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Sci Technol Adv Mater
Sci Technol Adv Mater
Science and Technology of Advanced Materials
1468-6996
1878-5514
Taylor & Francis

10.1080/14686996.2024.2404380
2404380
Version of Record
Research Article
Bio-Inspired and Biomedical Materials
Effects of the matrix-bounded nanovesicles of high-hydrostatic pressure decellularized tissues on neural regeneration
Sci. Technol. Adv. Mater.
M. KOBAYASHI et al.
Kobayashi Mako a b
Negishi Jun b c
Ishida Naoki b
Hashimoto Yoshihide b
Sasaki Yoshihiro d
Akiyoshi Kazunari d
Kimura Tsuyoshi b e
Kishida Akio b
a Department of Materials Processing, Graduate School of Engineering, Tohoku University , Sendai, Miyagi, Japan
b Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University , Chiyoda, Tokyo, Japan
c Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University , Ueda City, Nagano, Japan
d Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University , Kyoto, Japan
e Department of Biomedical Engineering, Toyo University , Asaka-city, Saitama, Japan
CONTACT Mako Kobayashi mako.kobayashi.e1@tohoku.ac.jp Department of Materials Processing, Graduate School of Engineering, Tohoku University, Aramaki-Aza Aoba, Aoba-Ku, Sendai 6-6-02, Japan
18 9 2024
2024
18 9 2024
25 1 2404380Integra18 9 2024
Integra18 9 2024
16 7 2024
04 9 2024
10 9 2024
© 2024 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Group.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 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. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Decellularized tissues have been used as implantable materials for tissue regeneration because of their high biofunctionality. We have reported that high hydrostatic pressured (HHP) decellularized tissue developed in our laboratory exhibits good in vivo performance, but the details of the mechanism are still not known. Based on previous reports of bioactive factors called matrix bound nanovesicles (MBVs) within decellularized tissues, this study aims to investigate whether MBVs are also present in decellularized tissues prepared by HHP decellularization, which is different from the previously reported methods. In this study, we tried to extract bioactive factors from HHP decellularized brain and placenta, and evaluated their effects on nerves in vitro and in vivo, where its effects have been previously reported. The results confirmed that those factors can be extracted even if the decellularization method and tissue of origin differ, and that they have effects on a series of processes toward nerve regeneration, such as neurite outgrowth and nerve fiber repair.

GRAPHICAL ABSTRACT

IMPACT STATEMENT

In this study, we evaluated the neuroregenerative effects of matrix-bounded nanovesicles extracted from decellularized tissue using a high hydrostatic pressure method. The results indicate that bioactive factors, including matrix-bounded nanovesicles, can be extracted regardless of the decellularization method and tissue origin.

KEYWORDS

Decellularized tissue
high hydrostatic pressure
extracted components
nerve regeneration
Creative Scientific Research of the Viable Material via Integration of Biology and Engineering from MEXT the Cooperative Research Project of the Research Center for Biomedical Engineering from MEXT This work was supported in part by Grants-in-Aid for Scientific Research, KAKENHI [21H04954, 19H04465, 21K20512, 23K17207], the Creative Scientific Research of the Viable Material via Integration of Biology and Engineering from MEXT, and the Cooperative Research Project of the Research Center for Biomedical Engineering from MEXT.
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pmc1. Introduction

Decellularized tissue is used as an implantable material for tissue regeneration since it maintains the structure of biological tissue and has high bio-acceptability, growth, and tissue regeneration potential. For example, it is used orthotopically in heart valves and blood vessels [1–6]. In addition, it is applied ectopically in powder or sheet form; in this case, the transplant site is eventually constructed with tissue suitable for the transplant site [7–12]. It is thought that bioactive factors, particularly those within the extracellular matrix (ECM), exert a bioadaptive effect through communication with ectopic tissues and cells. For instance, it is suggested that the constructing remodeling and wound healing have been achieved due to the presence of bioactive peptides [13–15], cytokines and growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β released during decellularized ECM degradation [16–19]. Recently, matrix-bound nanovesicles (MBVs), which are tightly bound to the ECM, have been reported as one of those bioactive factors [20,21]. MBVs are similar to exosomes in terms of particle size and lipid bilayer. However, since they do not express exosome markers, they have been defined as new extracellular vesicles. It has been reported that MBVs can be obtained from a variety of decellularized tissues, but their functions differ depending on the tissue of origin and preparation method [22,23]. Previously reported bioactive factors were mainly extracted from surfactant decellularized tissues, but these factors derived from high hydrostatic pressure (HHP) decellularized tissue, a physical method without surfactants, have not been characterized. Tissues decellularized with surfactants show no cell adhesion or proliferation in vitro, which is thought to be due to residual surfactant. In the presence of surfactants, it is crucial to consider both the stability of the MBV and the toxicity of the surfactant. Consequently, a direct comparison with MBV isolated using the HHP method may not be appropriate. Therefore, in this study, we focused exclusively on MBV isolated by the HHP method to determine whether MBV remains intact after high hydrostatic pressure treatment and to assess its bioactive functions. Therefore, we are attempting to extract those bioactive factors from decellularized tissues prepared by a decellularization method different from existing reports and evaluating its effects on various tissues. Our previous report indicated that bioactive factor containing MBVs can be collected from the small intestine submucosa, urinary bladder matrix, and liver, and that some tissues show bioactive functions in vascular endothelial cells [24]. In this study, we tried to extract bioactive factors from HHP decellularized brain and placenta, and evaluated their effects on nerves in vitro and in vivo, where its effects have been previously reported [25]. First, we attempted to extract bioactive components from the decellularized brain and placenta of miniature swine. The extracted factors are evaluated in vitro for PC12 and mouse dorsal root ganglion elongation. The effect of those factors on nerve in vivo is also examined using the rat sciatic nerve injury model, a commonly used experimental model for studying nerve repair and regeneration. Fibrin gel containing decellularized brain and placenta-derived factor is applied to the site of nerve injury for 6 wk, and the sciatic function index (SFI) of each group of animals is calculated to evaluate functional recovery.

2. Materials and methods

2.1. Materials

Fresh miniature swine brains and placentas were purchased from the Kagoshima Miniature Swine Research Center (Kagoshima, Japan). Magnesium chloride hexahydrate, 0.1 mol/L sodium citrate hydroxide solution, anhydrous ethanol, phosphate-buffered saline (PBS), and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Rat pheochromocytoma cells (PC12) were purchased from the Japanese Collection of Research Bioresources (Osaka, Japan). Liberase was purchased from Roche Diagnostics (Basel, Switzerland). Mice (C57BL/6JJmsSlc, 7-wk-old) and Sprague-Dawley rats (8-wk-old) were obtained from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan).

2.2. Decellularized tissue preparation

The purchased placentas were immediately placed in saline and washed several times by changing the solution to completely remove blood and clots. Following the identification of the remaining umbilical cord, the thin and transparent amniotic membrane was slowly detached from the placenta. The tissues were then cut into 5-cm-long lengths. Since the brain is a soft and fragile tissue, the brain stem section was lifted using tweezers and removed onto a stainless-steel bat. The thin membrane, that is, the arachnoid matter on the surface, was peeled off and the brain was divided vertically into two sections. The left and right brains were further divided into two sections and cut into approximately 5-cm squares. Trimmed placenta and brain fragments were packed into a plastic bag filled with saline and decellularized under HHP (600 MPa, 30°C for 10 min) (Dr. Chef, Kobelco, Tokyo, Japan). After completion of HHP, the tissues were rinsed with DNase-containing saline at 4°C for 1 wk, 80% ethanol/saline for 3 d, and 0.1 M citrate buffer containing 1% penicillin-streptomycin for 3 d while being shaken.

2.3. Extraction of components from HHP decellularized placenta and brain

The decellularized placenta and brain were then lyophilized (DRC-1100, FDU-2110, EYELA, Tokyo Rikakikai Co., Ltd, Tokyo, Japan) and ground into a powder using electric milling (Tube Mill Control, IKA Japan K, Osaka, Japan). Liberase (0.01 mg/mL) buffer (50 mm Tris (pH 7.5), 5 mm CaCl2, 200 mm NaCl) was added to 10 mg/mL of lyophilized tissue, and the mixture was stirred at 37°C for 24 h. Immediately after solubilization, each solution was centrifuged at 500 × g, for 10 min and 10,000 × g for 30 min (three times, 4 °C) to remove insoluble collagen fibrils and proteins. The supernatant was collected at each centrifugation step and subjected to an ultracentrifugation step using a Beckman Coulter Optima L-90K Ultracentrifuge (Beckman Coulter, Inc., Brea, CA, U.S.A.) at 100,000 × g and 4°C for 70 min. Following the wash process and ultracentrifugation at 100,000 × g and 4°C for 30 min, the collected pellets were resuspended in 500 µL of 1×PBS (-) and passed through a 0.22-µm filter (Merck Millipore, Darmstadt, Germany). The final pellets were stored at −20°C until evaluation.

2.4. Characterization of HHP decellularized tissue-derived pellets

TEM was used to observe the morphology and microstructure of MBVs. The ultracentrifuged pellet mixture was loaded onto a carbon-coated copper grid and stained with 2% uranyl acetate to enhance the contrast. The grids were then visualized at 80 kV using a JEM-1400 Flash (Japan Electron Optics Laboratory (JEOL), Tokyo, Japan). A NanoSight LM10 (Malvern Panalytical, Worcestershire, UK) was used to measure the size distribution and concentration of the nanovesicles. The ultracentrifuged pellet solution was diluted with Milli-Q water, and the images were captured five times for 60 s at camera level 13. The particle size and concentration were calculated based on a video analysis.

2.5. RNA isolation and characterization

The total RNA was extracted from the MBVs derived from the decellularized tissues using the miRNeasy Mini Kit with spin columns (QIAGEN, Venlo, The Netherlands) according to the manufacturer’s instructions. The isolated RNA was resuspended in RNase/DNase-free water, and the RNA yield was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific K, Tokyo, Japan). The quality and size of the RNA were determined through capillary electrophoresis using an RNA 6000 Pico Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, U.S.A.) following the manufacturer’s protocol.

2.6. Neurite growth assay of PC12

PC12 (4.0 × 103 cells/well) were seeded in a collagen-coated 96-well dish. Following incubation, the MBVs (14 µg/ml) were added to each well, and nerve growth factor (50 ng/ml) was added as a positive control. After 24 h, the cell morphology was observed under a microscope.

2.7. Dorsal root ganglion dissection and plating for culture

This study was approved by the Animal Care and Ethics Committee of Tokyo Medical and Dental University (approval no. A2020-181A). Euthanized mice (C57BL/6JJmsSlc, 7-wk-old) were laid on their faces and shaved with clippers to remove body hair. Their back skins were cut open using scissors to expose the backs. The spines were harvested by inserting a pair of scissors from the waist up to the neck. The vertebrae were then placed in a Petri dish filled with PBS (-) and rinsed gently to remove the blood. The connective tissues surrounding the spines were removed, and the spines were trimmed until white spinal cords were visible. In each case, the vertebral column was held using a tweezer, and the cranial aspect of the spinal cord was pulled from the cranial to the caudal using another tweezer. As the dorsal root ganglions (DRGs) are situated in the intervertebral foramina connected to the spinal cord, they were carefully pulled out of the intervertebral foramen. Following the complete disconnection of the spinal roots and nerves from the DRGs, the isolated DRGs were collected in a Petri dish filled with DMEM.

Subsequently, diluted fibrinogen solution was added to each well. The DRGs were placed in the fibrinogen solution using micro tweezers, one per well. A drop of thrombin solution equal to the volume of the fibrinogen solution was added to the border between the DRG and the fibrinogen solution. The plates were covered with lids and left to stand for 10 min to enable fibrin formation. Next, 0.9 ml of Neurobasal/B-27 solution was gently added to each well. To the negative control wells, 0.1 ml of PBS (-) was added. To the positive control wells, 0.1 ml of brain-derived neurotrophic factor (BDNF) (1 µl of 100 µg/ml BDNF and 99 µl of PBS) was added. The wells to which the samples were added were adjusted with the MBV solution in PBS to obtain the desired concentration (14 µg/ml). 0.1 mL of MBV solution were added to each well. The well plates were incubated in a 5% CO2 incubator at 37°C for 21 d.

2.8. DRG staining and neurite outgrowth evaluation

The culture plates were removed from the incubator 21 d after the culture was initiated. The medium was removed from each well, and 500 µl of 1×PBS (−) each was added; the wells were gently washed. After two washes with 1×PBS (-), each well was permeabilized with 500 µl of 0.25% Triton X-100 in PBS for 30 min. After washing twice with 1×PBS (-), 500 µl of 5% bovine serum/0.5% Triton X-100 in PBS was added, and the wells were immersed for 90 min for blocking. The primary antibody, β-tubulin (rabbit polyclonal, GTX101279, GeneTex), was diluted 1:500 in 2.5% BSA and 0.5% Triton X-100 in PBS. Thereafter, an antibody diluent was added and left overnight. After washing the solution with 1×PBS (-), it was diluted to a volume of 500 µl by adding 2.5% BSA and 0.5% Triton X-100 in PBS secondary antibody (goat anti-rabbit IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor 568, Thermo Fisher, 2 µg/ml) and DAPI (10 µg/ml). Following overnight incubation, the cells were washed with 1×PBS (−) and observed under a fluorescence microscope (BZ-X710, Keyence Corp., Osaka, Japan). A hybrid cell count (Keyence, Osaka, Japan) was used to calculate the area of neurite extension in the DRG. The area of the DRG body was subtracted from the combined area of the DRG body and neurite outgrowths to obtain the area of the neurite outgrowth only. These values were substituted into equation (1) to calculate the ratio of the neurite area.(1) Ratio of the neurite outgrowth area%=The area of neurite outgrowthThe area of DRG body and neurite outgrowth×100

2.9. Preparation of rat sciatic nerve injury model

This animal study was approved by the Animal Care and Ethics Committee of the Tokyo Medical and Dental University (approval no. A2020-134A). Sprague-Dawley rats (8-wk-old) were anesthetized with isoflurane mixed with oxygen. Under deep anesthesia, the rats were placed in a supine position. In each case, the right hind leg was shaved using a clipper and was placed on a cautery plate covered with wet gauze. The area was wiped and sterilized with iodine and 70% ethanol. The skin of the right thigh was removed using tweezers and dissected using a shear. The sciatic nerve was exposed by incising the muscular layer using surgical shears. After the tissue and membrane surrounding the sciatic nerve were separated, the tip of the monopolar was applied to the sciatic nerve and treated (for 2 s; 6 mm wide). In the untreated group, the muscle layer and skin were sutured with 5–0 Nylon (Monosof, SN-3695). For the other group, fibrin gel containing prepared PBS (-) or decellularized tissue-derived MBVs (14 µg/ml) was applied over the OASIS matrix (Cook Biotech, Inc., Indiana, US) using a two-component syringe. The OASIS matrix with samples containing fibrin gel was placed under the sciatic nerve and wrapped around it. The ends were sutured with 7–0 prolene (Ethicon, 8743 h), and the muscle layer and skin were closed by suturing with 5–0 Nylon.

2.10. Walking track analysis

The sciatic function index (SFI) was measured according to previously described procedures [26]. An acrylic box (30 × 30 × 30) was prepared, in which a rat could move freely, and a video camera was set under the box. The rat was placed in a box and the ceiling was covered with drapes. The rats’ walking was recorded on video for 10 min at weekly time points. Five screenshots were taken from the video recording, which clearly showed both the hind limbs and all the toes. The following three points were measured for the uninjured (normal; N) and injured (experimental; E) sides from each image using Fiji (ImageJ open source software): (1) paw length (PL; length between the tip of the center toe and the furthest point of contact on the back of the foot), (2) toe spread (TS; length between the first and fifth digits), and (3) intermediary toe spread (IT; length between the second and fourth digits). The measured values were substituted into the following formula to calculate the SFI [27], and its average value was obtained as follows:(2) SFI=−38.3×EPL−NPLNPL+109.5×ETS−NTSNTS+13.3×EIT−NITNIT−8.8

2.11. Luxol fast blue (LFB)/H-E staining of rat sciatic nerve

The combination of LFB staining, which stains the myelin blue, and HE staining, which stains the cytoplasm and nuclei, aids in the detection of myelin sheaths that are difficult to identify through only H-E staining and evaluation of the degree of damage and repair of the sciatic nerve. The dissected rat sciatic nerves were fixed with a neutral-buffered (pH 7.4) solution of 10% formalin in PBS and dehydrated with 70%, 80%, 90%, and 100% ethanol. The samples were then treated with xylene and embedded in paraffin, which was cut into 5-mm-thick sections for staining. The paraffin slices were re-placed in xylene and dehydrated using graded ethanol. Thereafter, they were immersed in distilled water, washed, and re-placed in 95% ethanol. After soaking in the LFB staining solution for 24 h (57 °C), the slices were immersed in 95% ethanol. Next, they were washed with distilled water and immersed in a 0.05% lithium carbonate solution for 5 s to obtain a clear stained image. Following immersion in 70% ethanol, the slices were washed gently and immersed in Mayer’s hematoxylin solution for 10 min. Following washing with distilled water, the samples were dehydrated using a graded ethanol series.

2.12. Statistical analysis

Figures 3 and 4 are expressed as mean ± standard deviation (SD). The Student’s t-test was used to compare the ratio of the neurite area of DRG with the untreated DRG (Figure 3c). The statistical significance was set at p < 0.05. Analysis of variance followed by Tukey’s multiple comparison test was used to determine statistical significance in the evaluation of the SFI (Figure 4d). The value of p < 0.05 was considered significant.

3. Results

To observe the morphology and microstructure of extracted MBVs, the TEM was performed on decellularized miniature swine brains and placenta-derived ultracentrifuged pellets (Figure 1a). Multiple vesicles with lipid membranes were observed in both pellets. Vesicles of various sizes were observed in the brain-derived pellets, ranging from approximately 200 nm to 500 nm. Multiple particles of approximately 200 nm in size were identified in the placenta-derived pellet. Figure 1b presents the results of the NTA. In the brain-derived pellets, two peaks were detected at diameters of approximately 200 nm and 400 nm. In contrast, a single peak was detected at a diameter of approximately 150 nm for the placenta-derived pellets. Both the NTA results correlate closely with the TEM images. RNA evaluation indicated peaks of 20–25 nt typical of miRNAs and small RNAs less than 200 nt in length, suggesting that the collected MBVs contained miRNAs and small RNAs (Figure 1c). These results implied MBVs can be extracted from HHP decellularized brain and placenta. Figure 1. Characterization of decellularized tissue-derived MBVs.

(a) Transmission electron microscopy (TEM) observation of MBVs derived from the decellularized brain and placenta. (b) Measuring size and concentration of MBVs derived from the decellularized brain (above) and placenta (below) using nano tracking assay (NTA). The average vesicle diameter and total concentration are written on each graph. (c) Bioanalyzer electropherogram of MBVs derived from the decellularized brain (above) and placenta (below).

The neuron-like characteristics of differentiated PC12 cells were used to evaluate the neuronal function of extracted components from HHP decellularized tissues. Figure 2c and 2d present images taken 24 h after the addition of pellets from the decellularized brain and placenta, respectively, to PC12. Although not as much as PC12 with NGF added (Figure 2b), neurite extensions were observed in the decellularized derived pellets added cells as compared with the untreated cells (Figure 2a). Figure 2. Neurite growth assay of PC12.

(a) Untreated, (b) exposed by nerve growth factor (NGF), decellularized pellets extracted from (c) decellularized brain and (d) decellularized placenta. Black arrows represent the neurites. Scale bar : 100 µm.

The DRGs were harvested from the spinal cord of C57BL/6JJmsSLC mice and embedded in fibrin gel. The nerve growth factor (NGF), decellularized brain, and placenta-derived pellets were added to the medium respectively and cultured for 21 d (Figure 3a). The results of DRG staining with β-tubulin after 21 d of incubation are presented in Figure 3b. All the DRGs survived over the observation period of 21 d. As no neurite outgrowth was observed in the nontreated DRGs and significant neurite outgrowth was observed at NGF- treated DRGs, the results of the sample set as control were properly analyzed. Neurite outgrowth was observed in both the decellularized brain and placenta-derived pellets treated DRGs as compared with the nontreated DRGs. A magnified view of the neurite extension from the cell body is presented in Figure 3b. Sharp neurite outgrowths similar to the NGF treated DRGs were observed in the DRGs exposed to the pellets extracted from HHP decellularized brain and placenta. Next, the ratio of the area of neurite outgrowths was determined from the microscopic images obtained using the hybrid cell counter, and calculated by the Equation (1) in the Materials and Methods section. Although the effect beyond the NGF was not confirmed, the addition of the decellularized tissue-derived pellets significantly increased the ratio of the neurite extension area when compared with nontreated (Figure 3c). Figure 3. Neurite outgrowth of dorsal root ganglion (DRG) neurons.

(a) Schematic illustration of DRG isolation and culture. (b) Neurite outgrowth observation of DRG stained with β-tubulin and DAPI. (c) The ratio of neurite area of DRG following 21 d of incubation with brain and placenta-derived nanovesicles. The asterisk (*) indicates the statistical significance in comparison with nontreated (*p < 0.05).

A rat sciatic nerve injury model was used to evaluate the effects of the decellularized brain- and placenta-derived pellets on nerve regeneration and functional recovery. On the right side of the lower limb, 6 mm of the sciatic nerve was burnt. A fibrin gel containing decellularized tissue pellets was applied to the OASIS extracellular matrix and wrapped around the nerve injury site (Figure 4a). A walking track analysis was performed every 2 wk. Figure 4b presents screenshots extracted from the video recordings of the walking track analysis for each group. At 2 wk postoperatively, although the rats from the injured group limped, the rats from the other groups walked with curled toes on the damaged side of the paws. By 4 wk postoperatively, all the groups except the injured group walked with their toes slightly extended. At 6 wk, the damaged toes in all the test groups were further widened, indicating nerve recovery. In addition, to assess motor function, the SFI was measured from the video recordings of the animals every 2 wk. The SFI scores were calculated by measuring three parameters for each paw on the normal and experimental sides, as shown in Figure 4c, and substituting them into Equation (2). Figure 4d describes the SFI values for all four groups. The SFI value approaches zero as the recovery trend progresses. No significant difference in recovery was observed between the groups 2 wk after the operation. At 4 wk postoperatively, the injured group had SFI values equivalent to those at 2 wk, whereas the other groups indicated a tendency toward recovery. At 6 wk postoperatively, the SFI values of all the groups were higher, indicating a more advanced recovery. Since the OASIS used for wrapping contained active factors such as growth factors, no significant neurodegenerative effects were observed when comparing fibrin gel alone to gel with MBV added. However, significant nerve regeneration was observed when compared to the damaged group. This suggests that increasing the amount of MBVs added may have a synergistic effect with OASIS. The optimal amount of MBV to be added will be investigated in future studies. Figure 4. Sciatic nerve injury model and sciatic function index (SFI).

(a) Schematic illustration of the sciatic nerve injury model preparation. The sciatic nerve damaged by coagulation was wrapped with the OASIS matrix filled with fibrin hydrogel and decellularized tissue-derived pellets. (b) Screenshots from video recordings of the rat every 2 wk after surgery. The red circles represent the experimental paws. (c) SFI score was calculated by measuring the PL, TS, and IT from the experimental (E) and normal (N) paws from the screenshots. (d) Average SFI score for each group at 2, 4 and 6 wk. The asterisk (*) indicates statistical significance as compared with the injured group at 6 wk after surgery (*p < 0.05).

Figure 5 shows the rat sciatic nerve immediately after cauterization and after 6 wk. Adhesion to the surrounding tissue was observed in the injured sciatic nerve. When fibrin gel alone or that containing decellularized tissue- derived pellets was applied to the OASIS matrix and wrapped around the sciatic nerve, no adhesion to the surrounding tissue was observed. Figure 5. Photographs of sciatic nerves immediately and 6 wk after surgery.

Figure 6 presents the results of Luxol fast blue (LFB)/H-E staining 6 wk after cauterization of the rat sciatic nerve. By staining the myelin sheath blue by LFB staining and the cytoplasm and nuclei purple by H-E staining, the degree of sciatic nerve damage and repair is assessed. The un-injured sciatic nerve can be seen to be neatly aligned in the direction of the fibers stained purple. In contrast, the injured sciatic nerve displayed disconnected nerve fibers. In the sciatic nerve of rats to which the decellularized tissue- derived pellets-mixed gel was applied, axons were elongated in the direction of the fibers, similar to the uninjured group. Figure 6. Luxol fast blue/H-E staining of sciatic nerve 6 wk after surgery.

4. Discussion

The surfactant method, a widely used decellularization method, has been reported to be highly functional in vivo [28–30] and products used in clinical practice also use this method [31,32]. HHP treatment, on the other hand, involves immersing biological tissue in a liquid and applying isotropic pressure to disrupt cell membranes, with decellularization completed by a subsequent washing process. The effects of these different decellularization methods on bioactive substances were considered. While the surfactant method dissolves the ECM and may be considered easier to extract bioactive substances in the ECM, there is concern that bioactive factors may leak out during the decellularization process because the ECM structure cannot be maintained due to dissolution and an intense washing process is required to remove surfactant residues. On the other hand, the HHP method has been shown to maintain the ECM structure compared to the surfactant method [6,33], suggesting that MBVs that are strongly bound to the ECM may be retained in an intact state. Therefore, we considered that HHP-decellularized tissue functions in vivo because the bioactive substances maintained in HHP-decellularized tissue are released slowly with ECM degradation over time in vivo. Based on this, the present study investigated whether HHP-decellularized tissue contains the same kinds of bioactive factors as surfactant-decellularized tissue, and analyzed the effects of HHP-decellularized tissue containing bioactive factors on neural regeneration in vitro and in vivo. First, MBV-like vesicles were tried to be extracted from both the decellularized brain and placenta, and evaluated by TEM, NTA, and RNA evaluation. The results of TEM and NTA indicated that vesicles of various sizes, ranging from 100 to 500 nm, were detected in the decellularized brain. In other words, vesicles larger than previously reported were present. The International Society for Extracellular Vesicles (ISEV) defines an extracellular vesicle as ‘a particle surrounded by a lipid bilayer without a nucleus (unable to replicate) that is released from a cell’ [34]. It is well known that although microvesicles (100–1,000 nm in diameter), which are a type of extracellular vesicle, have a different production mechanism from exosomes, they share several components and sizes with exosomes, making it difficult to completely separate them [35]. Considering this fact, although MBVs are considered to have different functions from liquid-phase EVs [36], it is possible that MBVs are not only small vesicles such as exosomes but also large vesicles such as microvesicles, similar to the general extracellular secretory granules described by the ISEV. In addition, in RNA evaluation, unlike the placenta-derived MBVs, a large peak was observed at the position of the small RNA of approximately 200 nt in brain-derived MBVs, suggesting that vesicle cargos and functions may differ not only by the tissue of origin but also by vesicle size. Future studies should include a more detailed investigation of the MBV’s secretory pathway, cargo, and mechanism of binding to the extracellular matrix.

Furthermore, the collected decellularized brain- and placenta-derived pellets were evaluated in vitro by adding them to PC12 and the DRGs. PC12 is derived from rat pheochromocytoma, and it extends neurites and morphologically resembles neurons when stimulated by neurotrophins [37,38]. The DRG is the most frequently used primary cultured neuronal cell and can be used to evaluate the functional properties of peripheral sensory neurons in vitro [39]. In vitro evaluation using these cells indicated that the addition of decellularized tissue-derived pellets led to neurite outgrowth in the PC12 and DRG. Exosomes derived from mesenchymal stem cells (MSCs) have been reported to deliver exogenous miRNAs to neurons and promote the neuronal differentiation and recovery of neuronal function [40,41]. Therefore, it is considered that the presence of functional substances such as miRNAs, which affect neurons, in the cargo of vesicles was observed to promote neurite outgrowth and neuronal migration. The specific predicted miRNAs are miR-1 [42], miR-124 [43], miR-133 [44,45], miR-210 [46,47], miRNA 222 [48], miR-340 [49] which are closely related to nerve cell proliferation, migration, and myelination. In the future, it will be necessary to verify the mechanism through which vesicles exert their effects on neurons.

Since the in vitro evaluation confirmed the effect of decellularized derived pellets on neurons, as an in vivo evaluation, nerve function recovery was observed using a sciatic nerve injury model. Damage to peripheral nerves causes motor paralysis and nerve disorders. The three types of peripheral nerve injuries are as follows: neurapraxia, axonotmesis, and neurotmesis [50]. The lowest degree of nerve injury is called neurapraxia, in which the nerve remains intact but it results in transient weakness. The second degree called axonotmesis, in which the axon is damaged but the surrounding connecting tissue remains intact. The most severe injury is called neurotmesis, in which both the axon and connective tissues are completely disrupted. Surgery for peripheral nerve injury involves compressed tissue or nerve detachment and neurorrhaphy, but after the surgery, the nerves adhere to the surrounding tissues, causing neuropathy such as numbness and pain [51,52]. In addition, it is well known that peripheral nerve regeneration takes time, leading to muscle atrophy and significant muscle weakness [53]. However, nerve induction materials intended for nerve protection and induction do not promote nerve regeneration. Therefore, in this study, a rat sciatic nerve cauterized injury model was prepared to investigate both the prevention of peripheral nerve adhesion to the surrounding tissues and the effect of decellularized tissue-derived pellets on nerve regeneration. We considered that by encapsulating decellularized tissue-derived pellets in fibrin gel and applying it to the damaged peripheral nerves, the nerve-induction-promoting effect of the pellets could be directly applied to the injured area. In addition, the fibrin gel acts as a scaffold material for nerve induction, which is expected to induce nerve regeneration in the early postoperative period, leading to the suppression of muscle atrophy. Although NeuroMend and Axoguard are commercially available nerve wraps [54,55], the fibrin gel containing decellularized tissue derived pellets, an additional bioactive substance applied to the OASIS extracellular matrix in this study, is expected to further accelerate the rate of nerve regeneration. Figure 4 describes that the evaluation of nerve function recovery over a 6-wk period indicates that decellularized tissue-derived pellets have a nerve recovery function. Significant functional recovery was observed in the other study groups when compared with the damage-only study group. However, the effect of decellularized tissue-derived pellets was not significantly different from the SFI values of the study group to which only fibrin gel had been applied. This may simply be attributed to the low content of bioactive factors including growth factors and MBVs. Other reasons include the fact that neurite outgrowth is known to be significantly affected by the complex relationships among several matrix properties such as stiffness [56,57], degradability [58,59], and porosity [60,61]. Thus, by modifying the properties of the hydrogel and wrapping material, it may be possible to create a nerve-wrapping device that induces nerve regenerative recovery and function. In addition, at the 6-wk follow-up, the wrapped nerve exhibited only mild perineural adhesions (Figure 5). This suggests that wrapping the nerve tended to prevent muscle atrophy because nerve damage owing to adhesion with the surrounding tissues was suppressed (data not shown). Although artificial nerves lack environmental factors necessary to induce regenerative axons, such as cytokines, wrapping decellularized tissue-derived pellets-containing gel around the nerve injury site has a nerve regeneration function, and it simultaneously prevents nerve adhesion by wrapping around the nerve.

These results indicate that the effects of HHP decellularized brain- and placenta-derived pellets on neural regeneration were similar, although differences were observed in their characterization. This suggests that the common parts of the brain- and placenta-derived pellets are strongly involved in the mechanism of nerve regeneration. Taken together, it is considered that one of the reasons for the high tissue regenerative potential of HHP decellularized tissue is due to the cooperative manner multi-faceted effects of MBVs.

5. Conclusions

Regardless of decellularization methods, nanosized and membranous vesicles containing miRNA and small RNA can be extracted from HHP-decellularized brain and placenta. The results of in vitro and in vivo analyses indicate that these decellularized tissue-derived factors affect the comprehensive sequence of events leading to nerve regeneration. This study also provides useful insights into the development of highly functional materials by compositing decellularized tissue-derived factors.

Acknowledgments

We deeply appreciate Mr. Hideki Saga (KM Biologics Co., Ltd) for his generous support and expertise that significantly assisted our research. We acknowledge Dr. Riley Suhar (Genentech, California, US) for helpful discussions and advice on the experimental design for the dorsal root ganglion dissection, rat sciatic nerve injury model fabrication, and walking track analysis. We acknowledge Yuriko Sakamaki for technical assistance with the TEM observation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

M.K., J.N., N. I., and H.S. performed all the experiments. M.K. and J.N. drafted the manuscript. A.K., Y.H and T.K revised and edited the manuscript. Y.H., Y.S., K.A., and T.K. supervised this study. J.N., H.S., M.K., and A.K. contributed to study conceptualization. All the authors have read and agreed to the published version of the manuscript.
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