Self-power-generating composite membrane for repairing critical-size bone defect and preparation method of self-power-generating composite membrane

Abstract

The invention provides a self-generating composite membrane for repairing critical-size bone defects and a preparation method of the self-generating composite membrane, and relates to the technical field of bone tissue defect repairing and medical biological materials. An electrode layer of the self-generating composite membrane is PPCs hydrogel, and the PPCs hydrogel comprises polyacrylamide (PAM), polydopamine (PDA) and silk fibroin derived peptide (Cs). The invention provides a degradable self-adhesion composite membrane for repairing critical-size bone defects, which can realize bionic electrical stimulation which is self-adjusted along with a motion load state, can be applied to bone defect repair of different parts, different sizes and different states, and has good adaptability.

Description

Technical Field

[0001] The present invention relates to the technical field of bone tissue defect repair and medical biomaterials, and in particular to a self-generating composite membrane for repairing critical-size bone defects and a preparation method thereof. Background Art

[0002] Currently, the rapid and effective repair of critical-size bone defects (CSBD) remains a major challenge. In clinical practice, autologous grafts, allografts, and inert artificial grafts are commonly used methods for the reconstruction of critical-size bone defects. However, issues such as immune rejection of grafts, limited sources of donor bone, and the non-degradability of grafts restrict their medical applications. Bone tissue engineering has been introduced to treat critical-size bone defects, but its therapeutic effect faces challenges such as low cell viability after implantation, short lifespan of growth factors, and insufficient self-adaptation and self-regulation capabilities of biomaterials.

[0003] Fundamentally, bone tissue exhibits a constant electrical potential (resting potential) under static physiological loads, whereas it exhibits a dynamic electrical potential (action potential) when the load caused by host movement is converted into electrical output. Electrical output under varying mechanical loads stimulates the regulation of downstream biochemical signals. Ultimately, this intrinsic “mechanical-electrical-biochemical” cascade can rapidly and effectively promote bone remodeling and healing. However, the structural discontinuity of critical bone defects renders these intrinsic mechanoelectrical properties ineffective at the defect site, thereby inhibiting the activation of biochemical signals and preventing the initiation of the CSBD healing process.

[0004] The development of piezoelectric and triboelectric materials has driven the advancement of self-powered stimulation strategies. These materials can convert mechanical stimulation into biofeedback electrical signals or directly generate resting potentials to promote local bone healing. However, known piezoelectric and triboelectric materials cannot simultaneously restore resting potential and action potential. In addition, due to the use of metal electrodes, the electrical output is limited during low-frequency or low-load host movement and lacks adhesion, which limits their ability to output in situ electrical stimulation at critical bone defect sites. In addition, their clinical application still faces problems such as potential toxicity, limited bioactivity, and poor degradability. Summary of the Invention

[0005] In view of the problems of existing composite membranes in terms of in situ electrical stimulation output capability, adhesion, potential toxicity, limited biological activity, and poor degradability, the purpose of the present invention is to propose a self-generating composite membrane for repairing critical-size bone defects and its preparation method.

[0006] In the first aspect, the present invention provides a self-generating composite membrane for repairing critical-size bone defects, comprising a friction layer, an electrode layer, and an external package, wherein the electrode layer is a PPCs hydrogel, and the PPCs hydrogel comprises polyacrylamide (PAM), polydopamine (PDA), and silk fibroin-derived peptide (Cs).

[0007] As an optional embodiment, the PPCs hydrogel comprises, by mass fraction:

[0008] polyacrylamide, 60%;

[0009] polydopamine, 1.6%;

[0010] Silk fibroin-derived peptide, 0.5% to 2%.

[0011] As an optional embodiment, the PPCs hydrogel comprises, by mass fraction:

[0012] polyacrylamide, 60%;

[0013] polydopamine, 1.6%;

[0014] Silk fibroin-derived peptides, 2%.

[0015] As an optional embodiment, the PPCs hydrogel further comprises, by mass fraction:

[0016] Ammonium persulfate (APS), 6%;

[0017] N,N′-methylenebisacrylamide (BIS), 0.2%.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned self-generating composite film, wherein the process of preparing the electrode layer comprises:

[0019] (1) Dopamine (DA) powder was dissolved in NaOH solution, and silk fibroin-derived peptide (Cs) powder was added and stirred continuously;

[0020] (2) Add acrylamide (AM), initiator and cross-linking agent and stir continuously;

[0021] (3) Adding a catalyst, pouring the solution into a mold, and preparing a hydrogel electrode layer with a predetermined shape.

[0022] As an optional embodiment, in terms of mass fraction:

[0023] polyacrylamide, 60%;

[0024] polydopamine, 1.6%;

[0025] Silk fibroin-derived peptides, 0.5%–2%;

[0026] initiator, 6%;

[0027] Cross-linking agent, 0.2%.

[0028] As an optional embodiment, in terms of mass fraction:

[0029] polyacrylamide, 60%;

[0030] polydopamine, 1.6%;

[0031] silk fibroin-derived peptides, 2%;

[0032] initiator, 6%;

[0033] Cross-linking agent, 0.2%.

[0034] As an optional embodiment, the initiator is ammonium persulfate (APS), and the cross-linking agent is N,N′-methylenebisacrylamide (BIS).

[0035] In a third aspect, a method for preparing an electrode layer of a composite membrane for repairing a critical-size bone defect is provided, the method comprising:

[0036] (1) Dopamine is subjected to a self-polymerization reaction in an alkaline aqueous solution to form a PDA chain; wherein a silk fibroin-derived peptide (Cs) is added during the dopamine polymerization process;

[0037] (2) Adding acrylamide to polymerize and generate PAM chains; wherein, PDA chains and PAM chains are randomly cross-linked by physical and covalent means to form a PDA-PAM polymer network.

[0038] As an optional embodiment, the mass fraction ratio of acrylamide, dopamine, and silk fibroin-derived peptide is 60:1.6:0.5-2, preferably 60:1.6:2.

[0039] The gel polymerization process was carried out in a water bath at 70°C.

[0040] The present invention provides a degradable self-adhesive composite membrane for repairing critical-sized bone defects, which can realize bionic electrical stimulation that self-regulates with the motion load state, can be applied to repair bone defects in different parts, sizes, and states, and has good adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 Schematic diagram of a composite membrane stack structure according to an embodiment of the present invention;

[0043] Figure 2 Schematic diagram of the static potential of the electrode layer surface with different concentrations of Cs added;

[0044] Figure 3 Schematic diagram of the electrode layer self-adhesion ability test;

[0045] Figure 4 The action potential output of the composite membrane at different frequencies;

[0046] Figure 5 Comparison of electrical output between PPCs and copper as electrode layers;

[0047] Figure 6 is the in vivo degradation performance of the composite membrane;

[0048] Figure 7 In vivo bone repair effect of composite membrane DETAILED DESCRIPTION

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0050] In order to solve the above problems of existing composite membranes, the present invention provides a fully implantable, self-adhesive and degradable biomimetic self-generating composite membrane for quickly and effectively repairing, for example, critical size bone defects (CSBD).

[0051] Figure 1 Schematic diagram of the composite membrane stack structure according to an embodiment of the present invention. Figure 1 As shown, the composite film includes a friction layer, an electrode layer, and an encapsulation layer. The friction layer includes an upper friction layer, a lower friction layer, and an intermediate spacer layer disposed between the two friction layers. The electrode layer is disposed on the lower surface of the lower friction layer. The encapsulation layer includes an upper encapsulation layer and a lower encapsulation layer. The upper encapsulation layer covers the upper friction layer, and the lower encapsulation layer is disposed on the lower surface of the electrode layer and has an opening exposing the lower surface of the electrode layer.

[0052] During host movement, the friction layer of the composite membrane converts the tiny host motion load into real-time biofeedback electrical signals, which are transmitted through the electrode layer to the bone defect area, manifesting as action potentials. Metal materials have long been used as electrode materials. However, as mentioned above, metal electrodes have limited electrical output and lack adhesion during low-frequency or low-load host movement, limiting their ability to deliver in situ electrical stimulation at critical bone defect sites. To this end, the composite membrane provided by the present invention utilizes hydrogels as composite membrane electrodes to significantly improve electrical output performance.

[0053] In one embodiment, the composite membrane electrode is a PPCs hydrogel comprising polydopamine, polyacrylamide, and silk fibroin-derived peptides.

[0054] As an example, in terms of weight fraction (wt%), the PPCs hydrogel includes:

[0055] Polyacrylamide (PAM), 60%;

[0056] polydopamine (PDA), 1.6%;

[0057] Silk fibroin-derived peptide (Cs), 0.5% to 2%.

[0058] The PPCs hydrogel can firmly adhere to the complex surface of bone defects through DOPA-mediated hydrogen and covalent bonds, enhancing the transmission efficiency of bioelectric stimulation. Furthermore, the composite membrane can respond to the host's motion state to provide biomimetic electrical stimulation, manifesting as a resting potential (-50mV to -100mV) on the electrode layer surface when the host is at rest. This is due to the grafted silk fibroin-derived peptide (Cs), an anionic polypeptide, which imparts a negative charge to the electrode layer surface. By adjusting the concentration (preferably 2% by mass), this charge matches the resting potential of bone tissue.

[0059] Furthermore, PPCs hydrogels can form an electrical double layer at the interface with, for example, copper wires, enabling the transfer of ions within the PPCs hydrogels along with the induced charges, ultimately enhancing the electrical output capability.

[0060] As a further example, the PPCs hydrogel also includes, by mass fraction:

[0061] Initiator: ammonium persulfate (APS), 6%;

[0062] Cross-linking agent: N,N'-methylenebisacrylamide (BIS), 0.2%.

[0063] In one embodiment, the upper friction layer is a PLGA film, the lower friction layer is a PCL film, and the middle spacer layer is a PCL material.

[0064] As an example, a PLGA film (friction layer) was prepared using a PLGA solution with a mass volume fraction of 7.5%, and a PCL film (friction layer) was prepared using a PCL solution with a mass volume fraction of 7.5%, with a thickness of 100 μm. The PLGA film had a surface nanostructured structure.

[0065] As an example, the intermediate spacer layer is prepared by using a PCL solution with a mass volume fraction of 7.5% and a thickness of 400 μm. In one embodiment, the encapsulation layer is a PLGA film.

[0066] As an example, the PLGA film (encapsulation layer) was prepared using a PLGA solution with a mass volume fraction of 15%.

[0067] The following describes the method for preparing the composite film.

[0068] According to an embodiment of the present invention, a method for preparing a self-generating composite film for repairing a critical-size bone defect is provided, the method comprising the following steps:

[0069] Step S1, preparing a friction layer;

[0070] The friction layer includes an upper friction layer and a lower friction layer. Taking the above friction layer as an example, its preparation process includes:

[0071] (1) Preparation of PLGA film;

[0072] PLGA was dissolved in dichloromethane at a concentration of 7.5% (w / v), and the solution was cast onto a glass plate. After the solution on the glass plate was air-dried, it was dried in a vacuum oven to remove residual solvent, thereby obtaining a PLGA film.

[0073] (2) creating surface nanostructures on PLGA films;

[0074] The obtained PLGA film was immersed in NaOH solution to create surface nanostructures, and then washed with deionized water and dried to obtain a PLGA membrane.

[0075] The lower friction layer was prepared using a 7.5% (w / v) PCL solution, and its preparation process was similar to that of the above-mentioned PLGA film.

[0076] Step S2, preparing an intermediate spacer layer;

[0077] The middle spacer layer was prepared using a 7.5% (w / v) PCL solution, and its preparation process was similar to that of the above-mentioned PLGA film.

[0078] Step S3, preparing an encapsulation layer;

[0079] The encapsulation layer includes an upper encapsulation layer and a lower encapsulation layer. Both encapsulation layers are composed of a 15% (w / v) PLGA film, and the preparation process thereof is similar to that of the above-mentioned PLGA film.

[0080] Step S4, preparing an electrode layer;

[0081] The process of preparing the electrode layer includes:

[0082] (1) Dopamine (DA) powder was dissolved in NaOH solution, and silk fibroin-derived peptide (Cs) powder was added and stirred continuously;

[0083] (2) Add acrylamide (AM), ammonium persulfate (APS) and N,N′-methylenebisacrylamide (BIS) and continue stirring.

[0084] (3) Tetramethylethylenediamine (TMEDA) is added and the solution is poured into a mold to prepare a hydrogel electrode layer with a predetermined shape.

[0085] The gel polymerization was carried out in a water bath at 70°C.

[0086] Dopamine (DA) undergoes self-polymerization in an alkaline aqueous solution to form PDA chains (polydopamine chains). During the PDA polymerization process, a silk fibroin-derived peptide (Cs) is added. The amino groups of Cs undergo a Michael addition reaction with the quinone structure of PDA, successfully grafting Cs onto the PDA chains. Acrylamide (AM) is then added and subsequently polymerizes to form polyacrylamide (PAM) chains. These PDA and PAM chains are randomly cross-linked through physical and covalent means to form a PDA-PAM polymer network. Simultaneously, with the help of another cross-linking agent, BIS, the viscosity of the reaction mixture gradually increases, a process initiated by ammonium persulfate (APS).

[0087] Furthermore, the subsequent addition of TEMED catalyzed the reaction process, thereby accelerating the polymerization process.

[0088] Step S5: packaging

[0089] The above structure is heat-sealed in the order of upper packaging layer, upper friction layer, middle spacer layer, lower friction layer, electrode layer and lower packaging layer to obtain a composite film.

[0090] It is understood that the materials and preparation methods of the above-mentioned friction layer and packaging layer are only examples, and other known composite film friction layer and packaging layer materials and processes can also be used for preparation, and the electrode layer provided by the present invention is also applicable.

[0091] Example 1 :

[0092] Step S1, preparing the friction layer

[0093] PLGA (75:25) was dissolved in dichloromethane at a concentration of 7.5% (w / v) and cast onto a glass plate (2 cm × 5 cm). The solution on the glass plate was air-dried for 2 hours and then dried in a vacuum oven at 37°C for 12 hours to remove residual solvent, resulting in a PLGA membrane. The resulting PLGA membrane was then immersed in a 1M NaOH solution for 20 minutes to create surface nanostructures. Finally, it was washed three times with deionized water and dried at 40°C.

[0094] The same method was used to prepare a 7.5% (w / v) PCL film as an additional friction layer, with a final thickness of approximately 100 μm.

[0095] Step S2: preparing the intermediate spacer layer

[0096] The middle spacer layer was prepared using 7.5% (w / v) PCL solution and had a thickness of about 400 μm.

[0097] Step S3: preparing the encapsulation layer

[0098] The encapsulation layer consisted of a 15% (w / v) PLGA film with a thickness of 200 μm.

[0099] Step S4, preparing the electrode layer

[0100] 0.4 g of dopamine (DA) powder was dissolved in 25 mL of 0.01 M NaOH solution, and 0.25 g (1% by mass) of silk fibroin-derived peptide (Cs) powder was added. The mixture was stirred continuously at 600 rpm for 20 minutes. In a 70°C water bath, 15.0 g of acrylamide (AM), 1.5 g of ammonium persulfate (APS), and 0.05 g of N,N′-methylenebisacrylamide (BIS) were added and stirred for 5 minutes. Subsequently, 60 μL of tetramethylethylenediamine (TMEDA) was added, and the solution was poured into a cylindrical mold with a diameter of 1 cm. Gel polymerization was performed in a 70°C water bath.

[0101] Step S5: packaging

[0102] The above four-layer structure is thermally packaged in the order of packaging layer, friction layer PCL, spacer layer PCL, friction layer PLGA, electrode layer, and packaging layer.

[0103] Example 2 :

[0104] Different from the above-mentioned embodiment 1, in step S4 of preparing the electrode layer, the mass fraction of the silk fibroin-derived peptide (Cs) powder added in this embodiment is 2% (wt%), ie, 0.5 g.

[0105] Example 3 :

[0106] Different from the above-mentioned embodiment 1, in step S4 of preparing the electrode layer, the mass fraction of the silk fibroin-derived peptide (Cs) powder added in this embodiment is 0.5% (wt%), ie, 0.125 g.

[0107] Comparative Example 1 :

[0108] Different from the above-mentioned embodiment 1, in step S4 of preparing the electrode layer, the mass fraction of the silk fibroin-derived peptide (Cs) powder added in this embodiment is 4% (wt%), ie, 1 g.

[0109] Comparative Example 2 :

[0110] Different from the above-mentioned embodiment 1, in this embodiment, in step S4 of preparing the electrode layer, no silk fibroin-derived peptide (Cs) powder is added.

[0111] Figure 2 Schematic diagram of the static potential of the electrode layer surface with different concentrations of Cs added. Figure 2 It can be seen that the electrode layer of the composite membrane containing 2% Cs can restore the endogenous potential (-50 to -100 mV) under static physiological conditions within the critical-sized bone defect. However, the electrode layer of the composite membrane containing more than 2% Cs (for example, 4%) or the composite membrane containing no Cs cannot restore the endogenous potential under static physiological conditions within the critical-sized bone defect.

[0112] Figure 3 Schematic diagram of the electrode layer's self-adhesion test. After pressing the prepared PPCs hydrogel electrode layer against a portion of Sprague-Dawley (SD) rat skull for 30 seconds, the electrode layer was able to lift a 1kg weight, demonstrating its excellent self-adhesion and ability to bond tightly to the bone.

[0113] Figure 4 The action potential output of the composite membrane at different frequencies is shown. Figure 5 The comparison of electrical output between PPCs and copper as electrode layers is shown. It can be seen that the PPCs electrode layer has better electrical output performance.

[0114] Figure 6 The in vivo degradation performance of the composite membrane is shown. As can be seen from the figure, after 12 weeks, the composite membrane in the rat skull can be completely degraded and absorbed.

[0115] Figure 7 The figure shows the in vivo bone repair effect of the composite membrane. As can be seen from the figure, after 8 weeks, the bone repair effect of the composite membrane of the present invention is significantly better than that of the known bone damage repair membrane.

[0116] Experimental results show that the present invention provides a biodegradable self-adhesive composite membrane that can be used to activate the "mechanical-electrical-biochemical" cascade effect in situ to repair critical-size bone defects. The composite membrane has self-adhesion ability and is tightly integrated with the bone, thereby achieving effective and continuous electrical signal transmission.

[0117] Furthermore, the composite membrane can detect subtle mechanical change signals and generate biofeedback electrical stimulation in situ, significantly accelerating critical bone defect repair. Specifically, through the contact separation of the friction layer, subtle, motion-induced loads can be converted into electrical signals, providing real-time biofeedback-driven, self-regulated electrical stimulation. At the same time, the electrical stimulation generated by the composite membrane directly regulates extracellular and intracellular biochemical signals, changing cell behavior. This mechanical-electrical stimulation further enhances the enrichment of osteogenesis-related extracellular biochemical factors such as calcium ions, phosphate ions, and growth factors in the bone defect area. In addition, it also activates the intracellular mechanosensitive protein Piezo1, promotes calcium influx, and initiates calcium signaling and mechanically driven intracellular transduction pathways, thereby enhancing the proliferation, migration, and osteogenic / angiogenic differentiation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells.

[0118] In summary, the present invention provides a biodegradable, self-adhesive composite membrane for repairing critical-sized bone defects. It enables biomimetic electrical stimulation that self-regulates with the load state of the exercise. Compared to existing technologies, the composite membrane proposed in this invention exhibits excellent biocompatibility and robust osteoinduction, making it suitable for repairing bone defects of varying locations, sizes, and conditions, demonstrating excellent adaptability.

[0119] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of the present invention. Therefore, the scope of the present invention shall be determined by the appended claims.

Claims

1. A self-generating composite membrane for repairing critical-size bone defects, comprising a friction layer, an electrode layer, and an external package, characterized in that: The electrode layer is a PPCs hydrogel, which includes polyacrylamide (PAM), polydopamine (PDA) and silk fibroin-derived peptide (Cs).

2. The self-generating composite membrane according to claim 1, characterized in that: In terms of mass fraction, the PPCs hydrogel comprises: polyacrylamide, 60%; polydopamine, 1.6%; Silk fibroin-derived peptide, 0.5% to 2%.

3. The self-generating composite membrane according to claim 1, characterized in that: In terms of mass fraction, the PPCs hydrogel comprises: polyacrylamide, 60%; polydopamine, 1.6%; Silk fibroin-derived peptides, 2%.

4. The self-generating composite membrane according to claim 2 or 3, characterized in that: In terms of mass fraction, the PPCs hydrogel further comprises: Ammonium persulfate (APS), 6%; N,N′-methylenebisacrylamide (BIS), 0.2%.

5. A method for preparing the self-generating composite membrane according to claim 1, characterized in that: The process of preparing the electrode layer includes: (1) Dopamine (DA) powder was dissolved in NaOH solution, and silk fibroin-derived peptide (Cs) powder was added and stirred continuously; (2) Add acrylamide (AM), initiator and cross-linking agent and stir continuously; (3) Adding a catalyst, pouring the solution into a mold, and preparing a hydrogel electrode layer with a predetermined shape.

6. The preparation method according to claim 5, characterized in that In terms of mass fraction: polyacrylamide, 60%; polydopamine, 1.6%; Silk fibroin-derived peptides, 0.5%–2%; initiator, 6%; Cross-linking agent, 0.2%.

7. The preparation method according to claim 6, characterized in that In terms of mass fraction: polyacrylamide, 60%; polydopamine, 1.6%; silk fibroin-derived peptides, 2%; initiator, 6%; Cross-linking agent, 0.2%.

8. The preparation method according to any one of claims 5 to 7, characterized in that The initiator is ammonium persulfate (APS), and the cross-linking agent is N,N'-methylenebisacrylamide (BIS).

9. A method for preparing an electrode layer of a composite membrane for repairing critical size bone defects, characterized in that: The method includes: (1) Dopamine is subjected to a self-polymerization reaction in an alkaline aqueous solution to form a PDA chain; wherein a silk fibroin-derived peptide (Cs) is added during the dopamine polymerization process; (2) Adding acrylamide to polymerize and generate PAM chains; wherein, PDA chains and PAM chains are randomly cross-linked by physical and covalent means to form a PDA-PAM polymer network.

10. The method for preparing an electrode layer according to claim 9, wherein: Calculated by mass fraction, the mass fraction ratio of acrylamide, dopamine and silk fibroin-derived peptide is 60:1.6:0.5-2, preferably 60:1.6:2.

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