Gradient biomimetic scaffold for guided oral soft and hard tissue combined restoration

Abstract

The present application is based on the anatomical features of oral periodontal alveolar bone tissue and the regeneration speed difference of soft and hard tissues, and provides a method for preparing a gradient biomimetic scaffold including a soft tissue layer, a barrier layer and a hard tissue layer by using coaxial electrospinning technology. The gradient biomimetic scaffold exhibits good mechanical properties and biocompatibility. Among them, the gradient distribution of the soft tissue layer can induce the adhesion and regeneration of fibroblasts, the barrier layer at the interface can prevent fibroblasts from invading the osteogenic layer, and the hard tissue layer can induce the differentiation and regeneration of osteoblasts. The gradient biomimetic scaffold prepared by the present application can be used as an excellent guided oral soft and hard tissue combined repair material, and has a broad application prospect in oral medicine.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of oral implantology, and particularly relates to a gradient biomimetic scaffold for guiding combined repair of oral soft and hard tissues. BACKGROUND

[0002] Guided bone regeneration (GBR) is an important medical technology for solving bone tissue defects. It spatially isolates soft tissue from the bone repair site, prevents connective tissue cells from invading, and ensures that the osteogenesis process is not disturbed, which has significant clinical significance for periodontal treatment and oral implantation. The existing GBR barrier membrane still has many limitations: uncontrollable in vivo degradation period, lack of osteogenic active factors, lack of antibacterial performance and inflammation risk, etc. Therefore, there is an urgent need for a gradient biomimetic scaffold for guiding combined repair of oral soft and hard tissues. SUMMARY

[0003] The application provides a gradient biomimetic scaffold, which comprises, from top to bottom, a soft tissue layer, a barrier layer and a hard tissue layer.

[0004] According to the embodiment of the application, the pore size distribution of each layer in the gradient biomimetic scaffold is: soft tissue layer > hard tissue layer > barrier layer.

[0005] According to the embodiment of the application, the soft tissue layer is gradient distributed. According to the embodiment of the application, the soft tissue layer is a loose porous layer. According to the embodiment of the application, the pore size of the soft tissue layer gradually decreases from outside to inside, and is gradient distributed.

[0006] According to the embodiment of the application, the barrier layer is a dense layer. The barrier layer can prevent fibroblasts from invading the osteogenic layer. The barrier layer can act as a barrier for soft tissue and provide a stable microenvironment for bone regeneration.

[0007] According to embodiments of the present application, the pore size of the soft tissue layer of the gradient biomimetic scaffold is 0.4 pm - 2.2 pm; preferably, the pore size of the soft tissue layer is 0.5 pm - 2.0 pm; for example 0.55 pm, 0.59 pm, 0.60 pm, 0.63 pm, 0.70 pm, 0.80 pm, 0.90 pm, 1.00 pm, 1.30 pm, 1.50 pm, 1.80 pm, 2.00 pm. According to embodiments of the present application, the soft tissue layer comprises a soft tissue layer outer layer, a soft tissue layer middle layer, a soft tissue layer inner layer. According to embodiments of the present application, the pore size distribution is: soft tissue layer outer layer > soft tissue layer middle layer > soft tissue layer inner layer. According to embodiments of the present application, the pore size of the soft tissue layer outer layer is 1.0 pm - 2.2 pm; preferably, the pore size of the soft tissue layer outer layer is 1.2 pm - 2.0 pm; for example 1.20 pm, 1.30 pm, 1.40 pm, 1.50 pm, 1.60 pm, 1.70 pm, 1.80 pm, 1.90 pm, 2.00 pm. According to embodiments of the present application, the pore size of the soft tissue layer middle layer is 0.5 pm - 1.0 pm; for example 0.60 pm, 0.63 pm, 0.65 pm, 0.68 pm, 0.70 pm, 0.75 pm, 0.80 pm, 0.90 pm. According to embodiments of the present application, the pore size of the soft tissue layer inner layer is 0.4 pm - 0.7 pm; for example 0.45 pm, 0.50 pm, 0.55 pm, 0.60 pm, 0.63 pm, 0.65 pm, 0.70 pm.

[0008] According to embodiments of the present application, the pore size of the barrier layer is 0.3 pm - 0.7 pm; for example 0.4 pm, 0.45 pm, 0.50 pm, 0.52 pm, 0.55 pm, 0.60 pm.

[0009] According to embodiments of the present application, the pore size of the hard tissue layer is 0.3 pm - 0.8 pm. According to embodiments of the present application, the hard tissue layer comprises a hard tissue layer outer layer, a hard tissue layer inner layer. According to embodiments of the present application, the pore size distribution is: hard tissue layer outer layer > hard tissue layer inner layer. According to embodiments of the present application, the pore size of the hard tissue layer outer layer is 0.4 pm - 0.8 pm, for example 0.45 pm, 0.50 pm, 0.53 pm, 0.54 pm, 0.55 pm, 0.60 pm, 0.65 pm, 0.70 pm. According to embodiments of the present application, the pore size of the hard tissue layer inner layer is 0.3 pm - 0.7 pm, for example 0.35 pm, 0.4 pm, 0.45 pm, 0.50 pm, 0.53 pm, 0.54 pm, 0.55 pm, 0.60 pm, 0.65 pm.

[0010] According to embodiments of the present application, the thickness of the soft tissue layer, the barrier layer, the hard tissue layer is 5 - 50 pm.

[0011] According to an embodiment of the present application, the surface structure of the gradient biomimetic scaffold comprises directional parallel shape, disordered cross shape, cross shape or radial shape.

[0012] According to an embodiment of the present application, the soft tissue layer contains an active factor capable of inducing fibroblast adhesion regeneration; preferably, any one of VEGF, PDGF. According to an embodiment of the present application, the soft tissue layer adopts the high molecular material-1; preferably, the high molecular material-1 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, methyl cellulose. According to an embodiment of the present application, the high molecular material-1 is selected from polylactic acid and collagen; preferably, the mass ratio of polylactic acid and collagen is 5-20:1, for example, 8:1, 10:1, 13:1, 15:1.

[0013] According to an embodiment of the present application, the barrier layer adopts the high molecular material-2; preferably, the high molecular material-2 is selected from any one, two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, methyl cellulose. According to an embodiment of the present application, the high molecular material-2 is selected from polylactic acid.

[0014] According to an embodiment of the present application, the hard tissue layer contains an active factor capable of inducing osteogenic differentiation of cells; preferably, any one of BMP-2, BMP-5, BMP-7. According to an embodiment of the present application, the hard tissue layer further contains an active factor capable of inducing fibroblast adhesion regeneration; preferably, any one of VEGF, PDGF. According to an embodiment of the present application, the hard tissue layer adopts the high molecular material-3; preferably, the high molecular material-3 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, methyl cellulose. According to an embodiment of the present application, the high molecular material-3 is selected from polylactic acid and collagen; preferably, the mass ratio of polylactic acid and collagen is 5-20:1, for example, 8:1, 10:1, 13:1, 15:1.

[0015] According to an embodiment of the present application, the soft tissue layer, the barrier layer, the hard tissue layer are prepared by electrospinning.

[0016] The present application also provides a preparation method of the above-mentioned gradient biomimetic scaffold, which comprises the following steps:

[0017] S1: preparing a soft tissue layer electrospinning solution, a barrier layer electrospinning solution, a hard tissue layer electrospinning solution;

[0018] S2: electrospinning the spinning solution in step S1 layer by layer in the order of soft tissue layer, barrier layer and hard tissue layer, to obtain the gradient biomimetic scaffold.

[0019] According to the embodiment of the present application, the parameter settings of electrospinning include: voltage of 12-40kV, flow rate of 1-3mL / h, and rotation speed of 200-2000r / min. According to the embodiment of the present application, the soft tissue layer and the hard tissue layer are coaxially electrospun.

[0020] According to the embodiment of the present application, the specific operation parameters of electrospinning the soft tissue layer are as follows: coaxial electrospinning is performed using a 20-gauge coaxial needle, the distance between the needle and the receiver is 20cm, the spinning voltage is 20kV, the spinning time is 1h, and the rotation speed of the drum is increased from 200r / min to 2000r / min.

[0021] Preferably, the shell layer has a pushing speed of 1mL / h.

[0022] Preferably, the core layer has a pushing speed of 0.5mL / h.

[0023] Preferably, the rotation speed of the drum is increased according to 200r / min, 500r / min, 1000r / min and 2000r / min.

[0024] According to the embodiment of the present application, the specific operation parameters of electrospinning the barrier layer are as follows:

[0025] A 20-gauge needle is used, the distance between the needle and the receiver is 20cm, the drum wrapped with aluminum foil is used as the receiving electrode, the pushing speed is 1mL / h, the rotation speed of the drum is 3000r / min, the spinning voltage is 20kV, and the spinning time is 1h.

[0026] According to the embodiment of the present application, the specific operation parameters of electrospinning the hard tissue layer are as follows:

[0027] Coaxial electrospinning is performed using a 20 / 15-gauge coaxial needle, the distance between the needle and the receiver is 20cm, the rotation speed of the drum is 200r / min, the spinning voltage is 20kV, and the spinning time is 1h.

[0028] Preferably, the shell layer has a pushing speed of 1mL / h.

[0029] Preferably, the core layer has a pushing speed of 0.5mL / h.

[0030] According to the embodiment of the present application, the temperature control during electrospinning is 26±3℃, and the humidity is 35±5%.

[0031] According to the embodiment of the present application, after the electrospinning is completed, the fiber membrane is peeled off from the aluminum foil, and is ventilated in a fume hood for 48h to remove the residual solvent, to obtain the gradient biomimetic scaffold.

[0032] According to an embodiment of the present application, the average diameter of the nanofibers of the gradient biomimetic scaffold formed is 5-80 nm; for example, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm.

[0033] According to an embodiment of the present application, the soft tissue layer is prepared by electrospinning of a soft tissue layer electrospinning solution.

[0034] According to an embodiment of the present application, the soft tissue layer electrospinning solution is prepared by dissolving the polymer material-1 and the surfactant-1 in the solvent-1, and then adding the active factor-1 solution, and stirring to obtain the soft tissue layer electrospinning solution.

[0035] According to an embodiment of the present application, the concentration of the polymer material-1 is 0.04-1 g / mL; preferably 0.05-0.5 g / mL; for example, 0.06 g / mL, 0.08 g / mL, 0.10 g / mL, 0.15 g / mL, 0.2 g / mL, 0.25 g / mL, 0.3 g / mL, 0.35 g / mL, 0.4 g / mL, 0.45 g / mL, 0.5 g / mL.

[0036] According to an embodiment of the present application, the concentration of the surfactant-1 is 1-20 μg / mL; for example, 5 μg / mL, 6 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 13 μg / mL, 15 μg / mL.

[0037] According to an embodiment of the present application, the surfactant-1 is selected from non-ionic surfactants, and is preferably a Span, for example, Span 80.

[0038] According to an embodiment of the present application, the active factor-1 is an active factor that can induce adhesion and regeneration of fibroblasts; and is preferably any one of VEGF and PDGF. According to an embodiment of the present application, the active factor-1 is VEGF. According to an embodiment of the present application, the concentration of the active factor-1 is 25-1000 ng / mL, and is preferably 50-500 ng / mL; for example, 100 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL.

[0039] According to an embodiment of the present application, the active factor-1 solution is a solution formed by dissolving the active factor in a bovine serum albumin-PBS solution (for example, 2% BSA-PBS).

[0040] According to an embodiment of the present application, the solvent-1 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N, N dimethylformamide, N, N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethylsulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid; preferably hexafluoroisopropanol.

[0041] According to an embodiment of the present application, the barrier layer is prepared by electrospinning of a barrier layer electrospinning solution.

[0042] According to an embodiment of the present application, the barrier layer electrospinning solution is prepared by dissolving the high molecular material-2 in the solvent-2, and stirring to obtain the barrier layer electrospinning solution.

[0043] According to an embodiment of the present application, the high molecular material-2 is selected from one, two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, methyl cellulose; preferably polylactic acid.

[0044] According to an embodiment of the present application, the concentration of the high molecular material-2 in the electrospinning solution is 0.04 g / mL to 1 g / mL; preferably 0.05 g / mL to 0.5 g / mL; for example 0.06 g / mL, 0.08 g / mL, 0.10 g / mL, 0.15 g / mL, 0.2 g / mL, 0.25 g / mL, 0.3 g / mL, 0.35 g / mL, 0.4 g / mL, 0.45 g / mL, 0.5 g / mL.

[0045] According to an embodiment of the present application, the solvent-2 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N, N dimethylformamide, N, N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethylsulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid; preferably hexafluoroisopropanol.

[0046] According to an embodiment of the present application, the hard tissue layer electrospinning solution comprises a core layer electrospinning solution and a shell layer electrospinning solution.

[0047] According to an embodiment of the present application, the hard tissue layer core layer electrospinning solution is prepared by dissolving the high molecular material-3 and the surfactant-2 in the solvent-3, and then adding the active factor-2 solution, and stirring to obtain the core layer electrospinning solution.

[0048] According to an embodiment of the present application, the high molecular material-3 is selected from any two or more of collagen, gelatin, chitosan, fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, methyl cellulose. According to an embodiment of the present application, the high molecular material-3 is selected from polylactic acid and collagen; preferably, the mass ratio of polylactic acid and collagen is 5-20: 1, for example 10: 1.

[0049] According to an embodiment of the present application, the concentration of the high molecular material-3 is 0.04 g / mL-1 g / mL; preferably 0.05 g / mL-0.5 g / mL; for example 0.06 g / mL, 0.08 g / mL, 0.10 g / mL, 0.15 g / mL, 0.2 g / mL, 0.25 g / mL, 0.3 g / mL, 0.35 g / mL, 0.4 g / mL, 0.45 g / mL, 0.5 g / mL.

[0050] According to an embodiment of the present application, the concentration of the surfactant-2 is 1 μg / mL-20 μg / mL, for example 5 μg / mL, 6 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 13 μg / mL, 15 μg / mL.

[0051] According to an embodiment of the present application, the surfactant-2 is selected from non-ionic surfactants, preferably Span, for example Span 80.

[0052] According to an embodiment of the present application, the active factor-2 is a cell osteogenic differentiation reactivation active factor; preferably any one of BMP-2, BMP-5, BMP-7. According to an embodiment of the present application, the active factor-2 is BMP-2.

[0053] According to an embodiment of the present application, the concentration of the active factor-2 is 25-1000 ng / mL, preferably 50 ng / mL-500 ng / mL, for example 100 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL.

[0054] According to an embodiment of the present application, the active factor-2 solution is a solution formed by dissolving the active factor in a bovine serum albumin-PBS solution (2% BSA-PBS). According to an embodiment of the present application, the active factor-2 is loaded on hydroxyapatite; preferably, 1 microgram is loaded on 25 mg of hydroxyapatite.

[0055] According to an embodiment of the present application, the solvent-3 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N, N dimethylformamide, N, N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethylsulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid; preferably hexafluoroisopropanol.

[0056] According to an embodiment of the present application, the preparation process of the hard tissue layer shell layer electrospinning solution is as follows:

[0057] The polymer material-4 and the surfactant-3 are dissolved in the solvent-4, and then the active factor-3 solution is added, and the shell layer electrospinning solution is obtained after uniform stirring.

[0058] According to an embodiment of the present application, the polymer material-4 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. According to an embodiment of the present application, the polymer material-4 is selected from polylactic acid and collagen; preferably, the mass ratio of polylactic acid and collagen is 5-20:1, for example, 10:1.

[0059] According to an embodiment of the present application, the concentration of the polymer material-4 is 0.04 g / mL-1 g / mL; preferably, 0.05 g / mL-0.5 g / mL; for example, 0.06 g / mL, 0.08 g / mL, 0.10 g / mL, 0.15 g / mL, 0.2 g / mL, 0.25 g / mL, 0.3 g / mL, 0.35 g / mL, 0.4 g / mL, 0.45 g / mL, 0.5 g / mL.

[0060] According to an embodiment of the present application, the concentration of the surfactant-3 is 1 μg / mL-20 μg / mL, for example, 5 μg / mL, 6 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 13 μg / mL, 15 μg / mL.

[0061] According to an embodiment of the present application, the surfactant-3 is selected from a non-ionic surfactant, preferably a Span, for example, Span 80.

[0062] According to an embodiment of the present application, the active factor-3 is an active factor that can induce fibroblast adhesion regeneration; preferably, any one of VEGF and PDGF. According to an embodiment of the present application, the active factor-3 is VEGF.

[0063] According to an embodiment of the present application, the concentration of the active factor-3 is 25-1000 ng / mL, preferably 50 ng / mL-500 ng / mL, for example 100 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL.

[0064] According to an embodiment of the present application, the active factor-3 solution is a solution formed by dissolving the active factor in a bovine serum albumin-PBS solution (for example 2% BSA-PBS).

[0065] According to an embodiment of the present application, the solvent-4 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N,N dimethylformamide, N,N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethyl sulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid; preferably hexafluoroisopropanol.

[0066] The present application also provides a gradient biomimetic scaffold prepared by the above preparation method.

[0067] The present application also provides the use of the above preparation method and the above gradient biomimetic scaffold in oral tissue engineering.

[0068] Advantages

[0069] The present application is based on the anatomical characteristics of oral periodontal alveolar bone tissue and the difference in regeneration speed of soft and hard tissues, and provides a method for preparing a gradient biomimetic scaffold including a soft tissue layer, a barrier layer and a hard tissue layer by using coaxial electrospinning technology. The gradient biomimetic scaffold exhibits good mechanical properties and biocompatibility. Among them, the gradient distribution of the soft tissue layer can induce the adhesion and regeneration of fibroblasts, the barrier layer at the interface can prevent fibroblasts from invading the osteogenic layer, and the hard tissue layer can induce the differentiation and regeneration of osteoblasts. The gradient biomimetic scaffold prepared by the present application can be used as an excellent guided oral soft and hard tissue combined repair material, and has a wide application prospect in oral medicine.

[0070] Compared with the commonly used GBR membrane, the gradient biomimetic scaffold prepared by using coaxial electrospinning technology and by controlling the spinning stock solution and spinning parameters has a biomimetic nano-morphology and a slow-release effect of active factors in the loose porous layer, which can significantly promote tissue regeneration; the barrier layer can play a barrier role for soft tissue and provide a stable microenvironment for bone regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1: Gradient biomimetic scaffold design: based on the anatomical features of oral tissue and the different regeneration rates of soft and hard tissues, a gradient biomimetic scaffold is constructed.

[0072] Figure 2 : Gradient biomimetic scaffold: the pore size of the soft tissue layer gradually decreases from outside to inside, and the pore size of the barrier layer is the smallest; the pore size of the hard tissue layer is smaller than that of the soft tissue layer and larger than that of the barrier layer.

[0073] Figure 3 : The pore size of each layer of the gradient biomimetic scaffold.

[0074] Figure 4 : After combining the soft tissue layer and the hard tissue layer, the mechanical strength is improved;

[0075] Figure 5 : Cell proliferation: compared with the control group, the composite film has no obvious killing effect on cells and has good cell compatibility.

[0076] Figure 6 : Cell penetration: the soft tissue layer can induce fibroblast penetration, and the barrier layer can effectively organize its invasion into the hard tissue layer.

[0077] Figure 7 : The gradient biomimetic scaffold can promote blood vessel regeneration.

[0078] Figure 8 : The gradient biomimetic scaffold can promote bone regeneration. DETAILED DESCRIPTION

[0079] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of the present application.

[0080] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0081] PLA: polylactic acid, purchased from NatureWorks, USA.

[0082] COL: collagen, purchased from Platinum Biotechnology (Shanghai) Co., Ltd.

[0083] HFIP: hexafluoroisopropanol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0084] Span80: Span80, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0085] VEGF: Vascular endothelial growth factor, purchased from PEPROTECH.

[0086] BSA-PBS solution: Bovine serum albumin-PBS solution, purchased from Beijing Gypsy Biotechnology Co., Ltd.

[0087] BMP-2: Human bone morphogenetic protein-2, purchased from PEPROTECH.

[0088] Fibroblast: purchased from Shanghai Cell Bank of Chinese Academy of Sciences.

[0089] Vascular endothelial cell: purchased from Shanghai Cell Bank of Chinese Academy of Sciences.

[0090] Osteoblast: purchased from Shanghai Cell Bank of Chinese Academy of Sciences.

[0091] Example 1

[0092] Preparation of soft tissue layer: 0.5 g of PLA and 0.05 g of COL were weighed and added to 5 mL of HFIP solution, and then 50 microliters of Span80 were added, and magnetic stirring was performed until a uniform solution was formed. 1 microgram of VEGF was dissolved in 500 microliters of 2% BSA-PBS solution, and then added to the above solution. The solution was stirred on a magnetic stirrer for 2 h to obtain a soft tissue layer electrospinning solution.

[0093] Preparation of barrier layer: 1 g of PLA was added to 10 mL of HFIP, and magnetic stirring was performed until the solution was clear, serving as a spinning solution for the barrier layer.

[0094] Preparation of hard tissue layer: 0.5 g of PLA and 0.05 g of COL were weighed and added to 5 mL of HFIP solution, and then 50 microliters of Span80 were added, and magnetic stirring was performed until a uniform solution was formed. 25 mg of BMP-2-loaded hydroxyapatite (1 microgram) was dispersed in 500 microliters of 2% BSA-PBS solution, and then added to the above solution. The solution was stirred on a magnetic stirrer for 2 h and ultrasonicated for 20 min to obtain a core layer electrospinning solution for the hard tissue layer. 0.5 g of PLA and 0.05 g of COL were weighed and added to 5 mL of HFIP solution, and then 50 microliters of Span80 were added, and magnetic stirring was performed until a uniform solution was formed. 1 microgram of VEGF was dissolved in 500 microliters of 2% BSA-PBS solution, and then added to the above solution. The solution was stirred on a magnetic stirrer for 2 h to obtain a shell layer electrospinning solution for the hard tissue layer.

[0095] Preparation of gradient biomimetic scaffold: The above spinning solutions were taken up with a 10 mL syringe, and then the syringe was placed on a push pump. The spinning solutions of different layers were electrospun layer by layer in the order of soft tissue layer, barrier layer, and hard tissue layer to obtain a gradient biomimetic scaffold (as shown in Figures 1-2 ).

[0096] (1) For the soft tissue layer, coaxial electrospinning was performed using a 20-gauge coaxial needle, the distance between the needle and the receiver was 20 cm, the shell layer pushing speed was 1 mL / h, the core layer pushing speed was 0.5 mL / h, the rotating drum rotating speed was increased according to 200, 500, 1000, 2000 r / min (each rotating speed was maintained for 15 min), the spinning voltage was 20 kV, and the spinning time was 1 h;

[0097] (2) For the barrier layer, a 20-gauge needle was used, the distance between the needle and the receiver was 20 cm, the rotating drum wrapped with aluminum foil was used as the receiving electrode, the injector pushing speed was 1 mL / h, the rotating drum rotating speed was 3000 r / min, the spinning voltage was 20 kV, and the spinning time was 1 h;

[0098] (3) For the hard tissue layer, coaxial electrospinning was performed using a 20 / 15-gauge coaxial needle, the distance between the needle and the receiver was 20 cm, the shell layer pushing speed was 1 mL / h, the core layer pushing speed was 0.5 mL / h, the rotating drum rotating speed was 200 r / min, the spinning voltage was 20 kV, and the spinning time was 1 h.

[0099] During the spinning process, the temperature was controlled at 26±3℃, and the humidity was 35±5%. After the spinning was completed, the fiber membrane was removed from the aluminum foil, ventilated in a fume hood for 48 h to remove the residual solvent, and the final gradient biomimetic scaffold was obtained.

[0100] The pore diameters of the layers of the gradient biomimetic scaffold prepared were detected by scanning electron microscopy, and the results are shown in Figure 3 . Among them, the pore diameter of the soft tissue layer gradually decreases from outside to inside, and the pore diameter of the barrier layer (isolation layer) is the smallest; the pore diameter of the hard tissue layer is smaller than that of the soft tissue layer and larger than that of the barrier layer.

[0101] Example 2

[0102] The mechanical strength of the gradient biomimetic scaffold (composite layer) prepared in Comparative Example 1 was compared with that of the soft tissue layer and the hard tissue layer, and the experimental method was as follows:

[0103] 1. The soft tissue layer, the hard tissue layer, and the composite scaffold were loaded on the universal mechanical testing machine, respectively;

[0104] 2. According to the setting program of the machine, stress test was performed until the scaffold was broken;

[0105] 3. The data were recorded and analyzed.

[0106] The experimental results are shown in Figure 4 . As can be seen from Figure 4 , the mechanical strength (including tensile stress and tensile strain) of the gradient biomimetic scaffold (composite layer) prepared in the application is significantly improved.

[0107] Example 3

[0108] The cell compatibility of the gradient biomimetic scaffold (composite layer) prepared in Research Example 1 was investigated, and the experimental method was as follows:

[0109] 1. Fibroblasts were inoculated into the soft tissue layer of the gradient biomimetic scaffold, and incubated at 37°C, with liquid changed every 2 days;

[0110] 2. Live cell staining was performed at 1, 4, and 7 days of culture, respectively;

[0111] 3. The proliferation of fibroblasts was observed by confocal microscopy.

[0112] The experimental results are shown in Table 1. Figure 5 As can be seen from Table 1, compared with the blank control group (culture medium without material), the gradient biomimetic scaffold (composite layer) prepared in the application had no obvious killing effect on L929 (fibroblasts), and had good cell compatibility. Figure 5 Example 4

[0113] The proliferation and penetration of fibroblasts on the gradient biomimetic scaffold were observed and detected, and the specific experimental process was as follows:

[0114] 1. Fibroblasts were inoculated into the soft tissue layer of the gradient biomimetic scaffold, and incubated at 37°C, with liquid changed every 2 days;

[0115] 2. Live cell staining was performed at 1, 4, and 7 days of culture, respectively;

[0116] 3. The proliferation and penetration of fibroblasts were observed by confocal microscopy.

[0117] The results are shown in Table 2.

[0118] As can be seen from Table 2, fibroblasts proliferated significantly and penetrated into the soft tissue layer of the gradient biomimetic scaffold, but did not penetrate into the hard tissue layer. The results show that, by gradient design of the pore size of each layer, fibroblasts can enter the inside of the soft tissue side of the scaffold, but cannot break through the barrier layer to invade the hard tissue layer. Figure 6 Example 5

[0119] The network formation of vascular endothelial cells on the gradient biomimetic scaffold was observed and detected, and the specific experimental process was as follows:

[0120] 1. Vascular endothelial cells were co-cultured with the gradient biomimetic scaffold (transwell), and incubated at 37°C, with liquid changed every 2 days;

[0121] 2. The network formation of vascular endothelial cells was observed at the set time point.

[0122] The results are shown in Table 3.

[0123] Figure 7 ​As shown in the figure: the gradient biomimetic scaffold can promote vascular endothelial cells to form vascular network.

[0124] Example 6

[0125] The regeneration of osteoblasts on the gradient biomimetic scaffold was observed and detected, and the specific experimental process was as follows:

[0126] 1. The osteoblasts were co-cultured with the gradient biomimetic scaffold, and incubated at 37°C, and the liquid was changed every 2 days;

[0127] 2. At the set time points of 7, 14 and 21 days, alkaline phosphatase reagent and alizarin red staining solution were used for staining, and then the osteogenic performance was observed.

[0128] The results are shown in the figure: Figure 8 The gradient biomimetic scaffold can promote the alkaline phosphatase activity of osteoblasts and the formation of calcium crystals.

[0129] The above describes the embodiments of the present application. However, the present application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A gradient biomimetic scaffold, comprising, in order from top to bottom, a soft tissue layer, a barrier layer, and a hard tissue layer. The gradient biomimetic scaffold is used for guiding combined repair of oral soft and hard tissues. In the gradient biomimetic scaffold, the pore size distribution of each layer is: soft tissue layer > hard tissue layer > barrier layer. The soft tissue layer is gradient distributed, and the pore size of the soft tissue layer gradually decreases from outside to inside. The barrier layer is a dense layer. The soft tissue layer contains active factors for inducing fibroblast adhesion and regeneration. The hard tissue layer contains active factors for inducing osteogenic differentiation and regeneration of cells. The soft tissue layer, the barrier layer, and the hard tissue layer are prepared by electrospinning.

2. The gradient biomimetic scaffold of claim 1, wherein, The pore size of the soft tissue layer of the gradient biomimetic scaffold is 0.4 μm-2.2 μm. The pore size of the barrier layer is 0.3 μm-0.7 μm. The pore size of the hard tissue layer is 0.3 μm-0.8 μm. The thickness of the soft tissue layer, the barrier layer, and the hard tissue layer is 5-50 μm.

3. The gradient biomimetic scaffold of claim 1, wherein, The soft tissue layer is a loose porous layer. The surface structure of the gradient biomimetic scaffold includes directional parallel shape, disordered cross shape, cross shape, or radial shape.

4. The gradient biomimetic scaffold of any one of claims 1-3, wherein, The soft tissue layer uses a high molecular material-1; the high molecular material-1 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. The barrier layer uses a high molecular material-2; the high molecular material-2 is selected from any one, two, or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. The hard tissue layer further contains active factors for inducing fibroblast adhesion and regeneration. The hard tissue layer uses a high molecular material-3; the high molecular material-3 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. 5.A method for preparing the gradient biomimetic scaffold of any one of claims 1-4, comprising the following steps: S1: preparing a soft tissue layer electrospinning solution, a barrier layer electrospinning solution, and a hard tissue layer electrospinning solution; S2: electrospinning the spinning solutions in step S1 layer by layer in the order of the soft tissue layer, the barrier layer, and the hard tissue layer, to obtain the gradient biomimetic scaffold.

6. The production method according to claim 5, characterized by, The parameter settings for electrospinning include: voltage of 12-40 kV, flow rate of 1-3 mL / h, and rotation speed of 200-2000 r / min. The soft tissue layer and the hard tissue layer are coaxially electrospun.

7. The production method according to claim 6, wherein The specific operation parameters for electrospinning the soft tissue layer are: coaxial electrospinning is performed using a 20-gauge coaxial needle, the distance between the needle and the receiver is 20 cm, the spinning voltage is 20 kV, the spinning time is 1 h, and the rotation speed of the drum is increased from 200 r / min to 2000 r / min. And / or, the specific operation parameters of electrospinning of the barrier layer are as follows: a 20-gauge needle is used, the distance between the needle and the receiver is 20 cm, an aluminum foil-wrapped rotating drum is used as the receiving electrode, the pushing speed is 1 mL / h, the rotating speed of the rotating drum is 3000 r / min, the spinning voltage is 20 kV, and the spinning time is 1 h; And / or, the specific operation parameters of electrospinning of the hard tissue layer are as follows: coaxial electrospinning is used, a 20 / 15-gauge coaxial needle is used, the distance between the needle and the receiver is 20 cm, the rotating speed of the rotating drum is 200 r / min, the spinning voltage is 20 kV, and the spinning time is 1 h; preferably, the pushing speed of the shell layer is 1 mL / h.

8. The preparation method according to claim 7, characterized in that, The pushing speed of the shell layer of electrospinning of the soft tissue layer is 1 mL / h; And / or, the pushing speed of the core layer of electrospinning of the soft tissue layer is 0.5 mL / h; And / or, the pushing speed of the shell layer of electrospinning of the hard tissue layer is 1 mL / h; And / or, the pushing speed of the core layer of electrospinning of the hard tissue layer is 0.5 mL / h.

9. The preparation method according to claim 5, characterized in that, During the electrospinning process, the temperature is controlled to be 26±3 ℃, and the humidity is controlled to be 35±5 %.

10. The method of claim 5, wherein, After the electrospinning is completed, the fiber membrane is peeled off from the aluminum foil, ventilated in a fume hood for 48 h to remove the residual solvent, and thus the gradient biomimetic scaffold is obtained.

11. The method of claim 5, wherein, The average diameter of the nanofibers of the formed gradient biomimetic scaffold is 5 nm-80 nm.

12. The method of claim 5, wherein, The soft tissue layer is prepared by electrospinning of a soft tissue layer electrospinning solution.

13. The method of claim 12, wherein, The preparation process of the soft tissue layer electrospinning solution is as follows: the high polymer material-1 and the surfactant-1 are dissolved in the solvent-1, the active factor-1 solution is added, and the mixture is stirred uniformly to obtain the soft tissue layer electrospinning solution. The active factor-1 is an active factor that can induce adhesion and regeneration of fibroblasts.

14. The method of claim 13, wherein, The concentration of the high polymer material-1 is 0.04 g / mL-1 g / mL; And / or, the concentration of the surfactant-1 is 1 μg / mL-20 μg / mL; And / or, the surfactant-1 is selected from non-ionic surfactants. And / or, the concentration of the active factor-1 is 25-1000 ng / mL; And / or, the solvent-1 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N,N dimethylformamide, N,N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethyl sulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, and acetic acid.

15. The preparation method according to claim 5, characterized in that, The barrier layer is prepared by electrospinning of a barrier layer electrospinning solution.

16. The method of claim 15, wherein, The preparation process of the barrier layer electrospinning solution is as follows: the high polymer material-2 is dissolved in the solvent-2, and the mixture is stirred uniformly to obtain the barrier layer electrospinning solution.

17. The method of claim 16, wherein the method further comprises, The high polymer material-2 is selected from one, two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. And / or, the concentration of the high polymer material-2 in the electrospinning solution is 0.04 g / mL-1 g / mL. And / or, the solvent-2 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N, N dimethylformamide, N, N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethyl sulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid.

18. The method of claim 5, wherein, The hard tissue layer electrospinning solution comprises a core layer electrospinning solution and a shell layer electrospinning solution.

19. The method of claim 18, wherein, The preparation process of the hard tissue layer core layer electrospinning solution is as follows: dissolving the high polymer material-3 and the surfactant-2 in a solvent-3, then adding a solution of the active factor-2, and stirring uniformly to obtain the core layer electrospinning solution. The active factor-2 is an active factor that can induce osteogenic differentiation and regeneration of cells.

20. The method of claim 19, wherein, The high polymer material-3 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. And / or, the concentration of the high polymer material-3 is 0.04 g / mL to 1 g / mL. And / or, the concentration of the surfactant-2 is 1 μg / mL to 20 μg / mL. And / or, the surfactant-2 is selected from a non-ionic surfactant. And / or, the concentration of the active factor-2 is 25 ng / mL to 1000 ng / mL. And / or, the solvent-3 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N, N dimethylformamide, N, N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethyl sulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid.

21. The method of claim 18, wherein, The preparation process of the hard tissue layer shell layer electrospinning solution is as follows: dissolving the high polymer material-4 and the surfactant-3 in a solvent-4, then adding a solution of the active factor-3, and stirring uniformly to obtain the shell layer electrospinning solution.

22. The method of claim 21, wherein, Preferably, the high polymer material-4 is selected from any two or more of collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, polycaprolactone, polylactic acid, polyvinyl alcohol, and methyl cellulose. And / or, the concentration of the high polymer material-4 is 0.04 g / mL to 1 g / mL. And / or, the concentration of the surfactant-3 is 1 μg / mL to 20 μg / mL. And / or, the surfactant-3 is selected from a non-ionic surfactant. And / or, the active factor-3 is an active factor that can induce adhesion and regeneration of fibroblasts. And / or, the concentration of the active factor-3 is 25 ng / mL to 1000 ng / mL. And / or, the solvent-4 is selected from one, two or more of hexafluoroisopropanol, cresol, chloroform, formic acid, N, N dimethylformamide, N, N dimethylacetamide, trifluoroacetic acid, acetone, tetrahydrofuran, dimethyl sulfoxide, trifluoroethanol, dichloromethane, saturated zinc chloride solution, saturated calcium chloride solution, ethanol, methanol, one of acetic acid.

23. A gradient biomimetic scaffold prepared by the preparation method of any one of claims 5-22.

24. Use of the preparation method of any one of claims 5-22 or the gradient biomimetic scaffold of any one of claims 1-4 and 23 in oral tissue engineering.

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