Degradation Pattern of a Biodegradable and Photocurable Sealants Based on Hyaluronic Acid : A Serial Magnetic Resonance Imaging Observational Study in Rat Craniectomy Model

Hyeseon Lee; Sijoon Lee; Seung Yun Yang; Dong Hwan Kim; Mahnjeong Ha; Kyoung Hyup Nam

doi:10.3340/jkns.2024.0138

Journal of Korean Neurosurgical Society > Epub ahead of print

Lee, Lee, Yang, Kim, Ha, and Nam: Degradation Pattern of a Biodegradable and Photocurable Sealants Based on Hyaluronic Acid : A Serial Magnetic Resonance Imaging Observational Study in Rat Craniectomy Model

Laboratory Research

Journal of Korean Neurosurgical Society 2024; jkns.2024.0138.

Published online: December 3, 2024

DOI: https://doi.org/10.3340/jkns.2024.0138

Degradation Pattern of a Biodegradable and Photocurable Sealants Based on Hyaluronic Acid : A Serial Magnetic Resonance Imaging Observational Study in Rat Craniectomy Model

Hyeseon Lee^1,^*

, Sijoon Lee^2,^*

, Seung Yun Yang^1,³

, Dong Hwan Kim⁴

, Mahnjeong Ha⁴

, Kyoung Hyup Nam⁴

¹Department of Biomaterials Science (BK21 Four Program), Life and Industry Convergence Institute, Pusan National University, Miryang, Korea

²Preclinical Research Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Korea

³SNvia Co., Ltd., Busan, Korea

⁴Department of Neurosurgery, Biomedical Research Institute, Pusan National University Hospital, Pusan National University School of Medicine, Busan, Korea

Address for reprints : Kyoung Hyup Nam Department of Neurosurgery, Pusan National University Hospital, 305 Gudeok-ro, Seo-gu, Busan 49241, Korea Tel : +82-51-240-7257, Fax : +82-51-244-0282, E-mail : goodnsdoctor@daum.net

^* Hyeseon Lee and Sijoon Lee contributed equally to this study as co-first authors.

Received: July 22, 2024 Revised: October 31, 2024 Accepted: December 2, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

The aim of this study is evaluating in vivo degradation of photocrosslinkable hyaluronic acid (HA)-based dural sealant (HA photosealant) using magnetic resonance imaging (MRI) and histopathological analysis to assess its biodegradability and effectiveness in preventing cerebrospinal fluid (CSF) leakage.

Methods

HA photosealants were applied to the incised dura in a rat craniectomy and durotomy. The HA photosealant quickly sealed the wound upon low-energy visible light exposure (405 nm, <5 seconds, < 1 J/cm²). The degradation of HA photosealants was tracked through serial MRI scans at 1, 2, and 4 weeks post-application. The remaining area of HA photosealants on the dura was measured using image processing program for volumetric analysis. Additionally, histopathological analyses were performed to evaluate the biocompatibility and effectiveness of the dural repair.

Results

The MRI and histopathological analyses showed that the HA photosealant achieved progressive degradation while successfully preventing CSF leakage without any adverse tissue reactions. The residual area of HA photosealants measured at 2 weeks ranged from 41.35% to 94.88%, with an average of 66.57%. At 4 weeks, a more distinct degradation pattern was observed compared to 2 weeks, showing a residual area of 10.28% to 56.11%. The HA photosealant maintained structural integrity until dural regeneration was completed.

Conclusion

HA photosealant showed gradual degradation in vivo while maintaining mechanical strength until the dura was repaired for preventing CSF leakage without inflammation and toxicity. HA photosealant has great potentials for clinical application for dural repair with biodegradable properties and biocompatibility.

Key Words: Dural sealant · Hyaluronic acid · Photocurable tissue adhesive · Magnetic resonance imaging · Biodegradation.

INTRODUCTION

As the incidence of central nervous system disorders in the brain and spine has increased in recent decades, the number of neurosurgical procedures has also increased. Cerebrospinal fluid (CSF) leakage has been reported as a complication of intraoperative durotomy, whether occurring intentionally or accidentally. CSF leakage may result in a various complications ranging from mild to severe, including dizziness, headaches, arachnoiditis, and meningitis. These complications may lead to prolonged hospitalization, increased medical costs, and even mortality in extreme cases [2,5,12,15,20,23,25].

Achieving adequate dural sealing is essential to avoid a CSF leakage. Despite applying various techniques used for leakage prevention [1,10,17,27], the prevalence of postoperative CSF leakage is within a range of 6.6% to 10% [5,7,15,24]. The traditional method involves suturing the dural tear, often in conjunction with a dural sealant or fascia. However, suture-based dural closure is time-consuming and may not completely prevent a CSF leakage due to loose sutures or inevitable pin holes [1,13,17,22,27]. Besides, challenges are more prominent when dural tears occur in anatomically challenging locations or involve large defects [4,9,13].

As an alternative to conventional methods, sealant-based dural repair is gaining popularity for dural healing owing to their promising potentials. In our previous study, photocrosslinkable hyaluronic acid (HA) has utilized and elaborated its advantages as a surgical sealant [3,17]. Photocrosslinkable HA-based dural sealants (HA photosealant) overcome unmet needs in clinics : 1) rapid and watertight closure of CSF leakage after a safe light exposure, 2) proper mechanical properties considering lesion, 3) adhesive layer maintenance until a complete dural regeneration, and 4) biocompatible and biodegradable properties.

In this study, we performed magnetic resonance imaging (MRI) observational study with rat craniectomy model for further understanding of in vivo efficacy and serial degradation pattern. We analyzed whether the CSF leak prevention effect was maintained during the approximately 1-month period of dural regeneration and subsequently biodegraded.

MATERIALS AND METHODS

The animal experiments were reviewed and approved based on the ethical procedures for scientific care by the Institutional Animal Care and Use Committee of the Preclinical Research Center of the Daegu-Gyeongbuk Medical Innovation Foundation (Daegu, Korea) (approval number : DGMIF-21101502-00).

Experimental materials

HA (average molecular weight : 150 kDa), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and visible light pen (405 nm) for photocuring were purchased from SNvia (Busan, Korea). Methacrylic anhydride (MAA), 4-pentenoic anhydride (PEA), ethanol, sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO) and brilliant blue FCF (blue dye) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1 mL Luer-Lok syringe was purchased from BD Korea (Seoul, Korea).

Preparation of glue-type HA photosealants

Photocrosslinkable HAs were synthesized according to a previously reported method [3,17]. Briefly, HA were dissolved in a mixture of distilled water and DMSO (water : DMSO = 10 : 2) and cooled to 5℃. Using a 1 M NaOH, pH was adjusted to 8.0. A mixture of 5 equivalents of MAA and PEA (3.5 : 1.5) was added dropwise into the HA solution for 1 hour. While mixing, the pH was maintained 8-10 by 1 M NaOH. The temperature and pH were maintained for another 12 hours. To obtain photocrosslinkable copolymeric HA (HAMA-PA) after reaction, a 94% ethanol was slowly added to precipitate. HAMA-PA was filtered and washed with ethanol to remove unreacted reagent. Then, obtained precipitate was lyophilized and stored at -20℃ until use. To prepare glue-type HA photosealants, HAMA-PA was dissolved in 0.1% LAP solution. Blue dye was added in the HA solutions at a concentration of 0.01% for the visualization of the HA photosealant. Then, 1 mL Luer-Lok syringe were filled with HA photosealants. Pre-filled syringe was kept in an opaque box until use to avoid an undesired polymerization by light exposure.

Animal model and study design

The study used male Sprague-Dawley (SD) rats weighing 400-450 g and aged 12 weeks. Forty-three SD rats were included in this study. Five were used to analyze HA degradation using MRI and 38 SD rats were used to analyze histopathological findings. The rats for histopathological evaluation were randomly divided into two groups as follows : sham group (n=19) and HA photosealant group (n=19). The sham group underwent craniectomy and durotomy without HA photosealant. The HA photosealant group underwent craniectomy and durotomy followed by application of HA photosealant to the incised dura mater.

Rats were anesthetized by the inhalation of isoflurane. The scalp of each rat was topically anesthetized by injection of lidocaine 0.5% solution and disinfected using povidone-iodine. The incision of scalp and dissection of the fascia were conducted by a scalpel. A craniectomy was performed by creating an 8 mm cranial defect with a burr, and the dura mater was fully exposed. Then, the meninges were incised via intentional durotomy until CSF leakage occurred. In the HA photosealant group, HA photosealant (0.1 mL) was applied and polymerzied after visible light exposure (405 nm, 200 mW/cm² for 5seconds) by visible light pen for water-tight sealing. The experimental procedure is shown in Fig. 1.

HA degradation analysis using serial MRI observation

All rats received post-operative care and were closely monitored for signs of infection, distress, or CSF leakage. Rats were euthanized and MRI was performed serially at 1, 2, and 4 weeks after the surgery. In vivo MRI was acquired on a 9.4 T small-animal MRI scanner (BioSpec 94/20USR; Bruker, Billerica, MA, USA) equipped with a 40 mm volume coil and a respiratory monitoring system. The RARE of T2-weighted sequence parameters were obtained as follows : TR=6500 ms, TE=40 ms, ETL : 4, matrix size : 256×256, slice thickness : 1 mm, FOV= 40×40 mm, scan time=13 minutes and 52 seconds. The main objective of these MRIs was to evaluate the degradation process of the HA photosealant and to monitor the integrity of the dural repair. The volumetric analysis of the white area estimated to be the photocured HA hydrogel was calculated using the Image J program (version 1.54; National Institutes of Health, Washington, DC, USA) (Fig. 2).

Histopathological evaluation

After euthanasia of rats, the brain and meninges with bone were collected in a mass by a grinder using a disc type blade (1, 2, 4, 8 weeks). Tissues were fixed in 10% neutral buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) after collection. For histopathological evaluation, a tissue processor (Thermo Fisher Scientific, Runcorn, UK) was used to prepare organs and tissues from the formalin-fixed samples for evaluation by fixing, staining, and dehydration. Paraffin-embedded tissue blocks were cut to a thickness of 4 µm and mounted on glass slides. Staining was performed with Hematoxylin and Eosin (H&E) and masson trichrome (MT) using an autostainer (Dako Coverstainer; Agilent, Santa Clara, CA, USA).

Statistical analysis

All the experiments were repeated at least three times and the data are reported as mean±standard deviation. Statistical significance was determined using t-test and one-way analysis of variance with Tukey’s analysis. Significant differences between the two groups are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001).

RESULTS

HA photosealant degradation pattern using MRI observation

The Fig. 3 shows coronal MRI images taken 1, 2, and 4 weeks after surgery. The area of residual HA photosealant was measured and calculated as a ratio compared to the area of HA photosealant in the MRI taken 1 week after surgery. Observation of the remaining HA photosealant in the MRI shows that it gradually disintegrates. Table 1 summarises the measurements of HA hydrogel area using image processing program (Image J; National Institutes of Health). The area of hydrogel measured 1 week after surgery was set at 100% and used as a standard to evaluate the degradation process. At 2 weeks, the area of hydrogel remaining ranged from 41.35% to 94.88%, with an average of 66.57% remaining. In the MRI images measured at 4 weeks, a more pronounced degradation pattern was observed compared to week 2, and the percentage of hydrogel remaining decreased further to a value between 10.28% and 56.11%. The difference in the area of remaining hydrogel between weeks 1 and 2 was measured from 5.12% to 58.65%, and the difference in the area of remaining hydrogel between weeks 2 and 4 was measured from 2.14% to 48.23% (Fig. 3). There were individual differences in biodegradability depending on the week for each individual, but the remaining hydrogel showed a gradual decrease. In addition, in the MRI performed at week 8, the remaining hydrogel was degraded in most rats and was barely visible. During the period of observation by MRI, there were no subjects with CSF leakage around the durotomy site and the mechanical strength of the HA photosealant as a function of dural repair was adequately maintained.

Histopathological evaluation

The in vivo dural repair effect and biological stability of HA photosealant were evaluated using a rat craniectomy and durotomy model, in which experiments were performed using methods such as MRI degradation. After checking for CSF leakage following durotomy, HA photosealant was applied and photocured after exposure to visible light (405 nm) for 5 seconds to ensure complete sealing. Rats were euthanised at 1, 2, 4, and 8 weeks after surgery and histological analysis was performed (Fig. 4). At week 1, tissues from four animals in the sham group and four animals in the HA photosealant group were collected and analysed. At 2, 4, and 8 weeks, histological analysis was performed on five animals in each group. The results of H&E staining showed no clear recovery of the skull or dura mater from 1 to 4 weeks, but new bone formation in the dura mater and surrounding skull was seen at 8 weeks. There were no side effects such as foreign body reaction, granulomatous inflammation or necrosis caused by HA photosealant. MT staining was performed to check the degree of dura mater recovery and no abnormal changes were observed. In all tissues, no inflammatory reaction, toxic reaction or adhesion was observed in the cerebral cortex below the dura mater. A comparison of the results at week 8 showed that the thickness of the dura had healed more than in the sham group.

DISCUSSION

When CSF leakage occurs after brain and spine surgery, it can lead to complications such as vertigo, headache, arachnoiditis and meningitis. In particular, when CSF leakage occurs after spinal surgery, it can leave serious sequelae due to neurological symptoms and infection [2,5,12,15,17,20,23,25,28]. Therefore, proper dural repair is essential to prevent CSF leakage, and a variety of methods have been used to perform dural repair [10,17,27]. In our previous research, we designed and tested the effectiveness of an ideal dural sealant [17]. Our team developed an HA photosealants based on photocurable and biodegradable HA. HA is a natural polysaccharide composed of the disaccharide of D-glucuronic acid and N-acetyl-glucosamine. HA is highly biocompatible and does not cause foreign body reaction [13,14]. In addition, it is used as an antiadhesive agent by reducing scarring and fibrosis in the early stages of wound healing [11,13,18]. Furthermore, HA is known to promote dural healing by suppressing astrocytic scarring that interferes with dural regeneration. Due to excellent biocompatibility and bioactivity of HA-based biomaterials [19,21], HA photosealants have potential for promoting regeneration and repair in the nervous system [19,21]. Therefore, we hypothesized that it would be a suitable material as a dural sealant [16,17]. However, the main disadvantages of HA-based products are their rapid degradation rate and poor mechanical properties [8,13]. The HA photosealant, enforced by multi-length networks, overcome existing shortcomings and unmet needs. HA photosealant was able to form a waterproof multi-network hydrogel upon exposure to low-energy visible light. In addition, HA photosealants showed excellent adhesion even under wet conditions and maintained adhesion at pressures 10 times higher than normal intracranial pressure (approximately 15 mmHg) [6,17]. Cellular experiments have also demonstrated that dura mater tissue recovery is superior to existing tissue adhesives, which can cause granulomatous inflammation and fibrosis [26].

This study highlights the potential of medical adhesive hydrogels as biodegradable materials and demonstrates that the rate of degradation is critical to their in vivo efficacy and safety. In our previous research, in rabbit experiments, dura mater recovery was faster than other dural sealants and no inflammatory cell infiltration was observed, resulting in superior biocompatibility compared to commercial tissue adhesives. In this study, HA photosealant was applied over incised dura in a healthy rat craniectomy model and monitored over a period of 1 month to investigate the serial degradation pattern. The biodegradation pattern of HA photosealant and regeneration of damaged dura were analyzed by MRI and histopathological evaluation. HA photosealant is expected to remain in place until the dura mater has healed and then be completely resorbed. The observed variation in degradation rates between subjects suggests that it is important to consider individual biological responses when designing and applying hydrogel-based wound closure and therapies. This is the first paper to analyze the biodegradability of HA photosealant in a rat craniectomy model using MRI. As an anticipated outcome of this result, perspectives may be given on elucidating in vivo efficacy and biodegradation patterns of applied HA photosealant.

CONCLUSION

In this study, the biodegradability of low-energy visible light-activated HA photosealant based on biocompatible natural polymer was analyzed using MRI images in a rat craniectomy model. HA photosealant was gradually biodegraded while maintaining mechanical performance during the period until dura recovery. Histological analysis showed gradual degradation on MRI images with excellent dura tissue recovery without granulomatous inflammation or toxicity. The HA photosealants developed in this study has excellent usability as it crosslinks rapidly within 5 seconds and is easily dispensed from a syringe widely used in clinical practice. Therefore, the new dural closure method using HA photosealants is expected to have excellent CSF leakage prevention effect and biodegradability, making it highly applicable and usable in clinical settings.

Notes

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Informed consent

This type of study does not require informed consent.

Author contributions

Conceptualization : SYY, KHN; Data curation : HL, SL, SYY, DHK, MH, KHN; Formal analysis : SL, SYY, DHK, MH, KHN; Funding acquisition : SYY, KHN; Methodology : HL, SL, SYY, MH, KHN; Project administration : SYY, KHN; Visualization : HL, SL; Writing - original draft : HL, SL, SYY, KHN; Writing - review & editing : SYY, KHN

Data sharing

None

Preprint

None

Acknowledgements

This study was supported by Biomedical Research Institute Grant (No. 202100220001), Pusan National University Hospital.

Fig. 1.

Experimental scheme of degradation pattern of a hyaluronic acid (HA)-based photocurable sealant. Illustration and photography showing the HA photosealant application in a rat craniectomy and durotomy model (left). Experimental timeline demonstrating specific analyses at each time point (right). MRI : magnetic resonance imaging.

Fig. 2.

Illustration of measuring hydrogel in brain magnetic resonance imaging of rat using image processing program (Image J, version 1.54; National Institutes of Health, Washington, DC, USA). The photocured hyaluronic acid hydrogel (white area) is indicated by a red dotted line.

Fig. 3.

Hyaluronic acid (HA) photosealants degradation pattern characterized by magnetic resonance imaging. A : Photographs of residual areas of HA photosealants in weeks 1, 2, and 4 at the craniectomy site. HA hydrogels (white area) are indicated by a red dotted line. B : Residual area of HA photosealants for each rat per week. C : Average residual area of HA photosealants per week.

Fig. 4.

Histological findings demonstrating the efficacy of hyaluronic acid (HA) photosealants on dural repair (scale bar : 1 mm). Left column : photographs of rat craniectomy and durotomy model at determined time point, middle column : Hematoxylin and Eosin (H&E) staining, right column : masson trichrome (MT) staining, white arrows indicate remained HA hydrogels.

Table 1.

Residual area of HA photosealants measured by image processing program^*

	1 week	2 weeks	4 weeks
Area (%)
Rat 1	100.0	41.35	10.28
Rat 2	100.0	68.35	56.11
Rat 3	100.0	94.88	46.65
Rat 4	100.0	82.18	42.51
Rat 5	100.0	46.07	43.93
Average (%)	100.0	66.57	39.90

^* Image J (version 1.54; National Institutes of Health, Washington, DC, USA).

HA : hyaluronic acid

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