CN114425102A

CN114425102A - Hydrophilic electrostatic spinning implant for inducing skin tissue regeneration

Info

Publication number: CN114425102A
Application number: CN202210114604.3A
Authority: CN
Inventors: 何红兵; 尹荣鑫; 周星宇; 杨莉; 苏岭; 闫侃
Original assignee: Shanghai P & P Biotech Co ltd
Current assignee: Shanghai P & P Biotech Co ltd
Priority date: 2022-01-30
Filing date: 2022-01-30
Publication date: 2022-05-03
Also published as: WO2023143335A9; WO2023143335A1

Abstract

The invention discloses a hydrophilic electrostatic spinning implant for inducing skin tissue regeneration, which is prepared by electrostatic spinning of raw materials containing a fibrinogen compound and a polylactic acid polycaprolactone copolymer, and a three-dimensional nano-network structure is arranged inside the implant. By fixing the implant of the present invention to a skin wound surface, regeneration of skin tissues and skin appendages such as hair follicles, sebaceous glands and sweat glands can be induced.

Description

Hydrophilic electrostatic spinning implant for inducing skin tissue regeneration

Technical Field

The invention relates to the technical field of medicine, in particular to a hydrophilic electrostatic spinning implant for inducing skin tissue regeneration.

Background

The skin is the largest organ of the human body. It combines with the external environment to protect mammals from various irritations, infections and dehydration, and also to regulate body temperature perception. Different skin cells perform these different functions. Skin wounds can be divided into two categories: acute (e.g., surgical and traumatic wounds, abrasions and burns) and chronic (e.g., diabetic foot ulcers and decubitus ulcers). According to the data of the national center for health and statistics, 4000 million hospitalized patients and 3150 million outpatients were subjected to surgery in 2000. In addition, there are approximately 4100 million trauma cases per year in the united states, creating an economic burden of 6700 billion dollars per year. 3500 tens of thousands of people worldwide are injured by fire and high-temperature burns each year, 290 of them are hospitalized, and about 238000 die.

Chronic wounds affect 650 million patients in the united states, costing 250 million dollars per year for treatment. This situation is even more severe in developing countries. In addition, 250 tens of thousands of decubitus patients are treated in emergency care facilities each year in the united states alone. The cost of treatment for a single full bed sore is about $ 70,000; the total annual U.S. expense for treating bedsores is estimated to be $ 110 billion. Thus, human skin wounds represent a major, rapidly growing threat to public health and economy.

Treatment of skin wounds depends in part on the size, depth and degree of destruction of the wound. Skin wounds can be classified as superficial (super), partial-thickness (partial-thickness) or full-thickness (full-thickness) wounds. In addition, skin wounds can be classified into burns, wounds, and chronic wounds. Burns and wounds may be superficial, partial thickness or full thickness. Chronic wounds are mostly full-thickness wounds. Superficial wounds, defined as defects in the epidermal and dermal papilla, heal by re-epithelialization of the surviving hair follicles and other skin appendages. Wound dressings help prevent infection and maintain an appropriate wound environment to heal superficial wounds. Some skin wounds are associated with the epidermis and the deep dermis. These lesions (e.g., partial thickness burns and deep ulcers) may not heal adequately due to an insufficient number of skin appendages that are unable to form regenerative buds. Treatment of partial thickness wounds may require dermal replacement (including decellularization and cell seed replacement) to promote migration of nearby cells to the defect site. Full-thickness wounds involve subcutaneous fat or deeper tissue in addition to the epidermis and dermis layers, and are more difficult to heal than superficial or partial-thickness wounds. Healing of these lesions often requires the use of skin autografts or artificial skin substitutes.

Skin grafting is one of the most promising methods for repairing large area wounds, and full thickness skin wounds larger than 4cm in diameter are difficult to heal without grafting. However, autografts are not always available, especially for burn patients, and suitable autografts tend to be of limited origin. For allografts, skin tissue requirements are heavily oversupplied and xenogeneic tissue presents potential immune rejection problems. Since the late 1980 s the formal introduction of tissue engineering technology, tissue engineered skin substitutes have become an attractive solution for the treatment of acute and chronic skin wounds. A commonly used method of manufacturing tissue engineered skin substitutes involves implanting biodegradable scaffolds with epidermal keratinocytes, dermal fibroblasts, and/or stem cells. In recent decades, simple epidermal substitutes have evolved into complex full-thickness skin, comprising two layers, the epidermal and dermal layers, which are also used in clinical commercial applications. However, in view of the current limitations in practice and therapy, there is still much room for improvement.

On the one hand, perfect skin with full function (protection, regulation and feel) remains challenging and has become a major target for wound healing. Although tissue engineered skin has been improved by the implementation of various appendages (including capillary networks, sensory innervation, adipose tissue, and pigmentation), it is still not possible to reliably reconstitute intact skin function, particularly functional hair follicles and sweat glands. The failure of cultured cells to regenerate hair follicles is due to the loss of trichogenicity during the culture propagation process. Therefore, establishing a system for maintaining the regenerative potential of cells in vitro is critical to effective regenerative medicine. On the other hand, the reconstitution of a full-thickness tissue engineered skin is very time consuming, since the production of different types of cells involves extensive cell culture processes. The epidermal and dermal components of double-layered tissue engineered skin, on the one hand, typically require 2-4 weeks of culture to obtain a sufficient number of cells, and then require an additional 3 weeks or more of culture to be ready for transplantation; on the other hand, the potential safety and ethical issues are greatly challenged by the lack of effective supervision and control of the corresponding immune system due to the proliferation and processing of cellular components in vitro.

Skin wound treatment mainly includes wound care and skin regeneration. The wound care mainly adopts various dressings and the combination of internal and external departments, and plays a positive role in treating chronic complicated severe skin injury. Skin regeneration is intended to achieve perfect healing of the function and structure of the defective skin by various methods. Among the various innovative skin rejuvenation treatment options, stents are an innovative strategy. Scaffolds combine the concepts of cell therapy, pharmacology and tissue engineering. Overcoming the limitations of the current wound healing technology. Among them is the electrospinning scaffold technology with great potential. Ultra-thin fibers can be produced with diameters from sub-micron to nanometer. The structure of the electrospinning plate is similar to a natural extracellular matrix (ECM) structure, and the electrospinning plate has controllable pore size, high specific surface area and appropriate gas permeability; to improve the wound healing process. It is highly reproducible and scalable at the industrial level, with great potential for development.

With the development and progress of society and medicine, the demand of people for facial rejuvenation is increasing. For facial depression with age, the current treatment of tissue filling and rejuvenation is mainly surgery and injection, and more materials are adopted, such as: hyaluronic acid, polytetrafluoroethylene bulks, silica gel prostheses, autologous fat and the like. After hyaluronic acid is injected, the metabolism of an organism is fast, the metabolism can only be kept for 9-12 months generally, and patients need to continuously inject hyaluronic acid to maintain the effect. The false body placed on the face can cause rejection reaction of the organism, various adverse reactions caused by the built-in object, uncomfortable feeling of the patient to the false body, unnatural appearance and the like.

Therefore, there is a need to develop new grafts that are effective in promoting regeneration of skin appendages.

Disclosure of Invention

The invention aims to provide a hydrophilic electrostatic spinning implant for inducing skin tissue regeneration in situ, a preparation method thereof and application thereof in inducing skin tissue regeneration.

In a first aspect, the present invention provides an implant for inducing skin tissue regeneration, having a three-dimensional nano-network structure inside, made of a raw material comprising a fibrinogen complex and a polylactic acid-polycaprolactone copolymer.

In some embodiments, the fibrinogen complex includes the following components in parts by weight: 0.1-20 parts of fibrinogen, 0.1-10 parts of arginine hydrochloride, 0.01-10 parts of sodium chloride and 1-10 parts of sodium citrate.

For example, the fibrinogen may be 0.1, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15, 18, or 20 parts, etc.

For example, arginine hydrochloride can be 0.1, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts, and the like.

For example, the sodium chloride can be 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts, and the like.

For example, sodium citrate can be 1 part, 1.2 parts, 1.5 parts, 1.8 parts, 2 parts, 2.2 parts, 2.5 parts, 2.8 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, or 10 parts, and the like.

In some embodiments, the fibrinogen complex includes the following components in parts by weight: 3-15 parts of fibrinogen, 0.5-5 parts of arginine hydrochloride, 0.3-5 parts of sodium chloride and 1-10 parts of sodium citrate.

In some embodiments, the fibrinogen is mammalian-derived fibrinogen; the mammal comprises human, pig, cattle, sheep or horse; further preferably, the fibrinogen is porcine blood-derived fibrinogen.

In some embodiments, the polylactic acid polycaprolactone copolymer has a molecular weight of 5 to 30 tens of thousands, and may be, for example, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, 130000, 140000, 150000, 160000, 170000, 180000, 190000, 200000, 210000, 220000, 230000, 240000, 250000, 260000, 270000, 280000, 290000, 300000, or the like.

In some embodiments, the mass ratio of the fibrinogen complex to the polylactic acid polycaprolactone copolymer is (0.48-1.1): 1, specifically, the mass ratio of the fibrinogen complex to the polylactic acid polycaprolactone copolymer can be 0.48:1, 0.5:1, 0.6:1, 0.67:1, 0.7:1, 0.8:1, 0.9:1, 0.96:1, 1:1, 1.1: 1.

In some embodiments, the implant is in the form of a membrane.

Preferably, the thickness of the implant is 0.51mm to 1.4mm, for example, 0.51mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, etc., more preferably 0.6mm to 0.8 mm.

Preferably, the protein content of the implant is 100-220mg/g, such as 100mg/g, 120mg/g, 130mg/g, 150mg/g, 160mg/g, 180mg/g, 200mg/g or 220 mg/g; the implant may have <12mg/g residual protein, for example 11mg/g, 10mg/g, 9mg/g, 8mg/g, 7mg/g, 6mg/g, 5mg/g, 3mg/g, 2mg/g, 1mg/g, or the like. Wherein, the protein content is the protein content measured in a test solution prepared by dissolving a sample in a sodium hydroxide solution; the residual protein content is the protein content measured in a test solution obtained by leaching a sample with water for injection or physiological saline.

Preferably, the tensile strength of the implant is 0.5-5 MPa, and more preferably 1-4 MPa.

Preferably, the implant has an elongation at break of 60% to 200%, such as 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, etc., more preferably 70% to 160%.

Preferably, the porosity of the implant is 41% to 80%, such as 41%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, etc., more preferably 45-70%.

Preferably, the water absorption of the implant is 100% to 250%, such as 100%, 120%, 140%, 160%, 180%, 200%, 250%, etc., more preferably 100% to 160%.

The implant prepared by the invention can be in a membrane shape, and can be subjected to superfine crushing and/or superfine crushing by adopting a proper crushing device without freezing treatment to be prepared into granules.

In some embodiments, the implant is in granular form.

Preferably, the diameter of the implant is 0.06-0.2mm, more preferably 0.07-0.1 mm.

Preferably, the protein content of the implant is 100-220 mg/g.

Preferably, the implant has a residual protein of <12 mg/g.

Preferably, the porosity of the implant is between 41% and 80%, more preferably between 45% and 70%;

preferably, the water absorption of the implant is 100% to 250%, more preferably 100% to 180%.

In some embodiments, the implants of the present invention are made using electrospinning.

When the implant is prepared, uniformly mixing a solution containing a fibrinogen compound and a solution containing a polylactic acid-polycaprolactone copolymer, adding the mixture into the same volumetric tube of an electrostatic spinning machine, and performing electrostatic spinning; or adding the solution containing the fibrinogen complex and the solution containing the polylactic acid-polycaprolactone copolymer into two different volumetric tubes of an electrostatic spinning machine respectively, and simultaneously carrying out electrostatic spinning.

In some embodiments, the solution containing the polylactic acid-polycaprolactone copolymer is prepared by dissolving the polylactic acid-polycaprolactone copolymer in a mixed solvent of one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran and acetone at a mass volume percentage concentration of 5% to 8% (i.e., 5 to 8g/100 mL). For example, the concentration of the polylactic acid polycaprolactone copolymer may be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%.

In some embodiments, the solvent has <0.55mg/g residual.

In some embodiments, the leach liquor is prepared according to ISO10993-12 requirements for use in connection with safety evaluations. Preferably, the pH value of the leaching liquor ranges from 6.0 to 8.0.

In some embodiments, the fibrinogen complex-containing solution is prepared by dissolving the fibrinogen complex in distilled water at a mass volume percent concentration of 8.0% to 29.0% (i.e., 8.0 to 29.0g/100mL), for example, the concentration of the fibrinogen complex may be 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%.

In some embodiments, the voltage difference of the electrostatic spinning is 15-140 Kv, and/or the electrospinning distance is 10-50 cm, and/or the electrospinning liquid advancing speed is 3-399 mL/h and 401-960 mL/h.

For example, the voltage difference of the electrospinning may be 15kV, 20kV, 30kV, 40kV, 50kV, 60kV, 70kV, 80kV, 90kV, 100kV, 110kV, 120kV, 130kV, 140kV, or the like.

For example, the electrospinning distance may be 10cm, 20cm, 30cm, 40cm, or 50cm, etc.

For example, the electrospinning liquid advancing speed may be 3mL/h, 10mL/h, 20mL/h, 50mL/h, 80mL/h, 100mL/h, 200mL/h, 300mL/h, 399mL/h, 401mL/h, 500mL/h, 600mL/h, 700mL/h, 800mL/h, 900mL/h, 960mL/h, and the like.

In a second aspect, the present invention provides a method of implanting the implant, the method comprising the steps of: the implant is applied to the desired site by needle injection or painting.

In a third aspect, the present invention provides the use of the implant in the manufacture of a material for repairing a defect in a body tissue.

In some embodiments, the material is a repair material for treating meninges, abdominal defects, pelvic floor organ prolapse, atria, ventricular septum, pericardial defects, tendon or ligament rupture, or parenchymal organ rupture.

In a fourth aspect, the invention provides the use of the implant in the manufacture of a medicament or material for dermal wound or cosmetic surgery.

In some embodiments, the skin wound is a chronic wound or a non-healing wound.

In some embodiments, the skin wound is not caused by a tumor.

In some embodiments, the skin wound comprises an abrasion wound, a puncture wound, a penetration wound, a gunshot wound, an explosive wound, an incision, a laceration wound, a avulsion wound, a surgical wound, an electrocautery wound, a radioactive wound, a chemical wound, a frozen wound, and a burn wound.

In some embodiments, the cosmetic treatment comprises a cosmetic treatment of the skin and a micro-cosmetic treatment.

In a fifth aspect, the invention provides an application of a hydrophilic electrostatic spinning biological composite scaffold material in preparing medicines or materials for skin wound or plastic and beauty, wherein the composite scaffold material is prepared by blending an aqueous solution of fibrinogen, L-arginine or hydrochloride thereof and a P (LLA-CL) solution and adopting an electrostatic spinning technology; wherein the mass ratio of the fibrinogen to the L-arginine or the hydrochloride thereof is 1.2: 1-12.5: 1;

the fibrinogen and the L-arginine or the hydrochloride aqueous solution thereof, wherein the solvent is selected from one or more of pure water, water for injection, a salt solution and a buffer solution; the salt solution is selected from sodium chloride solution and potassium chloride solution; the buffer solution is selected from phosphate buffer solution, Tris-HCl buffer solution, glycine buffer solution and D-Hank's solution.

In some embodiments, the skin wound is a chronic wound or a non-healing wound.

In some embodiments, the fibrinogen is mammalian-derived fibrinogen.

In some embodiments, the mammal is a human, pig, cow, sheep, or horse.

In some embodiments, the mass ratio of polylactic acid to polycaprolactone in P (LLA-CL) is 20:80 to 95: 5.

In some embodiments, the solvent in the P (LLA-CL) solution is selected from one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran, chloroform, or acetone.

In some embodiments, the fibrinogen, L-arginine, or hydrochloride salt thereof in an aqueous solution is blended with a P (LLA-CL) solution, wherein the mass ratio of fibrinogen to P (LLA-CL) is 0.2:1 to 2.1: 1.

In some embodiments, the hydrophilic electrospun biocomposite scaffold material has an equilibrium contact angle of less than 55 °.

In some embodiments, the hydrophilic electrospun biocomposite scaffold material has a total volume shrinkage of no greater than 20% after contact with an aqueous solution; the porosity is not less than 30%.

In some embodiments, the aqueous solution of fibrinogen, L-arginine, or hydrochloride thereof, is further loaded with an antimicrobial substance selected from one or more of penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, macrolides, glycosides, sulfonamides, quinolones, nitroimidazoles, lincomamines, fosfomycin, chloramphenicol, para-myxomycin B, bacitracin.

In some embodiments, the penicillin is selected from the group consisting of penicillin, ampicillin, carbenicillin; the cephalosporins are selected from cefalexin, cefuroxime sodium, ceftriaxone and cefpirome; the carbapenem is thiomycin; the aminoglycoside is selected from gentamicin, streptomycin, and kanamycin; the tetracycline is selected from tetracycline and chlortetracycline; the macrolides are selected from erythromycin and azithromycin; the glucoside is vancomycin; the sulfonamides are selected from sulfadiazine and trimethoprim; the quinolone is selected from the group consisting of pipemidic acid and ciprofloxacin; the nitroimidazoles are selected from metronidazole and tinidazole; the lincomycin is selected from lincomycin and clindamycin.

In some embodiments, the amount of the antimicrobial substance released is no less than 30% of the total loading within 15 minutes after implantation of the scaffold material in vivo.

The invention has at least one of the following beneficial effects:

the implant can be used as a hydrophilic electrostatic spinning biological composite stent graft for inducing the regeneration of the defected wound surface of skin tissue in situ. By affixing the implant to the skin wound, regeneration of the full-thickness skin tissue (including epidermis, dermis and sub-dermal layers) and skin appendages (including but not limited to hair follicles, sebaceous and sweat glands, etc.) is induced.

The implant of the invention does not contain bioactive factors (such as cell growth factors and the like) and living cells.

The implant is prepared by an electrostatic spinning method without adopting any chemical or biological crosslinking agent for crosslinking and fixing.

The invention takes fibrinogen and copolymer (Fg/PLCL) of degradable high molecular material L-lactide and caprolactone as raw materials, and after the raw materials are blended, the biodegradable composite reticular stent material with super-hydrophilicity is prepared by adopting an electrostatic spinning technology. We have surprisingly found that this material promotes rapid healing (<5 weeks) of the full-thickness wound (6cm x 4cm) in the thoracico-abdominal region of white pigs, whilst typical hair follicle microstructures are observed in the dermis of the healing skin. In addition, the electrostatic spinning technology is adopted to manufacture the implant material, so that the quality parameters of the product can be effectively designed and controlled by controlling raw materials (such as composition and proportion), equipment parameters (such as voltage, concentration, distance and the like) and the like, and the characteristics (such as mechanical strength, degradation rate, thickness, porosity, water absorption rate), induced functionality, remodeling regeneration rate and the like) of the implant are adjusted, so that the mass production and the popularization and application of innovative products are facilitated.

Drawings

Figure 1 shows the wound healing rate of a full-thickness skin defect at 1-5 weeks after surgery.

Figure 2 shows that the full-thickness skin defect wound surface of the chest and abdomen is basically completely healed after 5-8 weeks of operation.

Figure 3 shows HE staining and Masson staining at week 5, showing fusion of normal (left) and neonatal (right) tissues (x 20).

FIG. 4 shows the cell tissue response grading of the skin patch 1-8 weeks after surgery.

Fig. 5 shows hair follicles appearing in the neonatal dermal layer of the test group at 8 weeks after the operation.

Figure 6 shows Ki67 index changes for 1-8 weeks of full-thickness skin defect healing.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

In this example, a polylactic acid polycaprolactone/fibrinogen (PLCL/Fg) surgical bioprosthetic patch (for skin regeneration) was prepared using an electrospinning method.

Polylactic acid polycaprolactone copolymer (molecular weight 20 ten thousand) was dissolved in Hexafluoroisopropanol (HFIP) at a concentration of 8%, and fibrinogen complex (7.83 parts of fibrinogen, 1.8 parts of arginine hydrochloride, 2.2 parts of sodium chloride, and 6.17 parts of sodium citrate) was dissolved in distilled water at a concentration of 18%. The solution of the polylactic acid polycaprolactone copolymer and the solution of the fibrinogen compound (the mass ratio of the fibrinogen compound to the polylactic acid polycaprolactone copolymer is 0.67:1) are mixed and stirred, and the hydrophilic electrostatic spinning implant is prepared by an electrostatic spinning machine (model: NS1WS 500 Elmarco). The voltage difference of electrostatic spinning is 100Kv, the electrospinning distance is 20cm, and the advancing speed of the spinning solution is 200 mL/h. Sterilizing by adopting 25KGy electron beams, vacuum nitrogen-filled packaging, and storing at 2-8 ℃.

By adjusting the electrospinning parameters, the embodiment specifically provides 5 hydrophilic electrospun implants with different thicknesses, the obtained implants are in a membrane shape, the thicknesses are respectively 0.6mm, 0.7mm, 0.8mm, 0.9mm and 1.0mm, the corresponding porosity is in the range of 45-70%, the water absorption is in the range of 100-160%, and the tensile strength is in the range of 2.0-4.0 MPa.

Example 2

In this example, a polylactic acid polycaprolactone/fibrinogen complex (PLCL/Fg) surgical bioprosthetic patch (for skin regeneration) was prepared by electrospinning.

Polylactic acid polycaprolactone copolymer (molecular weight 30 ten thousand) was dissolved in Hexafluoroisopropanol (HFIP) at a concentration of 5%, and fibrinogen complex (fibrinogen 6.96 parts, arginine hydrochloride 2.1 parts, sodium chloride 1.9 parts, and sodium citrate 5.04 parts) was dissolved in distilled water at a concentration of 16%. The solution of polylactic acid polycaprolactone copolymer and the solution of fibrinogen compound (the mass ratio of the fibrinogen compound to the polylactic acid polycaprolactone copolymer is 0.96: 1) are mixed and stirred, and the hydrophilic electrostatic spinning implant is prepared by an electrostatic spinning machine (model: NS1WS 500Elmarco Czech). The voltage difference of electrostatic spinning is 100Kv, the electrospinning distance is 20cm, and the advancing speed of the spinning solution is 200 mL/h. Sterilizing by adopting 25KGy electron beams, vacuum nitrogen-filled packaging, and storing at 2-8 ℃.

The hydrophilic electrostatic spinning implant prepared is in a membrane shape by adjusting electrostatic spinning parameters, is subjected to freezing treatment, is subjected to superfine crushing by adopting a crushing device, and is prepared into granules, the diameter range of the obtained implant is 0.07 mm-0.1 mm, the porosity is in the range of 45% -70%, the water absorption is in the range of 100% -160%, and the tensile strength is in the range of 2.5-4.0 MPa.

Example 3

The same procedure as in example 1 was followed, except that the fibrinogen complex-containing solution and the polylactic acid-polycaprolactone copolymer-containing solution were fed to two different volumetric tubes of the electrospinning machine, respectively, and electrospinning was carried out simultaneously.

By adjusting the electrospinning parameters, the embodiment specifically provides 5 hydrophilic electrospun implants with different porosities, the obtained implants are in a membrane shape, the porosities are 41%, 45%, 50%, 70% and 80%, the corresponding thicknesses are in the range of 0.6mm to 1.0mm, the water absorption is in the range of 100% to 160%, and the tensile strength is in the range of 1.0 to 4.0 MPa.

Experimental example 1

1. Materials and methods

1.1 materials and reagents

Polylactic acid polycaprolactone copolymers (PLCL) are derived from Purace Biomaterials (Gorinchem, the Netherlands). Porcine fibrin (fibrinengen Fg) was prepared according to the formulation and process disclosed in CN 101759766A. All other reagents and chemicals, except where specifically noted, were purchased from Sigma-Aldrich (st louis, usa).

1.2 preparation of surgical BioPatch (for skin regeneration) samples

The polylactic acid polycaprolactone/fibrinogen (PLCL/Fg) surgical biological patch (for skin regeneration, hereinafter referred to as skin patch) is prepared by an electrostatic spinning method. The Fg complex (8.26 parts of fibrinogen, 2.7 parts of arginine hydrochloride, 2.3 parts of sodium chloride and 5.74 parts of sodium citrate) was dissolved in distilled water at a concentration of 19%, and PLCL was dissolved in Hexafluoroisopropanol (HFIP) at a concentration of 8% according to the procedure of example 1. The two solutions were mixed at 0.23: 0.77 and a sample for investigation was prepared by means of an electrostatic spinning machine (model: NS1WS 500Elmarco Czech). Sterilizing by adopting 25KGy electron beams, vacuum nitrogen-filled packaging, and storing at 2-8 ℃.

1.3 design of the experiment

The study was approved by my animal care and use committee. 20 experimental white pigs with mature skeletal development (female 11, male 9, age greater than 3-4 months; body weight 43.48.6 + -3.7 kg) were used. Divided into four groups according to predetermined post-operative observation times (1 week, 2 weeks, 5 weeks, 8 weeks), each group was randomly assigned 5 animals; the distribution of each component is shown in Table 1.

TABLE 1 full-thickness skin regeneration animal experimental design

Animals were fasted for 12 h. Sutai 50 (French Vickers, Lot. No.: 7VU4A)50mg/kg intramuscular injection, and xylazine hydrochloride (Jilin Hua Murray health products, Lot. No.: 20201118)5mg/kg intramuscular injection for anesthesia, optionally supplemented with an appropriate amount of isoflurane. After anesthesia, the animals were placed in the supine position, the four limbs were properly secured to the operating table, the chest and abdomen were prepped, and a conventional sterile drape was applied. Cutting rectangular full-layer skin defect wound surface along two sides of the median line of chest and abdomen, wherein the wound surface has length of 6cm and width of 4 cm. Self-pairing groups are respectively formed on the chest and the abdomen. Control and test groups were randomized. The control group adopts a self full-thickness skin sheet to cover the wound surface, and the test group adopts a skin patch; the skin sheet and the patch are respectively fixed with the skin at the wound edge by 3-0 non-absorbable suture line, and the wound surface is covered by pressurizing with sterile gauze. Antibiotic injections were given 3 consecutive days post-surgery and gauze was removed after 2 weeks.

1.4 wound healing Rate

Respectively taking pictures for recording the appearance of the wound surface after 1 week, 2 weeks, 3 weeks, 5 weeks and 8 weeks of operation, observing the healing condition of the skin of the wound surface, drawing the edge of the wound surface on a transparent plastic film, measuring the area of the wound surface by using image analysis software ImageJ, and calculating the healing rate of the wound surface by using the following formula 1.

1.5 histological staining and analysis

After general anesthesia, the wound surface of each group of animals is taken for 1 week, 2 weeks, 5 weeks and 8 weeks after operation, and a wound surface histological sample is obtained. Fixed in 10% neutral formalin for at least 48 hours. HE/Masson staining was performed, as well as CD31 and Ki67 immunostaining. Histological changes were observed under light microscopy. The pathological section is scanned by a KF-PRO-020 digital section scanner (KFBIO, Ningbo, China), and the pathological section is subjected to related analysis by K-Viewer software (KFBIO, Ningbo, China).

1.6 evaluation of cellular tissue response during healing of full-thickness skin Defect model

Tissue sections were stained with HE and Masson. Biological evaluation according to ISO10993-6 medical devices-part 6: the evaluation of cellular and tissue responses was performed using a semi-quantitative scoring protocol as described in the test of local effects after implantation. Inflammatory cell infiltration and necrosis were scored using the scoring protocol of table 2; these parameters are multiplied by a factor of 2, since inflammatory cell infiltration and necrosis are of greater importance. The tissue response scoring protocol in table 3 scores neovascularization, fibrosis and fat infiltration. To provide a weighting value compared to the parameters of neovascularization, fibrosis and fat infiltration. These values were added and the average score for the test and control groups was calculated. The average score of the control group was subtracted from the average score of the test group to determine the level of reactivity based on the scale in table 4.

TABLE 2 cell response score sheet

pfh Per high power field of view (400X)

TABLE 3 organization reaction scoring table

TABLE 4 grading of cell tissue reactions

Score of	Grading of cell tissue response
		0.0-2.9	Minimal or no reaction
3.0-8.9	Slight reaction
		9.0-15.0	Moderate reaction
>15.1	Severe reaction

1.7 immunohistochemical staining of CD31 and determination of microvascular density in the surgical field

Paraffin section specimens of 1 week, 2 weeks, 5 weeks and 8 weeks after operation are sequentially immersed in 0.3% H after dewaxing, rehydration and antigen retrieval₂O₂And 0.25% Triton-X100 solution; blocking by 10% horse serum, Avidin and Biotin solution, diluting the antibody by the horse serum, incubating overnight at 4 ℃, and developing according to the kit instructions; then, the cell nucleus is stained with hematoxylin, dehydrated, and sealed by neutral resin for observation. The content of skin capillaries is detected by using a rabbit anti-CD 31 polyclonal antibody. The primary antibody was diluted 1:10000 with rabbit anti-CD 31 polyclonal antibody. Positive control: positive control human tongue tissue specimens of known positive are used, localized to the cytoplasm; negative control: replacing primary antibody with normal dog serum, and obtaining negative result; blank control: primary antibody was replaced with PBS and the result was negative. Any brown-yellow endothelial cells or clusters of endothelial cells that do not connect to adjacent vessels, connective tissue within the selected area are counted as an independent microvessel. In each sample, a region with the maximum number of 3 microvessels, namely a 'hot spot', is selected under a low power microscope (x 20), the number of microvessels under a high power microscope (x 40) is counted in each region, and the mean value is taken as an MVD value to be used as the density of the microvessels.

1.8 Ki67 immunohistochemical staining and determination of Ki67 index

The primary antibody was diluted 1:1000 with rabbit anti-Ki 67 polyclonal antibody. The other processes are similar to those described above. Positive pairAccording to the following steps: positive control with known positive lymph node tissue, localized to the nucleus; negative control: replacing primary antibody with normal dog serum, and obtaining negative result; blank control: the primary antibody was replaced with PBS, and the result was negative. At 20 × (about 37 mm)²) In the hypo-visual field, the area of the section tissue with the highest positive expression (hot spot area) was examined, and the area was 400X visual field (about 0.1 mm)²) The hot spot area counted all cells and Ki67 stained positive brown cells. The Ki67 index is described by the percentage number of positive nuclei.

2. Results of the experiment

2.1 general examination results

The test animals were subjected to 8 consecutive weeks of pathological observation. Experiment 20 experimental animals except 1 animal which is killed by anesthesia accidentally in the observation process, the general conditions of other animals after operation are good, diet, weight, body temperature, respiration and defecation are all maintained in a normal range, all wound surfaces are not necrotic, and the healing progress is smooth. The change of the wound area at each observation point is shown in Table 5.

The wound healing rate was analyzed at each observation time point according to the data of table 5 and formula 1, and the results are shown in fig. 1. The healing rate of the full-layer skin defect test groups is 24.25 +/-24.7 cm after 1 to 5 weeks of operation²，74.97±18.37cm²，94.62±5.90cm²，100±0.00cm²(ii) a The control groups were 24.27. + -. 24.3cm, respectively²，68.77±14.15cm²，78.03±15.18cm²，100±0.00cm²。

TABLE 5 postoperative Change in area of skin wound at 1-5 weeks (cm)²)

It should be noted that, starting from week 5 after surgery, the wound surface of the full-thickness skin defect of the chest or the abdominal wall is treated with epidermal healing as shown in fig. 2. After all the wound surfaces of the chest and most of the wound surfaces of the abdominal wall are healed, a structure shown by arrows in figure 2 appears, the structure is wound on the chest and is vertical to the longitudinal axis of the spine, and the wound surfaces of the abdominal wall are parallel to the longitudinal axis of the spine. Obtaining pathological samples according to the initial defect area (4 multiplied by 6cm) of each wound surface at each observation time point; pathological sample sections were made according to the dotted line shown in FIG. 2, and the length was controlled to 2-3cm for easy sample handling.

2.2 HE and Masson pathological section analysis

2.2.1 specificity of tissue sections

The 1-week and 2-week neogenetic tissues have clear boundaries with the original surrounding normal tissues and are easily distinguished. 5 weeks and 8 weeks, due to normal tissue contraction, fusion with the neogenetic tissue, needs to be identified by careful histological characterization. FIG. 3 shows a comparison of normal and neogenetic tissue at week 5 in the test group (right) and the control group (left).

2.2.2 cellular tissue response grading

Biological evaluation according to ISO10993-6 medical devices-part 6: the evaluation of cellular and tissue responses was performed using a semi-quantitative scoring protocol as described in the test of local effects after implantation. The degree of neovascularization, fibrosis and fat infiltration is analyzed mainly by counting the cells such as multinucleated granulocytes, lymphocytes, plasma cells, macrophages and giant cells of epidermis, dermis and hypodermis of the test group and the control group after 1-8 weeks of operation, and then the degree is respectively scored according to tables 2 and 3. Referring to table 4, the difference between the scores of the paired test group and the control group was subjected to grading of cell tissue response.

After 1 week of operation, the superficial layer and the dermal layer cellular tissue reaction scores are 23 and 17 respectively, and are more than 15.1, and the serious reaction is obtained; the superficial and dermal layers at week 2 were 8.6 and 13.6, respectively, for moderate response; 3.4 in the dermis at week 5, judged as mild response; in addition, the reaction was classified into a minimum reaction or no reaction in the range of 0.0 to 2.9. The hypodermis was the reaction grade.

2.2.3 skin attachment Hair follicle

From week 5, the tissue sections were examined to find the appearance of hair follicle tissue in the neodermal tissue (see right side of FIG. 3). It may be a young cell mass, or may be expressed as a hair follicle tissue in the differentiation process, in which connective tissue hair follicles, hair papillae, outer root sheath, inner root sheath, hair bulbs, and the like appear. Figure 4 shows the mature hair follicle appearing at week 8 and essentially characterized by a large number of connective tissue cells in the surrounding dermal tissue, an abundant network of capillary vessels, Masson-stained collagen fibers that are significantly finer than normal tissue, appearing reddish-blue. In this experiment, hair follicle tissue was found to be in the differentiation process in the neodermis at 5 weeks 50% (5/10) and at 8 weeks 100% (8/8) in the test group, while substantially none was found in the control group (0/18).

2.3 immunological staining and analysis

2.3.1 staining with CD31

As can be seen from FIG. 5, the skin microvascular density of the control group (23.7 + -6.6) was higher than that of the test group (20.2 + -7.0P <0.05>0.01) at 1 week after the operation, and was higher than that of the control group (24.8 + -6.5, 25.2 + -8.2 and 27.7 + -11.3 at 2 weeks, 4 weeks and 8 weeks after the operation), which were all higher than that of the control group (13.5 + -2.2, 11.2 + -5.6 and 11.2 + -5.4 at 13.5 + -2, 11.2 + -5.6 and 11.2 + -5.4 (P <0.01, the difference was significant).

2.3.2 Ki67 staining

As can be seen from fig. 6, after Ki67 staining, the results of correlation analysis calculation show that the Ki67 index (0.29 ± 0.10) in the control group is higher than that in the test group (0.14 ± 0.06), and the Ki67 index in the test group is higher than that in the test group (0.45 ± 0.14, 0.37 ± 0.07 and 0.34 ± 0.15, and the Ki67 index in the test group is higher than that in the control group (0.19 ± 0.10, 0.21 ± 0.12 and 0.28 ± 0.08, respectively) (P <0.01, the difference is very significant) at 1 week after operation.

3. Results and discussion

As can be seen from the above experimental examples, the hydrophilic electrospun implant of the present invention can induce the regeneration of skin tissues (including but not limited to skin appendages such as hair follicles, sebaceous glands, and sweat glands) by being fixed to a skin wound surface.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. An implant for inducing skin tissue regeneration, which is characterized in that the implant has a three-dimensional nano-net structure inside and is prepared from raw materials comprising a fibrinogen compound and a polylactic acid-polycaprolactone copolymer.

2. An implant as claimed in claim 1, characterised in that the fibrinogen complex comprises the following components in parts by weight: 0.1-20 parts of fibrinogen, 0.1-10 parts of arginine hydrochloride, 0.01-10 parts of sodium chloride and 1-10 parts of sodium citrate;

preferably, the fibrinogen complex comprises the following components in parts by weight: 3-15 parts of fibrinogen, 0.5-5 parts of arginine hydrochloride, 0.3-5 parts of sodium chloride and 1-10 parts of sodium citrate;

more preferably, the fibrinogen is porcine blood-derived fibrinogen.

3. The implant of claim 1, wherein the polylactic acid polycaprolactone copolymer has a molecular weight of 5 to 30 ten thousand.

4. The implant of claim 1, wherein the mass ratio of the fibrinogen complex to the polylactic acid-polycaprolactone copolymer is 0.48-1.1: 1.

5. The implant of claim 1, wherein the implant is in the form of a membrane;

preferably, the thickness of the implant is 0.51mm to 1.4mm, more preferably 0.6mm to 0.8 mm;

and/or the protein content of the implant is 100-220 mg/g;

and/or the implant has <12mg/g residual protein;

and/or the tensile strength of the implant is 0.5-5 MPa, preferably 1-4 MPa;

and/or the elongation at break of the implant is 60-200%, preferably 70-160%;

and/or the porosity of the implant is 41-80%, preferably 45-70%;

and/or the water absorption rate of the implant is 100-250%, preferably 100-160%.

6. The implant of claim 1, wherein the implant is granular;

preferably, the diameter of the implant is 0.06-0.2mm, preferably 0.07-0.1 mm;

and/or the protein content of the implant is 100-220 mg/g;

and/or the implant has <12mg/g residual protein;

and/or the porosity of the implant is 41-80%, preferably 45-70%;

and/or the water absorption of the implant is 100-250%, preferably 100-180%.

7. An implant according to any of claims 1 to 6, wherein the implant is produced by electrospinning.

8. The implant according to claim 7, wherein the implant is prepared by uniformly mixing a solution containing the fibrinogen complex and a solution containing the polylactic acid-polycaprolactone copolymer, and then adding the mixture into the same volumetric tube of an electrospinning machine for electrospinning; or respectively adding the solution containing the fibrinogen compound and the solution containing the polylactic acid-polycaprolactone copolymer into two different volumetric tubes of an electrostatic spinning machine, and simultaneously carrying out electrostatic spinning;

preferably, the solution containing the polylactic acid-polycaprolactone copolymer is prepared by dissolving the polylactic acid-polycaprolactone copolymer in a mixed solvent of one or more than two of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran and acetone at a mass volume percentage concentration of 5-8%;

preferably, the solution containing fibrinogen complex is prepared by dissolving fibrinogen complex in distilled water at a mass volume percentage concentration of 8.0% to 29.0%.

9. Method for implanting an implant according to any of claims 1 to 8, characterized in that it comprises the following steps: the implant is applied to the desired site by needle injection or painting.

10. Use of an implant according to any one of claims 1 to 8 in the preparation of a material for repairing a defect in a body tissue.

11. Use according to claim 10, wherein the material is a repair material for the treatment of meninges, abdominal defects, pelvic floor organ prolapse, atria, ventricular septum, pericardial defects, tendon or ligament rupture, or parenchymal organ rupture.

12. Use of an implant according to any one of claims 1 to 8 in the manufacture of a medicament or material for dermal wound or cosmetic surgery;

preferably, the cutaneous wound is a chronic wound or a non-healing wound;

more preferably, the skin wound comprises an abrasion wound, a puncture wound, a penetration wound, a gunshot wound, an explosive wound, an incision, a laceration wound, a avulsion wound, a surgical wound, an electrocautery wound, a radioactive wound, a chemical wound, a frozen wound, and a burn wound;

more preferably, the cosmetic makeup includes skin makeup and micro-makeup.

13. The application of the hydrophilic electrostatic spinning biological composite scaffold material in preparing medicines or materials for skin wound or plastic and beauty is characterized in that the composite scaffold material is prepared by blending an aqueous solution of fibrinogen, L-arginine or hydrochloride thereof and a P (LLA-CL) solution and adopting an electrostatic spinning technology; wherein the mass ratio of the fibrinogen to the L-arginine or the hydrochloride thereof is 1.2: 1-12.5: 1;

the fibrinogen and L-arginine or hydrochloride aqueous solution thereof, wherein the solvent is selected from one or more of pure water, water for injection, salt solution and buffer solution; the salt solution is selected from sodium chloride solution and potassium chloride solution; the buffer solution is selected from phosphate buffer solution, Tris-HCl buffer solution, glycine buffer solution and D-Hank's solution.

14. Use according to claim 13, wherein the cutaneous wound is a chronic wound or a non-healing wound;

preferably, the skin wound surface comprises an abrasion wound, a puncture wound, a penetration wound, a gunshot wound, an explosion wound, an incision, a laceration wound, a avulsion wound, a surgical wound, an electrocautery wound, a radioactive wound, a chemical wound, a freezing wound and a burn wound.

15. Use according to claim 13, characterized in that the cosmetic makeup comprises dermocosmesis and micro-plastic.

16. Use according to any one of claims 13 to 15, wherein the fibrinogen is of mammalian origin.

17. The use according to claim 16, wherein the mammal is a human, pig, cow, sheep or horse.

18. Use according to any one of claims 13 to 15, wherein the mass ratio of polylactic acid to polycaprolactone in P (LLA-CL) is 20:80 to 95: 5.

19. Use according to any one of claims 13-15, wherein the solvent in the P (LLA-CL) solution is selected from one or more of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran, chloroform or acetone.

20. The use according to any one of claims 13 to 15, wherein the mass ratio of fibrinogen to P (LLA-CL) is 0.2:1 to 2.1:1 after blending the aqueous solution of fibrinogen, L-arginine or its hydrochloride with the solution of P (LLA-CL).

21. Use according to any one of claims 13 to 15, wherein the hydrophilic electrospun biocomposite scaffold material has an equilibrium contact angle of less than 55 °.

22. The use of any one of claims 13-15, wherein the hydrophilic electrospun biocomposite scaffold material has a total volume shrinkage after contact with an aqueous solution of no more than 20%; the porosity is not less than 30%.

23. Use according to any one of claims 13 to 15, wherein the aqueous solution of fibrinogen, L-arginine or its hydrochloride is further loaded with an antibacterial substance selected from one or more of penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, macrolides, glycosides, sulfonamides, quinolones, nitroimidazoles, lincomamines, fosfomycin, chloramphenicol, colistin B, bacitracin.

24. Use according to claim 23, wherein the penicillins are selected from the group consisting of penicillin, ampicillin, carbenicillin; the cephalosporins are selected from cefalexin, cefuroxime sodium, ceftriaxone and cefpirome; the carbapenem is thiomycin; the aminoglycoside is selected from gentamicin, streptomycin, and kanamycin; the tetracycline is selected from tetracycline and chlortetracycline; the macrolides are selected from erythromycin and azithromycin; the glucoside is vancomycin; the sulfonamides are selected from sulfadiazine and trimethoprim; the quinolone is selected from the group consisting of pipemidic acid and ciprofloxacin; the nitroimidazoles are selected from metronidazole and tinidazole; the lincomycin is selected from lincomycin and clindamycin.

25. Use according to claim 23, wherein the amount of said antibacterial substance released is not less than 30% of the total loading within 15 minutes after implantation of the scaffold material in the body.