Preparation method and application of a double-sided hard film sealant that can regulate fibroblast activity
By designing a double-sided dural sealant, side A promotes the activity of fibroblasts in the dural layer, while side B inhibits the activity of fibroblasts in the muscle layer. This solves the problem that dural sealant materials cannot simultaneously seal cerebrospinal fluid leakage and prevent epidural fibrosis, thus achieving simultaneous treatment of dural injury repair and fibrosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JILIN UNIVERSITY
- Filing Date
- 2026-06-09
- Publication Date
- 2026-07-10
AI Technical Summary
Existing dural sealing materials cannot simultaneously and effectively seal cerebrospinal fluid leakage and prevent epidural fibrosis, and existing strategies cannot simultaneously promote dural injury repair and prevent epidural fibrosis.
A double-sided hard membrane sealing adhesive is designed, with A facing the central part of the hard membrane layer to promote fibroblast activity and the surrounding part to provide good adhesion properties, and B facing the muscle layer to inhibit fibroblast activity. A double-sided structure with regulatory capabilities is formed by combining cross-linking agents and biomaterials.
It achieves the sealing of cerebrospinal fluid leakage and the prevention of epidural fibrosis, promotes the repair of dural injury and inhibits the excessive activation of fibroblasts, resolves the contradiction between dural injury and fibrosis, and provides reliable advantages for clinical application.
Smart Images
Figure CN122351561A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical biomaterials, and in particular to a method for preparing and applying a double-sided hard film sealant that can regulate fibroblast activity. Background Technology
[0002] Dural injury and epidural fibrosis are common surgical complications in spinal surgery. Dural injury can cause cerebrospinal fluid leakage, leading to intractable headaches, life-threatening central nervous system infections, and pseudodural cysts. Surgical procedures involving dural exposure often employ a posterior lumbar approach, which typically requires destruction of paraspinal muscles, laminae, and other epidural tissues to obtain a good surgical field. The inflammatory microenvironment created by tissue destruction can promote the proliferation and activation of fibroblasts, resulting in abnormally increased cell migration, enhanced cytoskeleton remodeling, and abnormal extracellular matrix deposition. These pathological processes collectively lead to epidural fibrosis, which can cause "back surgery failure syndrome," resulting in intractable back pain and radiating pain. Despite the continuously increasing risk of cerebrospinal fluid leakage associated with dural injury and its severe sequelae, current clinical management methods are inadequate. For example, the most widely used suturing technique can create pinholes in the dura mater, which the sutures cannot completely fill, allowing for continuous cerebrospinal fluid leakage. Autologous graft coverage is often limited by insufficient donor availability and carries risks of dead space effect and scar adhesion. Currently, various biomedical materials have been developed for dural defect repair and cerebrospinal fluid leakage sealing, with dural patches and dural sealing adhesives being the most widely used. Dural patches typically lack adhesive properties, while dural sealing adhesives suffer from insufficient adhesion in aqueous environments, are prone to swelling, and can cause nerve adhesion. Current treatment and prevention strategies for epidural fibrosis mainly fall into two categories: anti-fibroblast proliferation drugs and physical barrier materials. However, neither of these methods is suitable for preventing epidural fibrosis secondary to dural defects. Fibroblast inhibitors inevitably hinder the essential repair-promoting function of fibroblasts in dural repair, while most physical barrier materials lack adhesive properties and cannot effectively seal cerebrospinal fluid leakage. Therefore, clinically, there is still a lack of therapeutic products that can simultaneously achieve dural defect repair and epidural fibrosis prevention.
[0003] Fibroblasts are the most crucial cells in the repair of dural injuries and the formation of epidural fibrosis. In medical practice, we hope to both enhance fibroblast activity to promote dural repair and inhibit fibroblast activity to prevent epidural fibrosis. This contradiction has caused great difficulties for clinical and research work: existing dural sealing materials are often ineffective in sealing cerebrospinal fluid leakage and neglect the prevention of epidural fibrosis, while current clinical treatment strategies for epidural fibrosis are often unsuitable for situations following dural injury. Existing scientific research has also attempted to address both dural injury and epidural fibrosis simultaneously; however, the lack of targeted regulation of the core element, fibroblasts, casts a shadow over its application prospects. Summary of the Invention
[0004] The purpose of this invention is to solve the problems mentioned in the background art above, and to provide a method for preparing and applying a double-sided hard film sealant that can regulate fibroblast activity.
[0005] This invention is inspired by Janus, the two-faced god in Roman mythology. The Janus design concept is divided into two parts: A facing the dura mater and B facing the muscle layer. The central portion of A facing the dura mater enhances fibroblast activity, while the peripheral portion absorbs cerebrospinal fluid from the dura mater surface and provides good adhesion to the aqueous environment. The muscle layer portion of B inhibits fibroblast activity. This invention can simultaneously achieve cerebrospinal fluid leakage sealing and regulate fibroblast activity, thereby promoting dural injury repair and preventing epidural fibrosis.
[0006] A method for preparing a double-sided dura mater sealant capable of regulating fibroblast activity, wherein the double-sided dura mater sealant comprises an A-side facing the dura mater layer and a B-side facing the muscle layer, wherein the A-side facing the dura mater layer comprises a central portion and a peripheral portion; comprising the following steps: S1: Fabrication of the A-face hard film layer; S2: Prepare B-face muscle layer; S3: Assembly of double-sided hard film sealant.
[0007] In S1, the central portion of A facing the hard film layer is made of PR powder, and the surrounding portion is made of PG powder; The preparation process S1.1 for the PR powder used in the central part is as follows: S1.1.1: Disperse corn gluten powder in an aqueous solution of SDS (sodium dodecyl sulfonate) with a concentration of 250-350 mmol / L to form a stable corn gluten / SDS colloidal solution with a concentration of 120-200 mg / mL. S1.1.2: Dissolve the physical adhesion peptide RKDVY-COOH powder and the cell adhesion peptide RFFRGD-NH2 (abbreviated as R) in deionized water to obtain a transparent solution, such that the concentration of the physical adhesion peptide RKDVY-COOH powder dissolved in deionized water is 150-200 mg / ml, and the concentration of the cell adhesion peptide RFFRGD-NH2 dissolved in deionized water is 5-15 mg / ml; S1.1.3: Take the above transparent solution and corn protein / SDS colloidal solution and mix them at a volume ratio of 1:3 to prepare adhesive D; S1.1.4: The obtained adhesive D is freeze-dried and ground into powder, and named PR powder; The preparation process S1.2 for the PG powder used in the surrounding area is as follows: S1.2.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 250-350 mmol / L, so that the concentration of corn gluten powder in the SDS aqueous solution is 120-200 mg / mL, to form a stable corn gluten / SDS colloidal solution; S1.2.2: Dissolve the physical adhesion peptide RKDVY powder (RKDVY is thymopentin, abbreviated as TP-5 amino acid sequence) in deionized water to make the concentration of physical adhesion peptide RKDVY-COOH powder dissolved in deionized water 150-200 mg / ml, and obtain a physical adhesion peptide solution, named P solution (P is the abbreviation for peptide). S1.2.3: Mix solution P with corn protein / SDS colloidal solution at a volume ratio of 1:3 and stir to obtain adhesive E; S1.2.4: The obtained adhesive E is freeze-dried, ground into powder, and then mixed with genipin (G) powder at a mass ratio of 100:9.5 to obtain a powder, which is named PG powder.
[0008] In step S2, the preparation of the B-side muscle layer includes the following steps: S2.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 250-350 mmol / L, so that the concentration of corn gluten powder in the SDS aqueous solution is 120-200 mg / mL, to form a stable corn gluten / SDS colloidal solution. S2.2: Chitosan (C) and chitosan-Oligosaccharide (O) are co-dissolved in deionized water to achieve a chitosan concentration of 2.5-7.5 mg / mL and a chitosan-Oligosaccharide concentration of 0.05-0.15 mg / mL, forming a chitosan / chitosan-Oligosaccharide mixed solution; the chitosan / chitosan-Oligosaccharide mixed solution is mixed with corn protein / SDS colloidal solution at a volume ratio of 1.5:1, placed in a ring mold to form a film, labeled as CO membrane.
[0009] In S3, the assembly of the double-sided hard film sealant involves placing PR powder and PG powder on the upper surface of the CO membrane, with the PR powder located in the central region and the PG powder distributed around the PR powder; then, deionized water is dropped onto the surface of the PR powder and PG powder, and after freeze-drying, a double-sided hard film sealant that can regulate fibroblast activity is obtained.
[0010] The resulting double-sided hard film sealing adhesive, which can regulate fibroblast activity, can be stored for a long time after freeze-drying.
[0011] Application of the double-sided dura mater sealant with regulated fibroblast activity obtained according to the above preparation method in the preparation of dura mater sealants or epidural fibrosis prevention.
[0012] The applications of the double-sided dura mater sealant with modifiable fibroblast activity obtained according to the above preparation method in the preparation of dura mater sealants or epidural fibrosis prevention include: Products for preparing dura mater sealing and cerebrospinal fluid leakage sealing; Or prepare products for the prevention of epidural fibrosis after laminectomy; Or prepare products that promote the repair and healing of dural injuries; Or develop products to treat post-spinal surgery failure syndrome of the back; Or, to prepare products that simultaneously promote dural repair and prevent epidural fibrosis after dural injury in spinal and neurosurgical procedures.
[0013] The working process and working principle of this invention: This invention provides a method for preparing a double-sided dura mater sealant. The sealant prepared by this method consists of an A-side facing the dura mater layer and a B-side facing the muscle layer. The central portion of the A-side facing the dura mater layer uses SDS-modified corn protein as a cross-linking agent, which is cross-linked with the physical adhesion peptide RKDVY-COOH and the cell adhesion peptide RFFRGD-NH2 to form a PR component, significantly promoting the migration ability and ECM formation ability of fibroblasts. The peripheral portion of the A-side facing the dura mater layer is cross-linked with the physical adhesion peptide RKDVY-COOH and SDS-modified corn protein, and then with the covalent cross-linking agent genipin to form a PG component. This PG component provides excellent adhesion properties in an aqueous environment to seal cerebrospinal fluid leakage and prevent it from damaging the fibroblast ECM. Thus, the A-side facing the dura mater layer possesses the ability to comprehensively upregulate fibroblast activity and promote its repair function against dura mater damage. B-type fibroblasts, targeting the muscle layer, also use SDS-modified corn protein as a cross-linking agent. Simultaneously, they cross-link with chitosan and chitosan oligosaccharides to form a thin-film CO structure, inhibiting macrophage polarization towards the M1 type. Furthermore, through antioxidant effects, they comprehensively improve the inflammatory environment, preventing excessive activation of fibroblasts. This endows B-type fibroblasts with the ability to inhibit fibroblast activity in the muscle layer, providing an inhibitory effect on epidural fibrosis.
[0014] Peptide-based biomaterials combine the sequence diversity, residue tunability, and backbone similarity of protein molecules with the controllable synthesis and mass production capabilities of artificial molecules, making them an ideal choice for constructing biofunctional materials. Peptide-based biomaterials not only show promising prospects for individual applications, but also achieve excellent results when used in combination with other components. In particular, the combination of peptides and proteins, due to their similar composition and good biocompatibility, often exhibits a "1+1>2" effect. Zein is a high-yield, renewable, biodegradable protein with excellent mechanical properties, ease of production, low environmental impact, and sustainable development potential. It is a green candidate protein for biomaterial development, and its ability to form cross-links with arginine makes it highly suitable for the synthesis of peptide-based biomaterials.
[0015] This invention presents a double-sided dura mater sealing adhesive that regulates fibroblast activity, using peptides as the core component and employing various cross-linking methods. First, a non-swelling soft adhesive, corn protein / SDS, prepared from SDS-modified corn protein cross-linked physical adhesion peptides suitable for sealing dura mater injuries, is upgraded and assembled. The design features two sides, A and B, with side A facing the dura mater and side B facing the muscle layer. A novel peptide sequence, RFRRGD-NH2, containing the cell adhesion peptide sequence RGD, is added to the central portion of side A facing the dura mater. Compared to the traditional RGD component, the arginine R at the head end of RFRRGD-NH2 can form a cross-linking network with corn protein / SDS through electrostatic and hydrogen bonding interactions. The local hydrophobic environment created by the FF (phenylalanine-phenylalanine) fragment near R stabilizes these polar bonds, preventing premature dissociation in a highly polar aqueous environment, thus forming the final PR component. This portion covers the dura mater injury site during use to promote fibroblast repair of the dura mater. The portion surrounding the A-side dura mater layer incorporates genipin, a natural covalent cross-linking agent, into the adhesive E to form the PG component. PG can simultaneously form covalently cross-linked genipin with primary amine groups in collagen and chitosan, or lysine residues in peptide molecules, improving its adhesion strength in humid environments and providing cohesion for the components within the double-sided dura mater sealant, ensuring structural stability. When applied, PG covers the intact dura mater, forming the A-side dura mater layer, which simultaneously promotes fibroblast activity and provides reliable cerebrospinal fluid leakage sealing. The B-side muscle layer utilizes a cross-linked solution of SDS-modified corn protein and chitosan-chitosan oligosaccharide. Leveraging the film-forming properties of chitosan and the anti-inflammatory, antioxidant, and anti-fibrotic properties of chitosan oligosaccharide, a CO film is formed on the B-side muscle layer, inhibiting fibroblast activity. This film is tightly connected to the A-side dura mater layer through cross-linking with genipin in the PG component, forming a unified whole. It is stored and used via lyophilization, extending its shelf life and making application more convenient, demonstrating its advantages in clinical settings.
[0016] In vivo, various peptides are closely related to the regulation of fibroblast activity. For example, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can promote fibroblast activation and collagen synthesis to promote tissue repair. The glycine-histidine-lysine (GHK) sequence, first discovered in human plasma in the 1970s, has been shown to regulate the expression of fibroblast-related genes, thereby stimulating collagen synthesis and accelerating wound healing. Lysine-proline-valine (KPV) has been found to be added to hydrogel films (KPV@PPP_E) and regulate fibroblast proliferation and migration, stimulate collagen formation, enhance fibroblast ECM remodeling function, and improve tissue repair performance. RGD peptides, composed of a tripeptide sequence (Arg-Gly-Asp) of arginine (Arg), glycine (Gly), and aspartic acid (Asp), are core cell adhesion motifs ubiquitously present in the extracellular matrix (ECM). They are the smallest units mediating fibroblast adhesion activity, mediating key physiological processes such as cell adhesion, migration, and signal transduction by binding to integrin receptors on the cell membrane surface, and have wide applications in biomedicine and tissue engineering. RGD tissue engineering scaffolds can promote cell adhesion. When fixed on polymer scaffolds, they can activate fibroblasts and enhance their adhesion and proliferation functions. RGD can promote fibroblast adhesion to the matrix, prevent apoptosis, and promote the regeneration of new tissue attached to the matrix. Linking RGD to the carboxyl groups on the surface of graphene oxide (GO) results in good biocompatibility with fibroblasts, allowing fibroblasts to be evenly distributed on the material surface and exhibiting a well-structured cytoskeleton, with significantly improved adhesion and proliferation properties. RGD-modified biomaterials improve cell attachment, alignment, and proliferation while regulating the expression of type I and type V collagen and proteoglycans, demonstrating their ability to enhance tissue repair rate while ensuring effective tissue repair. Materials containing RGD-related components have been shown to significantly promote fibroblast activity, and RGD has a positive effect on nerve cell growth.
[0017] The beneficial effects of this invention are: This invention has the ability to bidirectionally regulate fibroblast activity, both increasing the activity of dural fibroblasts and inhibiting the activity of extradural fibroblasts, thus resolving the contradiction in the regulation of fibroblast activity during dural injury repair and extradural fibrosis prevention. At the same time, it can provide reliable cerebrospinal fluid leakage sealing performance, solving the problem of simultaneously achieving dural injury repair and extradural fibrosis prevention. Attached Figure Description
[0018] Figure 1 This is a schematic diagram of the preparation process of the method described in Embodiment 1 of the present invention.
[0019] Figure 2 This is a cross-sectional view of the double-sided hard film sealant prepared by the method described in Example 1 of the present invention.
[0020] Figure 3 This is a schematic diagram of the double-sided hard film sealing adhesive A facing the hard film layer prepared by the method described in Embodiment 1 of the present invention.
[0021] Figure 4 This is a physical image of the external burst pressure verification device in Embodiment 2 of the present invention.
[0022] Figure 5 This is a comparison chart of the external burst pressure verification results in Embodiment 2 of the present invention, where * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001, ns indicates P >0.05.
[0023] Figure 6 This is an optical microscope image showing the in vitro regulation of fibroblast migration ability by the components of the double-sided hard film sealing adhesive in Example 3 of the present invention.
[0024] Figure 7 This is a comparative graph showing the quantitative analysis of scratch test results in Example 3 of the present invention to evaluate the effect of double-sided hard film sealant on fibroblast migration performance, where * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001, ns indicates P >0.05.
[0025] Figure 8 These are fluorescence micrographs of the components of the double-sided hard film sealing adhesive used in Example 3 of this invention to regulate the ability of fibroblast ECM formation in vitro.
[0026] Figure 9 These are fluorescence micrographs showing the ability of each component of the double-sided hard film sealing adhesive to inhibit macrophage M1 polarization in vitro, as described in Example 4 of this invention.
[0027] Figure 10 In Example 4 of this invention, (a) is a quantitative analysis diagram of CD86 immunofluorescence staining in RAW264.7 macrophages, n=5; (b) is a diagram of SOD expression level detection in NIH3T3 fibroblasts, n=5, * indicates P <0.05, ** indicates P <0.01, *** indicates P<0.001, **** indicates P <0.0001, ns indicates P >0.05.
[0028] Figure 11 The image shows, under an optical microscope, the double-sided dura mater sealing adhesive and its components in Example 5 of this invention, and their ability to regulate fibroblast activity, repair dura mater damage, and prevent epidural fibrosis.
[0029] Figure 12 This is a gait analysis diagram of different groups of experimental animals at 8 weeks post-surgery in Example 5 of the present invention. Detailed Implementation
[0030] Please see Figures 1 to 3 As shown, this is Embodiment 1 of the present invention.
[0031] A method for preparing a double-sided dura mater sealant that can regulate fibroblast activity, wherein the double-sided dura mater sealant is composed of an A-side facing the dura mater layer 1 and a B-side facing the muscle layer 2, wherein the A-side facing the dura mater layer 1 is composed of a central portion 11 and a peripheral portion 12. Includes the following steps: S1: Prepare A-face hard film layer 1.
[0032] In S1, the central portion 11 of A facing the hard film layer 1 is made of PR powder, and the surrounding portion 12 is made of PG powder; The preparation process S1.1 of the PR powder used in the central part 11 is as follows: S1.1.1: Disperse corn gluten powder in a 300 mmol / L SDS aqueous solution to achieve a concentration of 150 mg / mL, forming a stable corn gluten / SDS colloidal solution; adjust the pH to 7.4. S1.1.2: The physical adhesion peptide RKDVY-COOH powder and the cell adhesion peptide RFFRGD-NH2 (abbreviated as R) were co-dissolved in deionized water to obtain a transparent solution, so that the concentration of the physical adhesion peptide RKDVY-COOH powder dissolved in deionized water was 180 mg / ml and the concentration of the cell adhesion peptide RFFRGD-NH2 dissolved in deionized water was 10 mg / ml; the pH value was adjusted to 7.4. S1.1.3: Take the above transparent solution and corn protein / SDS colloidal solution and mix them at a volume ratio of 1:3 to prepare adhesive D; S1.1.4: The obtained adhesive D is freeze-dried and ground into powder, and named PR powder; The preparation process S1.2 of the PG powder used in the surrounding part 12 is as follows: S1.2.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 300 mmol / L to achieve a concentration of 150 mg / mL in the SDS aqueous solution, thereby forming a stable corn gluten / SDS colloidal solution. S1.2.2: Dissolve the physical adhesion peptide RKDVY-COOH powder in deionized water to make the concentration of the physical adhesion peptide RKDVY-COOH powder dissolved in deionized water 180mg / ml, and obtain a physical adhesion peptide solution, named P solution; S1.2.3: Mix solution P with corn protein / SDS colloidal solution at a volume ratio of 1:3 and stir to obtain adhesive E; S1.2.4: The obtained adhesive E is freeze-dried, ground into powder, and then mixed with genipin (G) powder at a mass ratio of 100:9.5 to obtain a powder, which is named PG powder.
[0033] S2: Prepare B-face muscle layer 2.
[0034] In step S2, the preparation of B-face muscle layer 2 includes the following steps: S2.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 300 mmol / L to achieve a concentration of 150 mg / mL in the SDS aqueous solution, thereby forming a stable corn gluten / SDS colloidal solution. S2.2: Chitosan and chitosan oligosaccharide are co-dissolved in deionized water to make the concentration of chitosan 3.0 mg / mL and the concentration of chitosan oligosaccharide 0.1 mg / mL, forming a chitosan / chitosan oligosaccharide mixed solution; the pH is adjusted to 4.5; the chitosan / chitosan oligosaccharide mixed solution is mixed with corn protein / SDS colloidal solution at a volume ratio of 1.5:1, and placed in a ring mold with a radius of 1 cm and a thickness of 5 cm to form a film, which is labeled as CO film.
[0035] S3: Assembly of double-sided hard film sealant.
[0036] 50 mg of PR powder and 50 mg of PG powder were placed on the surface of a CO membrane, with the PR powder located in the central region with a radius of 5 mm, and the PG powder distributed around the PR powder. Deionized water was then dropped onto the surface of the PR and PG powders, and after freeze-drying, a double-sided hard membrane sealing adhesive that can regulate fibroblast activity was obtained.
[0037] The resulting double-sided hard film sealing adhesive, which can regulate fibroblast activity, can be stored for a long time after freeze-drying.
[0038] See Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown, this is Embodiment 2 of the present invention.
[0039] The double-sided hard film sealing adhesive prepared according to Example 1 is used in products for hard film sealing and cerebrospinal fluid leakage sealing.
[0040] The most basic function of double-sided dura mater sealing adhesive is to seal cerebrospinal fluid leakage, which requires it to withstand cerebrospinal fluid pressure when adhering to the dura mater. (Usage example...) Figure 4 The modified ASTM F2392-04 model shown was used for burst pressure testing of the double-sided hard film sealant and its components. Figure 5 As shown, the initial burst pressure of the PR component (A) facing the central portion 11 of the dura mater 1 was 22.44±0.59 cmH2O, and the burst pressure after 24 hours was 24.66±0.37 cmH2O. The initial burst pressure of the PG component (A) facing the peripheral portion 12 of the dura mater 1 was 25.84±0.95 cmH2O, and its burst pressure increased to 46.72±2.10 cmH2O after 24 hours. For the CO component (B) facing the muscle layer 2, its burst pressure was 3.54±0.288 cmH2O initially and 3.84±0.312 cmH2O after 24 hours. The burst pressures of the double-sided dura mater sealant were 21.38±1.26 cmH2O initially and 41.98±1.94 cmH2O after 24 hours. Intraoperative cerebrospinal fluid (CSF) pressure in the supine position is 8-18 cmH2O, while postoperative CSF pressure in the sitting and standing positions can reach 20-30 cmH2O. The double-sided dura mater sealant initially relies on the non-covalent forces of the P component for adhesion, which is rapid and has a burst pressure sufficient to withstand the supine CSF pressure. Subsequently, as the genipin cross-linking process gradually completes, the double-sided dura mater sealant, especially the PG component, withstands CSF pressures exceeding the highest CSF pressure in the sitting and standing positions, consistent with clinical application guidelines and serving as the main component providing reliable sealing performance. The CO membrane, facing the muscle layer 2, is non-adhesive due to the film-forming properties of chitosan, ensuring convenient intraoperative use and preventing adhesion to instruments. It also avoids physical adhesion between muscle tissue and the dura mater. The PR component, lacking genipin, maintains unchanged burst pressure both immediately and over 24 hours, preventing covalent bonding with nerve tissue at the site of dural injury and thus avoiding potential disturbance to nerve tissue. It is worth mentioning that a reliable cerebrospinal fluid leak seal can better seal dural injuries and avoid related complications, and can also prevent cerebrospinal fluid leakage from damaging the formation of fibroblast ECMs that repair damage, thus laying a good foundation for regulating fibroblast function.
[0041] See Figure 2 , Figure 3 , Figure 6 , Figure 7 and Figure 8As shown, this is Embodiment 3 of the present invention.
[0042] The double-sided dural sealing adhesive prepared according to Example 1 is a product that promotes dural injury repair and prevents epidural fibrosis after dural injury in spinal surgery and neurosurgery.
[0043] The ability of the double-sided hard film sealing adhesive prepared according to Example 1 to regulate fibroblast migration was verified by a scratch assay. NIH3T3 fibroblasts were seeded in 6-well plates and cultured to 80% confluence. A straight line was vertically drawn at the bottom of each well using a sterile 200 μL pipette tip to create a cell scratch. Exfoliated cells were washed away with PBS. The experimental groups were treated with PG, CO, or PR, respectively, while the blank group was untreated. The complete culture medium was replaced with DMEM medium containing 0.1% fetal bovine serum (FBS) to inhibit cell proliferation. The migration of NIH3T3 fibroblasts was observed under an optical microscope at 0, 8, 16, and 24 h after scratching. Images were acquired and analyzed using ImageJ software. The scratch healing rate was calculated using the following formula: Cell migration rate (%) = (cell migration area within scratch at 0h, 8h, 16h, or 24h / total scratch area at 0h) × 100%.
[0044] Under the combined influence of the PI3K / Akt and TGFβ / Smad pathways regulated by various components of the double-sided hard suture adhesive, the physiological activities of fibroblasts were subsequently altered. The migration ability of fibroblasts has a significant impact on their tissue repair function and is a crucial step in fibrosis formation. For example... Figure 6 As shown, the central component PR group (A facing the dura mater 1) showed significantly higher cell migration rates at 8, 16, and 24 hours compared to other groups. P <0.0001, demonstrating stronger cell migration ability after activation of related pathways, while the CO membrane facing muscle layer 2 showed lower migration ability than the control group ( P <0.0001), which is consistent with the expression level of the relevant pathway. Meanwhile, the PG component of the A-side, which only performs physical adhesion, showed no significant difference in cell migration rate at any time point compared to the control group. P> 0.05). The quality of the ECM formed by fibroblasts determines the quality of tissue repair and the degree of fibrosis, and Col-I is the most important component of the fibroblast ECM. Figure 8As shown, the Col-I expression level in the PR group was highest at 24, 48, and 72 hours, with a higher degree of cell density observed at 72 hours. This is related to the increased cell adhesion and activation of the aforementioned pathways resulting from the addition of the RGD sequence. In contrast, the ECM expression level in the CO group was lowest at all time points, while the PG group showed no significant difference compared to the control group.
[0045] Fibroblast repair of dural injuries requires continuous migration from both ends of the dura mater to cover the damaged area, forming an extracellular matrix (ECM) to anchor the cytoskeleton and create robust new fibrous tissue to cover the dural injury. Prevention of epidural fibrosis necessitates minimizing excessive fibroblast migration into the epidural space and avoiding the formation of high-intensity fibrotic scars that could lead to adhesions or compression. For layer A (dura mater 1), the central PR component exhibits the strongest cell migration ability, meaning it can mediate rapid fibroblast traversal of the dural injury site, accelerating the repair process. Higher-quality ECM formation results in denser, stronger new fibrous tissue, providing better dural repair. Conversely, layer B (copper oxide membrane) facing muscle layer 2 significantly inhibits both fibroblast migration and ECM formation, preventing excessive migration of fibroblasts from peripheral lesions like muscle into the epidural space and inhibiting the formation of excessively strong fibrous tissue that could cause adhesions and compression to the dura mater and nerve tissue. It is worth mentioning that although the PG component does not regulate the activity of fibroblasts, its excellent sealing performance can wrap the PR component 11 facing the central part of the dura mater 1 at the site of dura mater injury, preventing it from activating fibroblasts outside the dura mater, and also preventing the CO component facing the muscle layer 2 from inhibiting the activity of fibroblasts involved in dura mater repair.
[0046] See Figure 2 , Figure 3 , Figure 9 and Figure 10 As shown, this is Embodiment 4 of the present invention.
[0047] The double-sided dural sealing adhesive prepared according to Example 1 is used in the prevention or treatment of post-spinal surgery failure syndrome of epidural fibrosis after laminectomy.
[0048] The ability of the double-sided dura mater sealing adhesive prepared according to Example 1 to inhibit M1 macrophage polarization was verified. An in vitro inflammation model was established using LPS stimulation of RAW264.7 cells. In short, RAW264.7 cells were stimulated at 2 × 10⁻⁶... 6Cells were seeded at a density of cells / well in 6-well plates. The control group was cultured in complete medium, while the pro-inflammatory model group and treatment groups were cultured in complete medium containing 1 μg / mL LPS. Experimental groups were further treated with PG, CO, or PR, respectively, while the blank group received no treatment. Cells were incubated at 37°C for 48 h. After incubation, the supernatant was discarded, and cells were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. The paraformaldehyde was removed, and cells were blocked with BSA for 2 h. The blocking solution was discarded, and primary antibody diluted with 1% BSA in PBS was added. Cells were incubated at 37°C for 1.5 h. After washing three times with PBST, secondary antibody diluted with 1% BSA in PBS was added, and cells were incubated at 37°C for 1 h. After washing three times with PBST, DAPI anti-fluorescence quenching mounting medium was added, and cells were washed with PBS. The stained cells were then observed under an inverted optical microscope.
[0049] The inflammatory microenvironment of the epidural tissue can stimulate fibroblasts to become overactivated, thus exacerbating epidural fibrosis. Improving the inflammatory microenvironment through anti-inflammatory and antioxidant effects is one of the effective strategies for preventing epidural fibrosis. The chitosan oligosaccharide component in the CO membrane of the muscle layer of the double-sided epidural sealant (B-side) has been proven by multiple studies to have excellent anti-inflammatory and antioxidant properties. Figure 9 The results showed its regulatory effect on macrophage differentiation, especially in the inflammatory environment simulated by LPS stimulation. Both the control group and the PG group showed macrophage polarization to the M1 phenotype, while the PR group showed slightly lower polarization. Figure 10 Quantitative analysis of (a) showed no statistically significant difference. P> (0.05), in comparison, the CO membrane containing chitosan oligosaccharide significantly inhibited macrophage polarization towards the M1 phenotype due to its obvious anti-inflammatory properties. Facing oxidative stress in fibroblasts, the B-side CO membrane towards the muscle layer exhibited more significant antioxidant properties compared to other groups ( P <0.0001), Figure 10 (b) The SOD kit test results yielded the same conclusion.
[0050] See Figure 11 and Figure 12 As shown, this is Embodiment 5 of the present invention.
[0051] The double-sided dural sealing adhesive prepared according to Example 1 is a product that promotes dural injury repair and prevents epidural fibrosis after dural injury in spinal surgery and neurosurgery.
[0052] The ability of the double-sided dura mater sealing adhesive prepared according to Example 1 to regulate fibroblast activity, promote dural damage repair, and prevent epidural fibrosis was verified.
[0053] A rat model of L1 laminectomy and dural injury was established under isoflurane inhalation anesthesia. Preoperatively, the skin on the back was prepared and disinfected. A 3cm longitudinal incision was made along the midline of the back, centered on the L1 spinous process. The spinous process and lamina were exposed layer by layer. L1 segmental laminectomy was performed using microsurgical laminectomy forceps to expose the dura mater. A 5mm defect was created in the dura mater using microsurgical instruments; successful modeling was confirmed by the outflow of a mixture of cerebrospinal fluid and blood. Animal grouping and treatment: Rats were randomly divided into 5 groups according to different treatment strategies: blank group, PG / CO group (lacking PR component), PRG group (lacking CO membrane), PR / CO group (lacking PG component), and double-sided dural sealing adhesive group. Rats were housed in a dedicated experimental rat enclosure under clean, ventilated, temperature- and humidity-controlled conditions. Healthy rats were sacrificed by CO2 inhalation at 3 days, 2 weeks, and 8 weeks postoperatively. Spine tissues collected from SD rats were fixed in 4% paraformaldehyde buffer for 7 days, followed by decalcification in 10% EDTA PBS (pH 7.4) at 37°C for 4 weeks. After graded ethanol dehydration, the specimens were embedded in paraffin to prepare 4 μm thick tissue sections. The sections were stained with hematoxylin and eosin (HE) and Masson's stain, mounted with neutral resin, and observed using an inverted optical microscope.
Claims
1. A method for preparing a double-sided dura mater sealant capable of regulating fibroblast activity, wherein the double-sided dura mater sealant comprises an A-side facing the dura mater layer (1) and a B-side facing the muscle layer (2), wherein the A-side facing the dura mater layer (1) comprises a central portion (11) and a peripheral portion (12); characterized in that, Includes the following steps: S1: Prepare A-face hard film layer (1); S2: Prepare B-face muscle layer (2); S3: Assembly of double-sided hard film sealant.
2. The method for preparing a double-sided hard film sealing adhesive capable of regulating fibroblast activity according to claim 1, characterized in that: In S1, the central portion (11) of A facing the hard film layer (1) is made of PR powder, and the surrounding portion (12) is made of PG powder; The preparation process S1.1 of the PR powder used in the central part (11) is as follows: S1.1.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 250-350 mmol / L to form a stable corn gluten / SDS colloidal solution with a concentration of 120-200 mg / mL. S1.1.2: Dissolve the physical adhesion peptide RKDVY-COOH powder and the cell adhesion peptide RFFRGD-NH2 in deionized water to obtain a transparent solution, such that the concentration of the physical adhesion peptide RKDVY-COOH powder dissolved in deionized water is 150-200 mg / ml and the concentration of the cell adhesion peptide RFFRGD-NH2 dissolved in deionized water is 5-15 mg / ml. S1.1.3 Take the above transparent solution and corn protein / SDS colloidal solution and mix them at a volume ratio of 1:3 to prepare adhesive D; S1.1.4: The obtained adhesive C was freeze-dried and ground into powder, and named PR powder; The preparation process S1.2 of the PG powder used in the surrounding part (12) is as follows: S1.2.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 250-350 mmol / L to form a stable corn gluten / SDS colloidal solution with a concentration of 120-200 mg / mL. S1.2.2: Dissolve the physical adhesion peptide RKDVY-COOH powder in deionized water to obtain a physical adhesion peptide solution with a concentration of 150-200 mg / ml, which is named P solution. S1.2.3: Mix solution P with corn protein / SDS colloidal solution at a volume ratio of 1:3 and stir to obtain adhesive E; S1.2.4: The obtained adhesive E is freeze-dried, ground into powder, and then mixed with genipin powder at a mass ratio of 100:9.5 to obtain a powder, which is named PG powder.
3. The method for preparing a double-sided hard film sealing adhesive capable of regulating fibroblast activity according to claim 1, characterized in that: In S2, the preparation of B-facing muscle layer (2) includes the following steps: S2.1: Disperse corn gluten powder in an SDS aqueous solution with a concentration of 250-350 mmol / L to form a stable corn gluten / SDS colloidal solution with a concentration of 120-200 mg / mL. S2.2: Chitosan and chitosan oligosaccharide are co-dissolved in deionized water to make the concentration of chitosan 2.5-7.5 mg / mL and the concentration of chitosan oligosaccharide 0.05-0.15 mg / mL, forming a chitosan / chitosan oligosaccharide mixed solution; the chitosan / chitosan oligosaccharide mixed solution is mixed with corn protein / SDS colloidal solution at a volume ratio of 1.5:1, placed in a ring mold to form a film, labeled as CO membrane.
4. The method for preparing a double-sided hard film sealing adhesive capable of regulating fibroblast activity according to claim 1, characterized in that: In S3, the assembly of the double-sided hard film sealant involves placing PR powder and PG powder on the upper surface of the CO membrane, with the PR powder located in the central region and the PG powder distributed around the PR powder; then, deionized water is dropped onto the surface of the PR powder and PG powder, and after freeze-drying, a double-sided hard film sealant that can regulate fibroblast activity is obtained.
5. The application of the double-sided dura mater sealant with regulated fibroblast activity obtained by the preparation method according to any one of claims 1-4 in the preparation of dura mater sealants or epidural fibrosis prevention.
6. The application of the double-sided dura mater sealing adhesive with regulated fibroblast activity according to claim 5 in the preparation of dura mater sealing or epidural fibrosis prevention, characterized in that, include: Products for preparing dura mater sealing and cerebrospinal fluid leakage sealing; Or prepare products for the prevention of epidural fibrosis after laminectomy; Or prepare products that promote the repair and healing of dural injuries; Or develop products to treat post-spinal surgery failure syndrome of the back; Or, to prepare products that simultaneously promote dural repair and prevent epidural fibrosis after dural injury in spinal and neurosurgical procedures.