CN112295021A - Skin graft with improved topological structure - Google Patents
Skin graft with improved topological structure Download PDFInfo
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- CN112295021A CN112295021A CN202011119548.XA CN202011119548A CN112295021A CN 112295021 A CN112295021 A CN 112295021A CN 202011119548 A CN202011119548 A CN 202011119548A CN 112295021 A CN112295021 A CN 112295021A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
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- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
- D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
- D04H1/74—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being orientated, e.g. in parallel (anisotropic fleeces)
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- Health & Medical Sciences (AREA)
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Abstract
The invention provides a skin graft with an improved topological structure, and a manufacturing method thereof comprises the following steps: step 1: dissolving polylactic glycolic acid and fish collagen serving as raw materials in a solvent to obtain an electrospinning solution; step 2: placing the electrospinning solution in electrostatic spinning equipment, and spinning to obtain an electrospinning membrane; the electrospun membrane obtained in the step 2 is applied to serve as a skin graft. The skin graft with the improved topological structure provided by the invention is used as the skin graft, can promote undifferentiated keratinocytes to creep and cover a wound surface in a postoperative repair period (7 days after operation), can prevent new epidermis hyperkeratosis and promote normal healing of the wound, and is suitable for skin tissue repair.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a skin graft with an improved topological structure.
Background
Currently clinically available skin grafts include grafts of biological origin (autograft, allograft, xenograft), as well as synthetic grafts (loaded/unloaded with autologous or allogeneic cells). The electrospinning technology is increasingly used for constructing tissue engineering skin grafts in recent years because of the advantages of being capable of preparing nano-grade fibers, highly controllable in material topological structure and the like.
In the components for synthesizing the graft, the polylactic acid-glycolic acid copolymer (PLGA) is formed by randomly polymerizing two monomers, namely lactic acid and glycolic acid, is a degradable functional polymer organic compound, has good biocompatibility, no toxicity and good properties of capsulizing and film forming, and is widely applied to pharmaceutical and medical engineering materials. PLGA is certified by FDA and formally incorporated into the united states pharmacopeia as a pharmaceutical excipient.
On the aspect of preparing an electrospinning film with PLGA as a main component, research shows that the PLGA-I type collagen composite electrospinning film material added with collagen is beneficial to improving the mechanical property of the material, but factors such as the type, the using amount and the electrospinning method of the added collagen can influence the physical and biological properties of the spun collagen film, and the mature technology is a collagen and PLGA blended film for bone tissue regeneration. Due to medical requirements, the technology of the existing collagen and PLGA blended membrane for repairing bone tissues is mature, the requirements of skin and the bone tissues on the biological performance of the repairing membrane in the repairing process are different, and a skin graft with the repairing performance and the degradation performance matched with skin cells needs to be formulated.
Disclosure of Invention
Based on the above problems, it is an object of the present invention to provide a skin graft with an improved topology.
In order to achieve the above purpose, the invention adopts the technical scheme that: a skin graft having an improved topology, the method of making comprising:
step 1: dissolving polylactic glycolic acid and fish collagen serving as raw materials in a solvent to obtain an electrospinning solution;
step 2: placing the electrospinning solution in electrostatic spinning equipment, and spinning to obtain an electrospinning membrane;
the electrospun membrane obtained in the step 2 is applied to serve as a skin graft.
The skin graft with the improved topological structure avoids the use of collagen from mammals, the fish collagen with good biocompatibility, biodegradability, low antigenicity and low animal disease infectivity is utilized to prepare the composite electrospun membrane, and the fish collagen can interact with polylactic-co-glycolic acid (PLGA) molecular chains to form a molecular network structure, thereby being beneficial to increasing the mechanical property of materials. When the electrospun membrane provided by the invention is applied to a skin graft, the cell activity can be enhanced in a postoperative repair period (7 days after operation), undifferentiated cutin is promoted to quickly form epithelial cells to crawl to cover a wound surface, and the electrospun membrane can prevent new epidermal hyperkeratosis and promote normal healing of the wound, and is suitable for repairing the skin at the wound.
Optimally, in the step 2, a roller is adopted for collecting the electrospun membranes, and the collected electrospun membranes are in oriented morphology structures; the oriented structure electrospun material is better than the electrospun membrane with a random structure and a grid structure in the aspects of promoting skin defect healing, keratinization, reducing fibrosis degree and the like, presumably because the material has a contact guiding effect on cells, and the fibers of the oriented structure electrospun membrane change the cytoskeleton of skin cells (mainly comprising keratinocytes, fibroblasts and the like) along the fiber trend, provide a cell migration track, and contribute to improving the cell migration efficiency, thereby accelerating wound closure; the electrospun membrane using the fish collagen and the PLGA as raw materials is combined with an oriented membrane structure, so that type 2 immunoreaction can be induced, generation of type 2 immune cells (such as repair-promoting macrophages) at a wound surface is promoted, cell factors (such as Il10) are secreted, re-epithelialization of the wound surface is promoted, the fibrosis degree around the material is reduced, hyperkeratosis is prevented, and an excellent skin repair effect is achieved.
Further optimally, the concentrations of the polylactic glycolic acid and the fish gelatin in the electrospinning solution in the step 1 are respectively (19-21)% W/V and (1.8-2.2)% W/V, the intermolecular action of the polylactic glycolic acid and the fish gelatin is enhanced in the concentration range, the spun electrospinning film has high tensile strength, and when the electrospun film is used as a skin graft, the electrospun film can continuously promote the proliferation and migration of epidermal cells in the coagulation period, the inflammation period and the proliferation and repair period of skin defect healing, a little material is remained when the electrospun film heals for 28 days, and hyperkeratosis is avoided, so that the electrospun film is particularly suitable for the durable repair of skin.
Further optimally, the concentrations of the polylactic glycolic acid and the fish gelatin in the electrospinning solution in the step 1 are respectively (19-21)% W/V and (1.8-2.2)% W/V, and the oriented electrospinning film spun under the concentration condition is used as a skin graft, can promote the relevant gene expression of repair-promoting macrophages at the wound, and can reduce the fibrosis reaction around the material compared with grids and random shapes, thereby promoting the continuous repair of the wound epidermis. The mass ratio of lactic acid to glycolic acid in the polylactic-glycolic acid is 73: 27-77: 23.
And optimally, the parameters of the electrospinning device are as follows: applying a voltage of-2 Kv, 5 Kv; a 21G metal needle with an inner diameter of 0.5mm is used, and the tip of the needle is 16cm away from the collector; the injection rate of the electrospinning liquid is 0.068 mm/min; the rotating speed of the roller is 2700-2900 rpm, the electrospinning time is 3-4 hours, and the prepared electrospinning film has more excellent mechanical and biological properties by the parameters, particularly the rotating speed of the roller and the electrospinning liquid with the concentration range; specifically, the rotating speed of the roller is 2800rpm, the electrospinning time is 4 hours, and the electrospun membrane spun by the parameters has more excellent mechanical properties and the degradation rate is more adaptive to the normal period of skin repair.
Preferably, the electrospun membrane spun in step 2 is further subjected to the following treatment: vacuum drying at 37 deg.C for 72h, soaking in NHS/EDC solution at 4 deg.C for crosslinking for 24h, soaking in 75% ethanol for 10min to complete preshrinking, vacuum drying for 24h, and performing gamma ray irradiation sterilization at 15K Gy dose.
The invention has the beneficial effects that:
the skin graft with the improved topological structure provided by the invention is used as the skin graft, and the skin graft can promote undifferentiated keratinocyte to creep and cover a wound surface in a postoperative repair period (7 days after operation), prevent new epidermis hyperkeratosis and promote normal healing of the wound by taking polylactic-glycolic acid and fish collagen as raw materials to obtain an electrospun membrane through blending. In particular to an oriented electrospinning film spun by electrospinning liquid with the concentrations of polylactic glycolic acid and fish gelatin of 20% W/V and 2% W/V respectively, which induces 2-type immunoreaction, promotes the wound surface to generate 2-type immune cells (such as repair-promoting macrophages), secretes repair-promoting cytokines (such as Il10), reduces the fibrosis degree around the material, and is suitable for skin tissue repair.
Drawings
FIGS. 1 and 2 show the micro-morphology of the electrospun membranes of control 1;
FIGS. 3 and 4 are the micro-morphologies of the electrospun membranes of control 2;
FIGS. 5 and 6 show the microscopic morphology of the electrospun membrane of the experimental group;
FIG. 7 is a comparison table of tensile strength test results of electrospun membranes of control group 1, control group 2 and experimental group;
FIG. 8 is a comparison table of cell viability detection results of electrospun membrane L929 of control 1 group, control 2 group, experimental group and blank group;
FIG. 9 shows the morphology of control 1 group of electrospun membrane-seeded HOK cells under the observation of a scanning electron microscope;
FIG. 10 shows the morphology of control 2 groups of electrospun membrane-seeded HOK cells under scanning electron microscope observation;
FIG. 11 shows the morphology of HOK cells inoculated on the experimental electrospun membrane under the observation of a scanning electron microscope;
FIG. 12 is a graph showing the healing conditions of rats with dorsal skin defects in a blank control group, a control 1 group, a control 2 group and an experimental group;
FIG. 13 is a statistical comparison table of residual defect areas of electrospun membranes of control group 1, control group 2 and experimental group when used for dorsal skin defects of rats;
FIG. 14 is a comparison of staining of sections at 7 days of healing;
FIG. 15 comparison table of the ratio of the areas of coloring of undifferentiated/differentiated keratinocytes after 7 days of healing;
FIG. 16 is a table showing the expression of genes associated with repair-type macrophages;
FIG. 17 is a comparison of staining of sections at 14 days of healing;
FIG. 18 is a remaining gap width comparison table;
FIG. 19 is a chart of neonatal tissue thickness;
FIG. 20 is a table of comparison dermal thicknesses at defects;
FIG. 21 is a comparison of fiber encapsulation formation;
FIG. 22 is a fiber thickness statistical comparison table.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Examples
The present embodiment provides a skin graft with improved topology, and the manufacturing method thereof includes:
step 1: taking polylactic glycolic acid and fish collagen as raw materials, taking hexafluoroisopropanol as a solvent, firstly dissolving the fish collagen in the hexafluoroisopropanol, then adding the polylactic glycolic acid, and rotationally dissolving for 2.5h at 37 ℃ of a shaking table until no granular solute exists to obtain an electrospinning solution, wherein the concentrations of the polylactic glycolic acid and the fish gelatin in the electrospinning solution are respectively 20% W/V and 2% W/V;
step 2: placing the electrospinning solution in electrospinning equipment, and spinning to obtain an electrospinning membrane, wherein the parameters of the electrospinning equipment are as follows:
firstly, applying voltage of-2 Kv and 5 Kv;
secondly, a 21G metal needle (the inner diameter is 0.5mm) is used, and the tip of the needle is 16cm away from the collector;
③ the injection rate of the electrospinning solution is 0.068 mm/min;
the collector is divided into a metal flat plate, a metal grid and a 2800rpm roller, wherein the electrospun membrane collected by the metal flat plate is a control 1 group (shown in figures 1 and 2) with random morphology, the electrospun membrane collected by the metal grid is a control 2 group (shown in figures 3 and 4) with grid morphology, and the electrospun membrane collected by the roller is the orientation morphology of the experimental group (shown in figures 5 and 6);
electrospinning for 3-4 hours;
and step 3: and (3) respectively carrying out vacuum drying on the electro-spinning membranes with the three morphologies obtained in the step (2) for 72h, and then soaking in an NHS/EDC solution at 4 ℃ for crosslinking for 24 h. Soaking the material in 75% ethanol for 10min to shrink completely, vacuum drying for 24 hr, and sterilizing by irradiation.
The electrospun membranes of the control 1 group, the control 2 group and the experimental group obtained in the step 3 were tested as follows:
first, Scanning Electron Microscope (SEM) detection
Observing the microstructures of the three structural electrospun membranes, the nanofibers of the electrospun membranes of the control group 1, the control group 2 and the experimental group are respectively arranged into three structures, namely a random type, a grid type and an orientation type, the random type nanofibers are randomly and disorderly arranged in the direction, the grid type nanofibers are interwoven to form a larger grid space, and the orientation type nanofibers are arranged with a certain orientation.
Second, tensile Strength test
As shown in fig. 7, the tensile strength test showed that the oriented electrospun films of the experimental group had higher tensile strength than the electrospun films of the control 1 group and the control 2 group.
Third, cell viability assay
The electrospun membranes of the control 1, control 2 and experimental groups were cut into 5mm by 5mm squares and attached to the bottom of a 96-well plate, and the mouse skin fibroblast cell line (L929 cells) was inoculated in the well plate (cell density: 4 by 10^4 cells/ml) and cultured in RMPI medium containing 10% fetal bovine serum at 100. mu.1 per well. The culture was continued for 1, 3, 5, 7 days with liquid changes every 2 days. The cells were incubated with 10 μ 1CCK-8 solution per well for 1 hour (37 ℃, 5% CO2) in a cell incubator, the supernatant was aspirated, the absorbance (a) value of each well was read at a wavelength of 450nm on a microplate reader, and the cell viability was calculated as ═ 100% (treatment group a value-blank a value)/(control group a value-blank a value) (as shown in fig. 8), wherein no material was added to the blank control group and no fish collagen was added to the pure PLGA group as a component. The results show that the cell viability changes in a manner substantially consistent with that of the blank control group, and the cell viability is not affected by the visible test membrane material.
Fourthly, SEM observation of the growth and adhesion of cells on the surface of the membrane material
Human oral mucosa keratinocytes (HOK cells) were seeded on the electrospun membrane material of control 1, control 2 and experimental groups (3 x 10^4cells/cm2), and the culture results for 24h showed that the HOK cells grew well on the material and the cells of the experimental groups grew directionally along the fiber direction of the material (as shown in FIGS. 9 to 11).
Fifth, evaluation of defect healing area
The electrospun membranes of the control group 1, the control group 2 and the experimental group are implanted into the skin defect (circular excision defect with the diameter of 6 mm) on the back of a rat, the blank control group is not implanted with materials, Tegaderm (3M) covers the upper part of the defect, and the defect is fixed by a silica gel ring (with the inner diameter of 8mm, the outer diameter of 12mm, the edge width of 2mm and the thickness of 1mm) in a sewing manner, so that the back muscle is prevented from contracting and closing the wound, and the crawling healing capability of the skin wound is objectively evaluated. Based on the statistics of the defect healing area (shown in fig. 12, 13, 17, 18), the defects in the oriented group healed faster than those in the control 1 group, the control 2 group, and the blank control group.
Sixthly, evaluating healing effect of tissue section
1. The samples at the healing day 7 (repair period) and 14 days (reconstruction period) are subjected to HE and Masson staining, indexes such as residual gap width, new skin tissue thickness and the like (shown in figures 18-20) are measured semiquantitatively, and it can be seen that the experimental group can see the formation of the stratified new keratinized epithelium as early as the 7 th day, and the epithelium is covered more quickly and has a structure closer to a normal epithelium structure than other groups.
Keratin 5(Krt5) is characteristically expressed in undifferentiated keratinocytes near the basal layer of the epidermis, and Keratin 10(Krt10) is characteristically expressed in differentiated mature keratinocytes on the basal layer. Immunofluorescent staining is carried out on a sample at the healing day 7 (the repair period), the fluorescent staining areas of the new epidermis parts Krt5 and Krt10 are measured semiquantitatively, as shown in FIG. 15, an experimental group has a larger new epidermis area, and the ratio of the staining areas of undifferentiated/differentiated keratinocytes is large, which indicates that the oriented material promotes re-epithelialization and avoids epidermal hyperkeratosis, and indicates that the oriented material induces type 2 immunoreaction;
the tissue block RNA sequencing result shows that the repair-promoting macrophage-related genes (Cd68, Cd200r and Il10) of the experimental group are higher in expression, and thus the electrospun membrane of the experimental group can induce type 2 immune response when being used for skin repair (figure 16).
Seventhly, evaluating fibrosis degree of tissue section
The electrospun membranes of the control group 1, the control group 2 and the experimental group were implanted under the skin of rats, sampled on days 3, 7 and 14 for Masson trichrome staining, and the thickness of the fiber encapsulation around the material was measured to evaluate the fibrosis around the material, and the results showed that the electrospun membranes of the experimental group were beneficial to reducing the fibrosis around the material (fig. 21-22).
The tests show that the skin graft with the improved topological structure provided by the invention is used as the skin graft, can promote undifferentiated keratinocytes to creep and cover the wound surface in the postoperative repair period (7 days after operation), can prevent the hyperkeratosis of the new epidermis and can promote the normal healing of the wound by blending polylactic-glycolic acid and fish collagen which are used as raw materials to obtain the electrospun membrane. And meanwhile, 2-type immune reaction is induced, 2-type immune cells (such as repair promoting macrophages) are promoted to be generated at the wound surface, repair promoting cytokines (such as Il10) are secreted, the fibrosis degree around the material is reduced, and the wound repair promoting agent is suitable for skin tissue repair.
The above examples of the present invention are merely illustrative of the present invention and are not intended to limit the embodiments of the present invention. Variations and modifications in other variations will occur to those skilled in the art upon reading the foregoing description. Not all embodiments are exhaustive. All obvious changes and modifications of the present invention are within the scope of the present invention.
Claims (8)
1. A skin graft having an improved topology, the method comprising:
step 1: dissolving polylactic glycolic acid and fish collagen serving as raw materials in a solvent to obtain an electrospinning solution;
step 2: placing the electrospinning solution in electrostatic spinning equipment, and spinning to obtain an oriented electrospinning membrane;
the electrospun membrane obtained in the step 2 is applied to serve as a skin graft.
2. The skin graft with improved topology according to claim 1, wherein the concentration of poly (lactic-co-glycolic acid) and fish gelatin in the electrospinning solution of step 1 is (19-21)% W/V, (1.8-2.2)% W/V, respectively.
3. The skin graft with improved topology according to claim 2, wherein the concentrations of poly (lactic-co-glycolic acid) and fish gelatin in the electrospinning solution of step 1 are 20% W/V and 2% W/V, respectively.
4. The skin graft with improved topology according to claim 2, wherein the step 2 employs a roller to collect the electrospun membrane, and the collected electrospun membrane is an oriented morphology structure.
5. The skin graft with improved topology according to claim 4, wherein the electrospinning instrument parameters are: applying a voltage of-2 Kv, 5 Kv; a 21G metal needle with an inner diameter of 0.5mm is used, and the tip of the needle is 16cm away from the collector; the injection rate of the electrospinning liquid is 0.068 mm/min; the rotating speed of the roller is 2700-2900 rpm, and the electrospinning time is 3-4 hours.
6. The skin graft with improved topology according to claim 5, characterized in that the roller rotation speed is 2700-2900 rpm, the electrospinning time is 4 hours.
7. The skin graft with improved topology according to claim 1, wherein the electrospun membrane spun in step 2 is further subjected to the following treatments: vacuum drying at 37 deg.C for 72h, soaking in NHS/EDC solution at 4 deg.C for crosslinking for 24h, soaking in 75% ethanol for 10min to complete preshrinking, vacuum drying for 24h, and performing gamma ray irradiation sterilization at 15K Gy dose.
8. The skin graft with the improved topology structure of claim 1, wherein the mass ratio of lactic acid to glycolic acid in the poly (lactic-co-glycolic acid) is 73: 27-77: 23.
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| CN114272439A (en) * | 2021-06-02 | 2022-04-05 | 四川大学 | Skin graft with improved nano structure |
| CN115804867A (en) * | 2022-12-29 | 2023-03-17 | 广东旺合生物科技有限公司 | Fibrosis type III collagen nano-film, preparation method and application in skin regeneration |
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