Multi-channel nerve conduit having structural stability and manufacturing method therefor

Abstract

The present invention relates to a multi-channel nerve conduit having structural stability and a manufacturing method therefor. The multi-channel nerve conduit according to the present invention is a nerve conduit which is structurally stable and thus does not collapse. When implanted, the multi-channel nerve conduit having Schwann cells and endothelial cells loaded thereto has the advantage of making it easy to regenerate the nerve. In addition, the multi-channel nerve conduit according to the present invention can mount cells according to the improvement in the number of channels and thus provide a more elaborate microenvironment required for nerve regeneration, exhibiting an effect of excellent improved nerve regeneration ability. Therefore, when manufactured by applying 3D cell printing technology, the nerve conduit can be more precisely manufactured.

Description

Multi-channel nerve conduit with structural stability and method for manufacturing the same

[0001] The present invention relates to a multi-channel nerve conduit having structural stability and a method for manufacturing the same.

[0002] Traumatic spinal cord injury (SCI) can result in permanent loss of neural function and motor impairment. Following central nervous system injury, severed proximal and distal nerve endings undergo Wallerian degeneration, leading to axonal collapse and myelination. This is accompanied by a series of pathophysiological effects, including destruction of neural and vascular structures, as well as inflammation leading to ischemia. The injury microenvironment poses a major obstacle to nerve regeneration. Numerous research efforts have been devoted to understanding and promoting nerve regeneration. Sophisticated biomaterial conduits represent a viable solution to enhance nerve regeneration. Biomedical conduits require the manipulation of structured nerve guidance conduits (NGCs), demonstrating several properties, including cell-embedded structures and growth factor integration. Multichannel conduits offer several advantages over single-lumen NGC8-10 conduits, including a superior surface for cell adhesion and migration, as well as reduced axonal outgrowth dispersion.

[0003] Another promising therapeutic strategy is the transplantation of Schwann cells (SCs). SC transplantation reduces tissue loss, axonal regrowth, and myelination, improving sensorimotor function. SCs dedifferentiate into the basal lamina of distal nerve terminals after Wallerian degeneration, forming a vertically aligned neural bridge structure known as the Band of Bungner. This structure promotes reinnervation of the injured area and creates a microenvironment that promotes regeneration. Therefore, NGC-embedded SCs can serve as natural, living conduits, similar to the Band of Bungner, after Wallerian degeneration. Nerve injuries, including SCI, inevitably result in a hypoxic environment at the site of injury. Reactive oxygen species (ROS) generated under hypoxic conditions can accelerate the damage of neurons and oligodendrocytes at the site of injury. Therefore, improving ischemic conditions may support regeneration. Regarding ischemia mitigation, SC transplantation alone is less effective than combined strategies with vascularized SC transplantation. Moreover, angiogenesis influences SC migration along the vasculature and induces axonal regeneration. Therefore, promoting angiogenesis with endothelial cells (ECs) may attenuate secondary damage, limit tissue loss, and enhance axonal regeneration and functional recovery.

[0004] Previous reports aimed to fabricate multichannel NGCs using biocompatible hydrogels embedded with cells. More recently, 3D printing strategies have been used to fabricate NGCs with sophisticated microstructures. Extrusion 3D bioprinting is a representative technique for using biomaterials with different viscosities. NGCs produced through cartridges with desired structures can overcome limited printing resolution and speed. Furthermore, high-viscosity hydrogels prevent mixing during extrusion from filled cartridges. Extruded NGCs have the same structure as single-sided cartridges.

[0005] Accordingly, the inventors of the present invention developed angiogenic bands of Bungner NGC containing SC and EC, non-channel, 5-channel, and 9-channel, to resolve ischemia and Wallerian degeneration occurring during SCI, and confirmed the therapeutic effect on SCI according to the number of channels and the presence of cells, thereby completing the present invention.

[0006] An object of the present invention is to provide a multi-channel nerve conduit having structural stability and introducing nerve cells within the multi-channel, the multi-channel nerve conduit comprising a hydrogel containing collagen and alginate; and a biocompatible polymer; wherein collagen and alginate are each included in the hydrogel at a concentration of 1 to 10%. Another object of the present invention is to provide a nerve regeneration method comprising transplanting the multi-channel nerve conduit to a damaged nerve site.

[0007] Another object of the present invention is to provide a use of the multi-channel nerve conduit for treating nerve damage.

[0008] Another object of the present invention is to provide a method for manufacturing a multi-channel nerve conduit having structural stability and introducing neural cells into the multi-channels, comprising the steps of: a) mixing a hydrogel of collagen and alginate to manufacture a hydrogel of a matrix region; and b) loading the hydrogel into a biocompatible polymer equipped with a channel structure; wherein the diameter of the multi-channels is 500 to 1,000 μm.

[0009] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0010] Traumatic spinal cord injury (SCI) causes Wallerian degeneration, and the resulting vascular collapse leads to ischemia, impairing motor and sensory functions. Therefore, understanding the biological environment during regeneration is essential to overcome this phenomenon and promote neuronal regeneration. The Band of Bungner is a structure of aligned Schwann cells (SCs) that guide axonal elongation, providing a natural healing environment. During axonal elongation, SCs migrate along neovascularization (endothelial cells [ECs]) to promote axonal elongation. To model this, we developed a multi-channel conduit (MCC) using extrusion 3D bioprinting, utilizing collagen in the matrix region and sacrificial alginate to create the channels. The MCC was fabricated with a vertically aligned SC and EC structure to mimic the sophisticated recovery conditions of SCI. Furthermore, we fabricated MCCs with various channels. The aligned SCs and ECs of the 9-channel conduit (9MCC-SE; 9-channel conduit) were more biocompatible and induced greater proliferation in vitro than the 5-channel conduit (5MCC-SE; 5-channel conduit). Furthermore, 9MCC-SE exhibited greater healing effects than 5MCC-SE in vivo with regard to nerve regeneration, remyelination, inflammation, and angiogenesis. The above tissue recovery results led to the recovery of motor function. Therefore, the 9MCC-SE model represents a novel therapeutic strategy for SCI.

[0011] In this invention, we demonstrated the feasibility of fabricating implantable MCCs using high-viscosity hydrogels using extrusion-based 3D printing. The SCs and ECs embedded in the MCCs were structured similarly to the bands of Bungner and microvascular networks. Furthermore, the multi-channel architecture helped maximize the nerve regeneration effects of the implantable MCCs. The fabricated conduits address challenging SCI conditions, including Wallerian degeneration. The ECs embedded in the MCCs promote angiogenesis, resulting in healing effects against ischemia and inflammation. Specifically, as the number of channels increases, the SCs and ECs embedded in the MCCs accelerate nerve regeneration. Therefore, the innovative conduits not only offer sophisticated microstructures but also offer therapeutic benefits that improve prognosis and promote recovery from SCI.

[0012]

[0013] Hereinafter, the present invention will be described in detail.

[0014]

[0015] The present invention provides a multi-channel nerve conduit having structural stability and introducing neural cells within the multi-channel, the multi-channel nerve conduit comprising a hydrogel containing collagen and alginate; and a biocompatible polymer; wherein collagen and alginate are each included in the hydrogel at a concentration of 1 to 10%.

[0016] In the present invention, the diameter of each channel constituting the multi-channel may be 1,000 to 3,000 μm, preferably 1,000 to 2,000 μm, and more preferably 500 to 1,000 μm.

[0017] In the present invention, collagen and alginate may be included in the hydrogel at a concentration of 1 to 10% each, preferably at a concentration of 1 to 5% each, and more preferably at a concentration of 3.5% each.

[0018] In the present invention, collagen and alginate may be mixed in a volume ratio of 0 to 1:1 to 0 in the hydrogel, respectively, preferably in a volume ratio of 0.5 to 1:1 to 0 in the hydrogel, and more preferably in a volume ratio of 1:1 in the hydrogel. According to one embodiment of the present invention, it was confirmed that the structure of the multi-channel nerve conduit was stably maintained when collagen and alginate were mixed in a volume ratio of 1:1 in the hydrogel.

[0019] In the present invention, the number of multi-channels may be 5 to 9, and preferably, the number of multi-channels may be 9, but is not limited thereto. This means that, depending on the nerve to which the multi-channel nerve conduit of the present invention can be applied, the number of multi-channels can be flexibly applied depending on the diameter size of each nerve.

[0020] In the present invention, the neural cells may be at least one selected from the group consisting of Schwann cells and endothelial cells. Furthermore, preferably, the endothelial cells may be vascular endothelial cells.

[0021] In the present invention, the neural cells may be arranged in an aligned manner within a multi-channel nerve conduit. The multi-channel nerve conduit of the present invention has the advantage of being arranged with Schwann cells and endothelial cells to provide a bridge for neural cell attachment and axonal growth necessary for nerve regeneration, and the endothelial cells can supply oxygen and nutrients necessary for nerve regeneration along with vascular regeneration.

[0022] In the present invention, the biocompatible polymer may be selected from the group consisting of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, poly(lactic acid-caprolactone) copolymer, poly(hydroxybutyric acid-hydroxyvaleric acid) copolymer, polyphosphoester, and poly-L / D-lactide (PLDLA), but preferably, the biocompatible polymer is polycaprolactone. Meanwhile, according to an embodiment of the present invention, the polycaprolactone was used as a cartridge for manufacturing a hydrogel conduit by printing.

[0023] In addition, the present invention provides a nerve regeneration method comprising transplanting the multi-channel nerve conduit into a damaged nerve area.

[0024] In addition, the present invention provides a use of the multi-channel nerve conduit for treating nerve damage.

[0025] In the present invention, the nerve may be a peripheral nerve or a central nerve.

[0026] In addition, the present invention provides a method for manufacturing a multi-channel nerve conduit having structural stability and introducing nerve cells into the multi-channel, comprising the steps of: a) preparing a hydrogel of a matrix region by mixing a hydrogel of collagen and alginate; and b) loading the hydrogel into a biocompatible polymer equipped with a channel structure; wherein the diameter of the multi-channel is 500 to 1,000 μm.

[0027] Meanwhile, the corresponding features of the multi-channel nerve conduit described in the method for manufacturing the multi-channel nerve conduit described above can be replaced in the above-described part, and therefore, the description thereof is omitted.

[0028] The multi-channel nerve conduit according to the present invention is a nerve conduit that is structurally stable and does not collapse, and when Schwann cells and endothelial cells are loaded into the multi-channel nerve conduit and transplanted, it has the advantage of facilitating nerve regeneration. In addition, the multi-channel nerve conduit according to the present invention has an excellent effect of enhanced nerve regeneration ability by providing a more sophisticated microenvironment necessary for nerve regeneration due to the increased number of cells loaded into the channel. Therefore, when the nerve conduit is manufactured using 3D cell printing technology, it has the advantage of enabling more precise manufacturing.

[0029] Figure 1 illustrates extrusion-based 3D printing of multichannel conduits (MCCs) using dual biocompatible hydrogels and optimized printing hydrogels. (a) Mixing ratios of hydrogels for co-printing via extrusion. (b) Formation of pores by mixing hydrogels with implantable conduit shapes. (c-d) Rheological analysis of AC50 and Alg100 optimized for co-printing. (e) 3D cross-sectional images (left) and surface images (right) of printed MCCs without cells. Scale bar, 500 μm.

[0030] Figure 2 illustrates the biocompatibility and integrity of MCC structures for cell printing. Non-MCC-SE, 5, and 9MCC-SE represent Schwann cells (S) / endothelial cells (E) embedded in MCC, Non-MCC-SE represents S / E cells embedded in non-channels, 5MCC-SE represents S / E cells embedded in 5 channels, and 9MCC-SE represents S / E cells embedded in 9 channels. (a) Live / dead staining of S / E cells in the three groups. (b) Cell viability in the three groups. (c) Cell proliferation in the three groups (n = 3).

[0031] Figure 3 shows the vertically aligned SCs and ECs of MCCs mimicking the Bungner band structure. (a) Schematic illustration of vertically aligned SCs and ECs in a multichannel. (b) Expression of S100 (red), F-actin, CD31 (green), and S100 (red) in vertically aligned SCs and ECs in 5MCCs. (c) Expression of S100 (red), F-actin, and CD31 (green) in vertically aligned SCs and ECs in 9MCCs.

[0032] Figure 4 shows the behavior and nerve regeneration after biocompatible 9MCC-SE implantation. (a) Schematic diagram of the experimental time course. (b) BBB scores of the sham, SCI, 9MCC, 9MCC-SE, and 5MCC-SE groups. (c) Microscopic images of extracted spinal cord tissue and H&E staining (scale bar = 200 μm). (d) Immunofluorescence staining using DAPI (blue), MAP2 (white), MBP (red), and GFAP (green) at the lesion site (scale bar = 20 μm). (e) Quantification of MAP2, MBP, and GFAP-positive staining. (f) Immunofluorescence staining using DAPI (blue) and HuN (green) at the lesion site (scale bar = 20 μm). (g) Quantification of HuN-positive staining.

[0033] Figure 5 shows remyelination after biocompatible 9MCC-SE transplantation. (a) Eriochrome cyanine (EC) staining of myelin in cross-sectioned spinal cords (EC scale bar = 200 μm). (b) Quantification of myelin area, gray matter area, and total cord area in EC imaging. (c) Toluidine blue (TB) and transmission electron micrographs (TEM) of the lesion site (TB scale bar = 625 nm, TEM scale bar = 200 nm). (d) Number of myelinated axons and the G ratio of myelinated axon thickness to axon diameter. (f) Immunofluorescence staining using DAPI (blue), CNPase (green), and P0 (red) in the lesion site (scale bar = 20 μm). (g) Quantification of CNPase and P0 positive staining.

[0034] Figure 6 shows angiogenesis after biocompatible 9MCC-SE implantation. (a) Immunofluorescence staining using DAPI (blue), CD31 (green), and VEGF-A (red) at the lesion site (scale bar = 20 μm). (b) Quantification of CD31 and VEGF-A positive staining.

[0035] Figure 7 shows the reduction in inflammatory response after biocompatible 9MCC-SE implantation. (a) Immunofluorescence staining using DAPI (blue), CD68 (green), and IBA1 (red) at the lesion site (scale bar = 20 μm). (b) Quantification of CD68 and IBA1 positive staining (* p < 0.05, ** p < 0.01, and *** p < 0.001).

[0036] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0037]

[0038] Example 1: Materials and Methods

[0039]

[0040] 1.1. Hydrogel Preparation

[0041]

[0042] Sodium alginate (viscosity ≥ 2000 cP, 25°C) (Sigma-Aldrich, St. Louis, MO) was dissolved in distilled water at 25°C for 24 h. The final concentration of the sacrificial hydrogel alginate was 3.5% (Alg100).

[0043] To create the matrix portion of the MCC, a matrix portion hydrogel was prepared by blending collagen (MS-bio, Kyungki-do, Korea) and alginate (Sigma-Aldrich) hydrogel. Porcine freeze-dried atelocollagen sponge (MS bio) was dissolved in distilled water, and soluble collagen was mixed with neutral buffer (2.2 g NaHCO3, 4.77 g hydroxyethyl piperazine ethanesulfonic acid, 0.2 g NaOH (Sigma-Aldrich), 100 ml distilled water) and culture medium in a volume ratio of 8:1:1. Finally, 3.5% collagen (Col100) was mixed with 3.5% alginate (Alg100) in a volume ratio of 1:1 (AC50). Col100 was mixed with Alg100 in a volume ratio of 1:0.5 (AC25).

[0044] The rheological properties of two types of hydrogels (collagen (AC50)—matrix region and alginate (Alg100)—channel region) were evaluated using a rotational rheometer (ARES-G2, TA Instrument Ltd, New Castle, DE) equipped with clamps with a 25-mm diameter and a 1-mm gap. Rheological analyses were performed at 4°C. The extrusion conditions of AC50 and Alg100 could prevent AC50 gelation during the analysis. Next, the storage modulus of AC50 was measured under the same rheological analysis conditions to determine the mechanical properties after printing.

[0045]

[0046] 1.2. Cell culture

[0047]

[0048] Human NF1 Schwann-like cells (SC; CRL-2884, ATCC, Manassas, Virginia), a sNF96.2 cell line, were cultured in DMEM (ATCC) supplemented with 10% FBS (S-FBS-US-015, Serana science for life, Brandenburg, Germany) and 1% penicillin / streptomycin (15140122, Gibco, Paisley, UK). Human umbilical vein endothelial cells (HUVECs; ECs, CC-2519, Lonza, Basel, Switzerland) were cultured in EGM TM -2 SingleQuots TM Endothelial Cell Growth Medium Supplemented with Kit (CC-4176, Lonza) - 2 BulletKit TM (EGM TM -2, CC-3162, Lonza) were cultured at 37°C and 5% CO2. Cultures were detached from cell culture dishes using TrypLE (12604021, Gibco) and mixed with the hydrogel before printing.

[0049]

[0050] 1.3. Cell-free MCC and cell-embedded MCC printing

[0051]

[0052] To visualize the multichannel structure, fluorescent particles (R0200B, B0200, Thermo Fisher Scientific, Waltham, MA) were embedded in neutralized AC50 hydrogel and Alg100 hydrogel in acellular 3D MCCs and then printed. Cell-embedded MCCs were fabricated, and SCs and ECs were each incubated with 1 × 10 neutralized AC50 hydrogel and Alg100 hydrogel on ice. 7 cells / ml. Each combined AC50 hydrogel (acellular AC50 hydrogel, cell-embedded AC50 hydrogel) was centrifuged at 3500 rpm at 4°C for 15 minutes to remove air bubbles. The cell-embedded AC50 hydrogel was injected into the precursor cartridge of the matrix region, and the cell-free Alg100 hydrogel was then injected into the precursor cartridge of the hole region. The precursor cartridge of each filled hydrogel was connected to the pneumatic line of a 3D bioprinter (Baobab Root-1, Baobab Healthcare Inc., South Korea), and the sample was extruded into 80 mM CaCl2 solution (Sigma-Aldrich) at a feed rate of 17–20 kPa. After that, the printed sample was washed with PBS, cut, and transferred to pre-warmed culture medium. The cut samples (length: 5 mm, diameter: 3 mm) were cultured in a 24-well plate with culture medium at 37°C and 5% CO2. The printed samples were cut with a surgical blade to remove the sacrificial hydrogel (Fig. 1a).

[0053]

[0054] 1.4. Cell viability

[0055]

[0056] To evaluate the cytocompatibility and viability of 3D NGCs, a live / dead assay was performed using calcein AM and ethidium homodimer (Thermo Fisher Scientific). Cells encapsulated in 3D NGCs were incubated with 5 μM calcein AM and 20 μM ethidium homodimer-1 dissolved in phosphate-buffered saline (PBS) at 37°C for 30 min. Cell images were analyzed using a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany) using z-stacking (20 μm intervals) and tile scanning (2x2 tiles). Green signals indicate live cells, and red signals indicate dead cells. Cell viability was measured by counting cell numbers.

[0057]

[0058] 1.5. Cell proliferation

[0059]

[0060] To measure cell proliferation, 3D MCCs were processed using the Cell Counting Kit-8 (CCK-8 assay, Dojindo Molecular Technologies, Rockville, MD). The printed 3D MCCs were cut into 4–5 mm lengths, and each sample was treated with serum-free medium and CCK-8 reagent at a 10:1 ratio. After incubation at 37°C for 1 hour, the cells were analyzed using a multi-mode microplate reader (VERSAmax TM ; Molecular Devices, San Jose, CA) was used to measure the absorbance of the supernatant at 450 nm. The measured samples were cultured for 2, 5, and 8 days.

[0061]

[0062] 1.6. Immunofluorescence staining

[0063]

[0064] Immunofluorescence staining was used to measure the functions of cells and MCCs. Double-cell seeded ductal samples were fixed with 2.5% glutaraldehyde solution (G5882, Sigma-Aldrich) for 1 h and rinsed three times for 5 min each with 1X PBS. The fixed ducts were blocked and permeabilized with 2.5% bovine serum albumin (BSA, 9048-46-8, MP Biomedicals, LLC, Illkirch, France) blocking buffer containing 0.2% Triton-X100 (DAEJUNG, Siheung, Korea) for 1 h at room temperature (RT). The samples were then washed three times for 10 min each with 1X PBS. Primary antibodies were prepared at a 1:200 dilution in 2.5% blocking buffer: anti-S100 (1:100, ab52642, Abcam, Cambridge, UK), anti-CD31 (1:200, ab9498 Abcam), and incubated overnight at 4°C. Secondary antibodies were prepared at a 1:500 dilution in 1X PBS. Ductal samples were incubated for 1 hour with FITC-goat anti-mouse IgG (H+L) (ab150113, Abcam) or FITC-goat anti-rabbit IgG (H&L) (ab150092, Abcam). After three washes, the ductal samples were counterstained with DAPI (blue, 1:1000, Invitrogen, Waltham, MA) and phalloidin (green, 1:1000, ab176753, Abcam) and observed using a laser scanning confocal microscope (LSM700, Zeiss).

[0065]

[0066] 1.7. Spinal cord transection and transplantation

[0067]

[0068] In this study, adult male Sprague Dawley rats (220-250 g, OrientBio, Gyeonggi-do, South Korea) were used, and all animal experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC Approval No. 2019-0134). The rats were anesthetized with ketamine (100 mg / kg; Yuhan, Seoul, Korea) and Rompun (10 mg / kg; Bayer Korea, Seoul, Korea). A laminectomy was performed at the T7-9 thoracic vertebra level to expose the spinal cord. The exposed spinal cord was completely dissected to a length of 4-5 mm. After hemostasis, MCC was transplanted into the spinal fissure. Animals were randomly divided into five groups (Sham: posterior myectomy without spinal cord injury, SCI: non-MCC transplantation after spinal cord injury, 9-channel conduit without cells (9MCC), 9-channel conduit loaded with cells (Schwann cells and endothelial cells) (9MCC-SE), and 5-channel conduit loaded with cells (Schwann cells and endothelial cells) (5MCC-SE)). Each cell-loaded group received a total of 5X10 5 The SC and EC of the dog were used.

[0069] After MCC transplantation, the muscle and skin tissues were sutured. All rats were administered antibiotics (25 mg / kg; cefazolin, Chong Kun Dang, Seoul, Korea) for 5 days, and the bladders were emptied every 24 hours until bladder autonomy was restored. An immunosuppressant (20 mg / kg; cyclosporine, Chong Kun Dang) was administered daily until the end of the experiment (a total of 8 weeks).

[0070]

[0071] 1.8. Behavioral Assessment

[0072]

[0073] To assess behavioral recovery by MCC after SCI, the rats were tested once a week in the field for 8 weeks using the Basso, Beattie, and Bresnahan (BBB) ​​score, which assesses hindlimb motor function. After acclimating the rats to the open space for 5 minutes, their movements were observed for 15 minutes. Two different observers assigned scores from 0 to 21 based on standard conditions such as joint movement, paw position, weight bearing, fore / hind limb coordination, and hip and tail position.

[0074]

[0075] 1.9. Histological sample perspiration

[0076]

[0077] Eight weeks after transplantation, rats were anesthetized and their spinal cords were fixed by perfusion with saline and cold 4% paraformaldehyde (PFA, SAMCHUN, SEOUL, Korea). Spinal cords were harvested and immersed in cold 4% PFA for 48 hours before being transferred to cold 30% sucrose (DAEJUNG) for 1 week. Tissues were then embedded in frozen Section Media (3801480, Leica Biosystems, Wetzlar, Germany) and frozen at -80°C. Frozen samples were cryosectioned at 20 μm using a cryostat (CM1860, Leica Biosystems).

[0078]

[0079] 1.10. Histological analysis

[0080]

[0081] Hematoxylin and eosin (H&E) staining

[0082]

[0083] To compare inflammatory responses and tissue repair between groups, tissue sections were stained with hematoxylin and eosin (H&E). Samples were placed in acetone, air-dried for 10 minutes, and then stained with hematoxylin (Sigma-Aldrich) for 50 seconds. The stained samples were washed with distilled water for 5 minutes and then stained with eosin (Merck, Kenilworth, NJ) for 40 seconds. Finally, the stained samples were dehydrated in graded ethanol solutions (70%, 80%, 90%, and 100%) (64-17-5, Ansan, Korea) and mounted with permanent mounting solution (Fisher Scientific, Hampton, NH).

[0084]

[0085] Eriochrome cyanine (EC) staining

[0086]

[0087] Eriochrome cyanine staining (EC; MERCK) was performed to determine the extent of tissue repair and remyelination. Tissues were fixed in acetone for 10 minutes and air-dried for 10 minutes. After 10 minutes of staining with EC solution, cells were washed with distilled water, and gray and white matter were differentiated using 5% iron alum (MERCK). Subsequently, samples were thoroughly labeled using borax-ferricyanide solution (MERCK). The stained samples were dehydrated using graded ethanol (70%, 80%, 90%, and 100%) and immersed in mounting solution. EC-stained images were evaluated using ImageJ software.

[0088]

[0089] 1.11. Immunohistochemistry

[0090]

[0091] For immunohistochemical staining, tissue sections were washed three times with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (DAEJUNG) and blocked with 10% bovine serum albumin (9048-46-8, MP Biomedicals, LLC, Illkirch, France) in PBS containing 0.2% Triton X-100 for 1 h. The blocked sections were then incubated with primary antibodies overnight at 4°C.

[0092] Chicken anti-MAP2 (1:1000, ab5392, Abcam), rabbit anti-MBP (1:500, ab218011, Abcam), mouse anti-GFAP (1:500, ab4648, Abcam), mouse anti-HuN (1:100, ab191181, Abcam), mouse anti-CNPase (1:500, ab6319, Abcam), rabbit anti-P0 (1:500, ab31851, Abcam), mouse anti-CD31 (1:200, 550274, BD Biosciences San Jose CA USA), rabbit anti-VEGFA (1:500, ab46154, Abcam), mouse anti-CD68 (1:300, ab955, Abcam), and rabbit anti-Iba1 (1:500, ab178680, Abcam). Primary antibodies were diluted in 10% BSA and used to evaluate nerve regeneration, cell survival, remyelination, angiogenesis, and inflammatory responses. Sections were then washed three times with PBS containing 0.2% Triton X-100 and incubated with species-specific secondary antibodies for 1 h at room temperature. The secondary antibodies were FITC-goat anti-mouse IgG (H+L) (1:200, ab150113, Abcam), FITC-goat anti-rabbit IgG (H&L) (1:500, ab150092, Abcam), and FITC-goat anti-chicken IgY (IgG) (H+L) (1:600, 103-605-155, Jackson ImmunoResearch Labs, West Grove, PA). Sections were stained with DAPI (1:1000, Invitrogen) and mounted. Immunofluorescence images were analyzed using a scanning confocal laser microscope (LSM700, Carl Zeiss).

[0093]

[0094] 1.12. Toluidine blue staining and transmission electron microscopy (TEM)

[0095]

[0096] Spinal cords were fixed in 2% glutaraldehyde (MERCK) and 2% paraformaldehyde (MERCK) in 0.1 M phosphate buffer (pH 7.4) for 12 h. The fixed samples were washed with 0.1 M phosphate buffer and postfixed in 1% OsO4 (Polysciences, Philadelphia, USA) in 0.1 M phosphate buffer for 2 h. The samples were then dehydrated in graded ethanol solutions (50, 60, 70, 80, 90, 95, 100, and 100%) for 10 min and permeabilized with propylene oxide (Acros, Geel, Belgium) for 10 min. The samples were inserted into a Poly / Bed 812 kit (Polysciences) and incubated in an electron microscope oven (TD-700, DOSAKA, Japan) at 65°C for 12 h to polymerize. Sections were cut into 200 nm semi-thin sections using an Ultramichrome equipped with a diamond blade, stained with toluidine blue, and observed under a light microscope. The area to be analyzed was cut into 80 nm thin sections, placed on copper or gold grids, and then double-stained with 5% uranyl acetate (MERCK) for 30 min and 3% lead citrate (MERCK) for 7 min. Analysis was performed at an accelerating voltage of 80 kV using a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) equipped with a Megaview III CCD camera (Soft Imaging system-Germany).

[0097]

[0098] 1.13. Statistical Analysis

[0099]

[0100] All experimental data are expressed as the mean ± standard deviation (SEM). Significance was estimated and graphs were generated using GraphPad PRISM 8.3 (GraphPad Software Inc., San Diego, CA). One-way or two-way ANOVA tests were performed to compare statistically significant experimental differences between groups. P < 0.05 and *** p < 0.0001 indicate significant differences.

[0101]

[0102] Example 2: Experimental Results

[0103]

[0104] 2.1. Extrusion bioprinting of multichannel conduits

[0105]

[0106] Surgical resection of traumatic injury or cancers such as gliomas can result in large spinal cord defects. This leads to nerve loss and glial scarring at the defect site, often accompanied by functional abnormalities and neuropathic pain. To promote nerve regeneration in this hostile environment, we fabricated a multichannel conduit (MCC) using extrusion bioprinting technology using multi-hydrogel, MCC-structured cartridges (Figure 1a).

[0107] First, we optimized several hydrogels for implantable duct formation. Alg100 and AC25 ducts maintained their shape but were too rigid for implantation, and Col100 could not maintain its structure as an implantable duct. Therefore, AC50 hydrogel was used to create an MCC matrix (Fig. 1a, b). Alg100 hydrogel produced the most sustainable duct and was used as a sacrificial material to create the channels. We fabricated MCCs using 3D printing and characterized the hydrogels. We used rheological evaluation to confirm the co-printability of the hydrogels for channel-shaped duct formation. First, we measured the viscosities of AC50 and Alg100 hydrogels to confirm simultaneous extrusion. The viscosities of AC50 and Alg100 hydrogels were higher than 101, indicating similar viscosities across the initial shear rate range. These results indicate that the hydrogel coprinting has a viscosity (Fig. 1c). In addition, the AC50 hydrogel storage modulus (G') and loss modulus (G") gradually increased, indicating that the AC50 hydrogel has the elasticity of an implantable hydrogel conduit (Fig. 1d).

[0108] The selected hydrogels were then loaded into poly-ε-caprolactone cartridges equipped with a channel structure and extruded in an 80 mM calcium chloride solution. The hydrogels possess readily implantable mechanical properties due to the ionic reaction between calcium chloride and alginate. During incubation of the MCCs at 37°C, the matrix region of the AC50 hydrogel gelled, and the channel region of the Alg100 hydrogel was removed from the channels. To visualize the multi-channel structure, each segment was filled with AC50 hydrogel (red) and Alg100 hydrogel (sacrificial hydrogel, blue) containing different fluorescent particles, demonstrating that the MCCs were fabricated with the same structure as the cartridge. The cell-free 9-channel and 5-channel MCCs were composed solely of AC50 hydrogel with the Alg100 hydrogel removed (Fig. 1e). The printed MCCs were fabricated with a width equal to the cross-sectional diameter of the spinal cord. This determined the diameter of the cartridge nozzle.

[0109]

[0110] 2.2. Biocompatibility of multichannel conduits equipped with SC and EC

[0111]

[0112] Previous reports have demonstrated that MCCs mimic a tubular structure with a hollow lumen capable of supplying sufficient oxygen and nutrients to promote cell survival and proliferation, as well as mass transfer. In the present invention, we expanded upon this previous work by loading Schwann cells (SCs) and endothelial cells (ECs) into MCCs to create implantable conduits. Accurate axonal regeneration requires not only the MCC structure but also a leading SC and vascular bridge environment. Leading SCs guide axons to the correct location along the vascular bridge. Therefore, we evaluated the feasibility of loading SCs and ECs for MCC printing on AC50 hydrogel and confirmed that the MCC structure influences the survival and adhesion of loaded cells.

[0113] We printed non-MCC-SE, 5MCC-SE, and 9MCC-SE containing SC and EC and analyzed cell viability using live / dead staining. Non-MCC-SE showed a cell viability of approximately 35%, indicating that oxygen and nutrients did not diffuse sufficiently to the epicenter for cell survival. Notably, the cell viability of both 5MCC-SE and 9MCC-SE structures was twice that of the non-MCC-SE structure, indicating that the presence or absence of channels significantly affected cell survival (Fig. 2a, b). Cell proliferation assay results showed a similar pattern. Cells in the MCC structure exhibited higher adhesion compared to non-MCC-SE and were more adherent in 9MCC-SE than in 5MCC-SE (Fig. 2c). These results suggest that oxygen and nutrients are transported to cells through more channels. Furthermore, the number of channels is crucial for sufficient oxygen and nutrient diffusion. A greater number of channels provides more growth sites, increasing cell density. Furthermore, a greater number of aligned cell base channels can induce cell proliferation, thereby enhancing cell proliferation. Therefore, well-distributed SCs and ECs in 9MCCs can provide an enhanced microenvironment for nerve regeneration.

[0114]

[0115] 2.3. The vertical alignment of SC and EC of MCC mimics the band of Bungner structure.

[0116]

[0117] Precise axonal growth and nerve regeneration after nerve transection require an environment resembling the Bungner band. MCCs can be used to facilitate longitudinal guidance of neurite regeneration to the target tissue. MCCs also reduce axonal dispersion and provide a higher surface area for cell function. The high surface area of ​​the MCC multichannels enhances cell survival, and this effect is attributed to the channel diameter of 100–200 μm (the diffusion limit of oxygen and nutrients). SCs and ECs, which are important for axon guidance, elongation, and track regeneration in the target area, were loaded onto the printed MCCs because these cells mimic the Bungner band. Furthermore, stained S100 (SC), CD31, and phalloidin (EC) patterns revealed aligned SC and EC linings and bundle structures (Fig. 3a, b). These data demonstrate that the fabricated MCC-SE can mimic the band of Bungner structure and provide a natural living scaffold in the SCI microenvironment.

[0118]

[0119] 2.4. Improvement of behavioral function and nerve regeneration after MCC-SE transplantation

[0120]

[0121] To determine the therapeutic efficacy of a multichannel conduit containing SC and EC (MCC-SE) in the transected spinal cord, MCC-SE was implanted into the lesion site immediately after spinal cord transection. Behavioral assessments were then performed for 8 weeks, and histological analyses were performed (Fig. 4a). The non-conduited spinal cord (SCI group) maintained a low BBB score until the end of the study. In contrast, the 9-channel conduit without SC and EC (9MCC group) began to show some improvement after 3 weeks of implantation, and the BBB score improved by more than 8 points when using the 5-channel conduit with SC and EC and the 9-channel conduit with SC and EC (5 and 9 MCC-SE groups). Notably, the 9MCC-SE group reached the highest BBB score of 11±0.79 at the end of the experiment (Fig. 4b). Generally, improved behavioral function was associated with tissue recovery. Unlike the non-SCI group, which exhibited atrophy, large cavities, and a severed morphology, the 9MCC-implanted spinal cord exhibited some resolution of atrophy, reduced cavities, and lesion bridging morphology. Using H&E staining, we observed that 9MCC transplantation ligated the tissue and resolved atrophy in the presence or absence of cells.

[0122] However, more channels resolved atrophy, reduced pores, and promoted stable lesion connections (Fig. 4c). To investigate the molecular mechanisms of nerve regeneration using 9MCC-SE, immunofluorescence was performed. Nerve regeneration extends from the lesion site, forming a continuous neural bridge. To assess this, we assessed the expression of microtubule-associated protein (MAP2) at the SCI lesion site of transplanted 9MCCs. MAP2 expression decreased rapidly after SCI and was partially restored by 9MCC transplantation. Insertion of SCs and ECs into MCCs allowed the observation of more neurons, with a greater increase in 9-channel compared to 5-channel MCCs (Fig. 4d, e). The expression of MBP, a marker of oligodendrocytes responsible for axonal myelination, also decreased rapidly after SCI and was partially restored by 9MCC transplantation. MCC-SE induced myelination in more axons, and this effect was greater in 9-channel compared to 5-channel MCCs (Fig. 4d, e). At the SCI lesion site, reactive astrocytes can create scarring and impede nerve regeneration at the lesion site. GFAP expression, which was barely expressed in sham controls, was significantly increased after SCI. MCC transplantation reduced GFAP expression, which was restored to normal levels by the incorporation of SCs and ECs (Fig. 4d, e). To confirm whether these results indicate that the transplanted MCC-SE cells survive and differentiate or influence the transplanted host, HuN staining was performed. Based on the identification of a small number of transplanted SCs and ECs, regeneration was confirmed through the hydrogel and paracrine effects (Fig. 4f, g).

[0123] These results suggest that hydrogels are excellent materials for the treatment of neurodegeneration and improve behavior after spinal cord injury. Furthermore, these results demonstrate that porous collagen, composed of an extracellular matrix, offers numerous advantages for functional recovery due to its low immunogenicity, biocompatibility, and biodegradability. The structural characteristics of the porous collagen envelope can prevent compression of surrounding tissues and astrocyte infiltration, thereby minimizing scar formation and resolving atrophy. Furthermore, these results demonstrate that a cell-loaded multichannel structure can maximize these nerve regeneration effects.

[0124]

[0125] 2.5. Remyelination after MCC-SE transplantation

[0126]

[0127] Remyelination is crucial for neural signaling and function after spinal cord injury (SCI). Although peripheral nerve-derived cells (MCCs), previous reports have shown that adding SCs to MCCs can promote remyelination when transplanted into the injured spinal cord. Furthermore, ECs can form microvessels and act as amateur phagocytes by clearing myelin debris generated during SCI. Clearance of myelin debris is crucial for axonal regeneration, remyelination, and resolution of inflammation. This is because myelin debris contains molecules that potently inhibit axonal regeneration and remyelination at the injury site and actively participate in the inflammatory response. In addition to these cellular effects, the hydrogel in the conduit guides axonal growth at the injury site and, together with the included SCs' bands of Bungner, provides a framework for remyelination. To determine the effects of MCC-SE transplantation on myelination after SCI, we performed EC staining, toluidine blue (TB) staining, TEM image analysis, and immunohistochemistry.

[0128] Tissue atrophy is observed after SCI. As the overall neural architecture collapses, the boundary between gray and white matter is disrupted. EC staining clearly demonstrates this phenomenon. MCC-SE grafting ligated the tissue distal to the injury site, reducing atrophy and cavity size (Fig. 5a, b). MCC-SE grafting also significantly increased the bright blue-stained myelin area, with 9-channel performing slightly better than 5-channel (Fig. 5a, b). TB staining and TEM images revealed a significant increase in myelinated axons in 9MCC, particularly in MCC-SE, compared to the few myelinated axons observed in the SCI group (Fig. 5c, d). In particular, 9MCC-SE showed statistically significant higher myelination values ​​than 9MCC and 5MCC-SE, confirming that the presence of cells and increased channel number significantly influence axonal myelination.

[0129] We also measured the G-ratio of TEM images to confirm whether the regenerated myelinated axons were functioning properly. After spinal cord injury, myelinated axons can develop functional abnormalities, such as myelin loosening, thickening, and thinning. Appropriate myelin sheath thickness allows myelinated axons to have normal velocity and conduction. After MCC transplantation, the G-ratio was restored to a level close to that of the sham group in the 9MCC-SE, 5MCC-SE, and 9MCC groups, in that order. In addition, axons of various sizes were observed in all groups except the sham group (Fig. 5e). The G-ratio was calculated by dividing the measured inner (axonal) circumference by the measured outer (myelin) circumference.

[0130] Wallerian degeneration following SCI is caused by the apoptosis of oligodendrocytes. Most transplanted SCs, identified as P0, were unable to survive independently. However, this resulted in an increase in oligodendrocytes in the spinal cord, as confirmed by CNPase (Fig. 5f, g). Oligodendrocyte survival is particularly influenced by growth factors and cytokines. Hypoxia and increased ROS levels due to SCI also induce Wallerian degeneration. The MCC structure, which includes vascularized ECs, was an effective method to overcome this. Oligodendrocytes, which had almost disappeared after SCI, were observed in greater numbers after MCC-SE transplantation, and this increase was significantly greater in 9MCC-SE, which has a higher number of channels compared to 5MCC-SE (Fig. 5f, g). This suggests that 9MCC-SE has a more enhanced effect than 5MCC-SE due to its sophisticated structural features and the paracrine effect of the loaded cells. The high survival rate of the loaded cells upon transplantation can induce a higher paracrine effect after transplantation, which enhances axon regeneration and remyelination. These results confirm that when SCs and ECs exist together with MCCs, myelination by oligodendrocytes, regeneration of properly functioning myelinated axons, and tissue restoration can be enhanced.

[0131]

[0132] 2.6. MCC-SE transplantation promoted angiogenesis after SCI.

[0133]

[0134] After spinal cord injury, microvessels and the blood-spinal cord barrier are destroyed, leading to inflammation, ischemia (neuronal cell death), axonal degeneration, and neurological deficits. To overcome this phenomenon, angiogenesis must occur to overcome hypoxia and restore the blood-spinal cord barrier. MCC-SE can serve as an excellent source of angiogenesis. ECs contained in MCC-SE can act as a substance for angiogenesis and form bands of Bungner with SCs, and their channel structure can provide an excellent foundation for vascular cell lining. ECs identified by CD31 disappeared after SCI but were restored after MCC-SE transplantation. Notably, 9MCC-SE transplantation produced higher amounts of CD31 and VEGF-A, proteins that promote angiogenesis, compared to 5MCC-SE (Fig. 6a, b). The high cell viability of 9MCC-SE was attributed to enhanced angiogenic capacity and improved neuronal regeneration by enhanced paracrine factors of EC.

[0135] These results confirmed that cells in MCC-SE play an important role in angiogenesis and that a greater number of channels further promotes angiogenesis.

[0136]

[0137] 2.7. Reduction of inflammation following MCC-SE transplantation

[0138]

[0139] Previous reports have demonstrated that reactive astrocytes, myelin debris from myelin destruction, and vascular disruption can activate the inflammatory response after SCI50. Each of these factors not only recruits immune cells and releases inflammatory cytokines, but also can generate ROS51. We confirmed that MCC-SE transplantation reduced astrocyte reactivity, suppressed myelin debris production, and induced angiogenesis. Therefore, we expected that this would ultimately lead to a reduced immune response. Based on this, we identified CD68-expressing macrophages and IBA1-expressing microglia in the spinal cord. Many immune cells are recruited to the injury site after SCI. MCC transplantation suppressed immune cell recruitment, but 9MCC-SE was the only group with a suppressed immune response, similar to the sham group (Fig. 7a, b). In particular, the nerve conduit (9MCC) based on atelocollagen, in which antigenic telopeptides were removed by pepsin, showed an anti-inflammatory effect with the material itself without cell transplantation, and MCC-SE, a collagen hydrogel-based material with angiogenic ability, effectively engulfed myelin debris, reduced ROS, and increased phagocytosis, significantly reducing inflammation at the damaged site.

[0140] Based on these results, it was estimated that subacute inflammatory response was reduced, secondary damage was partially suppressed, and nerve regeneration and functional recovery were induced.

[0141]

[0142] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-channel nerve conduit having structural stability and introducing neural cells within the multi-channel, comprising a hydrogel containing collagen and alginate; and a biocompatible polymer; A multi-channel nerve conduit, wherein collagen and alginate are each contained in a concentration of 1 to 10% in the hydrogel.

2. In paragraph 1, A multi-channel nerve conduit, wherein each channel constituting the above multi-channel has a diameter of 1,000 to 3,000 μm.

3. In paragraph 1, A multi-channel nerve conduit, wherein collagen and alginate are mixed in a volume ratio of 0 to 1:1 to 0 in a hydrogel, respectively.

4. In paragraph 1, A multi-channel nerve conduit, wherein the multi-channels have a number of 5 to 9.

5. In paragraph 1, A multi-channel nerve conduit, wherein the above-mentioned nerve cells are mounted in an aligned array within the multi-channel nerve conduit.

6. In paragraph 1, A multi-channel nerve conduit, wherein the nerve cells are at least one selected from the group consisting of Schwann cells and endothelial cells.

7. In paragraph 1, A multichannel nerve conduit, wherein the biocompatible polymer is selected from the group consisting of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, poly(lactic acid-caprolactone) copolymer, poly(hydroxybutyric acid-hydroxyvaleric acid) copolymer, polyphosphoester, and poly-L / D-lactide (PLDLA).

8. In paragraph 1, The above multi-channel nerve conduit is a multi-channel nerve conduit that provides a bridge for nerve cell attachment and axon growth necessary for nerve regeneration, or supplies oxygen and nutrients necessary for nerve regeneration along with vascular regeneration.

9. A nerve regeneration method comprising transplanting a multi-channel nerve conduit according to any one of clauses 1 to 8 into a damaged nerve area.

10. Use of a multi-channel nerve conduit according to any one of Articles 1 to 8 for treating nerve damage.

11. A method for manufacturing a multi-channel nerve conduit having structural stability and introducing neural cells into the multi-channel, a) a step of preparing a hydrogel of a matrix region by mixing a hydrogel of collagen and alginate; and b) a step of loading the hydrogel into a biocompatible polymer equipped with a channel structure; A method for manufacturing a multi-channel nerve conduit, wherein the diameter of the multi-channel is 500 to 1,000 μm.

12. In paragraph 11, A method for manufacturing a multi-channel nerve conduit, wherein collagen and alginate are each included in a concentration of 1 to 10% in a hydrogel.

13. In paragraph 11, A method for manufacturing a multi-channel nerve conduit, wherein collagen and alginate are mixed in a hydrogel at a volume ratio of 0 to 1:1 to 0, respectively.

14. In paragraph 11, A method for manufacturing a multi-channel nerve conduit, wherein the multi-channels have a number of 5 to 9.

15. In paragraph 11, A method for manufacturing a multi-channel nerve conduit, wherein the above nerve cells are mounted in an aligned array within the multi-channel nerve conduit.

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