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EVALUATION OF THE EFFECTIVENESS OF SCAFFOLDS BASED ON HYALURONIC ACID GLYCIDYL METHACRYLATE AS A POSSIBLE PLATFORM FOR BRAIN TREATMENT
M.O. Novozhilova1*, T.A. Mishchenko1, A.G. Savelyev2, A.V. Sochilina2, E.V. Khaydukov2, M.V. Vedunova1
1 Institute of Biology and Biomedicine, National Research Lobachevsky State University of Nizhny Novgorod, 23 Gagarin ave., Nizhny Novgorod, 603950, Russia
2 Federal Scientific Research Center «Crystallography and Photonics» Russian Academy of Sciences, 59 Leninskiy Prospekt, Moscow, 119333, Russia
Abstract. 3D bioengineering constructs are currently a promising area of research in the regeneration of various tissues. In our work, several modifications of scaffolds based on hyaluronic acid glycidyl methacrylate are presented. Scaffolds have been tested for biocompatibility with nerve cells in an in vivo model of traumatic brain injury. Throughout the experiment, the neurological status of the animals was monitored, and at the end, a histological examination of the brain was carried out. It has been shown that scaffolds are non-toxic to nerve cells and reduce the development of neurological deficit in animals in the post-traumatic period. The possibility of using the scaffold with a lower biodegradation rate as a carrier of a therapeutic drug has also been demonstrated.
Keywords: neurotransplantation, scaffold, hyaluronic acid glycidyl methacrylate, temozolomide, brain trauma, mice, neurological deficit.
List of Abbreviations
TBI - traumatic brain injury BBB - blood-brain barrier SC - scaffold UV - ultraviolet radiation CNS - central nervous system TMZ - temozolomide
Introduction
Brain injuries are relatively common and stands at 3 to 6% of all accidents in developed countries (Karavaev, Kopysov, 2018). Annually, in Russia about 700 thousand people suffer from traumatic brain injury (TBI); 50 thousand of them die, and another 50 thousand become disabled (Likhterman, 2014). TBI is caused primarily by mechanical stress on a head (Zhou et al., 2020), and usually results from a severe blow or blow to the head with a blunt or penetrating object such as a bullet or sharp object (Nolan, 2005). The action of external physical force may lead to an extensive neuronal loss, which leads to impairments of cognitive or physical functions (Thurman et al., 1999). TBI is also can act as a key trigger for the development of neurodegenerative processes
and acute chronic diseases in the CNS (Menon et al., 2010; Hart et al., 2004).
Primary trauma (for example, intracranial hematomas, skull fractures, lacerations, bruises, and penetrating wounds) is a focus which spreads the mechanical damage to the surrounding brain tissues (Mustafa, Alshboul, 2013). It develops in a short period of time, approximately 100 milliseconds. (Smith-Seemiller et al., 1997). Intracerebral bleeding leads to hemorrhage and rupture of blood vessels in the brain parenchyma resulting in massive lesions (Mustafa, Alshboul, 2013).
Further activation of pathological molecular and cellular pathways leads to secondary brain damage (Kochanek et al., 2015; Pearn et al., 2016). Disruption in the ions flow triggers ax-otomy and demyelination. Altered cellular permeability also increases calcium influx, which causes mitochondrial dysfunction with a further energy deficit, followed by necrotic and apop-totic processes. These molecular and cellular changes may lead to the development of cyto-toxic or vasogenic cerebral edema and impaired self-regulation, resulting in an increase in intra-cranial volume due to vasodilation or water accumulation (Stocchetti and Maas, 2014).
The spread of cell membranes depolarization leads to activation of anaerobic metabolism and depletion of the energy substrate. Trauma affects the blood-brain barrier (BBB) directly and increases its permeability which, in turn, promotes the activation of the pro-inflammatory state (Corps et al., 2015). These complex series of events begins in a minutes after the injury but continues for weeks or even months, especially when inflammation develops.
TBI heterogeneity is usually graded according to clinical severity and is assessed by the Glasgow Coma Scale (Teasdale et al., 2014), which takes into consideration while selecting a therapeutic strategy. Current therapy of TBI mainly focuses on secondary trauma and is aimed at surgical removing the space-occupying intracranial lesion and intensive drug treatment (Hackenberg, Unterberg, 2016, Stein et al., 2017). However, full morpho-functional recovery of CNS after TBI is still difficult to achieve.
The most promising approach is associated with the use of bioengineering constructs (scaffolds). They represent bioactive matrices for neurotransplantation containing various biological compounds which stimulate the reparative processes of nerve tissue. A number of studies have already demonstrated the high efficiency of these structures for brain tissue reconstruction (Wong and Lo, 2015; Balyabin et al., 2016). It has been shown that transplantation of scaffold into the injury site promotes the maintenance of cell migration and their survival, and decreases inflammatory processes in the damaged area (Wang et al., 2005).
The aim of the study was to assess the outcomes of implantation of several types of scaffolds based on hyaluronic acid methacrylate in the reconstructive surgery in the modeled traumatic brain injury in vivo and access the possibility of scaffolds usage as a carrier for direct delivery of treatment.
Methods
Ethics statement. The experiments were carried out on adult male C57BL/6 mice (6-8 weeks of age, 25-28g). The animals were
housed in the SPF vivarium of Lobachevsky University.
All experimental procedures were performed in accordance with the Rules for the Work using Experimental Animals (Russia, 2010), International Guiding Principles for Biomedical Research Involving Animals (CIOMS and ICLAS, 2012), and the ethical principles established by European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 2006) and approved by the Bioeth-ics Committee of National Research Lobachev-sky State University of Nizhny Novgorod.
Scaffolds preparation
The following photocurable compositions (PCCs) were used to produce scaffolds. PCC1 composition of scaffold №1 (SC1) contained 20 wt % of low molecular weight (~100 kDa) hya-luronic acid glycidyl methacrylate (HAGM) synthesized with 43% degree of substitution according to the protocol (Sochilina, et al., 2019) as described above in poly (ethylene glycol) di-acrylate (PEGDA), Mn = 575 was added in the amount of 2.5 wt % to optimize the mechanical properties of the hydrogel. Flavin mononucleo-tide (FMN) was used as a photoinitiator with the concentration equal to 0.0004 wt%. Dissolution of PCC was carried out in sodium chloride 0.9% intravenous infusion in an ultrasonic bath until completion. PCC2 composition of scaffold №2 (SC2) was similar to PCC1, but the dissolution of precursors was performed in 0.04% collagen solution (Belcozin, Russia).
Scaffolds were produced using micromolding technique (Savelyev, et al., 2017) PCC was placed in a specially made silicone master mold and covered with a coverslip. The exposure was carried out for 30 min using the CW light-emitting diode at 365 nm wavelength (intensity 20 mW/cm2). After that the coverslip was carefully removed, and the cross-linked hydrogel scaffold was pulled out.
PCC3 of scaffold №3 (SC3) was produced similar to PCC1, but with increased concentration of FMN up to 0.001 wt %. PCC4 (SC3+TMZ) was prepared by mixing PCC3 with temozolomide-TEVA, (Nerparma, Italy)
A B
Fig. 1. Traumatic brain injury model. A - schematic representation of injury site (highlighted in orange square); B - head injury using the weight-drop method
in the 4:1 ratio. Photocuring of PCC3 and PCC4 was conducted in 1 mm height silicone spacer placed between two cover glasses. Spacer had a round hole 5 mm in diameter for loading PCC. Exposure was performed via irradiation for 15 min from the top and 15 min from the bottom.
Traumatic brain injury (TBI) model and scaffold transplantation
Before surgical procedures, each animal was anesthetized with an intraperitoneal injection of Zoletil 100 (70 mg/kg, Virbac Sante Animale, France) and Xylanite (0.02 mg/kg, NITA-PHARM, Russia). In the absence of proprioceptive reflexes, fur was removed from the top of the animal's head, and a soft tissue incision was carried out. Crani-otomy (2x2 mm in size and 1-2 mm thick) was performed in the right hemisphere near the central suture, to the right of the lambda-bregma intersection with a fine drill, leaving the dura mater intact (Fig. 1A).
Mechanical trauma was carried out using the weight-drop method (Jiasong, et al., 2009) (Fig. 1B). The animal was rigidly fixed on a stand. The damage site was placed under a hollow plastic stand with a height of 0.80 m. A weight with a blunt surface of 4 g was placed inside the stand and dropped on the abovementioned area.
To avoid a fracture of the jaw, the animals' head was fixed on a soft lining perpendicularly to the tip of the weight. The animal had a spontaneous breath. The skin was then tightly sutured with surgical threads (0.2 mm) and treated with an antiseptic solution. After procedure, all animals were allowed to recover after anesthesia before being returned to their home cages with postoperative care and ad libitum access to food and water.
On day 7 after TBI, experimental mice were again anesthetized and fixed on a stand. The sutures were removed with subsequent preparation of the operating field. The scaffold was placed directly to the injury site. The size of neurotransplantat was as close as possible to the injury volume. Then the incision was closed with surgical threads.
The animals were divided into the following groups: 1) intact animals were not subjected to surgical procedures; 2) sham-operated animals undergoing craniotomy without TBI; 3) TBI -the animals undergoing traumatic brain injury; 4) SC - the animals undergoing TBI with subsequent transplantation of scaffold.
Neurological status determination
Functional state of the CNS of experimental mice in the post-traumatic period was evaluated
using a scale for assessing the severity of neurological deficit modified for mice. The scale includes a number of tests to determine the motor activity of animal hanged by the tail, peculiarities of walking on a horizontal surface, coordination of movements, the severity of reflexes (an injection into the animal's footpad), muscle tone (pulling up on the bar), ptosis and exoph-thalmos (Prickaerts et al., 1999). Each test evaluates by a scoring system, where 2 points meant a lack of reaction. The obtained values were summarized and interpreted as severe CNS damage (10-20 points), moderate damage (6-9 points) and light CNS damage (1-5 points) (Beni-Adani et al., 2001).
In addition, the degree of sensorimotor disorders was identified according to Garcia scale which characterizes the spontaneous activity of animal in a home cage (5 min), the symmetry of forelimbs movement, the symmetry of body movements, ability to climb on a wire cage, reaction to touch on each body side and vibrissae (Garcia, 1995). The results from six tests are summarized where the maximum possible score is 18 (no damage), and the minimum score is 3 (severe CNS damage).
Morphological assessment. Histological studies were carried out on day 14 after scaffold transplantation. The brains of experimental animals were surgically removed and then fixed in 10% formalin solution at room temperature for 2 days. After the incubation period, the brain was placed in 15% (24-48 hours) and then in 30% (the next 24-48 hours) of sucrose solution at room temperature. Next, the samples were transferred on a platform of a Leica CM1520 freezing sliding cryostat (Leica, Germany) and gradually filled with cryogel (Leica, Germany).
Each brain was cut into 15 |im thin corona slices. Every fifth slice of each sample was placed on a slide and dried in the air within 24 h. The obtained slices were then stained according to a standard hematoxylin-eosin method (PanReac AppliChem, Germany). Next, the slices were dehydrated in alcohols of upward concentration, purified in xylols and embedded in a mounting medium (Thermo Fisher Scientific, USA).
The samples were examined using a Zeiss Primo Star light microscope (Zeiss, Germany) with integrated an Axio CamMRc camera (Zeiss, Germany).
Statistical analysis. The obtained data are presented as a mean ± a standard error of the mean (M ± SEM). The significance of differences between the experimental groups was determined using Microsoft Exel and Sigma Plot 12.5 software (Systat Software Inc., USA). Differences were considered significant at p < 0.05.
Results
Analysis of the functional state of the CNS after TBI and scaffolds transplantation
One of the main neuromonitoring approaches is an analysis of animals' neurological state. Any surgical intervention leads to a change in neurological status and requires evaluation. Experimentally, it is possible to consider the changes in the behavior and sen-sorimotor functions of animals before and after modeling TBI and scaffolds transplantation.
According to the neurological status assessment, it was shown that TBI leads to the development of severe neurological deficit (Fig. 2). In the "TBI" group, the animals showed marked disorders with the manifestation of grasping and protective reflexes. The parameters of the assessment of neurological deficit and the Garcia scale significantly differed from the «Intact» and «Sham-operated» groups. On day 14 after surgery, the animals with TBI showed a decrease in the general sensitivity to stimuli and overall activity.
Transplantation of scaffolds SC1 and SC2 decreased the development of neurological deficits in animals (Fig. 2). According to the assessment of neurological deficit and the Garcia scale, the animals from the «SC1» and «SC2» groups showed significantly better results than the «TBI» group.
Transplantation of SC1 scaffold decreases the negative impact of the consequences of brain trauma both on the parameters of neurological deficit (day 7 after transplantation: "SC1" 2.6 ± 0.5, «SC2» 3.6 ± 0.3) and the Garcia scale (day 7 after transplantation: «SC1 »
À
В
Fig. 2. Neurological status of mice after TBI and scaffold transplantation according to (A) Neurological impairment assessment and (B) Garcia scale. * - versus «Intact», # - versus «Control», • - versus «TBI», p < 0.05, Mann-Whitney test
14.6 ± 0.1; «SC2» 14.3 ± 0.1) more dynamically.
Morphological study of brain slices from
Of note, the animals with scaffold became more aggressive, which is possibly related to an increase in pain shock after TBI and transplantation. Nevertheless, the strong difference in the behavior of animals before and after transplantation of the scaffold into the TBI focus in the TBI group indicates that the matrices are definitely aimed at maintaining and improving the state of activity of the somatosensory system.
Morphological analysis of the murine brain in the posttraumatic period
During the TBI modelling, it was observed that the main loss of tissue structure occurs in the cerebral cortex; however, the injury site penetrates quite deeply (Fig. 3).
the «Intact» and «Sham-operated» groups showed the homogeneity of the nervous tissue. The neurons of the upper layers of the murine cerebral cortex are rounded-oval in shape with a well-defined nucleus and neat contours (Fig. 4). Simulation of traumatic brain injury leads to significant changes in the murine cerebral cortex. Massive osmotic edema of tissues at the site of TBI is observed. Cell bodies are deformed. Extensive foci of necrosis are also registered. There are apoptotic elements of cells, pronounced plethora and weak areas of tissue growth (Fig. 4B). The brain tissue has a complete loss of the structure of the substance in the vicinity of the TBI focus.
Fig. 3. Simulation of traumatic brain injury in C57Bl6 mice. A - representative photo of the brain after weight-drop; B - representative photo of the brain slice on day 7 after traumatic brain injury
A
B
Fig. 4. Representative images of histological samples of murine brain cortex on day 7 after traumatic brain injury. A - Intact group, B - Sham-operated, C, D, E - TBI. Hematoxylin-eosin staining, magnification x10 (C), x20 (A, B, D), x40 (E).
Accordingly, the main task of the scaffolds should be not only supporting the damaged area, but also its further functional restoration by gradually replacing the normal nerve tissue during scaffolds biodegradation.
Selection of optimal 3D construct for neurotransplantation
During the study, it was important to consider the biocompatible features of scaffold with the nervous tissue. After removal and fixation of the murine brain samples, we observed
how the scaffolds penetrate into the nervous tissue and occupy the TBI site (Fig. 5). Each matrix is embedded in the damaged area. Importantly, the construct should not cause additional disturbances of the damaged area; therefore, the dynamics of traumatic brain injury processes is necessary to be monitored.
Rapid degradation of 3D constructs and their low ability to destroy the nervous tissue are observed on day 7 after transplantation (Fig. 6). Histological samples obtained from the «SC1» group demonstrate minor damages of the brain
cortex. Mild osmotic edema of the cortex is observed. There are no obvious signs of transplant rejection. Morphological changes in neurons are predominantly necrobiotic. Non-pronounced signs of regeneration in 10 view fields were identified. It was shown that the closer the cell is to the TBI focus, the more elongated and less centrally located the nucleus. This also indicates the regenerative consequences of transplantation. Based on histological data, it can be assumed that SC1scaffolds are suitable for further study of nervous tissue repair processes, provided that the preservation of its mechanical properties must be improved.
On the other hand, the brain tissue at the site of transplantation of SC2 scaffold is significantly damaged. In the histological sections obtained from the «SC2» group, a sharp glial scar, traces of bleeding and tissue proliferation in both brain cortex and deeper layers are observed. The section shows extensive necrosis of brain cells in volume visible tissue, vasogenic edema of the cerebral cortex, absence of tissue structure, and the presence of apoptotic cellular elements. The absence of visible structural elements of the graft on day 7 after transplantation suggests that SC2 scaffold is not suitable for the implementation of the regenerative function in the nervous tissue.
Thus, it was shown that scaffold is an elastic structure capable of causing recovery processes in the brain due to its composition.
Scaffolds as bioactive platforms
Comparative analysis of the tested scaffolds showed that SC1 is more suitable for neurotransplantation. However, at the same time SC1 as a scaffold was rapidly degrading, so we changed its composition in order to improve mechanical properties and decrease the rate of biodegradation.
A scaffold should be a versatile composite for treatment of different brain damages, so its chemical structure should be targeted to resolve a specific issue. We decided to consider scaffold as a carrier for active substances. Therefore, after the modification of the scaffold composition, we conducted experiments with a focus on a specific brain injury (gliomas). We
have included in cytostatic drug temozolomide (TMZ) the scaffold composition in order to access the possibility to use this approach in further treatment of pathophysiological complications of brain tumors. First, we decided to determine whether the developed scaffold could be a platform for the delivery of treatment drugs. Taking into account the recent data on TMZ toxicity for normal nervous cells, we have used the concentration of 1-2 mM for scaffolds preparation.
Neurological and behavioral tests showed a decrease in animals activity after transplantation of scaffold with TMZ that points on tox-icity effects of the used concentration of drug for normal nervous cells (Fig. 7).
The neurological state of animals with transplantation of control scaffolds (without TMZ) is significantly higher than that of the «SC3 + TMZ» group. On day 7 after transplantation, the parameters of neurological deficit in the «SC3» and «SC3+TMZ» groups was 3.3 ± ± 0.33 and 6.3 ± 0.57 respectively. According to the Garcia scale, the use of SC3 scaffolds reduces nociceptive manifestation of the consequences of surgical intervention (day 1 after surgery: SC3 13.6 ± 0.88; SC3+TMZ 14.6 ± ± 0.33; day 7 after surgery: SC3 15.6 ± 0.33; SC3 + TMZ 13.0 ± 0.57).
Next, we conducted a histological analysis to accesses the biocompatibility of SC3 scaffolds for the nervous tissue. The design of SC3 scaffolds showed positive results in the formation of new connections between nervous cells (Fig. 8). In the «SC3» group, a growth of cell processes towards the scaffold, the formation of connections between cells was observed. The morphology of cells near the scaffold is similar to the intact group. The appearance of cells with an enlarged nucleus diameter, peripheral nuclei or binucleolar neurons is a morphological manifestation of regenerative processes. Such cells cover approximately 30% of the total number of cells in 10 view fields. Proliferating cells have been found near the hippocampus. Moreover, the presence of single cells with numerous processes and cellular conglomerates were detected on the surface of the scaffolds.
A
B
Fig. 7. Neurological status of mice after TBI and scaffold transplantation according to (A) Neurological impairment assessment and (B) Garcia scale. * - versus «Intact», # - versus «Control», • - versus «SC control», p < 0.05, Mann-Whitney test
SC3 A1
A2
SC3+TMZ
B1
B2
Fig. 8. Representative histological samples of the murine brain cortex on day 7 after scaffolds transplantation. (A1-A3) - SC3; (B1-B3) - SC3+TMZ. Hematoxylin and eosin staining. Magnification (A1-B1) X10, (A2-B2) X20, (A3-B3) X40 (a continuation of the pattern on the page 31)
Contrary to the histological samples from the «SC3» group, an uneven and tearing edge of the nerve tissue near the scaffold with TMZ
was shown. Multiple cellular debris and development of necrotic and apoptotic processes suggest that TMZ is actively released into the
A3 B3
Fig. 8. Representative histological samples of the murine brain cortex on day 7 after scaffolds transplantation. (A1-A3) - SC3; (B1-B3) - SC3+TMZ. Hematoxylin and eosin staining. Magnification (A1-B1) X10, (A2-B2) X20, (A3-B3) X40
intercellular space and caused a severe toxic effect.
Therefore, the developed SC3 scaffolds are non-toxic for the brain cells and could be considered as a carrier for biologically active substances and drugs. However, a selection of an optimal concentration of therapeutic agents in the scaffold composition is an exciting area for upcoming research.
Discussion
In recent years, tissue engineering techniques have advanced significantly. However, it is accompanied by a number of issues such as biocompatibility, immunogenicity, biodegradation rate and toxicity properties. Analysis of different polymer-based constructs has shown that copolymerized composite scaffolds are more efficient than with a single polymer since they allow copolymers to compensate for the disadvantages that a single polymer can have (Edgar et al., 2016). Immunogenic materials interfere with remodeling because they can cause inflammation. Thus it can reduce the efficiency and survival of the graft (Galler et al., 2011). The most successful scaffolds facilitate cell adhesion, growth, migration, and respond to molecular and physical signals in vivo (Bottcher-Haberzeth et al., 2010). The biodegradable properties of materials are factor specific to the desired function of engineered tissue. Slow biodegradation is preferred for long-term implants, while active and rapid biodegradation is
important to stimulate remodeling and replacement of biomaterials for tissue repair (Liao et al., 2014). To be clinically effective, these media should be as close as possible to the main characteristics of the native extracellular matrix at the cellular and subcellular levels (Pereira, Bartolo, 2015).
The current approach for neurotransplantation remains limited due to rejection and reaction from the immune system. As a solution, three-dimensional scaffolds were developed based on polymer biomaterials. These constructs can be used as carriers for various types of drugs and genes, with the release profile being tuned via modulating the morphology, porosity and composition of the scaffold (Sha-lumon, Chen, 2015). Scaffolds can be used as a platform for transferring various substances to the damaged area of the brain and participate in regenerative processes. In recent years, there has been a trend towards an increase in the combination of scaffolds and growth factors for creation a bioactive system providing biomimetic and biodegradable physical support for tissue growth, and activation of biological signals to modulate tissue regeneration (Shalumon, Chen, 2015).
In our study, comparative analysis of the effect of different types of scaffolds on the brains' morpho-functional characteristics in the modeled TBI showed that SC1 scaffolds demonstrated resistance for quick degradation and high biocompatibility with the nervous tissue
(the presence of regenerative processes, weak inflammatory reactions and tissue lysis), with preservation of animal activity. The modified SC1 scaffold was then taken as a basis for developing a platform for biologically active substances delivery to the damaged area of the brain. Behavioral and morphological studies revealed that the developed scaffolds SC3 are non-toxic for the nerve cells and can be used as a carrier for drug (temozolomide) delivery. However, a selection of an optimal concentration of the therapeutic agent in the scaffold composition is an exciting area for our upcoming research. Moreover, we propose to consider the possible symbiosis between scaffolds and neurotrophic factors BDNF and GDNF, since their effectiveness in cellular survival and
maintaining the functional activity of neuron-glial networks in stress conditions has already been confirmed earlier (Mishchenko et al., 2019; Mitroshina et al., 2019; Vedunova et al., 2015).
Acknowledgements
The study was supported by the Ministry of Science and Higher Education of the Russian Federation (project No. 0729-2020-0061) and partially by RFBR (project No. 18-29-01055) and grant of the President of the Russian Federation (MK-1485.2019.4). This research was carried out using The Core Facilities «Molecular Biology and Neurophysiology».
Authors declare no conflicts of interests.
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