Artificial dura mater material as well as preparation method and application thereof

Abstract

The invention provides an artificial dura mater material as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Aiming at the defects of the traditional artificially synthesized dura mater (spinal mater), the artificial dura mater (spinal mater) is processed by adopting the poly L-lactide-caprolactone (PLCL), adding the RAPA with a specific concentration and adopting an electrostatic spinning process, has the characteristics of low porosity and compact texture, and induces the rapid endothelialization of the dura mater in the early stage of craniocerebral trauma, and then, along with the release of the RAPA, the porosity of the material is increased, so that the endothelialization of the dura mater (spinal mater) in the early stage of craniocerebral trauma is improved. And proliferation and growth of new dura mater cells are facilitated. Meanwhile, the RAPA can prevent excessive proliferation of new endothelium and scar hyperplasia. Pathological proliferation can be selectively inhibited without damaging necessary lymphatic drainage; while the physical structure integrity of the dura mater is maintained, the nerve function repair can be promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an artificial dura mater material, a preparation method thereof, and an application thereof. Background Art

[0002] The dura mater 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 properties constitute an important natural protective barrier for the brain. Its primary function is to protect the brain and spinal cord, maintain normal neurological electrical processes, and participate in neuroimmune regulation through meningeal lymphatic vessels. The integrity and airtightness of the dura mater are crucial to fulfilling its protective role. However, in clinical practice, pathological factors such as trauma, inflammation, and tumor invasion, as well as invasive surgical procedures, can cause damage to the dura mater, severely weakening its protective function and leading to a series of serious complications such as cerebrospinal fluid leakage, epilepsy, and intracranial infection.

[0003] Dura mater repair can promote the restoration of the normal anatomical structure of the dura mater, reshape the airtightness of the cranial cavity, prevent blood and cerebrospinal fluid leakage, prevent intracranial infection, and reduce the occurrence of brain epilepsy. However, when the dura mater injury is located at the edge of the bone window, dura mater repair may become extremely difficult or even impossible to complete. Therefore, there is a need for a dura mater repair alternative material that can cover the dura mater defect and promote the formation of surrounding fibrous connective tissue. The repair alternative material for dura mater defects directly affects the incidence of complications such as intracranial infection, epilepsy, brain tissue protrusion, cerebrospinal fluid leakage after dura mater reconstruction, as well as the aesthetics of the subsequent plastic repair of skull injuries.

[0004] Currently available artificial dura mater materials include autologous tissue, allogeneic, xenogeneic, and synthetic materials. However, the clinical application of autologous and allogeneic materials is significantly limited by access area, ethical, and safety issues. Although xenogeneic materials currently dominate the market, the risk of allogeneic rejection and cytotoxicity caused by the cross-linking process during preparation have resulted in poor clinical efficacy. Compared to previous natural dura mater patches, synthetic dura mater patches created through chemical and materials science methods offer advantages such as unlimited material specifications, relatively low cost, and no potential for infection. However, synthetic materials lack the biological functions mediated by collagen, such as promoting cell migration and proliferation and the secretion of relevant cytokines, which hinders fibroblast migration and proliferation. Furthermore, existing synthetic dura mater materials primarily focus on strength and sealing, failing to address both dura mater restoration and the neuroprotective needs of neurological patients. Summary of the Invention

[0005] The present invention aims to provide an artificial dura mater material, its preparation method, and its application, which exhibits excellent mechanical properties, further improves the hydrophobicity of the polycaprolactone film, and simultaneously achieves the sustained and long-term release of RAPA. The artificial dura mater provided by the present invention has excellent anti-leakage properties, is degradable, and produces safe degradation products. The sustained-release RAPA drug carried by the artificial dura mater can induce dura mater regeneration and also has a neuroprotective effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing an artificial dura mater material, comprising the following steps: dissolving poly (L-lactide-caprolactone) in an organic solvent to obtain a solution 1; adding rapamycin to solution 1 to obtain a spinning solution; The spinning solution is spun into a nanofiber membrane through an electrospinning process, and the artificial dura mater material is obtained after removing the residual solvent.

[0007] Preferably, the poly (L-lactide-caprolactone) contains polylactic acid and polycaprolactone, and the mass ratio of the polylactic acid to the polycaprolactone is 60-80:40-20.

[0008] Preferably, the average molecular weight of the poly (L-lactide-caprolactone) is 40,000-60,000.

[0009] Preferably, the organic solvent is hexafluoroisopropanol or trifluoroethanol.

[0010] Preferably, the mass concentration of poly (L-lactide-caprolactone) in the solution 1 is 8% to 12%.

[0011] Preferably, the concentration of rapamycin in the spinning solution is 0.5% to 10%.

[0012] Preferably, the concentration of rapamycin in the spinning solution is 3-5%.

[0013] Preferably, the voltage of the electrospinning is 15-20 kV; The electrospinning was performed using a 20-24G metal needle, with a solution extrusion speed of 0.2-0.5 mm / min, at an ambient temperature of 18-25°C and a relative humidity of 30%-50%. The removal of the residual solvent is achieved by vacuum drying.

[0014] The present invention also provides an artificial dura mater material prepared by the preparation method.

[0015] The present invention also provides the use of the artificial dura mater material in the preparation or development of materials required for dura mater repair.

[0016] Preferably, the "preparation" includes: using the artificial dura mater material directly as an independent alternative material for repairing dura mater defects, or using it as a functional component in combination with other biocompatible materials to construct a composite repair system; The "development" mentioned above covers: developing new 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.

[0017] Beneficial effects of the present invention: To address the shortcomings of traditional synthetic dura mater (spinal cord) materials, the present invention utilizes poly(L-lactide-caprolactone) (PLCL) with a specific concentration of RAPA added and processed using an electrospinning process. The material provided by this invention has the following advantages: 1. Anti-adhesion effect. This invention utilizes the porous nature of the electrospun PLCL material to slowly release RAPA within the artificial dura mater. Leveraging the low porosity and dense texture of the electrospun PLCL composite material, this material induces rapid endothelialization of the dura mater in the early stages of craniocerebral trauma. Subsequently, as RAPA is released, the material's porosity increases, facilitating the proliferation and growth of newly formed dura mater cells. Furthermore, RAPA prevents excessive proliferation of the newly formed endothelium and scarring. 2. Promotes meningeal lymphatic regeneration. Although RAPA inhibits abnormal lymphangiogenesis, it preserves the integrity and marker expression of newly formed lymphatic vessels, as well as their ability to form ducts in vitro. This demonstrates that this invention selectively inhibits pathological proliferation without impairing essential 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 the present invention can promote nerve function repair while maintaining the physical structural integrity of the dura mater. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The preparation process of the artificial dura mater material is shown; Figure 2 The physicochemical properties of electrospun nanofibrous artificial dura mater with different rapamycin (RAPA) contents (0-8 wt%) are shown; Figure 3 The cytotoxicity evaluation and neuroprotective mechanism of PLCL-RAPA nanofiber artificial dura mater against oxygen-glucose deprivation / reperfusion (OGD / R) injury were demonstrated; Figure 4 It was shown that PLCL-RAPA nanofiber artificial dura mater inhibited the proliferation of fibroblasts and lymphatic endothelial cells in vitro; Figure 5 The results showed that the artificial dura mater material can provide neuroprotection, inhibit fibroblast proliferation, and maintain the integrity of meningeal lymphatic vessels in rats with traumatic brain injury. Figure 6 It was shown that this artificial dura mater material can regulate the Th17 / Treg balance in rats with traumatic brain injury; Figure 7 The artificial dura mater material was shown to alleviate brain atrophy, reduce adhesion to brain tissue, and mitigate neuronal loss in rats with traumatic brain injury. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present invention are 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

[0020] PLCL (PLA:PCL = 70:30; average molecular weight = 47,000) was dissolved in hexafluoropropanol (Energy Chemical Co., Ltd.) to a concentration of 10%. This solution was then mixed with rapamycin (RAPA) (North China Pharmaceutical Co., Ltd.) at a specific mass ratio to prepare a spinning solution with a 4% RAPA concentration. 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 any residual solvent. Example

[0021] PLCL (PLA:PCL = 70:30; average molecular weight = 47,000) was dissolved in hexafluoropropanol (Energy Chemical Co., Ltd.) at a concentration of 10%. This solution was then mixed with rapamycin (RAPA) (North China Pharmaceutical Co., Ltd.) at a specific mass ratio to prepare spinning solutions with RAPA concentrations of 0%, 1%, 2%, 4%, and 8%. 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.

[0022] Structural and surface characterization of electrospun PLCL artificial dura mater with different RAPA contents (0-8 wt%) confirmed the successful preparation of uniform nanofibrous scaffolds. SEM analysis showed that the addition of RAPA did not significantly change the fiber morphology, and all groups maintained consistent fiber diameters and interconnected porous structures (Figure 2A). This structural maintenance is essential for ensuring mechanical stability, promoting cell infiltration and nutrient transport. Surface wettability analysis revealed the pattern of hydrophilicity changing with concentration (Figure 2B, C). The water contact angle of the pure PLCL membrane was 119.15°±1.4°, while the addition of RAPA caused the contact angle to gradually increase to 121.84°±1.45° at 8% content, proving that the addition of RAPA enhanced the hydrophobicity of the material ( Figure 2 D). The drug-loaded films reached swelling equilibrium within 72 h, while the pure PLCL control film required 120 h. The equilibrium swelling ratio decreased from 103.5037% ± 18.0372% (PLCL) to 6.593567% ± 1.67105% (PLCL-8% RAPA), which may be due to the hydrophobicity of RAPA, which restricts water penetration and has a 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 continued carbonyl stretching vibration at 1753 cm-1 indicated that the ester bond in PLCL was not disturbed, while the new peaks at 1645 cm-1 (C=C stretching vibration) and 991 cm-1 (CH bending vibration) corresponded to the characteristic RAPA functional groups (16). XPS analysis was confirmed by the appearance of nitrogen 1s peak (399.8 eV) in the drug-loaded films ( Figure 2 F) Further evidence of RAPA incorporation, which is absent in the pure PLCL spectrum.

[0023] Comprehensive cytotoxicity assessment revealed differences in cellular responses to artificial dura mater extracts ( Figure 3 A). SH-SY5Y neuronal cells showed concentration-dependent sensitivity. Cells cultured with artificial dura mater extracts from PLCL, 1% RAPA, 2% RAPA, and 4% RAPA maintained >85% viability at all concentrations (10-90%) (in compliance with ISO 10993-5 standards). Notably, 8% RAPA extract had a certain cytotoxicity in neurons treated with OGD / R, with a cell viability of <70% (50%). Figure 3 A(a)).

[0024] Microglia (N9) demonstrated remarkable resilience, maintaining viability above 90% regardless of RAPA concentration (0-8%) or extract concentration, indicating inherent resistance to the immunomodulatory effects of rapamycin (Figure 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 (Figure 3A(c), Figure 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 (Figure 3A(c)).

[0025] Together, these findings suggest that while lower RAPA concentrations (1-2%) ensure broad biocompatibility, a 4% loading may offer the best balance between safety and therapeutic efficacy, particularly for neuronal and astrocyte populations.

[0026] PLCL-RAPA provides neuroprotection by regulating the Th17 / Treg balance To investigate the neuroprotective and immunomodulatory potential of rapamycin-loaded membranes, a sequential in vitro ischemia model ( Figure 3 B). Jurkat T lymphocytes were first treated with artificial dura mater extract containing different concentrations of rapamycin (0-8 wt%) for 24 hours to generate conditioned medium, which was then used to culture SH-SY5Y neurons under oxygen-glucose deprivation / reperfusion (OGD / R) ( Figure 3 B(a)). The neuroprotective ability of SH-SY5Y cells treated with OGD / R was evaluated by immunofluorescence and TUNEL staining ( Figure 3 B(b), 3C(a)). The conditioned medium of the 1-4% RAPA-treated group showed a dose-dependent neuroprotective effect, and its neuronal apoptosis (TUNEL+ cells) was reduced 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 membranes induced significant immunophenotypic shifts in Jurkat cells (Figures 3B(c,d), 3C(b,c)): (i) a decrease in the CD3+ / CD4+ cell population (p<0.001, Figures 3B(c), C(b); (ii) downregulation of Th17 subsets (p>0.05, Figures 3B(d), C(c)); and (iv) expansion of Treg cells (p<0.05, p<0.001, Figures 3B(d), C(c)). These immunomodulatory effects were concentration-dependent and absent in the 8% RAPA group (Figures 3B(c,d), 3C(b,c)). These results indicate that electrospun rapamycin (≤4% loading) can modulate: (1) Treg polarization and expansion, (2) inhibition of proinflammatory Th17 responses, and (3) Neuroprotection under ischemic conditions via paracrine immune modulation.

[0027] The results of this study demonstrate that rapamycin exerts multifaceted, dose-dependent effects on the dura mater repair process (Figure 4). Quantitative analysis of Ki67+ / Collagen I+ double-positive cells revealed a gradual decrease in fibroblast proliferation with increasing rapamycin concentrations (1-8% w / w) (Figure 4B, D(a)), demonstrating potent antifibrotic activity and the potential to prevent postoperative adhesions. Similarly, analysis of lymphatic endothelial cells revealed: (i) a decrease in Ki67+ / VEGFR3+ proliferating cells, while maintaining stable VEGFR3 expression (Figure 4C, D(b,c)); and (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 branch points or total tube length (Figure 4E). These results demonstrate that rapamycin selectively inhibits fibroblast and lymphatic endothelial cell proliferation while maintaining key lymphatic markers and angiogenic capacity—an ideal combination for preventing fibrosis while maintaining normal meningeal lymphatic drainage during dural repair. The present invention then evaluated the effects of the artificial dura mater material of the present invention on post-TBI sensorimotor function and its neuroprotective mechanisms in rats using a controlled cortical impact (CCI) TBI model. After three days of acclimation, rats were anesthetized with 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 at a point 2.5 mm posterior to bregma and 2.5 mm posterior to the midline to induce CCI injury using the following settings: a Rayward Brain Injury Impactor (Shenzhen Rayward Life Science Co., Ltd., China) with a 3 mm impactor tip, 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 alone, post-TBI dura mater repair with PLCL, post-TBI dura mater repair with PLCL + 1% RAPA, and post-TBI dura mater repair with PLCL + 2% RAPA on post-TBI sensorimotor function and dura mater regeneration in rats were compared. The rats were weighed on days 1, 3, 5, 7, and 14 after surgery to evaluate the safety of RAPA, and the sensorimotor dysfunction of the rats was assessed using the foot fault test.

[0028] Comprehensive in vivo evaluation of the present invention revealed that the rapamycin-loaded membrane had a concentration-dependent therapeutic effect in the treatment of TBI ( Figure 5 Survival analysis showed that there was a critical toxicity threshold, and the mortality rate (33%) in the 8% RAPA group was significantly higher than that in the other groups (p<0.05, Figure 5 B(b)), which established a safe upper limit for rapamycin concentration. Behavioral testing showed significant improvement in neurological function: the 2% and 4% RAPA groups had a reduced rate of foot faults compared to the TBI control group ( Figure 5B(d)), indicating that motor coordination was restored. Cognitive assessments in the Y-maze revealed that the 4% RAPA group performed better, characterized by increased exploration of the novel arm (p < 0.01, Figure 5B(f)), longer exploration duration (p > 0.05, Figure 5B(e)), greater movement distance (p > 0.05, Figure 5B(g)), and decreased exploration latency (p < 0.01, Figure 5B(h)). Electron microscopy analysis revealed that the swelling properties of the rapamycin-loaded membrane remained stable and showed no significant changes 21 days after implantation into rats with TBI (Figure 5C(b)). Immunofluorescence analysis of dura mater tissue revealed that the rapamycin-loaded membrane of the present invention had a significant therapeutic effect (Figure 5C(d, e, f)). Compared with the TBI control group, both the PLCL and 4% RAPA groups exhibited enhanced dura mater regeneration, as evidenced by an increase in collagen I+ cells (Figure 5C(d), Figure S3), indicating active extracellular matrix deposition. However, the 4% RAPA The 4% RAPA group showed excellent tissue remodeling characteristics, as shown by: (1) reduced dural thickness compared with the PLCL control group, indicating that pathological fibrosis could be prevented; and (2) significant restoration of the lymphatic network, accompanied by an increase in LYVE-1+ and VEGFR3+ cells, respectively (Figure 5C (e, f)). Notably, the lymphatic endothelial cells in the 4% RAPA group showed a more ordered spatial distribution and elongated morphology compared with the disordered pattern observed in the PLCL control group, indicating that rapamycin has the dual ability to simultaneously promote dural regeneration and maintain physiological lymphatic structure. These findings indicate that the 4% rapamycin loading optimally balances extracellular matrix deposition with anti-fibrotic activity and lymphatic network preservation during dural repair.

[0029] PLCL-RAPA regulates Th17 / Treg balance Flow cytometric analysis of the present invention showed that T cell subsets underwent significant changes after TBI, which were subsequently modulated by rapamycin treatment (Figure 6). Compared with the sham control group, both the TBI group and the PLCL group showed significant immune disorders, characterized by: (1) an increase in the CD3+ / CD4+ T cell ratio, indicating systemic T cell activation (Figure 6B, E(a)); (2) a polarized Th17 response, as manifested by an increase in CD4+ / IL-17A+ cells (Figure 6C, E(b)); and (3) impaired immunoregulatory function, as manifested by a decrease in CD4+ / FOXP3+ Treg cells (Figure 6D, E(c)). Notably, 4% RAPA treatment effectively normalized these pathological changes, restoring immune homeostasis by: (i) reducing overall T cell activation (CD3+ / CD4+, Figure 6B, E (a)); (ii) inhibition of Th17 response ( Figure 6 C, E (b); (iv) expansion of 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 suppressing harmful inflammatory responses in the TBI microenvironment.

[0030] Histopathological examination revealed that the artificial dura mater exhibited significant neuroprotective effects. While the TBI control group experienced right hemisphere atrophy and the PLCL group developed dural adhesions, animals treated with the artificial dura mater containing rapamycin (1-4%) exhibited better brain structure preservation (Figure 7A(a)). Quantitative analysis of HE-stained sections revealed a dose-dependent reduction in neuronal loss at the injury site (1-4% RAPA, Figure 7A(b), B(a)), with greater neuronal loss in the 8% RAPA group compared with the PLCL control group (p<0.01, Figure 7A(b), B(a)). Immunofluorescence analysis confirmed these findings, demonstrating upregulation of neurofilament expression in the 1-4% RAPA group (Figure 7A(c), B(b)), while decreased neurofilament expression in the 8% RAPA group (p<0.001, Figure 7A(c), B(b)). Together, these results establish 2–4% RAPA as the optimal therapeutic window of rapamycin concentration, providing: (1) significant functional recovery, (2) neural tissue preservation, and (3) prevention of dural adhesions, while clearly demonstrating neurotoxicity at a concentration of 8% RAPA across multiple evaluation parameters.

[0031] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing an artificial dura mater material, characterized in that: The following steps are involved: dissolving poly (L-lactide-caprolactone) in an organic solvent to obtain a solution 1; adding rapamycin to solution 1 to obtain a spinning solution; The spinning solution is spun into a nanofiber membrane through an electrospinning process, and the artificial dura mater material is obtained after removing the residual solvent.

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 the polylactic acid to the polycaprolactone is 60-80:40-20.

3. The preparation method according to claim 1, characterized in that The average molecular weight of the poly (L-lactide-caprolactone) is 40,000-60,000.

4. The preparation method according to claim 1, characterized in that The organic solvent is hexafluoroisopropanol or trifluoroethanol.

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

6. The preparation method according to claim 1, characterized in that The concentration of rapamycin in the spinning solution is 0.5% to 10%.

7. The preparation method according to claim 6, characterized in that The concentration of rapamycin in the spinning solution is 3-5%.

8. The preparation method according to claim 1, characterized in that The voltage of the electrospinning is 15-20 kV; The electrospinning was performed using a 20-24G metal needle, with a solution extrusion speed of 0.2-0.5 mm / min, at an ambient temperature of 18-25°C and a relative humidity of 30%-50%. The removal of the residual solvent is achieved by vacuum drying.

9. An artificial dura mater material obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the artificial dura mater material according to claim 9 in the preparation or development of materials required for dural repair surgery.

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