDRN did not show toxicity to HaCaT cells, but also showed no significant proliferative effect (Figures 4 and 5). Based on these results, it was concluded that PDRN at the above concentration (5-50 μg/ml) showed a proliferative effect on HDF cells without showing toxicity to HaCaT cells.

Effect on collagen production

To investigate the effect of P. pectinifera- derived PDRN on collagen production, increasing concentrations of P. pectinifera- derived PDRN were added to HDF. (0-200 μg/ml) for 1, 3, and 5 days, and Picro-Sirius red staining was performed. As a result, 50-200 μg/ml PDRN increased collagen production in HDF cells. These results demonstrate that P. pectinifera- derived PDRN affects high-concentration collagen production; However, no significant difference was seen between 50 and 200 μg/ml (Figure 6).

In vitro Cell migration promotion effect

In vitro cell migration analysis was performed to confirm whether PDRN promotes cell migration. As shown in Figure 7, the scratched wound area in the untreated group was not completely covered even after 72 hours, whereas PDRN promoted cell migration in a dose-dependent manner. Additionally, the wound area in the high concentration (50-200 μg/ml) PDRN-treated group showed no significant difference between concentrations. In particular, PDRN stimulated cell migration and collagen production without cytotoxicity. Taken together, a concentration range of 50 μg/ml or less was selected for further experiments.

Effect on wound healing-related protein expression

Western blot analysis was performed to confirm the effect of PDRN extracted from P. pectinifera on the expression of proteins related to wound healing such as type I collagen, type III collagen, and α-SMA in HDF cells. PDRN extracted from HDF cells from P. pectinifera (0-50 μg/ml) for 5 days. Figure 8A showed that PDRN significantly increased type III collagen expression in HDF cells and accelerated α-SMA expression in a dose-dependent manner. These results indicate that PDRN extracted from P. pectinifera promotes the expression of proteins related to wound healing.

Effects on Smad2/3 and MAPK pathways

To determine the mechanism of action of PDRN in protein expression related to wound healing and cell migration, PDRN extracted from P. pectinifera The effect on activation of MAPK and Smad2/3 pathways was assessed by Western blot analysis. PDRN significantly increased phosphorylation of ERK and Smad2/3 compared to untreated cells (Figure 8B). These results suggest that PDRN increases the expression of proteins related to wound healing by activating the phosphorylation of ERK and Smad2/3.

Example 2.

Preparation of PG and PGP nanofibers

Solutions of gelatin 10% (w/v) (G1890, Sigma-Aldrich, USA), PDRN 0.02% (w/v), and PCL 10% (w/v) were added at 1, 1, 1, 3, 3, 3- Each was prepared by dissolving it in hexafluoro-2-propanol (HFIP) (Sigma-Aldrich, USA). Electrospinning was performed using a 24-gauge needle with a syringe pump (KD Scientific Inc., Holliston, MA, USA), high-pressure power supply, stainless-steel rotating collector wrapped in aluminum foil, and different operating conditions (Table 3 and 4 and Figure 9). The manufactured nanofibers were stored in a -80°C freezer until use, and some of them were freeze-dried depending on the purpose.

[Table 3]

[Table 4]

Characterization of nanofibers

After sputtering coating the nanofibers with a silver thin film using Emi-Tech K500X, the surface morphology was confirmed using field emission scanning electron microscopy (FE-SEM, Hithachi S-2700, Japan) at 5 kV. Average fiber diameter and pore size were measured using image analysis software (ImageJ Wayne, Rsband).

The wetting behavior of nanofibers was evaluated using a contact angle analyzer (SEO Phoenix MT, Suwon, Gyeonggi-do, Korea). The volume of the droplet was 2 μl, and the contact angle was measured at five separate random locations and averaged. DMEM medium without serum was used as the test solution. Nanofibers of the same size were placed on the sample stage at ambient temperature (~296 K) and the contact angle was measured. Pictures were taken with a digital camera over time and analyzed using image processing software (Image Pro 300).

The tensile strength of nanofibers was measured using a universal tensile machine (LR5K Plus, Lloyd Instruments). Each sample was cut into dumbbell-shaped strips (15 mm I ordered it.

To evaluate the absorption capacity of the composite scaffolds, each different scaffold was cut into squares (15 mm To measure the in vitro expansion behavior of the nanofibers, excess water and surface moisture were removed with plotting paper, left for 1 minute to remove surface moisture, and the weight was measured. The equilibrium-swelling percentage of the nanofiber was calculated according to the equation (Swelling (%) = [(W _s -W _d )/W _d ] In the above equation, W _d and W _s mean the dry and expanded states of the nanofiber, respectively.

The functional groups of pure PCL, pure gelatin, pure PDRN, P nanofiber, PG nanofiber, and PGP nanofiber were analyzed using Fourier transform infrared (FT-IR) spectroscopy (FT-4100, JASCO). The IR spectrum showed an average of 30 scans at a frequency of 650-4000 cm ^-1 at a resolution of 4 cm ^-1 .

Thermogravimetric analysis (TGA) was performed using a Pyris 1 TGA analyzer (PerkinElmer TGA-7, Waltham, MA, USA) with a scan range of 30 °C to 700 °C and a constant heating rate of 20 °C under continuous nitrogen. Calorimetry was performed using differential scanning calorimetry (DSC) under nitrogen flowing at a rate of 10 ml/min. The specimen was pressed into a sealed aluminum pan. Heating cycles were performed until the glass transition temperature (T _g ) and melting temperature (T _m ) were reached. During the cycle, the sample was heated from 30°C to 180°C at a rate of 10°C. The sample was then cooled using nitrogen at an exponentially decreasing rate.

X-ray diffraction (XRD) analysis of the nanofibers was performed using X-ray diffraction (X'Pert3-Powder, PANalytical, Netherlands) with Cu-Kα radiation. Diffraction intensity was recorded at a scanning speed of 2.4° min ^-1 in the range of 5 to 90°.

Release profile of gallic acid-containing PG nanofibers

Gallic acid released from the gallic acid-containing PG nanofibers was released in a PBS solution (pH 7.4) at 37°C for 14 days and centrifuged at 4°C at 10,000 g for 10 minutes to remove residues. Then, gallic acid from gallic acid-containing PG nanofibers was analyzed via the Folin-Ciocalteu method. First, 20 μl of the film solution was mixed with 100 μl of 1 N Folin-Ciocalteu reagent and 80 μl of 7.5% sodium carbonate, incubated for 10 minutes at room temperature, and incubated at 765 nm with a PowerWave XS2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) was used to measure absorbance. A standard curve was constructed using gallic acid solution in PBS.

PDRN emission profile of PGP nanofibers

PDRN release from PGP nanofibers was performed in a PBS solution (pH 7.4) at 37°C for 14 days and centrifuged at 10,000 g for 10 minutes at 4°C to remove residues. Then, PDRN released from PGP nanofibers was analyzed by measuring the total DNA content. First, 1 μl of the released solution was mixed with 49 μl of DW, incubated at room temperature for 10 minutes, and absorbance was measured using a PowerWave XS2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 260 nm. did.

Direct and indirect cytotoxicity of nanofibers

For direct cytotoxicity testing, nanofibers (P, PG, and PGP) were exposed to UV light for at least 1 hour on each side before cell experiments. The biocompatibility of the nanofibers was evaluated using direct contact testing based on the ISO 10993-5 standard. To evaluate cell-nanofiber contact cytotoxicity, cells were cultured, seeded in 12-well plates, and incubated at 37 °C for 24 hours. The nanofibers were then cut into 8 mm diameter disks and placed on the surface of the cell monolayer. After further incubation for 24 hours, cells were analyzed for general morphology using light microscopy and compared to the no-contact group.

For indirect cytotoxicity, elution solutions of nanofibers (P, PG, and PGP) for in vitro experiments were prepared according to ISO10993-12:2014 ( Biological evaluation of medical devices - Sample preparation and reference materials ). First, nanofibers of 30 For sterilization, the solution was filtered through a 0.20 μm syringe filter (Sartorius, Gϕtingen, Germany) and stored in a 4°C chamber until use.

statistical analysis

All quantitative data are expressed as the mean ± S.D. of at least three independent experiments using fresh reagents. The statistical significance of the observed differences between groups was assessed by analysis of variance (ANOVA) followed by Duncan's multiple range test. All statistical analyzes were performed using SPSS Statistics 12.0 software (SPSS, Inc., Chicago, IL, USA). Each characteristic was considered to indicate a statistically significant difference.

<Result>

Surface morphology analysis of PCL/Gel nanofibers

The PCL/Gel mixed solution was electrospun into a stainless-steel drum under optimal electrospinning conditions (Table 3). Except for P2G8 and P0G10 nanofibers, nanofibers prepared with different blend ratios were successfully fabricated with dense and bead-free microstructures (Figure 10). For P2G8 and P0G10 nanofibers, significant loss in porosity and fiber morphology was observed. Increasing the gelatin ratio affected the fiber diameter differently (582.88 ± 202.65, 444.61 ± 124.64, 435.65 ± 149.87, 686.13 ± 218.35, 703 for P10G0, P8G2, P6G4, P4G6, P2G8, and P0G10 nanofibers, respectively. .01 ± 124.64 , and 1370.93 ± 492.57 nm).

Tensile strength of PCL/Gel nanofibers

The mechanical properties of the nanofibers, such as stress-strain curve (Figure 11A), tensile strength (Figure 11B), strain at maximum load (Figure 11C), and strain at maximum extension (Figure 11D), are shown in Figure 22. P0G10 nanofibers have the lowest flexibility and elasticity, the lowest tensile strength of 0.50 ± 0.17 MPa, the lowest strain at ultimate load of 3.54 ± 1.35 (%), and the lowest rupture at 12.66 ± 6.00 (%). showed an extension. The tensile strength of P8G2 and P6G4 increased to 2.95 ± 0.34 and 1.50 ± 0.10 MPa, respectively. The PCL/Gel blend ratio was changed to 4:6, and a sudden decrease in tensile strength (0.77 ± 0.14 MPa) was observed compared to P10G0 nanofiber (1.15 ± 0.06 MPa) after complete failure at maximum load and maximum extension.

Contact angle of PCL/Gel nanofibers

The surface wettability of nanofibers is an important property related to cell attachment, growth, proliferation and migration, and can be influenced by the material chemistry associated with the design and surface structure of the nanofibers. To confirm the hydrophilicity of the nanofibers, the contact angle between the nanofibers and the culture medium was measured. Images and graphs of contact angle over time are shown in Figure 12. The synthetic polymer PCL is naturally hydrophobic, but natural polymers such as collagen and gelatin are hydrophilic. P10G0 and P8G2 nanofibers have high initial contact angles (125.77 ± 5.00° and 128.53 ± 5.28°, respectively). P6G4 nanofibers had a high initial contact angle (120.09 ± 10.68°), which decreased significantly to 92.68 ± 13.97°. On the other hand, P4G6, P2G8, and P0G10 nanofibers have low initial contact angles (94.82 ± 8.71°, 88.99 ± 3.44°, and 40.43 ± 7.53°, respectively). In particular, the contact angle of P0G10 decreased close to 0 compared to other nanofibers (from 40.43 ± 7.53° to 15.16 ± 1.95°). These results indicate that the addition of gelatin to the nanofibers increased the surface wettability of the nanofibers, which led to the induction of cell attachment, proliferation, and migration. In summary, P6G4 (PG) nanofibers with mechanical properties, surface morphology, and surface wettability properties suitable for use in skin-tissue engineering for further experiments and P10G0 (P) nanofibers as a gelatin-free group to determine the effect of gelatin. Fiber was selected.

Drug release ability of gallic acid-containing PG nanofibers

To determine the content of PDRN in PG nanofibers, a drug release test was performed using gallic acid-containing PG nanofibers. The release rate of gallic acid from PG nanofibers continued to increase to 25% over 7 days, and gallic acid was steadily released at a rate of 3.57% at 37°C in a PBS solution at pH 7.4 (FIG. 13). Therefore, the content of PDRN in PG nanofibers was chosen as 200 μg/ml to match the concentration for in vitro experiments (50 μg/ml).

Surface morphology analysis of PGP nanofibers

The PCL/Gel/PDRN mixed solution was electrospun into a stainless-steel drum under optimal electrospinning conditions (Table 4). Nanofibers prepared at different mixing ratios were successfully fabricated into dense, bead-free nanostructures (Figure 14). As shown in Figure 25C, the surface morphology of PGP nanofibers (fiber diameter = 334.63 ± 98.09 nm) is denser and more integrated than P and PG nanofibers.

Tensile strength of PGP nanofibers

The mechanical properties of the nanofibers, such as stress-strain curve (Figure 15A), tensile strength (Figure 15B), strain at maximum load (Figure 15C), and strain at maximum extension (Figure 15D), are shown in Figure 26. P nanofibers had a tensile strength of 1.15 ± 0.06 MPa, the highest strain at maximum load of 475.85 ± 75.16%, and 620.84 ± 58.16%. P8G2 showed the highest strain at its maximum extension. Compared with P nanofibers, the tensile strength of PGP nanofibers increased to 1.80 ± 0.16 MPa, and the strain at maximum load (102.52 ± 3.61%) and strain at maximum extension (116.44 ± 0.50%) decreased. These results indicate that the addition of PDRN does not affect mechanical properties such as tensile strength, strain at maximum load, and strain at maximum extension.

PGP PDRN release ability of nanofibers

As shown in Figure 16, PDRN released from PGP nanofibers was similar to that of gallic acid-containing PG nanofibers. The release rate of PDRN from PGP nanofibers continued to increase over 7 days, and PDRN was steadily released at a rate of 3.09% in PBS at 37°C at pH 7.4. Therefore, it was confirmed that continuous release of PDRN from PGP nanofibers was maintained for up to 7 days.

FT-IR analysis

FT-IR spectra of pure PCL, pure gelatin, PDRN, and prepared nanofibers are shown in Figure 17. Significant peaks of asymmetric and symmetric CH stretching vibrations of PCL polymer appeared at 2994 and 2866 cm ^-1 , respectively. The strong peak at 1722 cm ^-1 represents the stretching vibration of the carbonyl band (C=O) in the ester group, and the peak at 1293 cm ^-1 is due to stretching of CO and CC in the crystal phase. Other significant peaks at 1239 and 1165 cm ^-1 correspond to asymmetric and symmetric vibrations of ether (COC), respectively (Figure 17a).

In Figure 17b, the amide I band representing the stretching vibration of C=O and CN groups in gelatin was recorded at 1630 cm ^-1 , and the weak band at 1440 cm ^-1 represents the aliphatic CH bending vibration. The bands at 1524 and 1237 cm ^-1 represent stretching vibrations of NH bending and CN stretching, respectively, and are attributed to the characteristic bands of amide II and amide III in gelatin, respectively. Additionally, the FT-IR spectrum of PDRN showed several characteristic peaks of DNA.

As can be seen in Figure 17c, the three main regions of the FT-IR spectrum of PDRN consist of a set of transmission peak components related to the following DNA molecular vibrations. The DNA nucleobase region (1800-1500 cm ^-1 ) is associated with DNA bases and contains three peaks observed at 1660, 1588, and 1532 cm ^-1 ; The base-sugar region (1500-1250 cm ^-1 ) contains three peaks observed at 1462, 1398, and 1287 cm ^-1 that correspond to the absorbance at the vibration of the base. The phosphate DNA framework region (1250-650 cm ^-1 ) represents the phosphate framework region and contains four peaks recorded at 1033, 1020, 981, and 788 cm ^-1 . The peak of the in-plane stretching vibration of the carbonyl group in the guanine base ring (C6=C6) was found at 1660 cm ^-1 , and the peak of the in-plane stretching vibration of the cytosine single-strand or double-strand was found at 1532 cm ^-1 . Additionally, the peak corresponding to the -C=N ring vibration of the imidazole ring in nucleic acids (guanine and adenine) appeared at 1588 cm ^-1 . In the region of base sugars, the peak at 1462 cm ^-1 represents deoxyadenosine N1=C6-N6 of A-type or B-type DNA, and the peak at 1398 cm ^-1 represents all bases representing the A-type DNA form. It refers to the vibration caused by the sugar of C3'-endo. The third peak shows vibration as the -PO ₂ vibration of the phosphodieter skeleton in nucleic acid and was recorded at 1287 cm ^-1 . The peak corresponding to the deoxyribose CO stretching vibration appeared at 1033 cm ^-1 . The strong peak at 1020 cm ^-1 represents furanose vibration, and the next strongest peak at 981 cm ^-1 is due to CC stretching of the DNA deoxyribose-phosphate backbone. The weak vibration appearing near 788 cm ^-1 is related to deoxy c3'-endo-OPO and represents the A-type DNA conformation. All characteristic peaks of pure PCL, pure gelatin and PDRN are shown in Table 5.

[Table 5]

Thermodynamic analysis

Figure 18 shows the TGA results of pure PCL, pure gelatin, PDRN, and manufactured nanofibers at 30 to 700 °C. Pure PCL showed only one decomposition step starting at approximately 380 °C. The TGA curve of pure gelatin showed two divisions of decomposition. The first degradation zone, occurring at about 80-90 °C, is due to the loss of absorbed and bound water, and the second degradation zone, at about 260 °C, is due to thermal degradation of different scales of gelatin, including the cleavage of peptide bonds in the protein chains. As shown in Figure 18, the thermogram of PDRN also showed two decomposition zones: a first zone below 100 °C and a second zone above 220 °C. The first section is due to the loss of loosely or tightly bound water, and the second section is due to the shortening of long DNA chains that begins at increased temperature. Moreover, the TGA results demonstrated that the proportions of pure gelatin, PDRN, PG, and PGP nanofibers remaining as residue at 700 °C were about 18.84%, 33.68%, 6.59%, and 7.38%, respectively. The addition of gelatin and PDRN to the nanofibers increased the residual weight and decreased the onset temperature of decomposition. Additionally, the temperature record of PGP nanofibers almost overlapped with that of PG nanofibers, indicating that adding a small amount of PDRN did not affect thermal decomposition.

The thermal effects of PCL, pure gelatin, PDRN, and the prepared nanofibers were confirmed by DSC measurements (Figure 19). During heating, a peak appeared at 55.3 °C, which represents the melting point of PCL. The crystallization rate X _c was 48.36% as a result of calculation according to the formula ₍ This figure represents the semi-crystalline nature of PCL. Similarly, the prepared nanofibers showed peaks at 56.1 °C, 55.1 °C, and 55.8 °C for P, PG, and PGP nanofibers, respectively. The crystallization percentage X _c of the blend was 48.45%, 30.16%, and 32.24% for P, PG, and PGP nanofibers, respectively. These findings demonstrate that the incorporation of Gel and PDRN does not significantly affect the melting point of the PCL matrix even though the crystallization rate X _c decreases.

XRD analysis

The diffraction patterns of pure PCL, pure gelatin, PDRN, and prepared nanofibers are shown in Figure 20. The XRD profile of PCL showed two characteristic peaks at angles of 2θ = 21.7° and 24.26°, which represent the (110) and (200) crystallographic planes of the semicrystalline nature of PCL, respectively. No distinct diffraction peaks were observed in pure gelatin. The XRD spectrum of PDRN shows a broad protrusion around the diffraction angle of 2θ = 20.5°, which corresponds to the base pair distance of the double helix. In the fabricated nanofibers, the peaks at angles of 2θ = 21.7° and 24.26° are due to the diffraction of PCL crystals, which have low intensity with increasing gelatin and PDRN, weakening of semi-crystalline and increasing its amorphous nature. It represents.

Contact angle of PGP nanofibers

To confirm the hydrophilicity of P, PG, and PGP nanofibers, the contact angle between the prepared nanofibers and the culture medium was measured. Representative images and contact angles over time are shown in Figure 21. PGP nanofibers showed a decrease in contact angle close to zero (90.17 ± 4.78° to 53.47 ± 15.35°) compared to P and PG nanofibers, indicating that PGP nanofibers are hydrophilic and have a highly interconnected pore structure. will be. These results indicate that cells can more easily attach to nanofibers due to cell attachment and rapid absorption of molecules in the surrounding fluid, and that PGP nanofibers can be applied to in vitro cell attachment, proliferation, and migration.

Swelling

During the wound healing process, exudate from the damaged area promotes cell migration and proliferation and increases the diffusion of essential wound healing factors that provide cellular metabolism and acid autolysis in damaged or necrotic tissue. Based on these facts, nanofibers with high water absorption capacity are suitable as dressings because they can absorb exudate from the wound bed and increase the supply of nutrients. It was confirmed that all manufactured nanofibers had a water absorption of more than 200% and achieved equilibrium within 3 hours of incubation (FIG. 22). The presence of gelatin and PDRN increased water absorption and retention time. However, the fibrous nanostructure and pore nature of the nanofibers allow for greater infiltration, limiting their expandability. Based on the characterization results of the prepared nanofibers, we hypothesized that PGP nanofibers could promote skin tissue regeneration more rapidly in vivo .

Direct cytotoxicity of manufactured nanofibers

The direct cytotoxicity of the prepared nanofibers against HDF and HaCaT was evaluated using the MTT assay. According to the ISO10993-5:2014 standard ( Biological evaluation of medical devices - in vitro cytotoxicity test ), the manufactured nanofibers were confirmed to have no effect on cell survival regardless of nanofiber type compared to the plated-culture group ( Figure 23).

Indirect cytotoxicity of nanofibers

According to the ISO10993-5:2014 standard ( Biological evaluation of medical devices - in vitro cytotoxicity test ), elution solutions of nanofibers prepared for 1 and 3 days were prepared and analyzed by MTT analysis and FDA and PI fluorescence in HDF and HaCaT. Indirect cytotoxicity was studied by performing live/dead cell staining. The MTT results showed that the elution solution of the prepared nanofibers was not cytotoxic (Figures 24, 25, and 26). The live cell/dead cell staining results also demonstrated no cytotoxicity, similar to the MTT analysis results.

Example 3

Using a circular full-thickness incision wound model In vivo Experiment

To confirm the wound healing ability of nanofibers (P, PG, and PGP), 7-week-old male ICR mice were used as an in vivo wound healing model. ICR mice were maintained in a controlled environment (relative humidity: 40-70%; temperature: 20-24 °C with a 12-h light/dark cycle. All animals were anesthetized by placing them in a chamber with 20% isoflurane in isopropanol for 20 min. To evaluate wound healing in ICR mice, the dorsal surface fur was shaved, the skin surface was disinfected with povidone-iodine and 70% ethanol, and a biopsy punch was used to make circular, full-circle marks on the dorsal skin. A thick skin incision wound (5 mm) was then placed into the wound and secured with bio glue at days 0, 4, 7, 11, 14, 17, and 21. Animals were sacrificed for imaging and tissue examination. The animal experiment process is shown in Figure 27.

In vivo experiments using a rectangular full-thickness incision wound model

To confirm the wound healing ability of nanofibers (P, PG, and PGP), 7-week-old male ICR mice were used as an in vivo wound healing model. ICR mice were maintained in a controlled environment (relative humidity: 40-70%; temperature: 20-24 °C with a 12-hour light/dark cycle.

All animals were anesthetized by placing them in a chamber with 20% isoflurane in isopropanol for 20 minutes and maintained through a facemask. To assess wound healing in ICR mice, dorsal rectangular full-thickness skin incision wounds (15 paid it Then, nanofibers (15 x 15 mm) were placed on the wound and secured with sutures. On days 0, 4, 7, 11, 14, 17, 21, and 28, wound closure was photographed and animals were sacrificed for histological examination. The animal experiment process is shown in Figure 28.

Histological analysis

Histological analysis was performed using hematoxylin and eosin (H&E). After 14 days, animals were sacrificed, and tissues were collected, formalin fixed, processed, impregnated, and dissected. The excised tissue was divided into 5-mm thick slides and stained with hematoxylin for 3 minutes. The slides were then stained with Eosin Y for 45 seconds. Afterwards, the sections were dehydrated using low to high concentration ethanol and cleared with xylene. Finally, the tissue was sutured with MM24 mounting medium (Leica Leica Biosystems, Wetzlar, Germany) and analyzed by light microscopy.

The excised tissue for Picro-Sirius Red staining was prepared as above, and the tissue slides were stained with hematoxylin for 8 minutes, washed with distilled water, and then stained with Picro-Sirius Red for 60 minutes. The sections were washed with acidified water, dehydrated using low to high concentration ethanol, and then cleared with xylene. Finally, tissue slides were sealed with MM24 mounting medium and analyzed under a light microscope with a polar filter.

<Result>

suturing of skin wounds

To evaluate the wound healing activity of PGP nanofibers, circular, full-thickness wounds of 5 mm diameter were created on the back of 8-week-old male ICR mice. All wounds treated or not treated with the manufactured nanofibers were almost completely healed within 3 weeks (FIG. 29). Closure of untreated wounds after 4, 7, 11, 14, 17, and 21 days was 70%, 42%, 26%, 21%, 14%, and 12%, respectively, compared with 67% and 35%, respectively, in the PGP group. %, 16%, 13%, 8% and 6%. Therefore, PGP nanofibers showed the best wound healing effect in the circular full-thickness model.

In addition, a rectangular full-thickness (15 mm Wounds were observed every 3 or 4 days for 4 weeks, and the wound area was measured. As a result, wounds were almost completely recovered in all groups, but after 4 weeks, initial wound healing and wound closure were promoted in the PGP group compared to the control group (Figure 30).

Re-epithelialization of skin wounds

On the 21st day after surgery, histological examination was performed on the mouse skin using H&E, Masson's trichrome, and Picro-Sirius Red staining to confirm the effect of nanofibers on skin tissue restoration at the wound site (FIG. 31). The results of H&E staining analysis showed that skin tissue restoration in the blank group was incomplete. The nanofiber-implanted group also showed more advanced skin restoration, with wounds closing more quickly and more blood vessels, hair follicles, and sebaceous glands forming than the untreated group, although it was not completely cured. In particular, the PGP group showed the best regenerative activity. According to Masson's trichrome and Picro-Sirius staining results that evaluated the histological appearance of collagen fibers in the wound area, the blank group showed a lower density of collagen fibers in the wound area compared to the PGP group. Moreover, the deposition of collagen fibers was smaller and thicker in the nanofiber-implanted group.

To confirm the effect of nanofibers on tissue restoration in the wound area, histological staining was performed on skin tissue with H&E, Masson's trichrome, and Picro-Sirius Red staining on the 28th day after surgery (FIG. 32). The results of H&E staining analysis showed that skin tissue restoration in the blank group was incomplete. The nanofiber-implanted group also showed more advanced skin restoration, with wounds closing more quickly and more blood vessels, hair follicles, and sebaceous glands forming than the untreated group, although it was not completely cured. In particular, the PGP group showed the best regenerative activity. According to Masson's trichrome and Picro-Sirius staining results that evaluated the histological appearance of collagen fibers in the wound area, the PGP group showed a higher density of collagen fibers in the wound area compared to other groups. Moreover, the deposition of collagen fibers was smaller and thicker in the nanofiber-implanted group. Therefore, these results indicate that PGP nanofibers can promote wound healing.

Expression of proteins involved in wound healing in a full-thickness wound model

Western blot analysis was performed on skin tissue at 21 and 28 days after surgery in round and rectangular full-thickness wound models, respectively, for the expression of proteins associated with wound healing, such as type I collagen, type III collagen, and α-SMA. The effect of PGP nanofibers was confirmed. As can be seen in Figure 33, the expression level of COL I was significantly increased in the PGP group compared to the other groups in the circular full-thickness wound model. However, no differences were seen in protein expression levels in the rectangular full-thickness wound model.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (8)

1. poly(ε-caprolactone; PCL);

   gelatin; and

   Polydeoxy-ribonucleotide (PDRN) derived from starfish ( Patiria pectinifera );

   A nanofiber scaffold comprising.
2. According to claim 1,

   The nanofiber scaffold is a nanofiber scaffold manufactured by electrospinning.
3. According to clause 1.

   A nanofiber scaffold comprising the polycaprolactone, gelatin, and polydeoxyribonucleotide in a weight ratio of 4 to 8:2 to 6:0.005 to 0.1.
4. Isolating polydeoxy-ribonucleotide (PDRN) from starfish ( Patiria pectinifera ) (step 1);

   Mixing the polydeoxyribonucleotide with polycaprolactone and gelatin (step 2); and

   Preparing a nanofiber mat by electrospinning the mixture (step 3);

   Method for manufacturing a nanofiber scaffold, including.
5. According to claim 4,

   In step 2, the polycaprolactone, gelatin, and polydeoxyribonucleotide are mixed at a weight ratio of 4 to 8: 2 to 6: 0.005 to 0.1.
6. A wound dressing comprising a nanofiber scaffold according to any one of claims 1 to 3.
7. A wound treatment method comprising: applying the wound dressing of claim 6 to the wound.
8. The method of claim 7, wherein the wound treatment method treats the wound by promoting regeneration of skin at the wound area.

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