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BY 4.0 license Open Access Published by De Gruyter Open Access October 22, 2022

Treatment of rat brain ischemia model by NSCs-polymer scaffold transplantation

  • Yue Qi , Tao Wu , Dongdong Yan , Manhui Li , Baodong Chen EMAIL logo and Yi Xiong EMAIL logo
From the journal Open Chemistry

Abstract

Neural stem cells (NSCs) transplantation is a promising therapeutic strategy for ischemic stroke. However, significant cell death after transplantation greatly limits its effectiveness. Poly (trimethylene carbonate)15-F127-poly (trimethylene carbonate)15 (PTMC15-F127-PTMC15, PFP) is a biodegradable thermo-sensitive hydrogel biomaterial, which can control drug release and provide permissive substrates for donor NSCs. In our study, we seeded NSCs into PFP polymer scaffold loaded with three neurotrophic factors, including brain-derived neurotrophic factor, nerve growth factor, and Neurotrophin-3. And then we transplanted this NSCs-polymer scaffold in rat brains 14 days after middle cerebral artery occlusion. ELISA assay showed that PFP polymer scaffold sustained releasing three neurotrophic factors for at least 14 days. Western Blot and fluorescence immunostaining revealed that NSCs-polymer scaffold transplantation significantly reduced apoptosis of ischemic penumbra and promoted differentiation of the transplanted NSCs into mature neurons. Furthermore, infarct size was reduced, and neurological performance of the animals were improved by the transplanted NSCs-polymer scaffold. These results demonstrate that PFP polymer scaffold loaded with neurotrophic factors can enhance the effectiveness of stem cell transplantation therapy, which provides a new way for cell transplantation therapy in ischemic stroke.

1 Introduction

Stroke is the leading cause of disability and death worldwide, which decreases patients’ quality of life and increases economic burden [1,2]. Approximately 62.4% of strokes are ischemic stroke, which is caused by cerebral ischemia [3,4]. At present, limited advances have been made in the therapeutic method of ischemic stroke, and thrombolysis or mechanical thrombectomy is the main acute treatment for ischemic stroke [5]. However, due to the narrow therapeutic window of thrombolytic therapy, only a finite number of patients receive treatment in time. Even worse is that ischemia-reperfusion injury may further increase the cerebral infarct size of patients, even if they have received effective thrombolytic therapy [6,7]. Therefore, it is critical to find an effective therapy against the neurological deficit caused by ischemia-reperfusion injury.

Neural stem cells (NSCs) have the capacity to differentiate into multiple cell lineages of brain. Numerous experimental studies have shown that transplanted NSCs contributed to repairing the damaged neural circuits and improving the behavioral recovery [8]. Despite promising results, a large scale of NSCs died soon after transplantation, which may be caused by the harsh microenvironment in the host’s damaged area [9,10]. Therefore, the effectiveness of cell-based therapies is limited. Thus, safe and effective ways are needed to improve the viability of grafted NSCs and potentiate neuronal differentiation.

The neurotrophic factors are essential for the survival and differentiation of transplanted NSCs. In recent years, brain-derived neurotrophic factor (BDNF) has been reported to be involved in neuroprotective effect for hypoxic-ischemic brain [11]. It could not only reduce cell apoptosis after ischemic stroke, but also govern NSCs to differentiate into neurons [12,13]. Consequently, BDNF has been widely used in the treatment of stroke, such as injecting BDNF into the injured brain [14], using drugs to increase the level of endogenous BDNF [15], and transplanting stem cells over expressing BDNF [16,17]. Nerve growth factor (NGF), the first discovered neurotrophic factor, also contributes to lessen apoptosis and promote the development of nerve cells [18,19,20], which has a great prospect in neural repair after stroke. Neurotrophin-3 (NT-3) is well known for its positive effect on neural repair, including inhibiting neuronal apoptosis [21], promoting neuronal differentiation [22], and inducing neurite growth [23]. For the above reasons, we chose these neurotrophic factors to enhance the therapeutic efficacy of NSCs transplantation for stroke.

Being biocompatible and nonimmunogenic, biomaterials are widely used as scaffolds for in vivo experiments [24,25]. As drug carriers, biomaterials can control drug release to maintain an effective drug concentration for a period of time. In addition, they can provide permissive substrates to increase survival and differentiation of donor cells [26,27,28,29,30,31]. Poly (trimethylene carbonate)15-F127-poly (trimethylene carbonate)15 (PTMC15-F127-PTMC15, PFP), as a biodegradable thermo-sensitive hydrogel biomaterial, can transform from liquid to gel at 37°C. The drug encapsulated in PFP can slowly continue releasing in vivo for up to 16 days [31]. Therefore, PFP is an ideal biomaterial for in vivo applications. In our study, we engineered a PFP carrier loaded with neurotrophic factors, including BDNF, NGF, and NT-3. NSCs were seeded into the PFP scaffold, and transplanted into the damaged brain area of rats. The purpose of our study was to determine whether the NSCs-polymer scaffold system helps to retain the viability of grafted NSCs, maintain their differentiation potential, and improve brain functional outcomes.

2 Materials and methods

2.1 Animals

Male Sprague–Dawley rats were purchased from Medical Experimental Animal Center of Guangdong Province. All rats were housed in separate cages with free access to food and water. The rooms were maintained at constant temperature and humidity and subjected to 12 h light/dark cycle. All animal experiments conducted were according to animal use protocols and approved by the Committee for the Ethics of Animal Experiments, Shenzhen-Peking University-The Hong Kong University of Science and Technology Medical Center.

2.2 Cell cultures and infection

NSCs were isolated from the hippocampus of the 3–5 days-old neonatal rats and cultured in Dulbecco’s modified Eagle’s medium (DMEM)-Ham’s F12 medium (Gibco, USA) with 20 g/L B27 (Gibco, USA), 20ug/L basic fibroblast growth factor (bFGF, PeproTech, USA) at 37°C in an incubator with 5% CO2. Half of the medium was replaced every 2 days, and cells were passaged once a week. After 3 weeks of culture, the NSCs were infected with adenovirus expressing GFP (multiplicity of infection was 10) and were cultured for 12 h. Then, half of the medium was replaced. After further culturing for 12 h, the GFP-infected NSCs (infection rate was 80%) were harvested for transplantation.

2.3 Preparation of NSCs-polymer scaffold

PFP was presented by Sun Yat-sen University. Specific synthesis method of PFP could be seen in the previous research [31]. PFP was dissolved in PBS buffer with or without 50 ng/mL neurotrophic factors (NGF, BDNF, and NT-3) to a final concentration of 5% (w/v). And then the solutions were stored at 4°C for 12 h. Neurospheres were dissociated mechanically and the number was counted. Before transplantation, 2 × 106 NSCs were seeded in the PFP scaffold.

2.4 Establishment of middle cerebral artery occlusion (MCAO) model

Adult male SD rats weighing 250–280 g were anesthetized with 10% pentobarbital sodium. The skin in the middle of the neck was cut, and the right common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were exposed. The MCAO-thread (3600, Guangzhou Jialing Biotech Co, China) was inserted from the ECA into the ICA until it blocked the origin of the middle cerebral artery (MCA). After 1 h, the MCAO-thread was pulled out slowly. The animals were returned to their home cage.

2.5 Transplantation

Fourteen days after the onset of stroke, the NSCs were transplanted by using a 10-μL Hamilton syringe attached to a stereotaxic apparatus. Four experimental groups were prepared: the PFP group (receiving 10 μL PFP), the PFP + NSCs group (receiving 10 μL PFP containing 1 × 106 NSCs), the PFP + NTs group (receiving 10 μL PFP containing neurotrophic factors), and the PFP + NSCs + NTs group (receiving 10 μL PFP containing neurotrophic factors and 1 × 106 NSCs). The rats were given 10 μL deposits at the coordinate: anterior-posterior, +0.5 mm; medial-lateral, −1.5 mm; and dorsal-ventral, −1.6 mm. Deposits were delivered at 1 μL/min, and the needle remained in situ for 5 min post-injection before being slowly removed.

2.6 Behavioral testing

A battery of behavioral tests was performed on the day of transplantation (baseline), and −14, 0, 28, and 56 days after transplantation, using the neurological severity scores (NSS) modified by Chen et al. [32] (Figure S1). The functional evaluation included motor, sensory, reflex, and balance tests. The total score ranged from 0 to 18 (0 = normal; 18 = maximal deficit). The higher the score, the more severe the injury.

2.7 Measurement of infarct area

Bregma as baseline, the brains were cut at every 2 mm (Figure 5a). The brain sections of coordinate −4 and −2 mm were stained by 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 15 min. The infarct areas were estimated as a percentage of the ipsilateral brain section area using the following formula: (area of contralateral brain section – area of remaining ipsilateral brain section)/area of contralateral brain section × 100%. The area of the infarct was quantified using Image Pro-plus.

2.8 Fluorescence immunostaining

For NSCs identification, NSCs were collected and fixed with 4% paraformaldehyde (PFA) in PBS for a night at 4°C, then permeabilized and blocked in blocking solution (PBS containing 4% goat serum, 1% bovine serum albumin, and 0.4% Triton X-100) for 1 h at room temperature. Subsequently, NSCs were incubated with primary antibody (anti-Nestin, 1:200, Abcam, mouse monoclonal antibody) overnight at 4°C, followed with secondary antibody (anti-mouse, CY3, 1:400, Jackson ImmunoResearch). Finally, NSCs were incubated for 5 min with DAPI and captured by a Zeiss LSM 710 confocal microscope with a 63× objective.

For tissue immunofluorescence staining, rats were perfused with cold PBS followed by 4% PFA in PBS. The rat brains were postfixed overnight in the 4% PFA solution at 4°C. Then, they were embedded in paraffin after dehydration and cut into 5 μm sections. The brain sections were blocked in blocking solution for 1 h at room temperature. The brain sections were subsequently incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Map2 (1:500, Sigma, mouse monoclonal antibody) or anti-Caspase3 (1:200, CST, rabbit monoclonal antibody). After three washes in PBS, the brain sections were incubated with secondary antibodies (anti-mouse, CY3, 1:400, Jackson ImmunoResearch) for 2 h. Finally, NSCs were incubated for 5 min with DAPI. Images were captured by a Zeiss LSM 710 confocal microscope with a 20× objective and analyzed with Zen 2012 (Zeiss) and Image-Pro Plus.

2.9 Western blot

The brain tissue of rats was treated with lysate. Equal amounts of protein samples (20 µg) were loaded per lane and separated through SDS-polyacrylamide gel electrophoresis using 10 or 12% running gels and transferred to a polyvinylidene difluoride filter membrane. After blocking with 5% nonfat milk in TBST for an hour, the membranes were incubated with primary antibodies (anti-Map2, 1:2,000, Sigma) overnight at 4°C. After three washes with TBST, the membranes were incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG, 1:20,000, cell signal) for 1 h. The protein bands were visualized using an enhanced chemiluminescence detection kit. The images were analyzed with Image-Pro Plus.

2.10 Enzyme-linked immunosorbent assay (ELISA)

The brain tissue samples in ischemic penumbra were extracted at 1, 7, 14, 28, 35 days after transplantation. ELISA was performed using ELISA kits (Beijing Ruierxinde, China) according to the manufacturer’s instructions, in order to examine the concentration of NGF, BDNF, and NT-3.

2.11 Statistical analysis

For the data, comparisons between two groups were performed with Student’s independent-samples T test. Comparisons among multiple groups were performed with one-way ANOVA, followed by LSD’s post hoc analysis. The software for analyzing data was SPSS 19.0. The data were presented as mean value ± SD. p < 0.05 was considered as statistically significant.

3 Results

3.1 In vitro culture and infection of NSCs

NSCs were isolated from the hippocampus of 3–5 days-old neonatal rats, and observed under microscope after 14 days of culture. NSCs were displayed as suspended cell spheres. To confirm whether these cell spheres were neurospheres, the cells were immunostained with anti-nestin antibody, a marker for neural precursors, while the nucleus were labelled with DAPI. The results showed that most of the cell spheres were nestin – positive (Figure 1a). For in vivo visualization, NSCs were labelled with GFP by adenovirus transduction (Figure 1b).

Figure 1 
                  NSCs identification and infection. (a) Cell spheres were immunostained with nestin antibody and nuclei were labeled by DAPI. The images were captured by a Zeiss LSM 710 confocal microscope with a 63× objective (Red, nestin; Blue, DAPI). Scale bar = 20 µm. (b) Stem cells were infected with adenovirus expressing GFP. The images were captured by fluorescent inverted microscope with a 10× objective. Scale bar = 200 µm.
Figure 1

NSCs identification and infection. (a) Cell spheres were immunostained with nestin antibody and nuclei were labeled by DAPI. The images were captured by a Zeiss LSM 710 confocal microscope with a 63× objective (Red, nestin; Blue, DAPI). Scale bar = 20 µm. (b) Stem cells were infected with adenovirus expressing GFP. The images were captured by fluorescent inverted microscope with a 10× objective. Scale bar = 200 µm.

3.2 Neurotrophic factors were released from PFP scaffolds steadily in vivo

To ensure that the transplanted NSCs have a stable nutritional supply, we examined the levels of NGF, BDNF, and NT-3 in the ischemic penumbra tissues from 1 to 35 days after transplantation by performing ELISA. The data showed that levels of the three neurotrophic factors were significantly higher in PFP carrier loaded neurotrophic factors groups than that without neurotrophic factors groups. For PFP scaffolds releasing neurotrophic factors groups, NGF levels decreased mildly for the first 14 days, and then more rapidly until 35 days (Figure 2a). In contrast, BDNF levels descended evidently within the first 7 days, followed by a period of slight decline (Figure 2b). NT-3 maintained a stable level for the first 14 days, and thereafter decreased slowly (Figure 2c). Taken together, the levels of three neurotrophic factors can be sustained for relatively long periods, which provided a good foundation for survival and differentiation of transplanted NSCs.

Figure 2 
                  Three neurotrophic factor levels in ischemic penumbra of model rats. (a) NGF (b) BDNF, and (c) NT-3 levels were examined by ELISA assay. The results are shown as the mean value ± SD (n = 3).
Figure 2

Three neurotrophic factor levels in ischemic penumbra of model rats. (a) NGF (b) BDNF, and (c) NT-3 levels were examined by ELISA assay. The results are shown as the mean value ± SD (n = 3).

3.3 Transplantation of NSCs-polymer scaffold reduced cell death

Apoptosis is one of the main pathways leading to cell death after cerebral ischemia-reperfusion [33]. We investigated whether transplantation of NSCs-polymer scaffold could reduce cell apoptosis in the ischemic penumbra. As shown in Figure 3a, 7 days after transplantation, we found that apoptosis in all three groups were remarkably reduced compared with the control PFP group, especially the PFP + NSCs + NTs group (Figure 3b and c). The result suggested that transplantation of NSCs-polymer scaffold loaded with neurotrophic factors can effectively improve the survival of cells in ischemic penumbra.

Figure 3 
                  Transplantation of NSCs-polymer scaffold reduced cells’ death 7 days after transplantation. (a) Brain tissues in ischemic penumbra indicated by the box were collected. (b and c) Fluorescent staining with caspase-3 antibody revealed the number of apoptosis. The images were captured by a Zeiss LSM 710 confocal microscope with a 20× objective (Red, caspase-3; Blue, DAPI). Scale bar = 20 µm. The results are shown as the mean value ± SD (* p < 0.05, ** p < 0.01 vs PFP group, n = 3).
Figure 3

Transplantation of NSCs-polymer scaffold reduced cells’ death 7 days after transplantation. (a) Brain tissues in ischemic penumbra indicated by the box were collected. (b and c) Fluorescent staining with caspase-3 antibody revealed the number of apoptosis. The images were captured by a Zeiss LSM 710 confocal microscope with a 20× objective (Red, caspase-3; Blue, DAPI). Scale bar = 20 µm. The results are shown as the mean value ± SD (* p < 0.05, ** p < 0.01 vs PFP group, n = 3).

3.4 PFP scaffold loaded with neurotrophic factors induced NSCs to differentiate into mature neurons

On day 56 after transplantation, Western Blot revealed that the Map2 protein level in PFP + NTs group and PFP + NSCs + NTs group was significantly increased compared with PFP group (Figure 4a). The result suggested that exogenous neurotrophic factors could improve the survival of neurons in ischemic penumbra and promote NSCs to differentiate into neurons. To further investigate whether transplantation of NSCs-polymer scaffold could promote grafted cell to differentiate into mature neurons, we counted the number of Map2+ donor cells by immunofluorescent staining. The number of mature neurons derived from grafted NSCs in PFP + NSCs + NTs group was about twice as many as that in PFP + NSCs group (Figure 4b and c).

Figure 4 
                  PFP scaffold loaded neurotrophic factors induced NSCs to differentiate into mature neurons on day 56 after transplantation. (a) The Map2 (a marker of mature neurons) levels in ischemic penumbra were tested by Western Blot (*p < 0.05, **p < 0.01 vs PFP group, n = 3). (b and c) Tissue samples were immunostained with Map2 antibody and nuclei were labeled by DAPI. The images were captured by a Zeiss LSM 710 confocal microscope with a 20× objective (Red, Map2; Green, GFP; Blue, DAPI). Fluorescent staining with GFP and Map2 revealed that the donor NSCs differentiated into mature neurons. Scale bar = 20 µm. The results are shown as the mean value ± SD (**p < 0.01 n = 3).
Figure 4

PFP scaffold loaded neurotrophic factors induced NSCs to differentiate into mature neurons on day 56 after transplantation. (a) The Map2 (a marker of mature neurons) levels in ischemic penumbra were tested by Western Blot (*p < 0.05, **p < 0.01 vs PFP group, n = 3). (b and c) Tissue samples were immunostained with Map2 antibody and nuclei were labeled by DAPI. The images were captured by a Zeiss LSM 710 confocal microscope with a 20× objective (Red, Map2; Green, GFP; Blue, DAPI). Fluorescent staining with GFP and Map2 revealed that the donor NSCs differentiated into mature neurons. Scale bar = 20 µm. The results are shown as the mean value ± SD (**p < 0.01 n = 3).

3.5 Transplantation of NSCs-polymer scaffold reduced infarct size and improved behavioral performance

We further investigated whether transplantation of NSCs-polymer scaffold could promote recovery of cerebral ischemia phenotypically. We first measured cerebral infarct size by TTC staining according to the method shown in Figure 5a and c. On day 28 after transplantation, the infarct size of brain section at −2 or −4 mm was significantly decreased in PFP + NTs and PFP + NSCs + NTs groups compared with the PFP group (Figure 5d and e). Reduction in infarct size was more evident on day 56 post transplantation. The infarcted area at −2 and −4 mm was reduced by approximately 60% in PFP + NTs group and 80% in PFP + NSCs + NTs group (Figure 5b, d, and e).

Figure 5 
                  Transplantation of NSCs-polymer scaffold reduced infarct size and improved behavioral performance. (a) Bregma as baseline, the brains were cut every 2 mm to stain by TTC. (b) The infarct size of brain slice at coordinate −4 mm (white) was measured by TTC staining 56 days after transplantation. (c) The schematic diagram of infarct size calculation. (d) Measurement of the infarct size with coordinate −2 mm level, 28 and 56 days after transplantation (n = 3). (e) Measurement of the infarct area at coordinate −4 mm level, 28 and 56 days after transplantation (n = 3). (f) Behavioral performances were tested using NSSs (n = 5). The results are shown as the mean value ± SD (*p < 0.05, **p < 0.01 vs PFP group).
Figure 5

Transplantation of NSCs-polymer scaffold reduced infarct size and improved behavioral performance. (a) Bregma as baseline, the brains were cut every 2 mm to stain by TTC. (b) The infarct size of brain slice at coordinate −4 mm (white) was measured by TTC staining 56 days after transplantation. (c) The schematic diagram of infarct size calculation. (d) Measurement of the infarct size with coordinate −2 mm level, 28 and 56 days after transplantation (n = 3). (e) Measurement of the infarct area at coordinate −4 mm level, 28 and 56 days after transplantation (n = 3). (f) Behavioral performances were tested using NSSs (n = 5). The results are shown as the mean value ± SD (*p < 0.05, **p < 0.01 vs PFP group).

Finally, the behavioral tests were performed at four time points using the NSS. On day 56, the rats from the PFP + NSCs + NTs group performed better than the PFP group (Figure 5f). However, significant behavioral improvement was not observed in the PFP + NTs group.

4 Discussion

Stem cell transplantation offers significant promise for stroke therapies with several challenges remaining. One of the keys to the success for cell-based therapies is to improve the survival of transplanted cells. In the present study, we seeded NSCs in PFP carrier loaded with neurotrophic factors. Subsequently, this NSCs-polymer scaffold was transplanted into the ischemic penumbra of stroke rat models. And we investigated whether this approach enhanced the grafted NSCs livability.

It has been reported that neurotrophic factors could protect donor cells [17,34]. In order to enhance the therapeutic effects of cell transplantation, many studies have tried to transplant gene-modified stem cells overexpressing neurotrophic factors [35,36,37,38]. Although neurotrophic factors gene modified stem cells exhibited enhanced survival, the safety of this method still requires further investigation. In our study, we used PFP as the delivery vehicle of neurotrophic factors. The PFP scaffold is biodegradable and nonimmunogenic [31], which meets the security requirements for the application in vivo. Furthermore, we confirmed that PFP could sustained release three neurotrophic factors and maintain suitable concentration in brain injury site of experimental animals. Taken together, PFP scaffold is a promising neurotrophic factors release system to support cell survival and differentiation.

In this study, our data showed that transplantation of PFP loaded with neurotrophic factors (BDNF, NGF, and NT-3) effectively reduced the apoptosis of ischemic penumbra in model rats, with or without grafted NSCs. The result suggests that these neurotrophic factors can protect brain cells after strokes, which is consistent with the role of neurotrophic factors reported in previous studies [17,34]. In addition, we found that NSCs transplantation without neurotrophic factors can also inhibit apoptosis of brain cells, which was also reported in other work [39]. Nevertheless, our results showed that the transplantation of NSCs-PFP carrying neurotrophic factors performed better in inhibiting apoptosis than without neurotrophic factors. Furthermore, we also found that neurotrophic factors released by PFP increased mature neuron numbers that were differentiated from donor NSCs.

Cerebral infarct size and neurological score are commonly used to evaluate the therapeutic effect of treatment on stroke. We discovered that the neurotrophic factors carried by PFP notedly diminished the infarct size, which was strengthened more when combined with NSC grafting. Moreover, transplantation of NSCs-polymer scaffold carrying neurotrophic factors promoted the functional recovery, while PFP + NTs group did not.

In conclusion, we confirmed that PFP scaffold could steadily release BDNF, NGF, and NT-3 to support survival and differentiation of grafted NSCs, resulting in improved effectiveness of cell-based therapy for ischemic stroke.


# These authors contributed equally to this manuscript.

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Acknowledgment

We would like to thank the Shenzhen Biomedical Research Support Platform for technical help. We thank Ms. Jiana Li for reagent preparation and experimental assistance. We also thank Dr. Alex Zhong for critical reading of the manuscript.

  1. Funding information: This work was supported by Shenzhen Science and Technology Program (JCYJ20140416144209745, JCYJ20160427185306518, JCYJ20170413161350892, and JCYJ2019001RC)

  2. Author contributions: Y.Q. and T.W. carried out the molecular and cellular studies. Y.Q., D.Y., and M.L. participated in the animal experiments. The manuscript was drafted by Y.Q. and revised by all authors. B.C. and Y.X. conceived the study and helped to draft the manuscripts. All authors have read and approved the final manuscript.

  3. Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

  4. Data availability statement: The dataset used and/or analyzed during the current study are available from the corresponding author on reasonable request.

  5. Ethical approval: The conducted research is not related to either human or animal use.

  6. Ethics statement: Adult male SD rats were used in our study. All procedures were approved by the Animal Use and Care Committee of Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center (SPHMC) (protocol number 2015-032). Considerations and procedures were taken to lower the number of animals to be used in the study and lower the pain of the animals.

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Received: 2022-04-09
Revised: 2022-08-08
Accepted: 2022-09-09
Published Online: 2022-10-22

© 2022 Yue Qi et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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