Abstract
Background/Aim: Dural reconstruction is a critical process after neurosurgical procedures. Improper dural repair leads to serious side-effects, such as cerebrospinal fluid leakage or infection. This is why it is important to properly repair the dura using a dural substitute, and research into dural substitutes is ongoing. The ideal dural substitute should be non-toxic, biocompatible, and capable of maintaining adequate tension and preventing cerebrospinal fluid leakage for extended periods in vivo. This study evaluated the biocompatibility and healing properties of Safe-Seal, poly-L-lactic acid synthetic bioabsorbable dural substitute produced by electrospinning technology. Materials and Methods: Safe-Seal, was created by electrospinning, which is a technique for nanofiberizing polymers into three-dimensional structures, and its cytotoxicity was evaluated. The animal study used 30 rats, divided into three groups assessed at two time points (4 and 12 weeks). The study groups were a negative control group with no treatment, an experimental group with Safe-Seal (TDM Co. Ltd., Gwangju, Republic of Korea) implantation, and a positive control group with a commercial product, Redura® (Medprin Biotech, Frankfurt, Germany) implantation. Results: Safe-Seal exhibited no cytotoxic or adverse effects in the in vivo animal study. Histologically, Safe-Seal displayed less inflammatory cell infiltration, less adhesion to brain tissue, and connectivity with the surrounding dura mater as compared to the negative control group and without any significant differences from Redura® in all evaluation criteria. Conclusion: Safe-Seal presented adequate biocompatibility in vivo and contributed to the healing of the dura mater at a similar level to that of Redura® when applied to dural defects.
The dura mater, the outermost layer of the meninges, can be damaged in various situations, including neurosurgical procedures, tumor intrusion, and other trauma (1, 2). After the damage has occurred, inappropriate repair causes serious side-effects, such as cerebrospinal fluid (CSF) leakage, intracranial infection, and adhesion of brain tissue because the dura mater has poor regenerative ability (1). Therefore, a dural substitute is needed to act as a support for the dural defect and as a barrier to CSF leakage. The ideal dural substitute should be non-toxic, biocompatible, and capable of maintaining adequate tension and preventing CSF leakage for extended periods in vivo (2).
Many studies have used autograft, allograft, xenograft, and synthetic materials to develop dural substitutes (1). Autografts have fewer side-effects than other transplant materials and are associated with the fastest expected recovery, but they have the disadvantage of creating additional defects (2). Allografts are not preferred due to the risk of transmission of Creutzfeldt-Jacob disease (3). Likewise, animal-derived xenografts cannot exclude concerns about transmission of diseases such as bovine transmissible spongiform encephalopathy (2). Therefore, development of inexpensive and biocompatible synthetic dural substitutes is necessary to overcome such limitations.
As a synthetic non-absorbable material, silastic has been used as a dural substitute, but after many years of follow-up, it was found to cause chronic inflammation and is no longer used (4, 5). Other non-absorbable synthetic materials used as dural substitutes include expanded polytetrafluoroethylene and polyurethane. These materials have the advantage of being easily produced and not adhering to brain tissue. Still, they have the disadvantage of allowing CSF to leak along the suture line (3). Among absorbable synthetic materials, polydioxanone, polyglactin 910, polycaprolactone, and poly-L-lactic acid (PLLA) are used as dural substitutes (6). However, the flexibility and duration of bioabsorption of these materials vary greatly depending on how they are fabricated, so it is essential to maintain adequate tension as they are being replaced by biological fibrous tissue while still allowing for convenient manipulation (7). Biomimetic design is important because excessively large fiber diameters can make manipulation challenging and result in longer absorption time, leading to foreign-body reactions. The synthetic dura mater should also be manufactured with appropriate pore size to have watertight properties to prevent CSF leakage.
This study was designed to evaluate the healing ability of Safe-Seal (TDM Co. Ltd., Gwangju, Republic of Korea), a newly developed bioabsorbable synthetic material for the dura mater. Safe-Seal is made of PLLA, a synthetic polymer that is drawing attention in the medical field for its properties, such as biodegradability, thermoplasticity, good mechanical strength, and excellent biocompatibility. In addition, its absorbent nature is not expected to cause long-term foreign-body reactions and is anticipated to provide structural support during proper healing of the dural defect. To increase biocompatibility, electrospinning technology has been used to nanofiberize polymeric materials to mimic the structure of the organism’s extracellular matrix (2, 6). Redura® (Medprin Biotech, Frankfurt, Germany), used as a positive control in this study to verify equivalence of Safe-Seal performance, is a commercially available product made from PLLA using electrospinning technology.
Materials and Methods
Preparation of dural substitutes. To prepare the PLLA solution for electrospinning, a solvent was prepared by mixing dichloromethane (DCM) with dimethylformamide (DMF) in an 8:2 ratio, then PLLA was added, and allowed to dissolve over 48 hours. Using the dissolved PLLA–DCM–DMF (5-10 wt%) solution, nanofiber patches were prepared by electrospinning with a voltage of 15-20 kV at a distance of 10-20 cm, using ESDL_V1 (Blueseal, Jeonju-si, Republic of Korea). Electron microscopy (JSM-5900; JEOL, Tokyo, Japan) confirmed that the fiber morphology was stable, with only few non-fiber-like beads in the form of lumps (Figure 1). The composition of the solvents used, DCM and DMF, was analyzed by gas chromatography, and no residual solvent was detected.
A: Visual appearance of the nanofiber patch, ‘Safe-Seal’. B: Scanning electron microscopic image confirmed that the fiber morphology was stable, scanned with JSM-5900 (JEOL, Tokyo, Japan). Scale bar: 10 μm. Reprinted with permission, Copyright (2022) by TDM Co. Ltd.
To verify the mechanical performance of Safe-Seal, a tensile strength test was conducted at the Korea Polymer Testing & Research Institute (Seoul, Republic of Korea) using a Universal Testing Machine Instron® 3367 (Instron, Norwood, MA, USA). A load cell of 500 N was used. The speed was 25 mm/min and the distance between the grips was 25 mm. The specimens provided were 2×6 cm (n=5). The maximum load averaged 11.9±0.3 N (n=5), the average tensile strength was 3.18±0.13 MPa (n=5), and the average elongation was 41.6±5.27% (n=5). All values are expressed as the mean±standard deviation.
Cytotoxicity test. The elution test on Safe-Seal was performed at the Korea Testing Certification Institute (Gunpo-si, Republic of Korea) using L-929 mouse fibroblast cells. The PLLA test substance (Safe-Seal), a negative control substance, and a positive control substance were eluted in 1× Minimum Essential Medium (MEM) containing 10% fetal bovine serum for 72±2 h at 37±1°C. Test substance eluate (test sample) was made of 60 cm2 of Safe-Seal with 10 ml of MEM. Negative control substance eluate (negative control) was made of 0.728 g of high-density polyethylene film (reference material-C) with 7 ml of MEM. Cytotoxic substance eluate (positive control) was made of 0.716 g of 0.25% zinc dibutyldithiocarbamate polyurethane film (reference material-B) with 7 ml of MEM. Blank test solution (solvent control) was MEM alone. Each eluate was applied to L-929 cells and the cells were incubated for 48±2 h at 37±1°C, in an incubator with 5±1% CO2. Cellular responses were graded by observation under a microscope and assessed quantitatively by counting cells.
In vivo animal study. This study was approved by the Institutional Animal Care and Use Committee of Chonnam National University in Korea (CNU-IACUC-YB-2022-65). A total of 30 10-week-old male rats (Samtako Bio, Osan-si, Republic of Korea), weighing 300-350 g, were used. They were group-housed in polycarbonate cages and had free access to water and food.
Safe-Seal, the test material to be implanted into the experimental group was a 0.2 mm-thick white, sheet-like material composed of electrospun PLLA fibers. The material to be implanted into the positive control group was the commercial product Redura®, which was constructed from the same PLLA material, and was also a 0.2-mm thick white, sheet-like material made of nanofibers. The implant samples were prepared by cutting them into a circular shape using a 6 mm-diameter sterilized biopsy punch.
The animals were randomly divided into three groups of 10 animals each and underwent creation of a skull bone/dura mater defect (see below). The negative control group had no implantation after the creation of a defect in the skull and dura mater. The experimental group had a Safe-Seal implant over the defect. Finally, the positive control group had Redura® implanted. Each of these three groups was divided into two subgroups and sampled after euthanasia at 4 and 12 weeks, respectively.
All animals were anesthetized with intraperitoneal injections of 80 mg/kg ketamine hydrochloride (Yuhan Ketamine 50 inj.; Yuhan Corp., Seoul, Republic of Korea) and 10 mg/kg xylazine hydrochloride (Rompun inj.; Bayer Korea, Seoul, Republic of Korea). Preoperative medication was administered by subcutaneous injection of 5 mg/kg tramadol hydrochloride (Maritrol amp.; Jeil Pharmaceutical Co. Ltd., Seoul, Republic of Korea), 5 mg/kg enrofloxacin (Baytril 25 inj.; Bayer Korea), 5 mg/kg ketoprofen (Eagle Ketoprofen 10% inj.; Eagelvet, Yesan-gun, Republic of Korea), and 0.1 mg/kg atropine sulfate (Jeil atropine sulfate; Jeil Pharmaceutical Co. Ltd., Seoul, Republic of Korea). After shaving the animal’s head, the surgical site was prepared using 10% povidone-iodine and 70% ethyl alcohol.
All surgical procedures were performed under general sterile conditions. The skin and periosteum of the rat head were incised to expose the skull. A bone defect of 5 mm diameter was formed using a trephine bur (THB50; Osung M&D, Kim-po-si, Republic of Korea). The dura mater was incised by a 26-G syringe needle to construct a defect of 5 mm diameter (Figure 2).
Surgical procedure of a rat cranial dura mater defect model. A: The skin was incised. B: After elevating the periosteum, a bone defect of 5 mm diameter was formed using a trephine bur. C: The dura mater was incised by a 26-G syringe needle to make a defect of 5 mm diameter.
After creating the dura mater defect, no dural substitute was implanted on the dural defect in the negative control group (n=10). The periosteum and the skin were each sutured. The Safe-Seal group (n=10) had a 6-mm diameter Safe-Seal patch implanted in the defect, and the Redura® group (n=10) had a 6-mm diameter Redura® implanted as an onlay in the defect (Figure 3). The periosteum and the skin were sutured by the same method.
Sample implantation in the dural defect. A: Negative control group with only the defect. B: Implanted Safe-Seal. C: Implanted Redura®.
After the surgical procedure, the animals were observed for neurological abnormalities. At 4 and 12 weeks after implantation, all animals were anesthetized with intraperitoneal injections of 80 mg/kg ketamine hydrochloride and 20 mg/kg xylazine hydrochloride. Subcutaneous injections of 5 mg/kg tramadol hydrochloride and 5 mg/kg ketoprofen were administered for analgesia. Exsanguination euthanasia was performed by perfusion of phosphate-buffered saline (PBS tablets; Gibco, Waltham, MA, USA) through the left ventricle of the heart. Afterwards, neutral buffered formalin (Maskform A; Dana Korea, Incheon, Republic of Korea) was perfused by the same route. After fixation via cardiac perfusion, the skulls and brains of the rats were collected.
Histological evaluation. Rat head tissues fixed in 10% neutral formalin were decalcified using commercially available 0.5 M EDTA (BE009b; Biosolution, Suwon-si, Republic of Korea), embedded in paraffin (HistoCore Arcadia; Leica, Wetzlar, Germany), and sectioned at 5-μm thickness using an automated rotary microtome (HistoCore AUTOCUT; Leica) to prepare histological specimens. All tissue specimens were stained with hematoxylin-eosin and Masson’s trichrome using a Leica Autostainer XL (ST5010; Leica) to obtain image data using a light microscope/digital slide scanner (Axio Scan.Z1; Zeiss, Oberkochen, Germany).
The grading system used for semiquantitative evaluation is illustrated in Table I. The grading system was modified from the histological evaluation criteria of Ozisik et al. (8), Kawai et al. (9). and MacEwan et al. (10). The scoring criteria were infiltrated cell types, formation of granulation tissue, collagen deposition, and adhesion to the pia mater, each of which was scored on a scale of 1 to 5. A higher score for each criterion indicated greater maturation of the dura mater. Four experimenters performed the evaluations, blinded to the treatment group, and the results are presented as the means±standard errors.
Grading system for quantification of histological findings.
Statistical analysis. All the experimental data are presented as the mean±standard error and were analyzed for significance by the Kruskal-Wallis test, followed by Bonferroni’s method. Data were analyzed using IBM SPSS Statistics for Windows, version 27.0 (IBM Corp., Armonk, NY, USA). Statistically significant differences were defined as having p<0.05.
Results
Cytotoxicity test. The microscopic qualitative evaluation of the cellular response confirmed that the cells with the blank test solution and negative control eluate were graded 0 (discrete intracytoplasmic granules; no cell lysis). The cells with the positive control eluate were graded 4 (nearly complete destruction of the cell layers), and the cells with the test material eluate were graded 2 (>20% - ≤50% of the cells were round and devoid of intracytoplasmic granules; no extensive cell lysis and empty areas between cells). Quantitative evaluation by cell counting revealed cell viability of 99.62% for cells treated with the negative control eluate, 0.00% for cells treated with the positive control eluate, and 72.80% for cells treated with the test substance eluate as compared to the blank eluate. Therefore, when the results of the negative and positive controls were determined, it was verified that the test procedure was appropriate and did not cause cytotoxicity as the cell viability was over 70%. As a result, the eluate of Safe-Seal showed no cytotoxicity in the qualitative and quantitative evaluations.
Histological evaluation. All animals of the 4- and 12-week groups did not show any neurological abnormalities and survived until the end of the experimental period. At necropsy, no abnormalities, such as inflammation or bleeding, were observed at the site of the defect and implantation.
Histological image data of coronal sections of the experimental sites were qualitatively and semi-quantitatively evaluated. Histology at 4 weeks is shown in Figure 4 and at 12 weeks is shown in Figure 5. Qualitative evaluations were made on the extent of dural repair and adhesion to brain tissues, the morphology of infiltrating cells in the graft material, and changes in the morphology of the samples in each experimental group. The semi-quantitative evaluation was conducted by a total of four people using the grading system shown in Table I.
Histological findings at 4 weeks after implantation of dural mater substitute as revealed by hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining. Representative slides from the negative control group, experimental group with Safe-Seal implantation and positive control group with Redura® implantation are shown. Arrow: Irregular connective tissue formed in the dural defect. Arrowhead: Connectivity between the existing dura and the dural substitute. Asterisk: Dural substitute. White arrowhead: Artifact. Scale bar: 200 μm.
Histological findings at 12 weeks after implantation of dural mater substitute as revealed by hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining. Representative slides from the negative control group, experimental group with Safe-Seal implantation and positive control group with Redura® implantation are shown. Arrow: Irregular connective tissue formed in the dural defect. Arrowhead: Connectivity between the existing dura and the dural substitute. Asterisk: Dural substitute. Scale bar: 200 μm.
The extent of dural repair for Safe-Seal indicated connectivity with the dura around the dural defect, suggesting that it may function to prevent the outflow of CSF. Considering adhesion to brain tissues, as compared to the negative control group, where irregular connective tissue was formed at the dura mater, Safe-Seal prevented the dura mater from adhering to the brain due to a lower degree of connective tissue formation at the interface between the brain tissue and the implant. The morphology of infiltrated cells inside the implants displayed multinucleated giant cells in both the Safe-Seal and Redura® groups at week 4, but by week 12, the proportion of multinucleated giant cells declined, and the proportion of fibroblasts was elevated in both the Safe-Seal and Redura® groups, indicating a low likelihood of a long-term foreign-body reaction.
The implanted dural substitutes exhibited changes in thickness due to cell infiltration. Unlike Redura®, which caused swelling in implant thickness due to the deposition of cells, Safe-Seal maintained a similar thickness at 4 and 12 weeks. Therefore, it was thought that Safe-Seal would have the advantages of not compressing brain tissue and not negatively affecting the regeneration of the skull.
As the implant is absorbed, fibroblasts produce collagen. When evaluating the density of collagen in the implant materials via Masson’s trichrome staining, the Safe-Seal and Redura® groups demonstrated a similar degree of collagen deposition, indicating proper healing of dural defects.
The results of the semi-quantitative analysis are explained in Figure 6. The Safe-Seal and Redura® groups scored significantly higher than the negative control group in the same week on all criteria. Both Safe-Seal and Redura® functioned as dural substitutes for restoring the dura mater. The granulation score was higher in the Redura® group than in the Safe-Seal group at both 4 and 12 weeks (Figure 6B), and the scores of other criteria, including cell type (Figure 6A), collagen deposition (Figure 6C), and lack of pia mater adhesion (Figure 6D) were higher in the Safe-Seal group than in the Redura® group, with no significant differences noted. Safe-Seal and Redura® were expected to perform similarly as there was no significant difference in the semi-quantitative analysis of dural repair ability. The negative control group scored lower in cell type, the Redura® group in granulation, and the Safe-Seal group in collagen deposition at week 12 as compared to week 4, but the differences were not significant. This lack of significance means that the degree of resorption of the implanted material and the degree of replacement with fibrous tissue were observed at a similar extent at week 4 and week 12.
Averages of semi-quantitative scoring for each group. Scoring data for cell types infiltrating the dural substitutes (A), granulation tissues around the implant materials (B), collagen deposition (C) and lack of pia mater adhesion (D). Data are expressed as the mean±standard error. Significantly different from the control in the same week at: *p≤0.05, **p≤0.01, and ***p≤0.001.
Discussion
After neurosurgical procedures, dural reconstruction is a critical process. An improper dural repair leads to serious side-effects, such as CSF leakage or infection (1, 11). Therefore, research is ongoing into effective methods and materials for suturing the dura for successful neurosurgery. The ideal dural substitute should be able to prevent CSF leakage, be biocompatible, pose no risk of infection, and be easy to handle during surgery. It should also minimize adhesions to brain tissue to reduce postoperative side-effects. For about 100 years, various materials have been studied and clinically used as dural substitutes (6). Autografts, such as skull periosteum, fascia lata, or temporal muscle fascia avoid immune rejection and disease transmission. However, they are difficult to apply to large defects because they require the formation of another defect (11). Allogenic materials, such as cadaveric dura mater, amniotic membrane, and dermis, can also be used as dural substitutes, and have achieved some positive results. However, allografts carry the risk of transmitting diseases such as Creutzfeldt-Jacob. Furthermore, there are not enough sources of allogenic materials readily available (7). Xenografts are mostly used as artificial dura mater, for they have good availability. There are two main types of xenogeneic dural substitutes: one that preserves the extracellular matrix and another that uses collagen extracted from animal tissues. However, there are still concerns about disease transmission. Non-absorbable synthetic materials such as expanded polytetrafluoroethylene and polyurethane have some advantages because of their inertness, including reduced infection risk and no adhesion with the surrounding tissues. However, they have limitations of possible CSF leakage through the suture line. Absorbent synthetic materials have many advantages for designing a substitute, such as adjustable watertightness and degradation rates. Absorbent synthetic dural substitutes provide adequate support to allow a patient’s dural defect to heal with the body’s own connective tissue before being completely absorbed, leaving no foreign-body reaction (7).
Among the techniques for fiberizing synthetic polymers into bio-applicable forms, a sheet must first be constructed with the appropriate diameter of fibers and pore size (6). Absorbent synthetic dural substitutes should be waterproof to prevent CSF leakage but also have a porous structure to allow for cell migration (6). Electrospinning techniques nano-fiberize synthetic polymers into shapes that mimic the structure of the extracellular matrix to induce cell adhesion and growth (2).
Sufficient mechanical strength of the dural substitute is critical for intraoperative maneuverability and prevention of CSF leakage. In this study, Safe-Seal displayed favourable mechanical properties, with a maximum load average of 11.9±0.3 N (n=5), an average tensile strength of 3.18±0.13 MPa (n=5), and an average stretching elongation of 41.6±5.27% (n=5). Deng et al. (2) measured the mechanical strength of a dural substitute made of PLLA and gelatin, and the average tensile strength of the composite substitute was 3.8±0.34 MPa (n=5), with an average stretching elongation of 84.87±8.45% (n=5). Control products with collagen ingredients are too fragile to be tested under the same conditions (2). Shi et al. (12) measured the mechanical strength of an approximately 0.3 mm-thick patch of PLLA components and determined that the average tensile strength was 4.14±0.18 Mpa (n=8), and the average stretching elongation was 60.5±13.2% (n=8) (12). Wang et al. reported that collagen-based dural substitutes have weaker tensile strength as compared to electrospun synthetic polymers (6). Based on the results of several studies, we are confident that Safe-Seal has sufficient mechanical strength to be used as a dura mater substitute, but additional data from comparisons under equivalent experimental conditions are needed.
In this research, animal testing of Safe-Seal, a PLLA sheet produced by electrospinning technology, was conducted, using a rat dural defect model (13). Visual and histological evaluations were performed at four and 12 weeks after implantation, respectively. None of the animals exhibited neurological abnormalities during post-implantation observation, and none of the animals presented with hematoma or inflammation at the implantation site based on visual evaluation at necropsy. Both Redura® and Safe-Seal contributed to dural repair at weeks 4 and 12, with histological connectivity to the surrounding dura mater, which can prevent CSF leakage. The implanted Safe-Seal had a biocompatible structure, which induced the infiltration of cells. However, few inflammatory cells were observed, and the infiltration of multinucleated giant cells was observed, which can occur naturally during the absorption of large biomaterial particles (14). In the case of non-absorbable material, the infiltration of multinucleated giant cells can be thought of as an unnecessary inflammatory response. However, in this experiment, as the graft was absorbed, the degree of multinucleated giant cell infiltration was less at week 12 than at week 4, suggesting a lower likelihood of long-term foreign-body reaction. Compared to Redura®, Safe-Seal was not significantly different in the semi-quantitative evaluation and scored higher than the control group for each criterion. Week 12 scores were higher for most criteria than at week 4, but there was no statistically significant difference between weeks 4 and 12. The absence of histologically significant differences may be due to the short observation period. Longer-term evaluation is needed to allow for complete absorption of the implanted dural substitute.
Moreover, the rat dural defect model was used, with a dural graft size of a 6 mm-diameter circle in this study. Because the size of the defect and the size of the dural graft were very small, the sutureless onlay method was chosen for application. In neurosurgery, depending on the type of dural substitute, a sutureless onlay may be possible, but only if the defect size is very small and the dural substitute can be widely covered around the defect. Because intracranial pressure changes widely in the immediate postoperative period, suture immobilization is often necessary to prevent CSF leakage. Therefore, additional tensile evaluation of suture stress, which was not performed in this experiment, will be required.
According to Kinaci et al., the average thickness of the dura mater in rats is 49±15 μm, whereas in humans, it is 564±50 μm (15). The dural substitute used in this experiment was about 200 μm-thick, which is very thick compared to the thickness of a rat’s dura mater, but thin compared to the thickness of a human dura mater. It will be necessary to evaluate whether Safe-Seal will maintain its tension after implantation, during the period when thick dural tissue is undergoing sufficient repair. Before the clinical application of this new dural substitute, a longer-term study in a large animal model should be conducted. Further studies on how the tensile strength and water tightness of the repaired dura change at different time points as the absorbable dura mater undergoes absorption and fibrosis after bioimplantation should also be considered.
Acknowledgements
This study was conducted with research funds from the Advanced Technology Center+ (Grant no. 20014237) dedicated to the Korea Evaluation Institute of Industrial Technology (KEIT), an affiliate of the Ministry of Trade, Industry, and Energy.
Footnotes
Authors’ Contributions
Writing – original draft preparation, review and editing: M.C., K.J., and S.E.K.; Sample preparation: S.S.P.; Conceptualization: M.C., S.S.P., S.S.K., K.J., and S.E.K.; Investigation and formal analysis: M.C., K.M.S., S.S.P., S.S.K., K.J., and S.E.K.. All Authors contributed to the experiments and approved the designed experiments and study protocol.
Conflicts of Interest
The Authors have no conflict of interest to declare in relation to this study.
- Received November 7, 2023.
- Revision received December 13, 2023.
- Accepted December 14, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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