CN111544653A - Bionic laminated tissue engineering skin adapting to multi-dimensional clinical requirements and preparation method thereof - Google Patents

Bionic laminated tissue engineering skin adapting to multi-dimensional clinical requirements and preparation method thereof Download PDF

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CN111544653A
CN111544653A CN202010369031.XA CN202010369031A CN111544653A CN 111544653 A CN111544653 A CN 111544653A CN 202010369031 A CN202010369031 A CN 202010369031A CN 111544653 A CN111544653 A CN 111544653A
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tissue engineering
fgf
hyaluronic acid
laminated tissue
layer
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王子健
胡伟康
陈熙
李毅祺
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Hubei Baina Huiyu Biotechnology Co ltd
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Hubei Baina Huiyu Biotechnology Co ltd
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Abstract

The invention discloses a bionic laminated tissue engineering skin adapting to multi-dimensional clinical requirements and a preparation method thereof, belonging to the field of biomedical materials and tissue engineering. The bionic laminated tissue engineering skin consists of a quaternized chitin layer and an FGF 2-hyaluronic acid layer, and is alternately adsorbed on the surface of a polylactic-co-glycolic acid (PLGA) nanofiber membrane. The quaternized chitin layer has broad-spectrum antibacterial property and in-situ antibacterial property; the FGF 2-hyaluronic acid layer has angiogenesis promoting activity; the synergistic effect of the two also has the functions of skin anti-inflammation and collagen synthesis promotion. The preparation method is simple, has little pollution and high industrial added value; the product has excellent clinical performance and can meet the requirements of various application environments such as infectious ulcer, diabetic foot, medical cosmetology and the like.

Description

Bionic laminated tissue engineering skin adapting to multi-dimensional clinical requirements and preparation method thereof
Technical Field
The invention belongs to the field of biomedical materials and tissue engineering, and particularly relates to a bionic lamination tissue engineering skin suitable for multi-dimensional clinical requirements and a preparation method thereof.
Background
Skin tissue and its appendages are important biological barriers for the body to resist external damage. When skin integrity is compromised, patients can develop symptoms of pain, bleeding, infection, and delayed healing. The skin healing process is divided into a hemostasis stage, an inflammation stage, a regeneration stage and a remodeling stage, and the key point of accelerating healing is to rebuild the microenvironment of a wound surface and adopt proper treatment measures. Tissue engineering skin such as hydrogel, 3D conformal printing support, etc. can be used for maintaining wound surface humid environment, preventing pathogen infection, and reducing secondary injury. However, the existing tissue engineering skin also has the defects of poor biological activity and biodegradability, strong immunogenicity and anaphylaxis, difficult cleaning, easy bleeding during replacement and the like, and can not completely meet the clinical requirements. The nanofiber material can be prepared by a high-voltage electrostatic spinning method, and has a three-dimensional structure similar to extracellular matrix (ECM), a high specific surface area and adjustable performance. Therefore, we prefer the nanofiber material as the substrate material for preparing the bionic tissue engineering skin.
Layer-by-layer self-assembly (LBL) is a modification technique in which a substrate material is sequentially immersed in a solution of oppositely charged polyelectrolytes to form a coating of polyelectrolyte complex. A number of studies have demonstrated that LBL technology is one of the versatile modification techniques for nanofiber materials. The qualitative and quantitative regulation and control of physical, chemical and biological functions of the material can be simply realized by increasing the number of self-assembled layers. The commonly used modified materials comprise silk fibroin, chitosan, sodium alginate and the like, and the prepared composite material has obviously improved biological functions such as antibacterial activity, biocompatibility, degradability and the like. At present, nanofiber materials based on LBL technology have been successfully applied to the field of skin tissue engineering. However, high performance tissue engineered skins that can simultaneously meet the clinical needs of multi-dimensionality have been reported.
The difficult healing wounds such as large-area defects, infectious ulcers, diabetic feet and the like put higher requirements on the biological activity of tissue engineering skin, particularly broad-spectrum antibacterial property and angiogenesis promoting activity. The existing commercial antibacterial skin is designed only for infection of gram-positive bacillus (s. aureus) and gram-negative bacteria (e. coli), wherein release of soluble antibacterial agents (antibiotics or silver ions) has potential toxic and side effects on the body. In recent years, the incidence of hospital acquired infection is rising year by year, and the conventional antibacterial dressing has poor treatment effect on hospital acquired infection (such as methicillin-resistant staphylococcus aureus). Therefore, the development of a novel tissue engineering skin which aims at drug-resistant bacterial infection and has an in-situ antibacterial effect is of great significance. Insufficient angiogenesis and poor blood supply are another important factor in delayed wound healing. The angiogenesis promoting activity is crucial to the repair of diabetic foot lesions. The number and maturity of new blood vessels is influenced by a range of physical and biological inducers. In particular, basic fibroblast growth factor (FGF 2) has a significant promoting effect on angiogenesis. Because of poor targeting of FGF2, the tissue engineering skin FGF2 has certain difficulty in realizing sustained and sustained release effect by eliminating inactivation during injection.
The invention aims to prepare the bionic laminated tissue engineering skin with in-situ antibacterial property and angiogenesis promoting activity by adopting an LBL (local breakout method), and aims to overcome the defects and be applied to treating various wounds which are difficult to cure. At present, similar reports are not found for the content and the application effect of the invention.
Disclosure of Invention
The invention firstly adopts a high-voltage electrostatic spinning method to prepare the polylactic acid-glycolic acid copolymer (PLGA) nanofiber membrane, and the polylactic acid-glycolic acid copolymer (PLGA) nanofiber membrane is used as a base material of tissue engineering skin. In the subsequent LBL process, the bionic laminated tissue engineering skin with a shell-core structure is prepared by using quaternized chitin as a positive component and FGF 2-hyaluronic acid solution as a negative component. We have proved that the quaternized chitin has excellent broad-spectrum antibacterial property and good antibacterial effect on methicillin-resistant staphylococcus aureus, gram-positive bacteria and gram-negative bacteria. Through LBL technology, the quaternized chitin is fixed on the surface of the tissue engineering skin, so that toxic and side effects caused by local drug release are effectively avoided. Hyaluronic acid is a macromolecular substance with excellent biocompatibility. FGF2 was slowly released from the product to achieve the effect of promoting angiogenesis.
According to the first aspect of the invention, a bionic laminated tissue engineering skin suitable for multi-dimensional clinical requirements is provided, which is characterized in that the tissue engineering skin contains a quaternized chitin layer and an FGF 2-hyaluronic acid layer; the quaternized chitin layer and the FGF 2-hyaluronic acid layer are adsorbed on the surface of the substrate through electrostatic action; the number of the quaternized chitin layer is the same as that of the FGF 2-hyaluronic acid layer, and the number of the quaternized chitin layer and the FGF 2-hyaluronic acid layer is not less than 1.
Preferably, the substrate is a polylactic-co-glycolic acid (PLGA) nanofiber membrane.
According to another aspect of the invention, a preparation method of the bionic laminated tissue engineering skin suitable for multi-dimensional clinical requirements is provided, and is characterized in that a base material is sequentially soaked in an FGF 2-hyaluronic acid solution and a quaternized chitin solution, so that the hydrophobicity of negatively charged FGF 2-hyaluronic acid and positively charged quaternized chitin is enhanced after adsorption through electrostatic interaction, and the bionic laminated tissue engineering skin with a shell-core structure is obtained.
Preferably, the base material is prepared by a high-voltage electrostatic spinning method, PLGA is dissolved in hexafluoroisopropanol to obtain a pre-electrospinning solution with the mass concentration of 5-15%, and the parameters of the spinning machine are as follows: the voltage between the plates is 10-15 kV, the receiving distance is 10-15cm, the flow rate is 0.5-1.5 mL/h, and the electrospinning time is 2-4 h; soaking the product in 0.5-5 wt% polyaniline hydrochloride solution for 30-120 min, taking out, and air drying.
Preferably, the mass concentration of the quaternized chitin solution is 2-8%; the mass concentration of the FGF 2-hyaluronic acid solution is 0.5-5%; the soaking time is 10-60 min.
Preferably, the concentration of FGF2 in the FGF 2-hyaluronic acid solution is 1-100 mg/mL.
Preferably, the number of successive soaking is not less than 1 cycle, and the successive soaking is performed in the FGF 2-hyaluronic acid solution and the quaternized chitin solution for 1 cycle.
Compared with the prior art, the invention has the following technical advantages and innovation points.
(1) The invention selects the quaternized chitin with positive charge and FGF 2-hyaluronic acid with negative charge as main active components, skillfully fixes the quaternized chitin and the FGF 2-hyaluronic acid on the surface of a PLGA nanofiber membrane by a layer-by-layer self-assembly technology, and prepares the novel tissue engineering skin with in-situ antibacterial property and angiogenesis promoting activity. The quaternized chitin, hyaluronic acid, PLGA and the like are biomedical polymer materials and meet the safety standard of FDA clinical application. Compared with the traditional material processing technologies such as chemical crosslinking and the like, the layer-by-layer self-assembly technology has the advantages of environmental protection, safety, controllable operation and the like. In the invention, the physicochemical and biological properties of the tissue engineering skin, including tensile strength, water contact angle, biocompatibility, in-situ antibacterial activity, in-vivo function and the like, can be easily regulated and controlled through the self-assembled layer number.
(2) Quaternized chitin is preferred as the main antimicrobial component in the present invention. In earlier studies, we have demonstrated that quaternized chitin has excellent broad-spectrum antibacterial effects against gram-positive bacteria, gram-negative bacteria, methicillin-resistant staphylococcus aureus, and the like. Furthermore, the quaternized chitin is fixed on the surface of the tissue engineering skin by adopting a layer-by-layer self-assembly technology, so that the in-situ antibacterial property is enhanced, and the toxic and side effects on organisms when antibacterial components are released are effectively avoided. FGF2 as another bioactive molecule is slowly released from the tissue engineering skin to the wound surface to achieve the function of promoting angiogenesis. The broad-spectrum antibacterial property, the in-situ antibacterial property and the angiogenesis promoting activity of the invention have therapeutic value on various skin lesions such as infected wound, diabetic foot, pressure sore in hospital and the like.
(3) The invention also has unexpected functions and application effects. In vivo animal experiments, the invention also can obviously reduce local inflammation of the wound (such as the expression of a CD45 inflammatory factor is regulated down), and promote the synthesis of skin collagen (such as the regulation of type I collagen and type III collagen). The anti-inflammatory effect may be attributed to the excellent in situ antibacterial and biocompatibility properties of the tissue engineered skin of the present invention. Collagen is one of the major components of skin tissue and has important significance in maintaining skin integrity, elasticity and defense function. The invention can promote the synthesis of collagen and has great application potential in the field of medical cosmetology.
(4) The preparation method is simple, the processing equipment is simple, the environmental pollution is small, the added value of the product is high, and the industrial production is expected to be obtained.
Drawings
Fig. 1 is a functional overview diagram of bionic laminated Tissue Engineering Skin (TESK).
FIG. 2 is the scanning electron microscope image and the water contact angle test image of the polylactic-co-glycolic acid (PLGA) nanofiber membrane and the bionic laminated Tissue Engineering Skin (TESK) obtained in example 1.
FIG. 3 shows the results of the cell flow and Transwell migration capability tests of the biomimetic laminated Tissue Engineered Skin (TESK) obtained in example 1.
FIG. 4 is a graph and a healing curve of the bionic laminated Tissue Engineering Skin (TESK) obtained in example 1 applied to diabetic skin lesion repair healing.
FIG. 5 shows the results of immunohistochemical quantitative analysis of biomimetic laminated Tissue Engineered Skin (TESK) obtained in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
Preparing polylactic acid-glycolic acid copolymer (PLGA) nanofiber membrane. And adding 10g of PLGA solid particles into 100 mL of hexafluoroisopropanol, and fully stirring and dissolving to obtain the pre-electrospinning solution. Adding the solution into a push injection pump of a high-voltage electrostatic spinning machine, and setting electrospinning parameters: the voltage between the plates is 12 kV, the receiving distance is 12 cm, the flow rate is 1.0 mL/h, and the electrospinning time is 3 h. And after the preparation is finished, taking down the nanofiber membrane, soaking the nanofiber membrane in 1% polyaniline hydrochloride solution, and airing for later use after 30 min. Preparing the bionic laminated tissue engineering skin. The PLGA nanofiber membrane serving as a base material is soaked in FGF 2-hyaluronic acid solution with the mass concentration of 2%, and is washed by physiological saline after 30 min. The concentration of FGF2 in the FGF 2-hyaluronic acid solution was 10. mu.g/mL. Soaking the chitosan in a quaternized chitin solution with the mass concentration of 5%, and cleaning with physiological saline after 30 min. The above soaking operation was recorded as 1 round, and the soaking operation was repeated for 10 rounds in total. The prepared bionic laminated tissue engineering skin is named as TESK, and the unmodified PLGA nanofiber membrane is used as a control group and is named as PLGA.
Example 2
The bionic laminated Tissue Engineering Skin (TESK) obtained in example 1 and the unmodified PLGA nanofiber membrane were dried and observed by a scanning electron microscope. And (3) detecting the hydrophilicity and hydrophobicity by adopting a microspur video recording system.
FIG. 2 is the scanning electron microscope image and the water contact angle test image of the polylactic-co-glycolic acid (PLGA) nanofiber membrane and the bionic laminated Tissue Engineering Skin (TESK) obtained in example 1. As can be seen, the PLGA nanofiber membrane exhibits an interwoven network nanofiber structure. After layer-by-layer self-assembly, FGF 2-hyaluronic acid and quaternized chitin are adsorbed on the surface of the obtained bionic laminated Tissue Engineering Skin (TESK) fiber, and the diameter of the TESK fiber is obviously increased. Compared with PLGA, the water contact angle of the TESK is obviously reduced, and the TESK is favorable for cell adhesion and migration in vivo application.
Example 3
The bionic laminated Tissue Engineering Skin (TESK) and the unmodified PLGA nanofiber membrane obtained in the embodiment 1 are sterilized by ultraviolet light, soaked in an RPMI-1640 complete culture medium and filtered after 24 hours to prepare a material leaching solution. Co-culturing fibroblast L929 and the material leaching liquor for 48h, detecting cell cycle distribution by adopting a flow cytometry, and detecting the migration capacity of cells by adopting a Transwell experiment.
FIG. 3 shows the results of the cell flow and Transwell migration capability tests of the biomimetic laminated Tissue Engineered Skin (TESK) obtained in example 1. As can be seen, the proportion of cells in the G0/G1 phase in the L929 cells is greatly reduced after the TESK treatment, which indicates that the cell proliferation activity is obviously improved. The migration capacity of the cells in the TESK treatment group is also obviously increased compared with that in the control group.
Example 4
The bionic laminated Tissue Engineering Skin (TESK) obtained in example 1 and the unmodified PLGA nanofiber membrane were cut into disks with a diameter of 20mm, and the skin repair effect was verified using a diabetic rat full-thickness skin injury model. The diabetic rats were purchased from Biotech Limited, Barkan, Wuhan, and were anesthetized by isoflurane inhalation and then the whole dorsal skin was excised, 20mm in diameter, and deep into the fascia. The experimental group applied TESK to the wound surface, the positive control group applied medical gauze to the wound surface, the negative control group applied PLGA to the wound surface, and the blank control group did not treat. And (5) counting a skin lesion healing curve, and collecting regenerated skin tissues for pathological detection.
FIG. 4 is a graph and a healing curve of the bionic laminated Tissue Engineering Skin (TESK) obtained in example 1 applied to diabetic skin lesion repair healing. As can be seen, the TESK treated group healed the fastest, with significant therapeutic effect particularly in the early stages of wound healing.
FIG. 5 shows the results of immunohistochemical quantitative analysis of biomimetic laminated Tissue Engineered Skin (TESK) obtained in example 1. As can be seen, TESK significantly reduces skin tissue inflammation (down-regulating CD45 inflammatory factor expression), promotes skin collagen synthesis (up-regulating type I collagen, type III collagen).
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (7)

1. A bionic laminated tissue engineering skin adapting to multi-dimensional clinical requirements is characterized in that the bionic laminated tissue engineering skin contains a quaternized chitin layer and an FGF 2-hyaluronic acid layer; the quaternized chitin layer and the FGF 2-hyaluronic acid layer are adsorbed on the surface of the substrate through electrostatic action; the number of the quaternized chitin layer is the same as that of the FGF 2-hyaluronic acid layer, and the number of the quaternized chitin layer and the FGF 2-hyaluronic acid layer is not less than 1.
2. A biomimetic laminated tissue engineered skin adapted to meet the multi-dimensional clinical requirement in accordance with claim 1, wherein the substrate is a poly (lactic-co-glycolic acid) (PLGA) nanofiber membrane.
3. A preparation method of a bionic laminated tissue engineering skin suitable for multi-dimensional clinical requirements is characterized in that a base material is sequentially soaked in an FGF 2-hyaluronic acid solution and a quaternized chitin solution, so that the hydrophobicity of FGF 2-hyaluronic acid with negative electricity and quaternized chitin with positive electricity is enhanced after the FGF 2-hyaluronic acid with negative electricity and the quaternized chitin with positive electricity are adsorbed through electrostatic action, and the bionic laminated tissue engineering skin with a shell-core structure is obtained.
4. The method for preparing a bionic laminated tissue engineering skin meeting the multi-dimensional clinical requirement as claimed in claim 3, wherein the substrate is prepared by a high-voltage electrostatic spinning method, PLGA is dissolved in hexafluoroisopropanol to obtain a pre-electrospinning solution with a mass concentration of 5-15%, and the parameters of a spinning machine are set as follows: the voltage between the plates is 10-15 kV, the receiving distance is 10-15cm, the flow rate is 0.5-1.5 mL/h, and the electrospinning time is 2-4 h; soaking the product in 0.5-5 wt% polyaniline hydrochloride solution for 30-120 min, taking out, and air drying.
5. The method for preparing bionic laminated tissue engineering skin meeting the multi-dimensional clinical requirement as claimed in claim 3, wherein the mass concentration of the quaternized chitin solution is 2-8%; the mass concentration of the FGF 2-hyaluronic acid solution is 0.5-5%; the soaking time is 10-60 min.
6. The method for preparing bionic laminated tissue engineering skin meeting the multi-dimensional clinical requirement as claimed in claim 5, wherein the concentration of FGF2 in the FGF 2-hyaluronic acid solution is 1-100 mg/mL.
7. The method for preparing a bionic laminated tissue engineering skin meeting the clinical requirement of multiple dimensions as claimed in claim 3, wherein the number of successive soaking cycles is not less than 1 cycle, and the successive soaking cycles are 1 cycle of successive soaking in FGF 2-hyaluronic acid solution and quaternized chitin solution.
CN202010369031.XA 2020-05-04 2020-05-04 Bionic laminated tissue engineering skin adapting to multi-dimensional clinical requirements and preparation method thereof Pending CN111544653A (en)

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