METHOD FOR PREPARATION OF EXTRACELLULAR MATRIX-MODIFIED DECELLULARIZED NERVE SCAFFOLD AND USE THEREOF

Abstract

An extracellular matrix-modified decellularized nerve scaffold and use thereof are provided. The extracellular matrix-modified decellularized nerve scaffold is prepared from a natural porcine optic nerve. The scaffold has a plurality of longitudinal channels and a plurality of transversal foramina intercommunicated with the longitudinal channels, which have relatively uniform diameters and relatively even distributions in the scaffold. The extracellular matrix-modified decellularized optic nerve scaffold of the present invention changes the poor microenvironment of existing decellularized material that lacks cell growth factors and nutrients, supports seeded cells to form neural networks in vitro or in vivo, and enables the connection of ascending nerve fibers or descending nerve fibers of the injured spinal cord to their target cells after transplantation.

Claims

1. An extracellular matrix-modified decellularized nerve scaffold, characterized in that the scaffold is derived from a natural porcine optic nerve, the scaffold has a plurality of longitudinal channels and a plurality of transversal foramina intercommunicated with the longitudinal channels, and the scaffold is loaded with extracellular matrix.

2. The extracellular matrix-modified decellularized nerve scaffold of claim 1, characterized in that the scaffold is prepared by decellularization, the plurality of longitudinal channels and the plurality of transversal foramina have relatively uniform diameters and relatively even distributions in the scaffold.

3. The extracellular matrix-modified decellularized nerve scaffold of claim 1, characterized in that the extracellular matrix is a cell growth factor and/or a cell nutritional factor; wherein the cell growth factor is selected from a group comprising neurotrophic factor-3 (NT-3), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and mixtures thereof; and the cell nutritional factor is selected from a group comprising fibroblast growth factor (FGF), insulin-like growth factor (IGF), or transforming growth factor (TGF) and mixtures thereof.

4. The extracellular matrix-modified decellularized nerve scaffold of claim 3, characterized in that the scaffold further comprises a cell secreting extracellular matrix, wherein the cell secreting extracellular matrix is selected from a group comprising Schwann cell and mesenchymal stem cell.

5. The extracellular matrix-modified decellularized nerve scaffold of claim 1, characterized in that the scaffold is seeded with seed cells to form a neural network-like structure which is able to be transplanted to an injured spinal cord and thus repair the injured spinal cord.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is a schematic view of an extracellular matrix-modified decellularized porcine optic nerve scaffold implanted with seeded cells, wherein reference numerals and symbols show: [0018] a: extracellular matrix-modified decellularized porcine optic nerve scaffold [0019] 1: longitudinal channels formed in scaffold [0020] 2: neuron [0021] 2-1: neuronal somata [0022] 2-2: nerve fibers

[0023] FIG. 2 shows an SEM (scanning electron microscope) picture of a decellularized porcine optic nerve scaffold, wherein arrows indicate longitudinal channels of the decellularized porcine optic nerve scaffold.

[0024] FIG. 3 shows an SEM (scanning electron microscope) picture of a decellularized porcine optic nerve scaffold, wherein arrows indicate transversal foramina of the decellularized porcine optic nerve scaffold.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

[0025] Embodiments of the present invention are further explained clearly as follows in conjunction with figures.

Experimental Materials

[0026] Instruments: Benchtop (Suzhou Purification Electronic Equipment Factory, China); low-speed multi-tube auto-balancing centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., China); 5% CO.sub.2 incubator (Queue, USA); inverted phase contrast microscope (Olympus, Japan); fluorescence microscope (Leica, Germany); scanning electron microscope (Philips, Netherlands); low temperature oven (Shanghai Yuejin Medical Equipment Factory, China); high temperature oven (Shanghai Yuejin Medical Equipment Factory, China); autoclave (Jiangyin Riverside Medical Equipment Factory, China); cryostat microtome (Shandon; UK); ultra-pure water purifier (Molsheim; France); shaker (Guangzhou Zhengyi Technology Co., Ltd., China); lyophilizer (Labconco, American).

[0027] Reagents: D-Hank's equilibrium liquid (self-prepared); 0.01 mol/L PBS (Zhongshan Jinqiao Biotech, China); Hoechst33342 (Sigma); goat serum (Zhonshan Jinqiao Biotech, China); sodium deoxycholate (Sigma); Triton X-100 (Shanghai Shenggong, China); -NGF (Peprotech); B27 (GIBCO); Matrigel (GIBCO), DMEM/F12 (Hyclone); Neurobasal (GIBCO).

Experimental Methods

[0028] 1. Preparation of Extracellular Matrix-Modified Decellularized Optic Nerve Scaffolds

[0029] Porcine optic nerves were supplied by the Experimental Animal Center of Sun Yat-sen University. Tissue was placed in an ice box, separated from all non-optic nerve connective tissues and membranes and then frozen at 20 C. for storage.

[0030] Surface adipose tissue and some dura mater cerebralis were removed, and the porcine optic nerves were cut into 5 mm segments. The optic nerve segments were bathed and agitated in distilled water for 6 h at 60 rpm, bathed and agitated in 30 mL/L aqueous Triton X-100 solution for at 12 h at 60 rpm, rinsed with distilled water three times, bathed and agitated in 40 g/L (4%) aqueous sodium deoxycholate solution for 24 h at 60 rpm, rinsed with distilled water three times, repeatedly treated with said Triton X-100-distilled water-sodium deoxycholate-distilled water process two times, stored in PBS, fixed in 4% paraformaldehyde for 24 h, dehydrated in gradient sucrose solution for 24 h, bathed in 75% alcohol for 15 min, rinsed with D-Hank's solution three times with 10 min each time. The obtained sterilized decellurarized optic nerve materials were placed into vials that had been autoclaved sterilization-treated, and lyophilized in a lyophilizer for 12 h. Scanning electron microscopy was carried out to ascertain the longitudinal channels and transversal foramina of decellularized porcine optic nerve scaffold. Scaffolds were firstly washed 3 times with PBS, fixed in 2.5% glutaraldehyde for 90 min, dehydrated with a series of graded ethanol, and then freeze dried for 24 hours. The dried samples were coated with gold and examined under a scanning electron microscope (Philips XL30 FEG). The lyophilized sterilized decellurarized optic nerve scaffolds were immersed in extracellular matrix, mixed for 3 h, cross-linked and then stored dry until use.

[0031] In one embodiment, the extracellular matrix is a cell growth factor and/or a cell nutritional factor; wherein the cell growth factor is selected from a group comprising neurotrophic factor-3 (NT-3), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and mixtures thereof; and the cell nutritional factor is selected from a group comprising fibroblast growth factor (FGF), insulin-like growth factor (IGF), or transforming growth factor (TGF) and mixtures thereof. In another embodiment, the extracellular matrix is NT-3/fibroin complex. In another embodiment, besides the extracellular matrix, the scaffold further comprises a cell secreting extracellular matrix, wherein the cell secreting extracellular matrix is selected from a group comprising Schwann cell, mesenchymal stem cell and fibroblast.

[0032] 2. Seeding of Neural Stem Cells (NSCs)

[0033] 2.1 Isolation and Cultivation of NSCs In Vitro

[0034] Brains of two one-day-old Sprague Dawley (SD) neonatal rats were taken out under sterile conditions, and were placed in cold D-Hank's solution. The hippocampus was isolated from the brains using an anatomical microscope, and cut into pieces by ophthalmic scissors, transferred into a centrifuge tube with D-Hank's solution, gently pipetted with a fine glass pipette several times until tissue fragments were not visible (the pipetting should be slow and gentle to avoid making bubbles), and centrifuged under 1000 rpm for 5 min to discarded the supernatant. The precipitations of NSCs after the centrifugation were suspended again with D-Hank's solution, said pipetting and centrifugation procedures were repeated. The obtained precipitations of NSCs were suspended with NSCs basal medium, then gently pipetted, diluted to control the cell density to about 110.sup.5 cells/mL. The cell suspension was transferred to a cultivation flask and incubated in an incubator under 37 C. and 5% CO.sub.2. Half of the liquid volume of the NSCs culture medium was renewed and gently pipetted every two days.

[0035] 2.2 Seeding of NSCs

[0036] NSCs that had been cultured for 5 days were seeded into the extracellular matrix-modified decellularized optic nerve scaffolds, Neurobasal medium were added. The NSCs-seeded extracellular matrix-modified decellularized optic nerve scaffolds were cultured for 7 days, during which the Neurobasal medium were renewed every two days.

[0037] 3. Seeding of Dorsal Root Ganglia (DRG)

[0038] 3.1 Isolation of DRG In Vitro

[0039] Spinal cords of one-day-old green fluorescent protein (GFP) transgenic neonatal rats were taken out under a dissecting microscope and then placed in a culture dish containing pre-cooled DMEM medium. All operations were done on an ice box. DRGs were cut off using a microscissors and were placed in a culture dish containing 2 mL of pre-cooled DMEM medium.

[0040] 3.2 Seeding of Dorsal Root Ganglia (DRG)

[0041] PLGA tubes each with a diameter of 3 mm and a height of 3 mm were placed on a 96-well plate which was coated with Matrigel for stabilizing the PLGA tubes. The prepared extracellular matrix-modified decellularized optic nerve scaffolds were inserted into the PLGA tubes with the transversal cross-sectional surfaces upwardly arranged, gently added with DRGs on the transversal cross-sectional surfaces, added with Neurobasal medium, incubated in an incubator under 37 C. and 5% CO.sub.2 for 7 days, during incubation the Neurobasal medium were renewed every two days.

[0042] 4. Detection of Cell Growth in Extracellular Matrix-Modified Decellularized Optic Nerve Scaffolds

[0043] The extracellular matrix-modified decellularized optic nerve scaffolds seeded with either of the two types of cells, i.e., NSCs and DRG cells, which had been cultured for 7 days, were transversely and longitudinally sectioned with a thickness of 20 m using a frozen microtome. The cell growths were observed under a fluorescence microscope.

[0044] Experiment Results

[0045] 1. Scaffolds Constructed from Extracellular Matrix and Decellularized Porcine Optic Nerves had a Plurality of Longitudinal Channels and a Plurality of Transversal Foramina.

[0046] FIG. 1 is a schematic view of the extracellular matrix-modified decellularized porcine optic nerve scaffold seeded with cells. The scaffold has a plurality of longitudinal channels and a plurality of transversal foramina intercommunicated with the longitudinal channels. On the one hand, the longitudinal channels are beneficial to the extension of synapses of the implanted neurons (seeded cells), the synapses may therefore reach and connect the neurons of host spinal cord through the longitudinal channels. On the other hand, the transversal foramina are beneficial to the communication of the implanted neurons (seeded cells) positioned in different longitudinal channels. Reference numerals and symbols: a: decellularized porcine optic nerve scaffold; 1: longitudinal channel formed in decellularized porcine optic nerve scaffold; 2: neuron; 2-1: neuronal somata; 2-2: nerve fibers.

[0047] FIG. 2 shows a plurality of longitudinal channels that are divided by cerebral leptomeninx.

[0048] FIG. 3 shows a plurality of transversal foramina intercommunicated with the longitudinal channels.

[0049] 2. Scaffolds Constructed from Extracellular Matrix and Decellularized Porcine Optic Nerves had a Plurality of Partitions

[0050] Referring to FIG. 1, the transversal cross-sectional area of the extracellular matrix-modified decellularized porcine optic nerve scaffold showed a plurality of longitudinal channels each having a circular transversal cross-sectional area. Transversal foramina connected neighbouring longitudinal channels. In this way, the seeded cells may be regionally planted.

[0051] 3. Three-Dimensional Cylindrical Shape of the Optic Nerve and Longitudinal Channels were Retained in the Decellularized Porcine Optic Nerve Scaffolds

[0052] HE staining of natural porcine optic nerves and the constructed extracellular matrix-modified decellularized optic nerve scaffolds showed that the transversal cross-sectional area of the natural porcine optic nerve had a plurality of circular channels filled with nerve fibers depicted by hematoxylin-stained neurons nucleus and eosin-stained cytoplasm and extracellular matrix, the longitudinal cross-sectional area of the natural porcine optic nerve had a plurality of parallel longitudinally-sectioned channels. After the removal of nerve fibers (neurons), epineurium around a whole optic nerve retained and effectively maintained the three-dimensional cylindrical shape of the decellularized porcine optic nerve scaffolds, cerebral leptomeninx extended into the interior of the optic nerve also retained and divided the interior of the optic nerve to form a plurality of channels intercommunicated with transversal foramina.

[0053] 4. Assessment of Residual DNA

[0054] Hoechst33342 staining showed that natural porcine optic nerves contained a large number of blue-colored nuclei under the fluorescence microscope, while no blue-colored nuclei were seen in decellularized porcine optic nerve scaffolds, indicating that the resulting scaffolds are sufficiently decellularized to obviate adverse host immune responses.

[0055] 4. Cytocompatibility

[0056] DRG cells with GFP gene were implanted and cultured for 7 days in the extracellular matrix-modified decellularized porcine optic nerve scaffold. Green DRG cells were seen attached to the surface of the scaffold under fluorescence microscopy. Green nerve fibers (cell protrusions) migrated inward along the longitudinal channels of the scaffold, and the nerve fibers contacted each other through the transversal foramina to form a network. The structure of the longitudinal channels and transversal foramina was conducive to long-distance cell growth and information exchange.

[0057] NSCs with GFP gene were implanted and cultured for 7 days in the extracellular matrix-modified decellularized porcine optic nerve scaffold. NSCs shown under fluorescence microscopy were distributed evenly in the longitudinal channels whose walls are formed by cerebral leptomeninx and were connected through the transversal foramina. The cerebral leptomeninx were conducive to cell implantation compartmentally, providing a good structural basis for implantation of different cells in different regions of the scaffold.

[0058] The above-mentioned embodiments are the preferred embodiments of the present invention. Variations and modifications are allowed within the scope of the invention. Those skilled in the art will appreciate that the features described above can be combined in various ways to form multiple variations of the invention. As a result, such variations fall within the scope of the protection to the present invention.