An artificial dura mater material and a preparation method and application thereof

Abstract

This invention provides an artificial dura mater material, its preparation method, and its applications, belonging to the field of biomedical technology. Addressing the shortcomings of traditional synthetic dura mater, this invention utilizes poly(L-lactide-caprolactone) (PLCL) with a specific concentration of RAPA, processed using electrospinning. It exhibits low porosity and a dense texture, inducing rapid endothelialization of the dura mater in the early stages of traumatic brain injury. Subsequently, with the release of RAPA, the material's porosity increases, facilitating the proliferation and growth of newly formed dura mater cells. Simultaneously, RAPA can prevent excessive proliferation of new endothelial cells and scar hyperplasia. It can selectively inhibit pathological proliferation without impairing necessary lymphatic drainage; while maintaining the physical structural integrity of the dura mater, it can promote neurological function repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an artificial dura mater material, its preparation method, and its application. Background Technology

[0002] The dura mater (spinal cord) is a double-layered membranous tissue located between the inner side of the skull (vertebrae) and the surface of the brain (spinal cord). Its thick and tough nature constitutes an important natural protective barrier for the brain tissue. Its main functions are to protect the brain and spinal cord, maintain the normal functioning of neural electrical processes, and participate in the regulation of neuroimmunity through the meningeal lymphatic vessels. The integrity and airtightness of the dura mater are crucial for performing its protective function. However, in clinical practice, pathological factors such as trauma, inflammation, and tumor invasion, as well as invasive surgical procedures, can all lead to dura mater damage, severely weakening its protective function and resulting in a series of serious complications such as cerebrospinal fluid leakage, epilepsy, and intracranial infection.

[0003] Dural repair can promote the restoration of the normal anatomical structure of the dura mater, reshape the cranial cavity's closure, prevent blood and cerebrospinal fluid leakage, and prevent intracranial infection and reduce the incidence of epilepsy. However, when the dural injury is located at the edge of a bone window, dural repair can become extremely difficult or even impossible. Therefore, a dural repair substitute material that can cover the dural defect and promote the formation of surrounding fibrous connective tissue is needed. The repair substitute material for dural defects directly affects the incidence of complications such as intracranial infection, epilepsy, brain tissue protrusion, and cerebrospinal fluid leakage after dural reconstruction, as well as the aesthetics of subsequent cranioplasty repair.

[0004] Currently available artificial dura mater materials include autologous tissue repair materials, allogeneic repair materials, xenogeneic biological repair materials, and synthetic repair materials. However, the clinical application of autologous tissue repair materials and allogeneic biological materials is greatly limited due to issues such as the area of ​​tissue harvesting and ethical and safety concerns. Although xenogeneic biological repair materials are currently the mainstream in the market, their clinical application is also unsatisfactory due to the risk of allogeneic rejection and the cytotoxic effects caused by the cross-linking process during preparation. Compared with the previous types of natural dura mater patches, the advantages of using chemical and materials science methods to synthesize artificial dura mater patches are that the material specifications are not limited, the price is relatively low, and there is no potential risk of infection. However, synthetic materials lack the biological functions mediated by collagen, such as promoting cell migration and proliferation and promoting the secretion of related cytokines, which is not conducive to the migration and proliferation of fibroblasts. Moreover, the existing synthetic dura mater materials mainly focus on their strength and sealing performance, and cannot simultaneously address the needs of dura mater function repair and neuroprotection for neurological patients. Summary of the Invention

[0005] The purpose of this invention is to provide an artificial dura mater material, its preparation method, and its applications. This material possesses excellent mechanical properties, further improves the hydrophobicity of the polycaprolactone film, and simultaneously achieves sustained-release of RAPA. The artificial dura mater provided by this invention exhibits good leak-proof performance, is biodegradable with safe degradation products, and its loaded sustained-release RAPA drug can induce dura mater regeneration and also provides neuroprotective effects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing an artificial dura mater material, comprising the following steps:

[0008] Poly(L-lactide-caprolactone) was dissolved in an organic solvent to obtain solution 1;

[0009] Rapamycin was added to solution 1 to obtain a spinning solution;

[0010] The spinning solution is spun into a nanofiber membrane by electrospinning, and the artificial dura mater material is obtained after removing the residual solvent.

[0011] Preferably, the poly-L-lactide-caprolactone contains polylactic acid and polycaprolactone, and the mass ratio of polylactic acid to polycaprolactone is 60~80:40~20.

[0012] Preferably, the average molecular weight of the poly-L-lactide-caprolactone is 40,000 to 60,000.

[0013] Preferably, the organic solvent is hexafluoroisopropanol or trifluoroethanol.

[0014] Preferably, the mass concentration of poly(L-lactide-caprolactone) in solution 1 is 8% to 12%.

[0015] Preferably, the concentration of rapamycin in the spinning solution is 0.5% to 10%.

[0016] Preferably, the concentration of rapamycin in the spinning solution is 3-5%.

[0017] Preferably, the voltage for electrospinning is 15~20 kV;

[0018] The electrospinning is carried out using a 20-24G metal needle, with a solution extrusion speed of 0.2-0.5 mm / min, under ambient temperature of 18-25℃ and relative humidity of 30%-50%.

[0019] The removal of residual solvent is achieved through vacuum drying.

[0020] The present invention also provides an artificial dura mater material prepared by the above preparation method.

[0021] The present invention also provides the application of the above-mentioned artificial dura mater material in the preparation or development of materials required for dura mater repair surgery.

[0022] Preferably, the "preparation" includes: using the artificial dura mater material directly as an independent alternative material for dura mater defect repair, or using it as a functional component to construct a composite repair system with other biocompatible materials;

[0023] The term "development" encompasses: developing novel neuroprotective devices using the artificial dura mater material as a carrier platform, or constructing a local drug delivery system based on its sustained-release properties.

[0024] The beneficial effects of this invention are:

[0025] To address the shortcomings of traditional synthetic dura mater, this invention utilizes poly(L-lactide-caprolactone) (PLCL) with a specific concentration of RAPA, processed using electrospinning. The material provided by this invention has the following advantages: 1. Anti-adhesion effect. This invention utilizes the porous properties of electrospun PLCL material to load a sustained-release RAPA into the artificial dura mater. The low porosity and dense texture of the electrospun PLCL composite material induce rapid endothelialization of the dura mater in the early stages of traumatic brain injury. Subsequently, as RAPA is released, the material porosity increases, which is beneficial for the proliferation and growth of newly formed dura mater cells. Simultaneously, RAPA can prevent excessive proliferation of new endothelial cells and scar hyperplasia. 2. Promotion of meningeal lymphatic vessel regeneration. Although RAPA inhibits abnormal lymphatic vessel formation, it preserves the integrity of newly formed lymphatic vessels, marker expression, and in vitro lumen formation ability. This indicates that this invention selectively inhibits pathological proliferation without impairing necessary lymphatic drainage. 3. Neuroprotective effect. RAPA is an FDA-approved immunosuppressant that can reduce inflammatory responses in the nervous system and has potential neuroprotective effects. The RAPA added in this invention can promote the repair of nerve function while maintaining the physical structural integrity of the dura mater. Attached Figure Description

[0026] Figure 1 The preparation process of this artificial dura mater material is shown;

[0027] Figure 2 The physicochemical properties of electrospun nanofiber artificial dura mater with different rapamycin (RAPA) contents (0-8 wt%) were shown;

[0028] Figure 3The study demonstrated the cytotoxicity assessment and neuroprotective mechanism of PLCL-RAPA nanofiber artificial dura mater against oxygen-glucose deprivation / reperfusion (OGD / R) injury.

[0029] Figure 4 The PLCL-RAPA nanofiber artificial dura mater was shown to inhibit the proliferation of fibroblasts and lymphatic endothelial cells in vitro.

[0030] Figure 5 The study demonstrated that the artificial dura mater material could provide neuroprotection, inhibit fibroblast proliferation, and maintain the integrity of meningeal lymphatic vessels in rats with traumatic brain injury.

[0031] Figure 6 The study demonstrated that this artificial dura mater material can modulate the Th17 / Treg balance in rats with traumatic brain injury.

[0032] Figure 7 The study demonstrated that this artificial dura mater material can reduce brain atrophy, decrease adhesion to brain tissue, and reduce neuronal loss in rats with traumatic brain injury. Detailed Implementation

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example

[0034] PLA:PCL (PLA:PCL = 70:30; average molecular weight = 47,000) was dissolved in hexafluoropropanol (Energy Chemical Company) to a concentration of 10%. This solution was then mixed with rapamycin (RAPA) (North China Pharmaceutical Company) at a mass ratio to prepare a spinning solution with a RAPA concentration of 4%. The spinning solution was loaded into a 10 mL syringe and extruded at 18 kV using a 22G needle at a feed rate of 0.35 mm / min. The manufacturing environment was maintained at 20°C and 40% relative humidity. The resulting patch was vacuum dried at room temperature to remove residual solvent. Example

[0035] PLA:PCL (PLA:PCL = 70:30; average molecular weight = 47,000) was dissolved in hexafluoropropanol (Energy Chemical Company) to a concentration of 10%. This solution was then mixed with rapamycin (RAPA) (North China Pharmaceutical Company) at a mass ratio to prepare spinning solutions with RAPA concentrations of 0%, 1%, 2%, 4%, and 8%. The spinning solutions were loaded into 10 mL syringes and extruded at 18 kV using a 22G needle at a feed rate of 0.35 mm / min. The manufacturing environment was maintained at 20°C and 40% relative humidity. The resulting patches were vacuum-dried at room temperature to remove residual solvent.

[0036] Structural and surface characterization of electrospun PLCL artificial dura maters with different RAPA contents (0-8 wt%) confirmed the successful fabrication of uniform nanofiber scaffolds. SEM analysis showed that the addition of RAPA did not significantly alter the fiber morphology; all groups maintained consistent fiber diameter and interconnected porous structures (Figure 2A). This structural preservation is crucial for ensuring mechanical stability, promoting cell infiltration, and nutrient transport. Surface wettability analysis revealed the variation of hydrophilicity with concentration (Figures 2B and 2C). The water contact angle of the pure PLCL membrane was 119.15° ± 1.4°, while the addition of RAPA led to a gradual increase in the contact angle to 121.84° ± 1.45° at an 8% content, demonstrating that the addition of RAPA enhanced the hydrophobicity of the material. Figure 2 D). The drug-loaded membrane reached swelling equilibrium within 72 hours, while the pure PLCL control membrane required 120 hours. The equilibrium swelling rate decreased from 103.5037% ± 18.0372% (PLCL) to 6.593567% ± 1.67105% (PLCL-8%RAPA), which may be due to the hydrophobicity of RAPA limiting water permeation and its potential plasticizing effect on the polymer matrix. Molecular characterization by FTIR confirmed the successful incorporation of RAPA while maintaining the structural integrity of PLCL (Figure 2E). The persistent carbonyl stretching vibration at 1753 cm⁻¹ indicates that the ester bond in PLCL is undisturbed, while the new peaks at 1645 cm⁻¹ (C=C stretching vibration) and 991 cm⁻¹ (CH bending vibration) correspond to the characteristic RAPA functional group (16). XPS analysis showed the presence of a nitrogen 1s peak (399.8 eV) in the drug-loaded membrane. Figure 2 F) further confirms the incorporation of RAPA, while this peak is not present in the pure PLCL spectrum.

[0037] Comprehensive cytotoxicity assessments showed variability in cellular responses to the artificial dura mater extract. Figure 3 A). SH-SY5Y neurons exhibited concentration-dependent sensitivity; cells cultured in PLCL, 1% RAPA, 2% RAPA, and 4% RAPA artificial dura mater extract maintained a survival rate >85% at all concentrations (10-90%) (compliant with ISO 10993-5). Notably, in neurons treated with 8% RAPA extract, the cell survival rate was <50% < 70%, indicating some cytotoxicity. Figure 3 A(a)).

[0038] Microglia (N9) exhibited remarkable resilience, maintaining viability above 90% regardless of RAPA concentration (0–8%) or extract concentration, indicating inherent resistance to the immunomodulatory effects of rapamycin (Fig. 3A(b)). In contrast, C8-D1A astrocytes exhibited complex concentration thresholds: while all formulations were biocompatible at 10% extract concentration, only 2% and 4% RAPA maintained viability above 75% at 50% concentration (Fig. 3A(c), Fig. S2C). Surprisingly, the 4% RAPA group uniquely maintained astrocyte viability (72.3 ± 3.1%) even at 90% extract concentration—the only formulation to reach this threshold (Fig. 3A(c)).

[0039] These findings collectively suggest that while lower RAPA concentrations (1–2%) ensure broad biocompatibility, a 4% loading may achieve the optimal balance between safety and therapeutic efficacy, particularly for neuronal and astrocyte populations.

[0040] PLCL-RAPA provides neuroprotection by modulating the Th17 / Treg balance.

[0041] To investigate the neuroprotective and immunomodulatory potential of rapamycin-loaded membranes, this invention establishes a sequential in vitro ischemia model ( Figure 3 B). Jurkat T lymphocytes were first treated with artificial dura mater extracts (0-8 wt%) containing different concentrations of rapamycin for 24 hours to generate conditioned medium, which was then used for the culture of SH-SY5Y neurons under oxygen-glucose deprivation / reperfusion (OGD / R). Figure 3 B(a)). The neuroprotective capacity of OGD / R-treated SH-SY5Y cells was assessed by immunofluorescence and TUNEL staining. Figure 3 B(b), 3C(a)). The conditioned medium in the 1-4% RAPA treatment group showed a dose-dependent neuroprotective effect, with reduced neuronal apoptosis (TUNEL+ cells) compared with the DMEM control group (p<0.05, p<0.001). Figure 3 B(b), 3C(a)). 8% RAPA medium failed to provide significant protection, confirming the therapeutic concentration threshold ( Figure 3B(b), 3C(a)). Multiparameter immunofluorescence analysis showed that, compared with the DMEM control group, conditioned medium containing 1–4% RAPA membrane induced significant immunophenotypic changes in Jurkat cells (Fig. 3B(c,d), 3C(b,c)): (i) a decrease in CD3+ / CD4+ cell population (p<0.001, Fig. 3B(c), C(b); (ii) downregulation of the Th17 subset (p>0.05, Fig. 3B(d), C(c)); and (iv) Treg cell expansion (p<0.05, p<0.001, Fig. 3B(d), C(c)). These immunomodulatory effects were concentration-dependent and absent in the 8% RAPA group (Fig. 3B(c,d), 3C(b,c)). These results indicate that electrospun rapamycin (≤4% loading) can modulate: (1) Treg polarization expansion, (2) inhibition of pro-inflammatory Th17 responses, and (3) Neuroprotection is achieved under ischemic conditions through paracrine immune regulation.

[0042] The results of this invention demonstrate that rapamycin exhibits multifaceted dose-dependent effects on the dura mater repair process (Figure 4). Quantitative analysis of Ki67+ / Collagen I+ double-positive cells showed that fibroblast proliferation gradually decreased with increasing rapamycin concentration (1-8% w / w) (Figure 4B, D(a)), indicating its potent anti-fibrotic activity and ability to prevent postoperative adhesions. Similarly, lymphatic endothelial cell analysis showed: (i) a decrease in Ki67+ / VEGFR3+ proliferating cells, while maintaining stable VEGFR3 expression (Figure 4C, D(b,c)); (ii) a parallel decrease in Ki67+ / LYVE-1+ cells, without affecting LYVE-1 expression (Figure 4C, D(d,d)). Notably, tube formation assays confirmed that lymphatic function was preserved at all concentrations, with no significant differences in branching points or common tube length (Figure 4E). These results indicate that rapamycin selectively inhibits the proliferation of fibroblasts and lymphovascular endothelial cells while maintaining key lymphatic markers and angiogenesis capacity—an ideal combination for preventing fibrosis and maintaining normal meningeal lymphatic drainage during dural repair.

[0043] Subsequently, this invention applied a controlled cortical impingement (CCI) TBI model to evaluate the effects of the artificial dura mater material repair treatment of this invention on sensorimotor function and its neuroprotective mechanism after TBI injury in rats. After 3 days of adaptation, rats were anesthetized using isoflurane. The rats were placed in a stereotaxic apparatus, and a midline longitudinal incision was made to expose the skull. A right craniotomy was performed using a dental drill, and CCI injury was induced 2.5 mm posterior to the anterior fontanelle and 2.5 mm posterior to the midline using the following setup: a 3 mm tip of a Reward craniocerebral injury impactor (Shenzhen Reward Life Science Co., Ltd., China), a 40 g weight, and a free fall height of 25 cm. After injury, the artificial dura mater material was applied to the brain tissue at the skull defect site. The effects of TBI modeling alone, post-TBI PLCL dura mater repair, post-TBI PLCL + 1% RAPA dura mater repair, and post-TBI PLCL + 2% RAPA dura mater repair on sensorimotor function and dura mater regeneration in rats after TBI injury were compared. Rats were weighed on days 1, 3, 5, 7, and 14 post-surgery to assess the safety of RAPA, and sensorimotor dysfunction was assessed using the footfault test.

[0044] Comprehensive in vivo evaluations of this invention revealed the concentration-dependent therapeutic effects of rapamycin membranes in TBI treatment. Figure 5 Survival analysis revealed a critical toxicity threshold; the mortality rate in the 8% RAPA group (33%) was significantly higher than in other groups (p<0.05). Figure 5 B(b)), which establishes the safe upper limit for rapamycin concentration. Behavioral tests showed significant improvement in neurological function: compared with the TBI control group, the 2% and 4% RAPA groups had reduced foot error rates ( Figure 5B(d) indicates that motor coordination has been restored. Cognitive assessment in the Y-maze showed that the 4% RAPA group performed better, characterized by increased new arm exploration (p<0.01, Fig. 5B(f)), longer exploration duration (p>0.05, Fig. 5B(e)), greater movement distance (p>0.05, Fig. 5B(g)), and reduced exploration latency (p<0.01, Fig. 5B(h)). Electron microscopy analysis showed that the rapamycin-loaded membrane remained stable in swelling characteristics 21 days after implantation in rats with traumatic brain injury, without significant changes (Fig. 5C(b)). Immunofluorescence analysis of dura mater tissue showed that the rapamycin-loaded membrane of the present invention has a significant therapeutic effect (Fig. 5C(d, e, f)). Compared with the TBI control group, both the PLCL group and the 4% RAPA group showed enhanced dura mater regeneration, manifested by an increase in collagen I+ cells (Fig. 5C(d), Fig. S3), indicating active extracellular matrix deposition. However, the 4% RAPA group showed... The group exhibited superior tissue remodeling characteristics, manifested as: (1) reduced dura mater thickness compared to the PLCL control group, indicating a potential prevention of pathological fibrosis; and (2) significant restoration of the lymphatic network, accompanied by increases in LYVE-1+ and VEGFR3+ cells, respectively (Fig. 5C(e, f)). Notably, compared to the disordered pattern observed in the PLCL control group, the lymphatic endothelial cells in the 4% RAPA group showed a more ordered spatial distribution and elongated morphology, indicating that rapamycin possesses the dual ability to simultaneously promote dura mater regeneration and maintain physiological lymphatic structure. These findings suggest that a 4% rapamycin loading optimally balances extracellular matrix deposition with antifibrotic activity and lymphatic network preservation during dura mater repair.

[0045] PLCL-RAPA regulates the Th17 / Treg balance.

[0046] Flow cytometry analysis of the present invention showed that T cell subsets changed significantly after TBI, which were subsequently modulated by rapamycin treatment (Fig. 6). Compared with the sham-operated control group, both the TBI group and the PLCL group showed significant immune dysregulation, characterized by: (1) an increased CD3+ / CD4+ T cell ratio, indicating systemic T cell activation (Fig. 6B, E(a)); (2) Th17 response polarization, manifested as an increase in CD4+ / IL-17A+ cells (Fig. 6C, E(b)); and (3) impaired immunomodulatory function, manifested as a decrease in CD4+ / FOXP3+ Treg cells (Fig. 6D, E(c)). Notably, 4% RAPA treatment effectively normalized these pathological changes by restoring immune homeostasis in the following ways: (i) reducing overall T cell activation (CD3+ / CD4+, Figure 6 B, E(a)); (ii) Inhibition of Th17 response ( Figure 6 (C, E(b)); (iv) Expanding regulatory T cells ( Figure 6 Immunofluorescence validation confirmed these findings, showing that the proportion of CD4+ / FOXP3+ (Treg) cells in the 4% RAPA group was significantly higher than that in the TBI and PLCL control groups (p < 0.05, Figure F (a, c)), indicating that rapamycin can simultaneously enhance protective immunity while inhibiting harmful inflammatory responses in the TBI microenvironment.

[0047] Histopathological examination revealed that the artificial dura mater had a significant neuroprotective effect. The TBI control group showed right hemisphere atrophy, the PLCL group showed dura mater adhesions, while the brain structure of the animals in the artificial dura mater containing rapamycin (1-4%) was better preserved (Fig. 7 A(a)). Quantitative analysis of HE-stained sections showed a dose-dependent decrease in neuronal loss at the injury site (1-4% RAPA, Fig. 7 A(b), B(a)), with the 8% RAPA group showing more significant neuronal loss than the PLCL control group (p<0.01, Fig. 7 A(b), B(a)). Immunofluorescence analysis confirmed these findings, showing upregulated neurofilament expression in the 1-4% RAPA group (Fig. 7 A(c), B(b)), while decreased neurofilament expression in the 8% RAPA group (p<0.001, Fig. 7 A(c), B(b)). These results collectively establish 2–4% RAPA as the optimal therapeutic window for rapamycin concentrations, providing: (1) significant functional recovery, (2) nerve tissue preservation, and (3) prevention of dural adhesions, while clearly demonstrating the neurotoxicity of 8% RAPA concentrations through multiple evaluation parameters.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an artificial dura mater material, characterized in that, Includes the following steps: Poly(L-lactide-caprolactone) is dissolved in an organic solvent to obtain solution 1, wherein the average molecular weight of the poly(L-lactide-caprolactone) is 40,000 to 60,000; and the organic solvent is hexafluoroisopropanol or trifluoroethanol. Rapamycin was added to solution 1 to obtain a spinning solution, wherein the concentration of rapamycin in the spinning solution was 3-5%. The spinning solution was spun into a nanofiber membrane by electrospinning, and the artificial dura mater material was obtained after removing the residual solvent. The electrospinning voltage is 15~20 kV; the electrospinning is carried out using a 20~24G metal needle, the solution extrusion speed is 0.2~0.5 mm / min, and the process is conducted at an ambient temperature of 18~25℃ and a relative humidity of 30%~50%. The removal of residual solvent is achieved through vacuum drying.

2. The preparation method according to claim 1, characterized in that, The poly-L-lactide-caprolactone contains polylactic acid and polycaprolactone, and the mass ratio of polylactic acid to polycaprolactone is 60~80:40~20.

3. The preparation method according to claim 1, characterized in that, The mass concentration of poly(L-lactide-caprolactone) in solution 1 is 8% to 12%.

4. An artificial dura mater material prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the artificial dura mater material according to claim 4 in the preparation or development of materials required for dura mater repair surgery.

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