Double-layer GBR membrane for promoting soft and hard tissue repair as well as preparation method and application of double-layer GBR membrane

Abstract

The invention discloses a double-layer GBR membrane for promoting soft and hard tissue repair and a preparation method and application thereof, and is applied to the field of medical materials. The double-layer GBR membrane mainly comprises an acellular dermal matrix obtained by decellularizing fetal calfskin and a piezoelectric layer prepared by electrostatic spinning of poly-L-lactic acid and chitosan, and the acellular dermal matrix and the piezoelectric layer are further combined through a polydopamine coating prepared on the surface of the piezoelectric layer. During application, the acellular dermal matrix side faces the soft tissue side, and the piezoelectric layer faces the bone tissue side, so that the acellular dermal matrix skin side which is compact in structure is in contact with the soft tissue to play a barrier role, and the relatively loose piezoelectric layer is in contact with bone defects to play a role in guiding osteoblasts to adhere; functionally, soft tissue repair is promoted through the angiogenesis promoting effect of the acellular dermal matrix and the similarity of the tissue of the acellular dermal matrix, and bone regeneration of a bone defect area is promoted through the piezoelectric osteogenesis and angiogenesis effects of the piezoelectric layer.

Description

Technical Field

[0001] This invention relates to the field of medical materials, specifically to GBR technology, and particularly to a bilayer GBR membrane for promoting soft and hard tissue repair in oral GBR technology, its preparation method, and its application. Background Technology

[0002] Periodontitis, periapical periodontitis, trauma, or tumors can all lead to dentition or alveolar bone defects, resulting in insufficient bone volume during implantation. Furthermore, alveolar bone defects are often accompanied by soft tissue defects or deficiencies. Long-term chronic periodontitis, along with alveolar bone resorption, often involves partial loss of keratinized gingiva, which worsens after tooth extraction. This leads to a situation where, in addition to bone defects, soft tissue deficiencies are frequently encountered during implant surgery, a situation particularly pronounced in the anterior aesthetic zone.

[0003] Guided bone regeneration (GBR) is a widely used bone augmentation technique in oral treatment. It involves placing a barrier membrane in the bone defect area to prevent the ingrowth of epithelial and connective tissue, while simultaneously providing space for osteoblast proliferation and growth. This technique has been widely used in periodontal and implant surgeries, demonstrating significant clinical efficacy in improving vertical alveolar bone resorption and increasing alveolar bone volume. Current research on GBR membranes focuses primarily on enhancing their osteogenic and angiogenic functions and antibacterial properties. Adding osteogenic-related growth factors to the GBR membrane can promote bone growth, but controlling the direction and rate of factor release remains a challenge. Therefore, further research is needed to develop materials that can consistently and stably promote bone growth. Furthermore, because bone defects are often accompanied by soft tissue defects, this combined defect often leads to wound dehiscence, membrane exposure, and subsequent infection after GBR surgery due to excessive soft tissue tension, ultimately resulting in surgical failure.

[0004] Bone and blood vessels both possess piezoelectric properties, which can exert a stable and continuous bone-promoting effect. Bone and blood vessels can convert externally applied mechanical stress into electrical signals, thereby regulating cell metabolism and growth. When piezoelectric materials are implanted into bone defects and subjected to pressure, they generate electrical signals that promote tissue healing. This effect is gentle and continuous, avoiding the uncontrollable problems of the speed and direction of factor release. Acellular dermal matrix is ​​a biological GBR membrane that promotes soft tissue healing. Acellular dermal matrix refers to tissue material that has been processed to remove cellular components but retain collagen fibers. It has the same structure as soft tissue, can promote fibroblast proliferation, rebuild collagen structure, and promote angiogenesis. Due to its excellent soft tissue repair capabilities, acellular dermal matrix is ​​widely used in soft tissue-related research, showing significant effects on gingival and keratinized gingival augmentation. Studies have shown that acellular dermal matrix can also promote the transformation of thin gingiva into thick gingiva. Therefore, acellular dermal matrix has a significant effect on promoting soft tissue recovery.

[0005] Polylactic acid (PLA) membranes are synthetic polymers and are widely used in clinical absorbable gamma-ray barrier membranes (GBRs). Its isomer, poly-L-lactic acid (PLL), can be prepared into nanofibers using electrospinning technology, which possess piezoelectric properties and have been shown to induce osteogenic differentiation. Chitosan, a natural polymer, possesses good hydrophilicity and antibacterial properties, and is widely used in drug delivery systems and can function as a component of GBR membranes. Due to its non-centrosymmetric molecular structure, it also exhibits piezoelectric characteristics. PLA has high hardness and poor hydrophilicity. The addition of chitosan enhances the piezoelectric properties of PLA while increasing the material's softness and hydrophilicity to some extent. However, the modified material still lags behind decellularized dermal matrix in terms of biocompatibility.

[0006] To address the aforementioned clinical challenges, those skilled in the art desire a GBR membrane that simultaneously possesses the ability to continuously and stably promote bone function and soft tissue healing, thereby achieving simultaneous regeneration of both hard and soft tissues. The GBR membrane is located at the junction of soft and bone tissues, directly contacting both sides. If a biomimetic gradient structure is constructed, allowing the soft tissue side of the membrane to closely adhere to the soft tissue structure and promote soft tissue healing, while the bone tissue side exerts its osteogenic effect to promote bone defect healing, then it can exert effects on both soft and hard tissues simultaneously.

[0007] Therefore, this invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to prepare a double-layer GBR membrane that promotes the repair of soft and hard tissues, as well as its preparation method and application, in order to provide a new approach for the repair of oral soft and hard tissues.

[0009] In a first aspect, the present invention provides a bilayer GBR membrane for promoting the repair of soft and hard tissues, characterized in that the bilayer GBR membrane comprises a stacked decellularized dermal matrix layer and a polydopamine-modified piezoelectric layer. The piezoelectric layer comprises a membrane formed by spinning chitosan and poly-L-lactic acid; the decellularized dermal matrix layer and the piezoelectric layer are combined by a polydopamine coating.

[0010] In this invention, to further enhance the biocompatibility of the piezoelectric membrane and promote its adhesion to decellularized dermal matrix and cells, polydopamine is introduced as a coating for surface modification of the piezoelectric membrane. Polydopamine is polymerized from dopamine monomers and possesses good biodegradability. The dopamine molecules and other functional groups can form hydrogen bonds, giving polydopamine high adhesion strength to both organic and inorganic surfaces, making it an excellent choice for material surface modification as an adhesive coating.

[0011] As a preferred embodiment of the present invention, the decellularized dermal matrix layer is prepared by a decellularization method based on sodium deoxycholate and nuclease.

[0012] Preferably, the mass ratio of poly-L-lactic acid to chitosan in the piezoelectric layer is (8-12):1, for example, it can be 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1 or 12:1, etc.; preferably 10:1.

[0013] Preferably, the polydopamine coating is prepared by using a dopamine hydrochloride solution of 1 mg / ml to 3 mg / ml, for example, 1 mg / ml, 1.5 mg / ml, 1.8 mg / ml, 2 mg / ml, 2.5 mg / ml, 2.8 mg / ml or 3 mg / ml.

[0014] In a second aspect, the present invention provides a method for preparing a bilayer GBR film as described in the first aspect, the method comprising the following steps: (1) Preparation of decellularized dermal matrix; (2) After preparing poly-L-lactic acid and chitosan into a spun film, polydopamine is used for modification to form a polydopamine coating on the surface of the spun film, thereby obtaining a piezoelectric layer; (3) The decellularized dermal matrix obtained in step (1) is tightly bonded and compacted with the piezoelectric layer obtained in step (2), and dried to obtain the double-layer GBR film.

[0015] As a preferred technical solution of the present invention, in the preparation of the decellularized dermal matrix in step (1), sodium deoxycholate, deoxyribonuclease I and α-galactosidase are used for decellularization; Preferably, the method for preparing the decellularized dermal matrix is ​​as follows: Add 1%~5% (e.g., 1%, 2%, 3%, 4%, or 5% by mass) of sodium deoxycholate solution, 0.01 mg / ml~0.05 mg / ml (e.g., 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, or 0.05 mg / ml) of deoxyribonuclease I (DNase I) solution, and 0.1~0.5 units / g (e.g., 0.1 units / g, 0.2 units / g, 0.3 units / g, 0.4 units / g, or 0.5 units / g) of tissue α-galactosidase solution to the pretreated animal tissue. Shake at 150 rpm~220 rpm for 15 h~24 h, and wash with PBS solution to obtain the decellularized dermal matrix.

[0016] The decellularized dermal matrix can also be prepared using acid-base methods or pancreatic enzyme-hyper-hypotonic methods; however, the acid-base method results in significant structural damage to the decellularized dermal matrix, with serrated fibers and incomplete cell removal; the pancreatic enzyme-hyper-hypotonic method also leaves a large amount of cellular components remaining. Therefore, this invention recommends using a combination of sodium deoxycholate, DNase I, and α-galactosidase for the preparation of the decellularized matrix.

[0017] This invention utilizes sodium deoxycholate and nuclease to prepare a decellularized dermal matrix composite piezoelectric membrane via electrospinning, thus creating a bilayer composite guided bone regeneration membrane material designed to promote the joint repair of soft and hard tissues. This composite membrane, with its bilayer structure, provides a foundation for the joint promotion of soft and hard tissue repair, further enhancing the therapeutic effect of GBR membranes.

[0018] As a preferred embodiment of the present invention, the animal tissue is fresh fetal bovine hide.

[0019] Preferably, the pretreatment step is as follows: removing the hair from the back of the fresh fetal calf hide, removing the fat layer, thinning it, and immersing the resulting tissue in a peracetic acid solution for disinfection.

[0020] As a preferred technical solution of the present invention, in step (2), poly-L-lactic acid and chitosan are first dissolved in hexafluoroisopropanol, and then mixed to obtain a spinning solution containing 8 wt%-12 wt% (for example, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, etc.) poly-L-lactic acid and 0.5 wt%-1.5 wt% (for example, 0.5 wt%, 0.6 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.3 wt%, 1.5 wt%, etc.) chitosan.

[0021] Preferably, the spinning solution is prepared into a spinning membrane by electrospinning.

[0022] Preferably, during the electrospinning process, the injection pump flow rate is 0.2 mm / min-0.5 mm / min (e.g., 0.2 mm / min, 0.3 mm / min, 0.4 mm / min, or 0.5 mm / min, etc.), the spinning voltage is 10 kV-15 kV (e.g., 10 kV, 11 kV, 12 kV, 13 kV, 14 kV, or 15 kV, etc.), the spinning distance is 8 cm-12 cm (e.g., 8 cm, 9 cm, 10 cm, 11 cm, or 12 cm, etc.), and the receiver rotation speed is 4000 rpm-8000 rpm (e.g., 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, or 8000 rpm, etc.).

[0023] As a preferred technical solution of the present invention, the modification method is to immerse the spinning membrane in a dopamine hydrochloride solution and stir at room temperature for 4 h-8 h (for example, it can be 4 h, 5 h, 6 h, 7 h or 8 h, etc.).

[0024] Preferably, the concentration of the dopamine hydrochloride solution is 1 mg / ml to 3 mg / ml, for example, it can be 1 mg / ml, 1.5 mg / ml, 1.8 mg / ml, 2 mg / ml, 2.5 mg / ml, 2.8 mg / ml or 3 mg / ml, etc.

[0025] As a preferred technical solution of the present invention, in step (3), the decellularized dermal matrix and the piezoelectric layer are compacted by a heavy object and then placed in an environment of -18℃ to -20℃ for 20 h to 48 h (for example, it can be 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h or 48 h, etc.) to achieve compaction and tight adhesion.

[0026] The preparation method of the double-layer GBR film described in this invention can be carried out by the following steps: (1) Preparation of decellularized dermal matrix: Remove the hair and fat layer from the back of the fresh calf hide, thin it out, and then disinfect the resulting tissue by soaking it in a peracetic acid solution. Add 1 wt%-5 wt% sodium deoxycholate solution, 0.01 mg / ml-0.05 mg / ml deoxyribonuclease I solution, and 0.1 unit / g-0.5 unit / g tissue α-galactosidase solution to the pretreated animal tissue. Shake at 150 rpm-220 rpm for 15 h-24 h, and wash with PBS solution to obtain the decellularized dermal matrix. (2) Preparation of a piezoelectric layer modified with polydopamine coating: Poly-L-lactic acid and chitosan were dissolved separately in hexafluoroisopropanol and then mixed to form a spinning solution of 8 wt%-12 wt% poly-L-lactic acid and 0.5 wt%-1.5 wt% chitosan. The spinning solution was used to prepare a spun membrane by electrospinning, wherein the injection pump flow rate was 0.2 mm / min-0.5 mm / min, the spinning voltage was 10 kV-15 kV, the spinning distance was 8 cm-12 cm, and the receiver rotation speed was 4000 rpm-8000 rpm; the spun membrane was then immersed in a 1 mg / ml-3 mg / ml dopamine hydrochloride solution and stirred at room temperature for 4 h-8 h. (3) The decellularized dermal matrix and the piezoelectric layer are compacted by a heavy object and then frozen in an environment of -18℃ to -20℃ for 20 h to 48 h to achieve compaction and tight bonding, thereby obtaining the double-layer GBR film.

[0027] Thirdly, the present invention provides the application of a double-layer GBR membrane as described in the first aspect or a preparation method as described in the second aspect in the preparation of medical repair materials.

[0028] The present invention features a tightly bonded decellularized dermal matrix layer and piezoelectric layer, possessing a surface structure that matches the application scenario, good hydrophilicity and piezoelectric properties, and has significant application potential in promoting the repair of soft and hard tissues, especially in osteogenic processes.

[0029] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the characteristics of the soft and hard tissue interface in the maxillofacial region, this invention combines decellularized dermal matrix and a piezoelectric membrane with the assistance of polydopamine. Utilizing the soft tissue repair function of the decellularized dermal matrix and the piezoelectric osteogenesis function of the piezoelectric membrane, a bilayer composite GBR membrane material is prepared that promotes wound soft tissue healing and gently and continuously promotes bone tissue regeneration. Simultaneously, the bilayer composite GBR membrane material exhibits good biocompatibility and will not impair normal physiological functions.

[0031] Animal experiments showed that after 4 and 8 weeks of use, the residual defect volume of the rat maxillary bone wound was significantly reduced, the bone volume fraction of the wound was increased, and the soft tissue thickness and the area of ​​new bone formation were significantly increased. Therefore, the double-layer GBR membrane provided by this invention has a significant repair effect compared with decellularized matrix and single-layer piezoelectric material. Attached Figure Description

[0032] Figure 1 A schematic diagram of the structure of a double-layer GBR membrane to promote the repair of soft and hard tissues; 1-decellularized dermal matrix layer, 101-epidermal side of decellularized dermal matrix, 102-dermal side of decellularized dermal matrix; 2-piezoelectric layer of dopamine coating after co-spun chitosan and poly-L-lactic acid.

[0033] Figure 2 The images show the cross-sectional structures of the bilayer GBR membranes observed by scanning electron microscopy; where A represents the epidermal side of the decellularized dermal matrix, B represents the dermal side of the decellularized dermal matrix, C represents the pure poly-L-lactic acid spun structure, D represents the poly-L-lactic acid and chitosan co-spun membrane structure, and E and F represent the cross-sectional structures of the bilayer GBR membranes at different scales.

[0034] Figure 3 This is a statistical chart of water contact angle data (inversely proportional to the hydrophilicity of the material).

[0035] Figure 4 The results are from the electrometer readings of the material; Figure A shows the charge Q-time, Figure B shows the voltage U-time, and Figure C shows the current I-time.

[0036] Figure 5 This is a statistical chart of the test results for the piezoelectric constant D33.

[0037] Figure 6 The results are from piezoelectric detection using atomic mechanical microscopy; Figure A shows the amplitude, and Figure B shows the phase.

[0038] Figure 7 The images show the HE staining results of the internal organs of rats in each group 4 weeks after surgery, including the heart, liver, spleen, lungs, and kidneys.

[0039] Figure 8 The images show the recovery results of the maxillary bone wound in rats at 4 and 8 weeks post-surgery; where A represents the MicroCT results; B represents the statistical results of the remaining defect volume of the wound; and C represents the statistical results of the bone volume fraction of the wound.

[0040] Figure 9 The images show the HE staining results and soft tissue thickness analysis of the rat maxilla; Image A shows the staining results, and Image B shows the soft tissue thickness comparison.

[0041] Figure 10 The images show the Masson staining results and area comparison of the newly formed bone region in the rat maxilla; Figure A shows the staining results, and Figure B shows the area comparison of the newly formed bone region. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0043] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art. Unless otherwise specified, all experimental methods and techniques used are conventional methods and techniques in the art.

[0044] Experimental data were statistically analyzed using SPSS 26.0 software, and plotted using GraphPad Prism and Inage Pro software. One-way ANOVA (normal distribution) or Kruskal-Wallis H test (non-normal distribution) was used to compare pairs between groups. Statistical significance was assessed by P-value. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) were considered statistically significant.

[0045] Example 1 This embodiment provides a bilayer GBR membrane that promotes the repair of soft and hard tissues, and the preparation method is as follows: 1. Preparation of acellular dermal matrix (ADM) (1) Material pretreatment: Remove the hair from the back of the fresh fetal calf hide, remove the fat layer, and retain the dermis, basement membrane and a small amount of epidermis, and thin it to 0.5 mm.

[0046] The treated tissue was then immersed in a 2% peracetic acid solution at a ratio of 1:10 for overnight sterilization. PBS was used for washing, with six washes lasting one hour each.

[0047] (2) Decellularization procedure: 2% sodium deoxycholate solution, 0.04 mg / ml deoxyribonuclease I (DNase I) solution, and 0.2 units / g tissue α-galactosidase solution were added sequentially. The mixture was shaken on a fixed-track shaker for 20 h at a frequency of 150 rpm. PBS was used for washing each time the liquid was changed, and the washing process lasted for a total of 6 h.

[0048] (3) ADM storage: Store the processed ADM in PBS solution containing 5% glycerol and 15 mM ethylenediaminetetraacetic acid (EDTA) for later use.

[0049] 2. Fabrication of piezoelectric layers (1) Material dissolution: 2 g of poly-L-lactic acid (purchased from Shenzhen Jusheng Biotechnology Co., Ltd., intrinsic viscosity 0.87) and 0.2 g of chitosan (purchased from Shanghai Aladdin Reagent Co., Ltd., analytical grade AR) were dissolved in 20 ml and 10 ml of hexafluoroisopropanol (purchased from Shanghai Aladdin Reagent Co., Ltd., purity 99.5%) respectively, and stirred with a magnetic stirrer until completely dissolved to form a homogeneous solution.

[0050] (2) Preparation of spinning solution: The poly-L-lactic acid-hexafluoroisopropanol solution was slowly added to the chitosan-hexafluoroisopropanol mixture and stirred until the liquid evaporated to 20 ml, forming a spinning solution containing 10% poly-L-lactic acid and 1% chitosan.

[0051] (3) Electrospinning: The spinning solution was drawn up using a 20 ml syringe and fixed on the electrospinning machine. The flow rate of the syringe pump was set to 0.3 mm / min, the spinning voltage to 15 kV, the spinning distance to 10 cm, and the receiver rotation speed to 5000 rpm. Poly(L-lactic acid) / chitosan spun membrane was obtained in this way.

[0052] (4) Post-treatment: The obtained poly(L-lactic acid) / chitosan spun film was placed in a vacuum drying oven and vacuum treated for 24 hours to remove residual hexafluoroisopropanol.

[0053] (5) Coating preparation: The prepared piezoelectric layer was immersed in a 2 mg / ml dopamine hydrochloride solution and stirred at room temperature for 6 h to contact with oxygen to form a small amount of light gray PDA viscous coating.

[0054] 3. Preparation of composite materials The coated piezoelectric layer (poly-L-lactic acid + chitosan + PDA) was tightly bonded to the ADM dermal leather side, pressed with a steel plate, and then placed in a -20℃ freezer for 24 hours.

[0055] The frozen composite membrane (poly-L-lactic acid + chitosan + PDA + ADM) was placed in a vacuum dryer for 24 hours to obtain the final double-layer piezoelectric composite membrane material.

[0056] The structure of the material obtained in Example 1 can be as follows: Figure 1 As shown, 1 represents the decellularized dermal matrix layer, 2 represents the piezoelectric layer of the dopamine coating after co-spun chitosan and poly-L-lactic acid; further, 101 represents the epidermal side of the decellularized dermal matrix, and 102 represents the dermal side of the decellularized dermal matrix.

[0057] The surface morphology and structure of the material obtained in Example 1 were observed using a scanning electron microscope.

[0058] like Figure 2As shown, the two sides of the decellularized dermal matrix exhibit different structural features. Among them, Figure A shows the epidermal side of the decellularized dermal matrix, Figure B shows the dermal side of the decellularized dermal matrix, Figure C shows the pure poly-L-lactic acid spun structure, Figure D shows the poly-L-lactic acid and chitosan co-spun membrane structure, and Figures E and F show the cross-sectional structure of the double-layer GBR membrane.

[0059] As can be seen, the dense surface of the epidermal side helps prevent cell invasion, while the dermal side, where it intersects with the piezoelectric layer, has a relatively loose porous structure, facilitating cell adhesion and bonding with the piezoelectric layer. The overlapping fibers and loose surface of the piezoelectric layer promote the ingrowth of bone cells and vascular cells, serving as a bone tissue regeneration layer. This structure helps improve the repair effect of both soft and hard tissues.

[0060] Example 2 This embodiment provides a bilayer GBR membrane that promotes the repair of soft and hard tissues. The difference from Example 1 is that the concentration of dopamine hydrochloride solution in the coating preparation step is adjusted to 0.5 mg / ml in this embodiment; the remaining steps are the same as in Example 1.

[0061] Example 3 This embodiment provides a bilayer GBR membrane that promotes the repair of soft and hard tissues. The difference from Example 1 is that the concentration of dopamine hydrochloride solution in the coating preparation step is adjusted to 5 mg / ml in this embodiment; the remaining steps are the same as in Example 1.

[0062] In Examples 2 and 3, the concentration of dopamine hydrochloride solution was adjusted. However, at lower concentrations, the piezoelectric layer and the decellularized dermal matrix layer did not bond tightly enough, leading to easy delamination. At higher concentrations, dopamine would excessively aggregate and stack, forming a dense, heterogeneous cross-linked network. This resulted in PDA-rich areas or large particles forming inside and on the surface of the membrane, causing structural inhomogeneity. The membrane surface would change from relatively smooth to highly rough, even exhibiting nanospheres or nodular structures. This might be beneficial for bilayer adhesion, but it easily affects the smoothness of the membrane.

[0063] Comparative Example 1 This comparative example uses decellularized dermal matrix, prepared in the same manner as in Example 1. Details are as follows: After dehairing and degreasing, fetal bovine hides were thinned to approximately 0.5 mm. Then, 2% sodium deoxycholate solution, 0.04 mg / ml deoxyribonuclease I (DNase I) solution, and 0.2 units / gram of α-galactosidase solution were added sequentially. The mixture was then shaken on a fixed-track shaker for 20 hours to prepare a decellularized dermal matrix.

[0064] The resulting decellularized matrix has a dense structure on the epidermal side, which plays a barrier role, while the dermal side is loose and conducive to adhesion. The residual cytokines in it are beneficial to angiogenesis, and after decomposition, they can become part of the soft tissue, promote healing, and increase the thickness of the soft tissue to a certain extent. However, decellularized dermal matrix alone is unlikely to play a significant role in bone defects.

[0065] Comparative Example 2 This comparative example directly uses poly-L-lactic acid to prepare the piezoelectric layer. The specific steps are as follows: 2 g of poly-L-lactic acid was dissolved in 20 ml of hexafluoroisopropanol to form a hexafluoroisopropanol spinning solution containing 10% poly-L-lactic acid. Poly-L-lactic acid spun membrane was prepared by electrospinning (setting parameters: injection pump flow rate of 0.3 mm / min, spinning voltage of 10 kV, spinning distance of 10 cm, and receiver rotation speed of 5000 rpm).

[0066] The resulting absorbable electrospun membrane has good piezoelectric properties, but poor hydrophilicity and lack of good biocompatibility, which is not conducive to cell adhesion and material bonding.

[0067] Comparative Example 3 This comparative example uses poly(L-lactic acid) and chitosan to prepare a piezoelectric layer. The specific steps are as follows: 2 g of poly-L-lactic acid and 0.2 g of chitosan were dissolved in 20 ml and 10 ml of hexafluoroisopropanol, respectively. The poly-L-lactic acid-hexafluoroisopropanol solution was slowly added to the chitosan-hexafluoroisopropanol mixture and stirred until the liquid evaporated to 20 ml, forming a spinning solution containing 10% poly-L-lactic acid and 1% chitosan. Poly(L-lactic acid) / chitosan spun membranes were prepared by electrospinning (setting parameters: injection pump flow rate of 0.3 mm / min, spinning voltage of 15 kV, spinning distance of 10 cm, and receiver rotation speed of 5000 rpm).

[0068] In this comparative example, the addition of chitosan improved the piezoelectric properties of the absorbable electrospun membrane, but also improved its hydrophilicity and biocompatibility. However, it still did not match the decellularized dermal matrix.

[0069] Comparative Example 4 In this comparative example, a piezoelectric layer was prepared using poly-L-lactic acid, chitosan, and polydopamine, following the same preparation method as in Example 1. Details are as follows: 2 g of poly-L-lactic acid and 0.2 g of chitosan were dissolved in 20 ml and 10 ml of hexafluoroisopropanol, respectively. The poly-L-lactic acid-hexafluoroisopropanol solution was slowly added to the chitosan-hexafluoroisopropanol mixture and stirred until the liquid evaporated to 20 ml, forming a spinning solution containing 10% poly-L-lactic acid and 1% chitosan. Poly(L-lactic acid) / chitosan spun membranes were prepared by electrospinning (setting parameters: injection pump flow rate of 0.3 mm / min, spinning voltage of 15 kV, spinning distance of 10 cm, and receiver rotation speed of 5000 rpm). A dopamine coating was prepared by placing the spun membrane in a 2 mg / ml dopamine hydrochloride solution to obtain a piezoelectric layer (poly-L-lactic acid + chitosan + PDA).

[0070] The piezoelectric layer prepared in this comparative example has good piezoelectric properties and hydrophilicity. Its biocompatibility is better than that of decellularized dermal matrix and it can be well combined with decellularized dermal matrix. However, it cannot perform well in repairing soft tissue defects on its own.

[0071] Comparative Example 5 The difference between this comparative example and Example 1 is that tannic acid is used instead of polydopamine as the coating in this comparative example; the remaining steps are the same as in Example 1.

[0072] Comparative Example 6 The difference between this comparative example and Example 1 is that sodium alginate is used instead of chitosan in this comparative example, and it is used with poly-L-lactic acid to prepare the piezoelectric layer; the remaining steps are the same as in Example 1.

[0073] Test Example 1 Performance Evaluation 1. Hydrophilic The water contact angle of the material was measured using a surface tension meter to confirm its hydrophilicity, which reflects the material's biocompatibility.

[0074] In this test case, the hydrophilicity of the materials prepared in Example 1 and Comparative Examples 1-4 was tested. The hydrophilicity of both sides (decellularized dermal matrix side and piezoelectric side) of the bilayer membrane prepared in Example 1 was tested.

[0075] The results are as follows Figure 3 As shown, pure poly-L-lactic acid (Comparative Example 2) has poor hydrophilicity, while the addition of chitosan (Comparative Example 3) improves the hydrophilicity of poly-L-lactic acid; and the polydopamine coating on the surface of the poly-L-lactic acid / chitosan layer to obtain a piezoelectric layer (Comparative Example 4) further enhances the hydrophilicity of the piezoelectric layer and significantly strengthens the interlayer bonding force and cell adhesion between the piezoelectric layer and the decellularized dermal matrix.

[0076] 2. Piezoelectricity The piezoelectric properties of the materials prepared in Examples 1 and Comparative Examples 1-4 were tested using an electrometer, a d33 piezoelectric coefficient test, and an atomic mechanical microscope.

[0077] The electrometer test results are as follows Figure 4As shown in the figure, graphs A, B, and C correspond to the charge (Q), voltage (U), and current (I) at different times. The piezoelectric constant D33 detection results are as follows. Figure 5 As shown. The piezoelectric detection results using atomic mechanical microscopy are as follows. Figure 6 As shown, Figure A represents amplitude; Figure B represents phase.

[0078] The experimental results show that the co-spun film of L-polylactic acid (PLA) and chitosan provides the piezoelectric properties of the composite material. Through electrospinning, PLA forms an ordered fibrous structure, with chitosan particles uniformly distributed within it; the addition of chitosan enhances the piezoelectricity.

[0079] The present invention also performs cell biocompatibility testing and immunohistochemical staining experiments on the GBR barrier membrane material prepared in Example 1.

[0080] The results show that the GBR composite membrane material prepared in Example 1 can support the growth and function of L929, BMSCs, and VECs, meet the basic requirements for soft and hard tissue repair, and exhibit excellent biocompatibility.

[0081] Meanwhile, the piezoelectric effect of the piezoelectric layer in the composite membrane material, excited by ultrasound, can effectively promote the adhesion, migration, and osteogenic processes of BMSCs, as well as the angiogenic capacity of VECs, demonstrating its unique advantages in bone tissue repair. The ADM layer in the composite membrane material acts as a barrier to L929 cells but can promote VEC attachment and significantly promote angiogenesis, providing important support for soft tissue and vascular repair.

[0082] In addition to cell experiments, a series of immunohistochemical staining experiments were also performed in this test case, including immunohistochemical staining of CD31, OPN, and COL-1, to observe the expression differences among the groups. The results can prove that the material promotes osteogenic and angiogenic properties.

[0083] Test Example 2: Animal Experiment In animal experiments, the piezoelectric effect of materials is stimulated by the transmission of chewing pressure, thereby promoting osteogenic properties.

[0084] This embodiment uses 6-8 week old SD rats to conduct animal experiments to confirm the biocompatibility and defect repair effect of the bilayer GBR membrane material. The specific steps are as follows: (1) A peach-shaped bur was used to locate and determine the depth on the palatal side of the maxillary premaxilla in the rat's mouth, and a 3 mm diameter spherical bur was used to grind away the soft and hard tissues to prepare a circular defect with a diameter of 3 mm.

[0085] (2) Apply intermittent pressure with gauze to stop bleeding and prevent rats from suffocating.

[0086] (3) The blank group only prepared defects and did not place materials; In the remaining experimental groups, materials (4 mm in diameter) were placed at the bone and soft tissue defects, and then the materials were fixed in the surrounding soft tissue by cross sutures to avoid closing the wound.

[0087] (4) Four weeks after the operation, the internal organs of each group of animals were taken for HE staining to observe tissue changes and determine the biocompatibility of the material in vivo; (5) At 4 and 8 weeks post-operation, the maxilla of experimental rats was taken and Micro-CT and histological analysis were used to compare the repair effects of bone and soft tissue defects in different groups.

[0088] HE staining results of viscera in rats 4 weeks post-surgery are as follows: Figure 7 As shown, the rat heart, liver, spleen, lungs, and kidneys were included. It can be seen that there were no significant differences in tissue structure among the groups.

[0089] As shown in the figure, the cellular structure of all organs remained normal, with no edema, inflammatory cell infiltration, or tissue necrosis observed. Particularly in vital organs such as the heart, liver, and spleen, the tissue structure was normal, and the cells were arranged regularly, indicating that the material used did not exhibit significant toxic effects in animals and did not impair their normal physiological functions. Therefore, the material showed good biocompatibility in rats, and no toxic reactions to internal organs were observed.

[0090] The repair effect of maxillary bone wound in rats at 4 and 8 weeks post-surgery is as follows: Figure 8 As shown: Among them, the Micro CT results (Figure A) clearly show that the wound of the rat is significantly reduced. The statistical results of the remaining bone volume of the wound (Figure B) and the statistical results of the bone volume fraction of the wound (Figure C) show that the composite group (i.e., Example 1) has the most obvious repair effect.

[0091] HE staining results and soft tissue thickness analysis of rat maxillae are as follows: Figure 9 As shown in the figure, Figure A shows the staining results, and Figure B shows the comparison of soft tissue thickness. Masson staining results and the area of ​​newly formed bone, as shown in... Figure 10 As shown, Figure A shows the staining results, and Figure B shows the area comparison of the newly formed bone region.

[0092] Combination Figures 8-10 The results show that, compared with the decellularized matrix and the single-layer piezoelectric material, the double-layer GBR membrane prepared in Example 1 has significantly increased soft tissue thickness and new bone formation area, and has obvious repair effect.

[0093] Furthermore, this invention has attempted to use other materials, such as tannic acid to replace dopamine (Comparative Example 5), or sodium alginate to replace chitosan (Comparative Example 6). However, the tannic acid coating may desorb or be lost due to the disruption of non-covalent bonds under physiological conditions or long-term immersion, leading to the failure of the functional layer. Simultaneously, the excessively strong hydrogen bonding of tannic acid may cause excessive aggregation, collapse, or densification of the electrospun fiber membrane, disrupting its designed pore structure and affecting nutrient transport and cell ingrowth. Chitosan has antibacterial activity, while sodium alginate itself has almost no antibacterial properties, potentially requiring the addition of an antibacterial agent. Moreover, the unmodified sodium alginate surface is anti-adhesive, which severely hinders the attachment and growth of target cells such as osteoblasts and fibroblasts, affecting tissue integration and regeneration.

[0094] In summary, the bilayer GBR membrane for promoting soft and hard tissue repair provided by this invention comprises two parts: a decellularized dermal matrix obtained from decellularized fetal bovine hide and a piezoelectric layer prepared by electrospinning poly-L-lactic acid and chitosan. These two parts are further bonded together by a polydopamine coating prepared on the surface of the piezoelectric layer. In application, the decellularized dermal matrix side faces the soft tissue side, and the piezoelectric layer faces the bone tissue side. Structurally, the dense epidermal side of the decellularized dermal matrix acts as a barrier in contact with the soft tissue, while the relatively loose piezoelectric layer guides osteoblast attachment in contact with the bone defect. Functionally, the angiogenesis-promoting effect of the decellularized dermal matrix and its similarity to the surrounding tissue promote soft tissue repair, while the piezoelectric osteogenic and angiogenic effects of the piezoelectric layer promote bone regeneration in the bone defect area.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double-layered GBR membrane for promoting soft and hard tissue repair, characterized in that, The double-layer GBR membrane comprises a stacked acellular dermal matrix layer and a polydopamine modified piezoelectric layer; The piezoelectric layer is a membrane formed by spinning of chitosan and poly-L-lactic acid; the acellular dermal matrix layer and the piezoelectric layer are combined by a polydopamine coating.

2. The bilayer GBR membrane according to claim 1, characterized in that, The acellular dermal matrix layer is prepared based on a deoxycholic acid sodium and nuclease acellular method; The mass ratio of poly-L-lactic acid and chitosan in the piezoelectric layer is (8-12):1; The polydopamine coating is prepared by using a 1 mg / ml-3 mg / ml dopamine hydrochloride solution.

3. The method for preparing a double-layer GBR membrane according to claim 1 or 2, characterized by, The preparation method comprises the following steps: (1) preparing an acellular dermal matrix; (2) after poly-L-lactic acid and chitosan are prepared into a spinning membrane, the spinning membrane is modified by polydopamine to form a polydopamine coating on the surface of the spinning membrane, thereby obtaining a piezoelectric layer; (3) the acellular dermal matrix obtained in step (1) is tightly combined with the piezoelectric layer obtained in step (2) and compacted, and then dried to obtain the double-layer GBR membrane.

4. The production method according to claim 3, characterized by, In the preparation of the acellular dermal matrix in step (1), deoxycholic acid sodium, deoxyribonuclease I and α-galactosidase are used for acellulization; Preferably, the preparation method of the acellular dermal matrix comprises the following steps: adding 1 wt%-5 wt% deoxycholic acid sodium solution, 0.01 mg / ml-0.05 mg / ml deoxyribonuclease I solution and 0.1 unit / g-0.5 unit / g tissue of α-galactosidase solution into the pretreated animal tissue, oscillating at 150 rpm-220 rpm for 15 h-24 h, and then cleaning with a PBS solution to obtain the acellular dermal matrix.

5. The preparation method according to claim 4, characterized in that, The animal tissue is fresh fetal bovine hide; Preferably, the pretreatment step comprises the following steps: removing the hair on the back of the fresh fetal bovine hide, removing the fat layer, thinning the hide, and then soaking the hide in a peroxoacetic acid solution for disinfection.

6. The production method according to claim 3, characterized by, In step (2), poly-L-lactic acid and chitosan are dissolved in hexafluoroisopropanol respectively, and then mixed to obtain a spinning solution containing 8 wt%-12 wt% poly-L-lactic acid and 0.5 wt%-1.5 wt% chitosan; Preferably, the spinning solution is prepared into a spinning membrane by electrospinning. Preferably, in the electrospinning process, the flow rate of the injection pump is 0.2 mm / min-0.5 mm / min, the spinning voltage is 10 kV-15 kV, the spinning distance is 8 cm-12 cm, and the rotation speed of the receiver is 4000 rpm-8000 rpm.

7. The production method according to claim 3, characterized by, The modification method comprises the following steps: soaking the spinning membrane in a dopamine hydrochloride solution, stirring at room temperature for 4 h-8 h, and then drying to obtain the piezoelectric layer. Preferably, the concentration of the dopamine hydrochloride solution is 1 mg / ml-3 mg / ml.

8. The production method according to claim 3, characterized by, In step (3), after the acellular dermal matrix and the piezoelectric layer are compacted by a weight, they are frozen in an environment at-18 ℃~-20 ℃ for 20 h-48 h to realize compaction and tight combination.

9. Use of the double-layer GBR membrane of claim 1 or 2 or the preparation method of any one of claims 3-8 in the preparation of a medical repair material.

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