CN115804867B - Fibrotic type III collagen nanomembrane, preparation method and application thereof in skin regeneration - Google Patents
Fibrotic type III collagen nanomembrane, preparation method and application thereof in skin regeneration Download PDFInfo
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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- Materials For Medical Uses (AREA)
Abstract
The invention discloses a fibrosis III type collagen nano-film, a preparation method and application thereof in skin regeneration, and belongs to the technical field of collagen nano-fiber films. The invention adopts an electrostatic spinning method to spin III type collagen and polylactic acid to prepare and form a fibrous III type collagen nano-film, and the fibrous III type collagen nano-film has certain mechanical property and good biocompatibility, and can be applied to the fields of wound healing and wound skin regeneration.
Description
Technical Field
The invention relates to the technical field of collagen nanofiber membranes, in particular to a fibrosis III type collagen nanofiber membrane, a preparation method and application thereof in skin regeneration.
Background
The collagen material is mainly applied to food health care, medical face-lifting, cosmetics, implantable medical devices, tissue engineering and the like by utilizing the degradation resistance, biocompatibility, low immunity and excellent physical and chemical properties of the collagen. In promoting skin repair, kishimoto Y et al (Kishimoto Y, 2010) prepared desquamated collagen as a scaffold sheet material, a crosslinked collagen material with abundant micropores, was applied as a scaffold for skin and epidermis repair, revealed scaffold material properties, encapsulate and infiltrate cells, stimulate interactions between molecules such as growth factors and external regulatory variables such as stress, strain and vibration, and show potential therapeutic benefits in recent studies on patients with vocal scars and grooved vocal cords. Hiroyuki M et al (Hiroyuki M, 2013) developed crosslinked frog-derived distressing collagen sponges and used rabbit models to evaluate the in vivo efficacy of Osteogenic Protein (OP) -1 containing frog-derived collagen sponges on cartilage regeneration. Regenerated cartilage tissue enriched in proteoglycans and type 2 collagen was found at week 12. Zhang Yin et al (Zhang Yin, 2014) used bone marrow stem cells for the operation of the composite treatment with a skin-removed collagen material, studied the treatment effect of the composite material on the degeneration of the intervertebral disc of New Zealand white rabbits, and achieved good results. Kim J et al (Kim J, 2015) used a combination of atelopeptide collagen and fibrin glue in patients with osteoporosis lesions ((OCL) talus to enhance microfracture techniques.
Although collagen, particularly type III collagen, has such a great number of applications, it still has problems in tissue or skin repair that inflammation is easily generated and biocompatibility is to be improved, etc.
Disclosure of Invention
In view of the above, the present invention aims to provide a fibrillated type III collagen nanomembrane, a preparation method and an application thereof in skin regeneration.
The invention provides a preparation method of a fibrosis III type collagen nano-film, which comprises the following steps:
preparing an electrostatic spinning solution, wherein the electrostatic spinning solution comprises type III collagen and polylactic acid;
carrying out electrostatic spinning by utilizing the electrostatic spinning solution;
and (3) performing glutaraldehyde steam crosslinking on the electrostatic spinning film to form the fibrillated III type collagen nano film.
Further, the electrospinning solution further comprises a water-insoluble dietary fiber.
Further, the preparation method of the electrostatic spinning solution comprises the following steps: the preparation method comprises the steps of preparing a Col solution with 15wt% and a PLA solution with 5wt% by using tetrafluoropropanol, and mixing the Col solution and the PLA solution to obtain a spinning solution for electrostatic spinning.
Further, mixing the Col solution and the PLA solution in equal volume, and enabling the mass ratio of the Col to the PLA in the mixed solution to be 3:1, and fully stirring and dissolving the mixed solution until the mixed solution is uniform, so as to be used as the spinning solution for electrostatic spinning.
Further, mixing the Col solution and the PLA solution in equal volume, and enabling the mass ratio of the Col to the PLA in the mixed solution to be 5:1, and fully stirring and dissolving the mixed solution until the mixed solution is uniform, so as to be used as the spinning solution for electrostatic spinning.
Further, the preparation method of the electrostatic spinning solution comprises the following steps: the preparation method comprises the steps of preparing a Col solution with 15 weight percent, a PLA solution with 5 weight percent and a water-insoluble dietary fiber solution with 5 weight percent by using tetrafluoropropanol, and mixing the Col solution, the PLA solution and the water-insoluble dietary fiber solution to serve as a spinning solution for electrostatic spinning.
Further, the volume ratio of Col solution to solid matters of water-insoluble dietary fibers to PLA in the mixed solution is 6:1:2, and ultrasonic is adopted for fully and uniformly mixing to be used as spinning solution for electrostatic spinning.
Further, the volume ratio of Col solution to solid matters of water-insoluble dietary fibers to PLA in the mixed solution is 7:2:2, and ultrasonic is adopted for fully and uniformly mixing to be used as spinning solution for electrostatic spinning.
The invention provides a fibrosis III type collagen nano-film prepared by the preparation method, which has a tensile strength of 1.22-2.66 MPa and an elongation at break of 117.8-165.9%.
The invention also provides application of the fibrosis III type collagen nano-film prepared by the preparation method in skin regeneration.
Compared with the prior art, the invention has at least one of the following beneficial effects:
the invention adopts an electrostatic spinning method to spin III type collagen and polylactic acid to prepare and form a fibrous III type collagen nano-film, and the fibrous III type collagen nano-film has certain mechanical property and good biocompatibility, and can be applied to the fields of wound healing and wound skin regeneration.
The fibrillated III type collagen nano film is formed by compounding nano fibers, has tensile strength of 1.22-2.66 MPa and elongation at break of 117.8-165.9%, and has excellent mechanical properties. In addition, the fibrillated III type collagen nano film has higher hydrophilic property and higher water absorption rate.
The fibrous III type collagen nano-film is subjected to double-sided co-culture on human surface keratinocytes and human dermal fibroblasts, so that three-dimensional culture, growth and proliferation of the human surface keratinocytes and the human dermal fibroblasts can be promoted, and excellent biocompatibility and in-vivo applicability are suggested.
Furthermore, the invention also applies the fibrosis III type collagen nano-film to repair the wound surface of a mouse, the transplanted mouse wound surface can be covered by epidermis on 21 st day, the defect disappears, the wound surface is closed, and after the fibrosis III type collagen nano-film provided in examples 1-4 regenerates the wound surface, the scar on the skin surface is smaller, which indicates that the fibrosis III type collagen nano-film provided by the invention has application prospect as a skin regeneration film or preparation. In addition, through protein expression analysis in the tissue of the repaired wound skin of the mice, the fiber III type collagen nano-film plays an important role in signal transduction through activating integrin beta 1 to mediate integrin signal transduction and activating FAK through autophosphorylation of tyrosine-397 locus in the interaction of integrin mediated cells and extracellular matrixes.
Drawings
Fig. 1 is an FE-SEM image of a fibrillated type III collagen nanomembrane provided in example 1 of the present invention.
Fig. 2 is an FE-SEM image of the fibrillated type III collagen nanomembrane provided in example 2 of the present invention.
Fig. 3 is an infrared spectrum of a fibrillated type III collagen nanomembrane provided by embodiments 1 to 4 of the present invention.
FIG. 4 is a graph of the wound surface of mice in the negative group (left) and the control group (right) on day 21 of the animal experiment of the present invention.
Fig. 5 is a graph of the wound surface of mice treated in examples 1 to 4 in the test group of the animal experiment of the present invention on day 21.
Fig. 6 is a graph of the wound surface of mice treated in comparative examples 1 to 4 in the test group of the animal experiment of the present invention on day 21.
FIG. 7 is a fluorescent staining pattern of HLA-ABC and CK10 on day 21 of the animal experiment of the present invention for the wound tissues of mice treated in examples 1 to 4.
FIG. 8 is a fluorescent staining pattern of HLA-ABC and CK10 on day 21 of the animal experiment of the present invention on the wound surface tissue of mice treated in comparative examples 1 to 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The reagents not specifically and individually described in the present invention are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
Collagen (Col) has its abundant supply and unique biological properties. The combination of Col and electrospinning technology has been widely studied due to the unique three-dimensional structure of electrospun fibrillated type III collagen nanomembranes. However, the beta-sheet secondary structure of Col appears to hinder the electrospinning process, and the mechanical properties of pure Col electrospun nanofibers are not ideal. Polycaprolactone (PCL), while having unique mechanical properties and excellent spinnability, has limited application due to its high hydrophobicity and low bioactivity. Therefore, in recent years, a plurality of scholars prepare the Col/PCL composite nanofiber through mixed electrostatic spinning, so that the Col/PCL composite nanofiber not only has good biological performance, but also has excellent mechanical performance, and has good application prospects in biomedical fields such as nerve tissue, bone tissue, cartilage tissue and skin tissue engineering.
The invention adopts an electrostatic spinning method to spin III type collagen and polylactic acid to prepare and form a fibrous III type collagen nano-film, and the fibrous III type collagen nano-film has certain mechanical property and good biocompatibility, and can be applied to the fields of wound healing and wound skin regeneration.
In some embodiments, the method for preparing the fibrillated type III collagen nanomembrane comprises the steps of preparing an electrostatic spinning solution, spinning by using the electrostatic spinning solution, and crosslinking a film obtained by spinning by using glutaraldehyde. The method comprises the following specific steps:
1. extraction of insoluble dietary fibers
(1) After the wheat bran is finished and decontaminated, crushing the wheat bran, sieving the crushed wheat bran with a 100-mesh sieve, soaking the wheat bran with water with the weight of 10 times of that of the wheat bran for about 30min, removing the water, and washing the wheat bran with warm water for 2-3 times to remove the soluble sugar and part of pigment substances in the wheat bran powder.
(2) Adding the wheat bran powder treated by the method into a sulfuric acid solution with the mass fraction of 60% which is 15 times of the weight of the wheat bran powder, placing the wheat bran powder into a constant-temperature water bath with the temperature of about 80 ℃ for hydrolysis for 2 hours, filtering the wheat bran powder while the wheat bran powder is hot, and washing filter residues with hot water to be neutral.
(3) Adding the wheat bran powder treated by the method into 40 times of sodium hydroxide solution with the pH value of 13.5, treating the wheat bran powder in a constant-temperature water bath at about 85 ℃ for 3 hours, vacuum filtering, neutralizing the wheat bran powder with 1% hydrochloric acid solution, and washing the wheat bran powder with 80 ℃ hot water to be neutral; dehydrating with absolute ethanol, degreasing at 60deg.C by Soxhlet extraction to obtain final solid of water insoluble dietary fiber.
In the embodiment of the invention, the content of the water-insoluble dietary fiber in the solid is measured by adopting a neutral elution method (Ouyang Linghua and the like. Research on the preparation process of lemon peel dietary fiber [ J ]. Food research and development, 2005, 26 (6): 80-83). The water insoluble dietary fiber content of the solids was calculated to be 16.8%.
2. Extraction of water-soluble dietary fibers
After the wheat bran is finished and decontaminated, crushing the wheat bran, sieving the crushed wheat bran with a 100-mesh sieve, soaking the wheat bran with water with the weight of 10 times of that of the wheat bran for about 30min, removing the water, and washing the wheat bran with warm water for 2-3 times to remove the soluble sugar and part of pigment substances in the wheat bran powder.
And adding the wheat bran powder subjected to the treatment into a sulfuric acid solution with the mass fraction of 60% and the weight of 10 times, adjusting the pH value of the feed liquid to be 1.5-2.0, placing the feed liquid in a constant-temperature water bath with the temperature of about 85 ℃ for hydrolysis for 1.5 hours, filtering while the feed liquid is hot, washing filter residues with hot water for 2-3 times, and collecting and combining the filtrates. Adding saturated aluminum sulfate solution (volume ratio is 5:1) into the filtrate, regulating the pH value of the feed liquid to be 4-5 by ammonia water, reacting for 30min, centrifuging, adding a certain amount of desalting resin (ion exchange desalting resin LX-160, blue technology) into the precipitate, stirring, desalting and washing for 3-5 times, centrifuging, washing the precipitate to be neutral by low-concentration alcohol solution, decolorizing, vacuum drying under 50 ℃, pulverizing and sieving to obtain the water-soluble dietary fiber. The content of galacturonic acid in the extract was determined by ultraviolet spectrophotometry as the content of water-soluble dietary fiber therein. The content of the water-soluble dietary fiber in the water-soluble dietary fiber is 14.7 percent through detection.
3. Preparing spinning solution
In one example 1, an appropriate amount of human Collagen III polypeptide (ab 101725, abcam, simply referred to as Col) was accurately weighed and formulated with tetrafluoropropanol to a Col solution of 15 wt%; accurately weighing a proper amount of polylactic acid (CAS number: 26680-10-4,Mw 18000~24000,Sigma-Aldrich, hereinafter simply referred to as PLA), and preparing PLA solution with a mass percent of 5wt% by using tetrafluoropropanol; and mixing the Col solution and the PLA solution in equal volumes, so that the mass ratio of the Col to the PLA in the mixed solution is 3:1, and fully stirring and dissolving the mixed solution until the solutions are uniform, thereby being used as the spinning solution for electrostatic spinning.
In one example 2, an appropriate amount of Col was accurately weighed and formulated with tetrafluoropropanol as a Col solution of 15 wt%; accurately weighing a proper amount of the solid matters of the water-insoluble dietary fiber prepared in the embodiment, uniformly mixing the solid matters in tetrafluoropropanol under ultrasonic conditions, and preparing a water-insoluble dietary fiber solution with the mass percent of 5 wt%; accurately weighing a proper amount of polylactic acid, and preparing PLA solution with the mass percentage of 5wt% by using tetrafluoropropanol; mixing the Col solution, the water-insoluble dietary fiber solution and the PLA solution to ensure that the volume ratio of the Col solution to the solid matters of the water-insoluble dietary fiber to the PLA in the mixed solution is 6:1:2, and fully and uniformly mixing by adopting ultrasound to be used as the spinning solution of electrostatic spinning.
In example 3, the composition of the dope was the same as in example 1, except that the mass ratio of Col and PLA was 5:1.
In one example 4, the components in the dope were the same as in example 2, except that the mass ratio of Col, solid matter of water-insoluble dietary fiber, and PLA was 7:2:2.
In a comparative example 1, a proper amount of hexafluoroisopropanol was used to dissolve a human Collagen III polypeptide solution to obtain a Col solution of 15wt%, a proper amount of hexafluoroisopropanol was used to prepare polylactic acid to prepare a PLA solution with a volume ratio of 5% w/v, the Col solution and the PLA solution were mixed in equal volumes so that the mass ratio of Col to PLA in the mixed solution was 3:1, and the mixed solution was sufficiently stirred and dissolved until the solution became uniform to obtain a spinning solution for electrospinning.
In comparative example 2, the components in the dope were the same as in example 2, except that hexafluoroisopropanol was used as a solvent.
In a comparative example 3, an appropriate amount of Col was accurately weighed, and a Col solution of 15wt% was prepared with tetrafluoropropanol; accurately weighing a proper amount of solid matters of the water-soluble dietary fiber prepared in the embodiment, uniformly mixing the solid matters in pure water by utilizing ultrasonic conditions, and preparing a water-soluble dietary fiber solution with the mass percent of 5 wt%; accurately weighing a proper amount of polylactic acid, and preparing PLA solution with the mass percentage of 5wt% by using tetrafluoropropanol; mixing the Col solution, the water-soluble dietary fiber solution and the PLA solution to ensure that the volume ratio of the Col solution to the solid of the water-soluble dietary fiber to the PLA in the mixed solution is 6:1:2, and fully and uniformly mixing by adopting ultrasound to be used as the spinning solution for electrostatic spinning.
In a comparative example 4, an appropriate amount of Col was accurately weighed, and a Col solution having a mass ratio of 15wt% was prepared with hexafluoroisopropanol; accurately weighing a proper amount of solid matters of the water-soluble dietary fiber prepared in the embodiment, uniformly mixing the solid matters in pure water by utilizing ultrasonic conditions, and preparing a water-soluble dietary fiber solution with the mass percent of 5 wt%; accurately weighing a proper amount of polylactic acid, and preparing PLA solution with the mass percent of 5wt% by using hexafluoroisopropanol; mixing the Col solution, the water-soluble dietary fiber solution and the PLA solution to ensure that the volume ratio of the Col solution to the solid of the water-soluble dietary fiber to the PLA in the mixed solution is 6:1:2, and fully and uniformly mixing by adopting ultrasound to be used as the spinning solution for electrostatic spinning.
4. Preparation of fibrillated III type collagen nano film by electrostatic spinning
The electrostatic spinning process is carried out on electrostatic spinning equipment (electrostatic spinning machine E02-001, buddha light son precision measurement and control technology Co., ltd.).
The spinning solutions prepared in example 1 and comparative example 1 were sucked into a 10 mL syringe equipped with an 8-gauge needle, and the air bubbles were discharged and then placed on a syringe pump and connected to a high-voltage power supply. The electrospinning conditions included: the high-voltage static electricity 16 kV, the solution filling speed is 1 mL/h, the distance between the receiving plates is 15cm, the spinning solution volume is 36 mL in order to ensure that the thickness and the area of the film are equivalent, and 6 injectors are utilized, and each injector is 6 mL to fill together. After spinning, glutaraldehyde vapor is crosslinked to form a fibrous III-type collagen nano film, and the film is taken down or put into a vacuum drying oven together with aluminum foil to be dried at normal temperature above 24 and h.
5. Physicochemical properties of fibrotic type III collagen nanomembrane
(1) Field emission scanning electron microscope (FE-SEM)
Three pieces of the fibrillated III type collagen nano film prepared in each of examples 1 to 4 and comparative examples 1 to 4 were randomly taken to be about 5mm 2 A sample of the size was used as an observation sample. The samples were then gold sprayed after being adhered flat to the sample stage by means of conductive glue, then observed and photographed under a field emission scanning electron microscope (ZEISS Σsigma, germany). 100 fibers in each sample were randomly measured using Image-J software and SEM photographs and averaged.
As shown in fig. 1-2, all fibrous membranes have a distinct 3D structure. The single-layer fibrous membrane prepared in example 1 had an average fiber diameter of about 1043±238 nm, while the fibrous bundles of the fibrillated type III collagen nanomembrane prepared in example 2 had some entanglement and formed irregular protrusions, and the average fiber diameter (898±353 nm) was low.
Infrared analysis
The surface groups of the straw before and after modification were characterized using a fourier transform infrared spectrometer (Ex-calibur 3100, varian, usa). 1mg of the sample was ground with 100mg of KBr under an infrared lamp and then compressed into tablets on a tablet press with a pressure of 10 MPa, resolution of 4cm in an infrared spectrometer -1 Scanning range is 600-4000 cm -1 The number of scans was 64.
As shown in FIG. 3, 1756cm of the fibrillated type III collagen nano-film prepared in examples 1 and 3 -1 、1661cm -1 、1580cm -1 、1455cm -1 、1452cm -1 、1382cm -1 、1234cm -1 、646cm -1 Wherein 1756cm -1 、1455cm -1 And 1382cm -1 Belongs to the characteristic absorption peak of polylactic acid, is 1661cm -1 、1580cm -1 、1452cm -1 、1234cm -1 And 646cm -1 Belongs to the characteristic absorption peak of the III type collagen peptide, and the surface of the fibrous III type collagen nano-film is a composite material. The fibrillated type III collagen nanomembranes prepared in examples 2 and 4 were 2924cm in addition to the above -1 An enhanced absorption peak appears, indicating that it is complexed with dietary fiber.
(3) Tensile Strength and elongation at break
1 piece of film sample is taken, cut into 1.0 cm wide strip-shaped test pieces, immersed into physiological saline at 37+/-1 ℃ for 10min, and respectively fixed on two chucks of a material sample machine at the stretching speed of 10 mm/min until the test pieces break, wherein the stretching strength of the skin-removing collagen film is not less than 0.1 MPa. Table 1 shows the tensile strength and elongation at break of the fibrillated type III collagen nanomembranes prepared in each example and comparative example, and multiple comparisons and significant difference labeling were performed for each column of data. As a result, the tensile strength and the elongation at break of the fibrillated type III collagen nanomembranes prepared in examples 1 to 4 were significantly higher than those of the comparative examples.
TABLE 1
| Description of the embodiments | Tensile Strength (MPa) | Elongation at break (%) |
| Example 1 | 1.27±0.25b | 125.4±7.6b |
| Example 2 | 1.87±0.79a | 154.7±11.2a |
| Example 3 | 1.35±0.33b | 129.2±5.8b |
| Example 4 | 1.92±0.57a | 158.1±7.5a |
| Comparative example 1 | 0.89±0.18d | 79.8±2.4d |
| Comparative example 2 | 0.96±0.25cd | 82.4±3.9cd |
| Comparative example 3 | 1.16±0.32c | 89.3±4.2c |
| Comparative example 4 | 1.09±0.27c | 87.5±3.8c |
(4) Water absorption and swelling
Weighing 1 piece or 0.1 piece g piece of membrane, immersing in physiological saline at 37+ -1deg.C for 10min, slightly clamping one corner with forceps in saturation state, standing on water surface for 1 min, weighing water, and taking the weight of the membrane according to the weight of the water. Weight testing was periodically performed according to a certain soak time to detect swelling of the fibrillated type III collagen nanomembrane.
The measurement was performed by using a contact angle meter (SL 200C, china). The fibrillated type III collagen nanomembranes prepared in examples 1 to 4 and comparative examples 1 to 4 were dried, cut into strips of 6cm by 2cm in the longitudinal and transverse directions, and contact angles at 5 different positions of each sample film were measured, respectively, and the results were averaged.
In addition, each sample of the fibrillated type III collagen nano film is cut into 4cm multiplied by 4cm and then immersed in deionized water for 24 h, taken out after swelling balance is achieved, the water on the surface of the film is quickly absorbed by filter paper and then weighed, dried at 45 ℃ to constant weight and then weighed, and the water absorption rate is calculated according to the percentage of the difference of the weight of the film before and after drying to the weight of the film after drying.
TABLE 2
| Description of the embodiments | Contact angle (°) | Water absorption (%) |
| Example 1 | 2.6±0.2 | 78.9±3.8a |
| Example 2 | 3.2±0.3 | 64.4±2.7b |
| Example 3 | 3.1±0.2 | 80.3±4.3a |
| Example 4 | 2.9±0.1 | 66.7±3.7b |
| Comparative example 1 | 2.8±0.3 | 74.5±2.6a |
| Comparative example 2 | 2.9±0.2 | 75.7±3.1a |
| Comparative example 3 | － | － |
| Comparative example 4 | － | － |
After the above experiments, the fibrillated type III collagen nanomembrane prepared in comparative examples 3 and 4 was swelled, and the surface thereof was also formed with macroscopic holes, and the thin film structure thereof was destroyed. The fibrillated III-type collagen nanomembranes provided in examples 1-4 and comparative examples 1-2 all show obvious water absorption, do not form holes, have no damage to the film structure, and have strong hydrophilicity and water absorption.
6. Co-culture test of fibrous III type collagen nano membrane cells
Human surface keratinocytes (abbreviated as Fbs), cat: CP-H113, plaxol. Human dermal fibroblasts (EEKCs for short), cat: CP-H103, pranoprofen.
(1) The fibrillated type III collagen nanomembranes prepared in examples 1 to 4 and comparative examples 1 to 4 were sterilized with ethylene oxide, and a sterile filter (SwinnexFDR-355-OlOC) having a diameter of 13 mm was modified to support the fibrillated type III collagen nanomembranes, and nanofibers prepared in examples 1 to 4 and comparative examples 1 to 4 after the sterilization treatment were placed so that both sides of the fibrillated type III collagen nanomembranes could be contacted with different cell culture solutions.
(2) Will contain 10 5 The l.5m1 culture of Fbs cells was added to one side of a fibrous III-type collagen nanomembrane in a sterile filter at 37℃with 5% CO 2 Culturing in an incubator for 2 days.
(3) The filter is then turned over and the filter is then turned over to contain 3X 10 5 100 mu L of culture solution of EEKCs cells is added to the other side of each bracket.
(4) The CO-cultivation device changes corresponding culture solution every 3 days, maintains 37 ℃ and 5% CO 2 Culturing in an incubator.
In order to study the effect of the fibrotic type III collagen nanomembranes prepared in examples 1-4 and comparative examples 1-4 on cell proliferation, the cytoskeleton and phenotype of EKCs and Fbs cells were also examined in this experiment. The detection indexes include F-actin (F-actin), ki67 and focal adhesion protein (Vinculin).
The detection process comprises the following steps: the co-cultured fibrillated III type collagen nano film is fixed for 10min at 4 ℃ by 4% paraformaldehyde, and is washed for 3 times by PBS at 4 ℃ for 5min each time. The membrane was permeabilized with 0.2% Triton for 20min at room temperature and washed 3 times with PBS. Blocking, namely blocking goat serum at room temperature for 30min, and washing for 1 time by PBS. Primary antibody after 1:100 dilution of primary antibody (F-actin antibody, ab205, abcam; ki67 antibody, ab15580, abcam; vinculin antibody, ab219649, abcam), each sample was incubated overnight at 500. Mu.L at 4℃and then rinsed 3 times with PBS. After 1:800 dilution of the secondary antibody (Alexa Fluor), 500. Mu.L of each sample was added, incubated at room temperature for 1h in the absence of light, and then rinsed 3 times with PBS. The nuclear staining is carried out by adding 1 ug/ml DAPI, adding 500 mu L of each sample, incubating for 10min at room temperature in dark place, and then flushing 3 times by PBS. And (5) diluting glycerol for 1 time, sealing the tablets, and storing the tablets in a dark place. Observation analysis observations were made under a laser scanning confocal microscope and analyzed with Image J software.
Table 3 shows the positive rate of Ki67 in Fbs, the MFI values of F-actin and Vinculelin proteins, table 4 shows the positive rate of Ki67 in EEKCs, the MFI values of F-actin and Vinculelin proteins, and multiple comparisons and significance differential markers were performed on each data, and "-" indicates undetected, ki67 positive rate was calculated by dividing the number of Ki67 positive cells by the total number of cells.
As can be seen from tables 3 and 4, the fibrotic type III collagen nanomembranes prepared in comparative examples 3 to 4 did not detect Fbs and EEKCs cells, indicating that they were unfavorable for both growth. The Ki67 positive rate (%), F-actin (MFI) and Vinculin (MFI) of the fibrotic type III collagen nanomembrane provided in examples 1-4 are all significantly higher than those of comparative examples 1-2, which indicates that the fibrotic type III collagen nanomembrane prepared in the examples is more beneficial to the growth and proliferation of Fbs and EEKCs cells, and has more excellent biocompatibility. Comparing the processes of the previous comparative examples 1-4 and comparative examples 1-4 for preparing the fibrillated type III collagen nano-films, respectively, it was found that comparative examples 1 and 2 use hexafluoroisopropanol as a solvent of their electrostatic spinning solutions, resulting in limited growth and proliferation of Fbs and EEKCs cells; comparative examples 3 and 4 not only used hexafluoroisopropanol as the solvent of the electrostatic spinning solution, but also used water-soluble dietary fiber, and found according to the above-mentioned water absorption test that after the addition of the culture medium, the film formed macroscopic through holes, which was unfavorable for the growth of Fbs and EEKCs cells, so that no positive cells were detected.
TABLE 3 Fbs cells
| Description of the embodiments | Ki67 Positive Rate (%) | F-actin(MFI) | Vinculin (MFI) |
| Example 1 | 76.2±2.1a | 224.2±27.4a | 205.9±22.1a |
| Example 2 | 81.5±1.8a | 235.1±32.2a | 224.3±19.4a |
| Example 3 | 78.4±1.6a | 226.7±22.9a | 196.8±20.7a |
| Example 4 | 83.5±2.1a | 237.5±21.7a | 231.2±21.5a |
| Comparative example 1 | 26.4±1.5b | 152.7±19.7b | 157.6±18.4b |
| Comparative example 2 | 24.7±1.4b | 164.8±25.4b | 162.5±23.1b |
| Comparative example 3 | － | － | － |
| Comparative example 4 | － | － | － |
TABLE 4 EEKCs cells
| Description of the embodiments | Ki67 Positive Rate (%) | F-actin(MFI) | Vinculin (MFI) |
| Example 1 | 79.4±1.6a | 249.3±21.5a | 212.3±25.2a |
| Example 2 | 82.8±2.4a | 252.2±34.8a | 221.2±26.7a |
| Example 3 | 77.0±1.9a | 247.2±29.4a | 209.7±23.4a |
| Example 4 | 85.1±2.6a | 251.2±26.4a | 235.6±20.8a |
| ComparisonExample 1 | 31.4±1.2b | 163.2±17.5b | 163.5±12.8b |
| Comparative example 2 | 33.2±2.1b | 161.4±23.4b | 165.9±21.4b |
| Comparative example 3 | － | － | － |
| Comparative example 4 | － | － | － |
Animal test
Test animals
BALB/c nude mice healthy, SPF grade male nude mice of 6 weeks old, strain number: c000103, division of common caners laboratory animals, inc.
Test sample
The method provided by each of examples 1-4 and comparative examples 1-4 was used to prepare a fibrous III-type collagen nanomembrane as a test film, and the film thickness was 50 [ mu ] m.
(3) Skin regeneration of nude mice with skin defects
Bare mice were anesthetized with 2% sodium pentobarbital (1 mL/kg). Skin preparation and disinfection, namely after anesthesia is effective, the back hair of the mouse is shaved, and alcohol is disinfected for 3 times.
Model group: the skin with the diameter of 10mm is removed from the back of a BALB/c nude mouse to form a wound surface. Average division is as follows: control (skin removal from the remaining back for autologous skin grafting), test (fibrous type III collagen nanomembrane grafting provided in examples 1-4 and comparative examples 1-4, respectively), and negative (no treatment).
After Fbs are inoculated to a wound surface, the films provided in examples 1-4 and comparative examples 1-4 are respectively covered on the wound surface, EKCs are inoculated to the films, and after 7 days of culture, the wound is photographed and the materials are obtained.
(4) Skin regeneration effect
Fig. 4-6 show the wound surface diagrams of the mice in each group of 21-th test. Except for the groups of comparative examples 3-4 in the negative group and the test group, the wound surface is covered by the epidermis after 21d, the defect disappears, the wound surface is closed, and the fibrous III-type collagen nano-film provided in examples 1-4 has smaller scars on the skin surface after the wound surface is regenerated, which indicates that the fibrous III-type collagen nano-film provided by the invention has application prospect as a skin regeneration film or preparation.
(5) Immunofluorescence detection
Immunoenzymatic staining with anti-human HLA-ABC type antigen and CK10 antibody was used for identification by direct immunofluorescence. If the healed skin tissue is positive for immunofluorescence against human HLA-ABC and CK10 antigens, it is demonstrated that the new epidermis is formed by transplanted human epidermal cells, not autologous cells derived from the wound margin.
As shown in fig. 7 and 8, the positive ratio of HLA-ABC type and CK10 in the wound skin tissue treated with the fibrillated type III collagen nanomembrane provided in examples 1 to 4 was higher than that in comparative examples 1 to 4.
(6) Western-blotting detection
The present experiment also examined the expression levels of active integrins β1, FAK, pFAK in wound skin tissue of mice of each group (Western-blotting).
(1) Total protein extraction from samples
Skin tissue after healing of skin wound of each group of mice. Ice lysate, 20ug/ml protease inhibitor skin inhibitor, 1mM PMSF,5mM sodium fluoride, 1mM sodium orthovanadate were added. Homogenizing, centrifuging, namely collecting tissue homogenate, centrifuging at 15000g for 10min at 4 ℃, and collecting supernatant. BSA was removed from-20 ℃ and slowly thawed on ice, and BSA solutions of different concentrations were prepared according to the following table and allowed to stand at ambient temperature for 30min before use. BSA solutions of 0, 2.5, 5, 10, 20, 30, 40 and 50 μg/mL were prepared with 0.15M sodium chloride solution, respectively.
(2) SDS-PAGE electrophoresis
5% of concentrated glue: 30% polyacrylamide solution 0.25mL, tris-HCl (1 mol/L pH 6.8) 0.19mL, 10% SDS 0.015mL, 10% ADS 0.015mL, distilled water 1.03m1, TEMED 0.015 mL.
10% of separation gel: 30% polyacrylamide solution 1.333m1, tris-Hel (1.5 mol/L pH 8.8) 1.0m1, 10% SDS 0.04mL,10% ADS 0.04m1, distilled water 1.6m1,TEMED 0.0025m1.
Pouring the separating gel into an electrophoresis tank, keeping the distance from the teeth of the comb to about 0.5cm, adding ddH 20.5 cm for sealing, observing for 30min, removing ddH20 after solidification, adding concentrated gel, inserting the comb, and after waiting for 1h, pulling out the comb, avoiding the comb Kong Bianxing, flushing a gel hole for 2 times by using ddH20, and removing residual gel.
Uniformly mixing the protein samples with the buffer solution of 5X according to the volume ratio of 4:1, and putting the mixture into a 100C water bath preheated in advance to be boiled for 3 min. The samples were loaded at 40 ug/porin and 3u1 Marker. After the dye is supplied according to the voltage of 80V for about 30min, when the dye appears at the edges of the separating gel and the concentrating gel, the voltage is changed to 120V, and the electrophoresis is continued for 1.5h, so that the dye enters the position about 1cm away from the bottom of the gel plate.
(3) Transfer film, seal, imaging
The target protein is cut from the gel, and the gel and filter paper are soaked in a transfer buffer for one time, PVDF is cut to gel size, soaked in absolute ethyl alcohol for about 1 min, and then transferred into an electrophoresis buffer for about 15min. Transfer was performed from negative electrode to positive electrode in the order of filter paper, gel, PVDF film, filter paper.
Table 5 WB relative expression levels of proteins
| Description of the embodiments | Active integrin beta 1 | FAK | pFAK |
| Example 1 | 0.59±0.022a | 0.52±0.021a | 0.39±0.015a |
| Example 2 | 0.67±0.023a | 0.59±0.019a | 0.41±0.026a |
| Example 3 | 0.66±0.027a | 0.57±0.016a | 0.38±0.012a |
| Example 4 | 0.64±0.026a | 0.53±0.018a | 0.36±0.019a |
| Comparative example 1 | 0.34±0.016b | 0.33±0.014b | 0.28±0.014b |
| Comparative example 2 | 0.36±0.015b | 0.34±0.012b | 0.26±0.013b |
| Comparative example 3 | 0.28±0.017c | 0.26±0.017c | 0.25±0.014b |
| Comparative example 4 | 0.26±0.022c | 0.27±0.015c | 0.24±0.016b |
| Negative group | 0.26±0.013c | 0.24±0.016c | 0.22±0.011b |
| Control group | 0.25±0.015c | 0.23±0.013c | 0.23±0.012b |
To gain insight into the mechanism by which the prepared skin regeneration membrane promotes basement membrane remodeling and re-epithelialization in vivo, the present test conducted expression level detection of active integrins β1, FAK and pFAK, the results are shown in table 5, and multiple comparisons and significant differential markers were performed for each column. The results show that comparative examples 3 and 4 have no significant difference in the expression levels of active integrins β1, FAK and pFAK in the wound skin regeneration tissues of mice relative to the negative and control groups, respectively. The skin regeneration films provided in examples 1-4 showed significantly higher expression levels of active integrins β1, FAK and pFAK in skin tissues after the wound surface of mice was treated, compared with the negative group and the control group. These results indicate that the skin regeneration membrane may play a role by activating integrin β1 mediated integrin signaling, integrin β1 focal adhesion kinase (focal adhesion kinase, FAK) in integrin mediated cell-extracellular matrix interactions, FAK activation by autophosphorylation of its tyrosine-397 site.
In conclusion, the III-type collagen and the polylactic acid are spun by adopting the electrostatic spinning method to prepare the fibrous III-type collagen nano-film, and the fibrous III-type collagen nano-film has certain mechanical property and good biocompatibility and can be applied to the fields of wound healing and wound skin regeneration.
The fibrillated III type collagen nano film is formed by compounding nano fibers, has tensile strength of 1.22-2.66 MPa and elongation at break of 117.8-165.9%, and has excellent mechanical properties. In addition, the fibrillated III type collagen nano film has higher hydrophilic property and higher water absorption rate.
The fibrous III type collagen nano-film is subjected to double-sided co-culture on human surface keratinocytes and human dermal fibroblasts, so that three-dimensional culture, growth and proliferation of the human surface keratinocytes and the human dermal fibroblasts can be promoted, and excellent biocompatibility and in-vivo applicability are suggested.
Furthermore, the invention also applies the fibrosis III type collagen nano-film to repair the wound surface of a mouse, the wound surface of the mouse is transplanted, the wound surface can be covered by epidermis, the defect disappears, the wound surface is closed, and after the fibrosis III type collagen nano-film provided in the embodiments 1-4 regenerates the wound surface, the scar on the skin surface is smaller, which indicates that the fibrosis III type collagen nano-film provided by the invention has application prospect as a skin regeneration film or preparation. In addition, through protein expression analysis in the tissue of the repaired wound skin of the mice, the fiber III type collagen nano-film plays an important role in signal transduction through activating integrin beta 1 to mediate integrin signal transduction and activating FAK through autophosphorylation of tyrosine-397 locus in the interaction of integrin mediated cells and extracellular matrixes.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (5)
1. The preparation method of the fibrillated III type collagen nano film is characterized by comprising the following steps of:
preparing an electrostatic spinning solution, wherein the electrostatic spinning solution comprises a solid matter of type III collagen, polylactic acid and water-insoluble dietary fiber;
carrying out electrostatic spinning by utilizing the electrostatic spinning solution;
performing glutaraldehyde steam cross-linking on the electrospun film to form the fibrillated III type collagen nano film;
the preparation method of the electrostatic spinning solution comprises the following steps: preparing a type III collagen solution, a polylactic acid solution and a water-insoluble dietary fiber solution, wherein the weight percentage of the type III collagen solution, the weight percentage of the polylactic acid solution and the weight percentage of the water-insoluble dietary fiber solution are 15wt%, and mixing the type III collagen solution, the polylactic acid solution and the water-insoluble dietary fiber solution to be used as spinning solution for electrostatic spinning;
wherein the 5wt% of the water-insoluble dietary fiber solution is prepared from a solid matter of the water-insoluble dietary fiber, and the preparation method of the solid matter comprises the following steps:
(1) After the wheat bran is finished and decontaminated, crushing the wheat bran, sieving the crushed wheat bran with a 100-mesh sieve, soaking the wheat bran with water with the weight of 10 times of that of the wheat bran for about 30min, removing the water, and washing the wheat bran with warm water for 2-3 times to remove soluble sugar and partial pigment substances in the wheat bran powder;
(2) Adding the wheat bran powder treated by the method into a sulfuric acid solution with the mass fraction of 60% which is 15 times of the weight of the wheat bran powder, placing the wheat bran powder into a constant-temperature water bath with the temperature of about 80 ℃ for hydrolysis for 2 hours, filtering the wheat bran powder while the wheat bran powder is hot, and washing filter residues with hot water to be neutral;
(3) Adding the wheat bran powder treated by the method into 40 times of sodium hydroxide solution with the pH value of 13.5, treating the wheat bran powder in a constant-temperature water bath at about 85 ℃ for 3 hours, vacuum filtering, neutralizing the wheat bran powder with 1% hydrochloric acid solution, and washing the wheat bran powder with 80 ℃ hot water to be neutral; dehydrating with absolute ethanol, degreasing at 60deg.C by Soxhlet extraction to obtain final solid of water insoluble dietary fiber;
wherein the water-insoluble dietary fiber content in the solid matter is 16.8%.
2. The preparation method of claim 1, wherein the volume ratio of the type III collagen solution, the solid matter of the water-insoluble dietary fiber and the polylactic acid in the mixed solution is 6:1:2, and the mixed solution is fully mixed by adopting ultrasonic to be used as a spinning solution for electrostatic spinning.
3. The preparation method of claim 1, wherein the volume ratio of the type III collagen solution, the solid of the water-insoluble dietary fiber and the polylactic acid in the mixed solution is 7:2:2, and the mixed solution is fully mixed by adopting ultrasonic to be used as a spinning solution for electrostatic spinning.
4. The fibrillated type III collagen nanomembrane prepared by the preparation method of any one of claims 1 to 3, wherein the fibrillated type III collagen nanomembrane has a tensile strength of 1.22 to 2.66mpa and an elongation at break of 117.8% to 165.9%.
5. Use of the fibrillated type III collagen nanomembrane prepared by the preparation method of any one of claims 1 to 3 in skin regeneration products.
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