• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    preparation of manipulated tissue nerve grafts with modified extracellular matrix for repair of peripheral nerve injury. a tissue nerve graft manipulated to repair a peripheral nerve defect comprises a nerve conduit and a cellular matrix (ecm), in which the cellular matrix is obtained by decellularization after autologous or allogeneic cells are secreted and formed. the method of preparing the manipulated tissue nerve graft comprises constructing a manipulated tissue nerve graft containing support cells and constructing a manipulated tissue nerve graft with a modified cell matrix.
    公开号:BR112015017174B1
    申请号:R112015017174-5
    申请日:2013-05-27
    公开日:2020-09-24
    发明作者:Xiaosong Gu；Fei Ding；Yun Gu；Chengbin Xue；Yumin Yang；Yongjun WANG；Leilei Gong
    申请人:Nantong University；
    IPC主号:
    专利说明:

    [0001] [001] The present invention, which belongs to the technical field of medical biomaterials used for transplantation in the human body, relates to the preparation of manipulated tissue nerve grafts with extracellular matrix (ECM) for repair of peripheral nerve damage. BACKGROUND
    [0002] [002] With the progress of social modernization and the acceleration of the pace of people's daily lives, an increasing number of accidents, resulting from traffic, industry, sports, local wars, violent events and natural disasters (for example, earthquakes), will lead injury to peripheral nerves. When the nerve defect formed at mid and long distance cannot be treated with a tip-to-tip suture at the clinic, the peripheral nerve graft must be applied to connect the nerve defect. Although nerve grafts have been researched and developed for over a hundred years, great efforts are still expended in the development of ideal nerve grafts to replace autologous nerve grafts in clinical practice. Despite being a gold standard for peripheral nerve repair, autologous nerve grafting cannot be widely used in the clinic due to its various limitations, such as deficient supply of nerve donors, no combination of the injured nerve and the donated nerve in structure and size, as well as morbidity of the donor site and secondary deformities.
    [0003] [003] The emergence and advancement of the field of tissue manipulation provides a unique opportunity to generate manipulated tissue for nerve grafts as a promising alternative to autologous nerve grafts. Existing manipulated tissue grafts mainly include two important types. One type is the acellular allogeneic nerve, that is, acellular nerve graft (ANG), in which cells in the allogeneic tissue are removed, but the original neural architecture is maintained. ANG meets the basic requirements for nerve grafts in the manipulation of peripheral nervous tissue and becomes a tissue for manipulated tissue nerve grafting derived from modified ECM. For example, a research article indicated that ANG induces the differentiation of adult rat BMSCs into Schwann cells (He Hongyun, Deng Yihao, Ke Xiaojie, et al, Morphologic Study of Bone Marrow Stromal Cells of Rat Differentiating into Schwann Cells By Acellularnerve Grafts, Chinese Journal of Neuroanatomy, 2007; 23 (6)). Another study investigates the protective effects of ANG implanted in motor neurons in the anterior horn of the spinal cord (Liu Jinchao, Lin Xiaoping, Ke Xiaojie, et al. The Protective Effects of Acellularnerve Matrix Allografts on the Motor Neurons of the Anterior Horn of Spinal Cord Progress of Anatomical Sciences, 2005 (3): 206-209) and indicates that the use of ANGs to connect peripheral nerve defects has had excellent protective effects on the survival of the cellular body of motor neurons. Although there are a large number of studies on ANG preparation technology making faster progress, several preparation methods have complicated procedures and the delicate structure and mechanical properties of biomaterials will be impacted during processing.
    [0004] [004] Another type is based on a nervous conduit prepared with an appropriate mold and which has ECM or support cells coated on its internal and external surfaces. For example, a research article reports on the use of a binding graft made of olfactory sheath cells (OECs) - Schwann cells (SCs) derived from ECM and poly (DL lactide-co-glycolide) (PLGA) to protect the neurons of peripheral target organs and spinal cord after sciatic nerve injury (You Hua, Jiao Shusheng, Feng Shuainan, et al, The Protective Effects of Tissue Engineering Artificial Nerves on Peripheral Target Organs and Spinal Cord Neurons after Sciatic Nerve Defect Chinese Journal of Trauma, 2010 Vol.26 No.3 P.265-269). As another example, a Chinese patent (order No. CN03134541.7 and order publication No. CN1589913) entitled "A tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method", describes a used manipulated tissue nerve for the repair of a peripheral nerve defect. The manipulated tissue nerve consists of a nervous conduit made of biodegradable materials added with glial cells or stem cells that have the ability to differentiate into glial cells, which are used as cell seeds, and modified with microspheres for controlled release neurotrophic factors and ECM molecules.
    [0005] [005] Currently, support cells used in manipulated tissue nerves include Schwann cells and various stem cells, which are allogeneic cells and can cause immunogenicity, which is not suitable for clinical applications. On the other hand, the fate in vivo and the biological effects of support cells after they are implanted in the body are not entirely clear and they can be inactivated in the body's environment, thus failing to achieve the expected biological effects. All of the above problems limit the development of manipulated tissue nerve grafts. SUMMARY
    [0006] [006] The present invention aims to manipulate nerve grafts of tissue manipulated with modified cell-derived ECM for repair of peripheral nerve damage. Nerve grafts are beneficial for cell adhesion and probably to promote axon regeneration, thus overcoming the disadvantages of existing technologies. The present invention is based on tissue manipulation methods and adopts a rotational cell reactor to grow support cells in a nerve conduit and lumen filaments and then applies decellularization to stimulate the secretion of ECM by the support cells, thus generating nerve grafts modified ECM manipulated tissue derived from support cells for repair of peripheral nerve injury. The present invention can overcome the limitations of autologous nerve grafting, avoids the immunogenicity of allogeneic nerve grafts, provides ECM beneficial to neural cell adhesion and establishes a suitable environment for de novo axonal growth. Therefore, the present invention assists in promoting nerve regeneration and restoring function and will be developed into a possible clinical therapy.
    [0007] [007] The technical scheme of the present invention is as follows.
    [0008] [008] A nerve graft of tissue manipulated to repair nerve damage consists of a nervous conduit and an ECM that is secreted by autologous or allogeneic support cells and obtained by decellularization. Support cells can be isolated or in combination with Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells or induced pluripotent stem cells. The nerve conduit is made of biodegradable materials and is preferably made alone or with a combination of silk fibroin, chitosan, collagen, polylactic acid or polyglycolic acid.
    [0009] [009] The nervous conduit can simply be a graft of manipulated tissue nerve commonly prepared and used in the technique. A chitosan-based nerve conduit with a porous surface is preferable, which has a porosity of 50 to 90%, a pore size of 50 to 300 μm, high tensile strength, internal diameter of 0.5 to 8 mm and wall thickness from 0.1 to 3 mm and can be prepared with respect to the method described in the patent (application No. CN20110324474.8, entitled "Tissue Engineering Nerve Graft and its Functions"). Another nerve conduit on a manometric scale based on silk fibroin, can be prepared specifically with reference to the method described in the patent (application No. CN200910034583.9 and application No. CN101664346, entitled "Artificial Nerve Graft Prepared by Electrospinning, Its Preparation Method, and Related Devices "). The third nerve conduit of choice has a composite structure, consisting of a chitosan based conduit with a porous surface or a silk fibroin based conduit (or any nerve conduit that can be prepared with reference to the above patents) with the introduction of 120 filaments of silk fibroin as luminous charges.
    [0010] [0010] The present invention also provides a method of preparing the manipulated tissue nerve graft modified by a natural cellular matrix for the repair of peripheral nerve defects, which prepares a manipulated tissue nerve graft containing support cells and then it is decellularized to obtain a decellularized manipulated tissue nerve graft. The manipulated tissue nerve graft that contains support cells can be prepared by cell culture to cause the support cells to adhere to the inner and outer surface of a nerve conduit, where a three-dimensional microgravity cell culture method is preferred. The present invention uses a RCMW three-dimensional microgravity culture system type Synthecon.
    [0011] [0011] The preferred technical scheme for the preparation of manipulated tissue nerve grafts containing support cells and decellularized grafts is as follows:
    [0012] [0012] (1) Preparation of manipulated tissue nerve grafts containing support cells. 100 ml of complete medium is slowly placed in a culture flask and then 2.5 x 107 cells and a sterile nerve conduit are added, followed by processing with a peristaltic pump to ensure the final cell density of 1 x 105 / ml. Then, the air is evacuated from the culture flask, a perfusion circulation culture in rotational microgravity is started with a rotary bioreactor placed in an incubator with CO2 at 37 ° C to ensure full contact and adhesion of the cells over the nervous conduit suspended in a culture solution by adjusting the rotation speed after 24 h. After cultivating for 2 days, the medium is switched to a differentiation medium to promote ECM secretion and allowed to grow for another two weeks before the nerve conduit coated with the supporting cells is removed. The optimized composition of the differentiation medium is H-DMEM + FBS 15% + HRG 50 ng / ml + forsklin 2 μg + Vc 50 μg / ml.
    [0013] [0013] (2) Preparation of manipulated tissue nerve grafts modified with ECM. The manipulated tissue nerve graft that contains a support cell undergoes decellularization. It is washed with phosphate buffered saline (PBS), placed in sterile deionized water at 37 ° C for hypotonic treatment for 10 minutes, followed by lysis of the cell in a non-ionic detergent for 10-15 minutes at 37 ° C, and then added in a non-ionic detergent with the addition of DNase I (4 mg / ml) for digestion for 30 min at 37 ° C to remove DNA. The acellular product is kept at -80 ° C for use. The optimized composition of the non-ionic detergent is phosphate buffered saline consisting of 0.5% TritonX-100 and 20 mM aqueous ammonia.
    [0014] [0014] The support cells used in the above procedures are alone or in combination with Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells and induced pluripotent stem cells, while the nervous conduit it is made with biodegradable materials, which are alone or are a combination of silk fibroin, chitosan, collagen, polylactic acid and polyglycolic acid. A chitosan-based nerve conduit with a porous surface is preferred, having a porosity of 50 to 90%, a pore size of 50 to 300 μm, high tensile strength, internal diameter of 0.5 to 8 mm and wall thickness of 0.1 to 3 mm and can be prepared with respect to the method described in the patent (application No. CN20110324474.8, entitled "Tissue Engineering Nerve Graft and its Functions"). Another nerve conduit on a manometric scale based on silk fibroin, can be prepared specifically with reference to the method described in the patent (application No. CN200910034583.9 and application No. CN101664346, entitled "Artificial Nerve Graft Prepared by Electrospinning, Its Preparation Method, and Related Devices "). The third nerve conduit of choice has a composite structure, consisting of a chitosan based conduit with a porous surface or a silk fibroin based conduit (or any nerve conduit that can be prepared with reference to the above patents) with the introduction of 120 filaments of silk fibroin as luminous charges.
    [0015] [0015] The present invention has the following advantages:
    [0016] [0016] (1) ECM is generally composed of collagen (Col), laminin (LN), fibronectin (FN), hyaluronic acid and proteoglycan (such as chondroitin sulphate, proteoglycan heparan sulphate). ECM provides mechanical support and physical resistance for the integrity of tissues, organs and the entire organism. Evidence shows that LN, FN and Col can provide adequate "adhesion" to nerve growth, cause axons to grow along a matrix bridge and guide the targeted growth of nerve fibers. One or more ECM components, such as LN and FN, have been widely adopted in the current tissue manipulation technique. Commercially available ECM, synthesized, however, is expensive and its components are relatively unique. The natural cell-derived ECM, obtained in the present invention after decellularization of cultured cells, can maintain several important components and the ECM structure and is more beneficial for the adhesion of nerve cells and has a certain guide effect in oriented axonal regeneration, promoting thus nerve regeneration. In addition, it is cheaper than synthesized ECM and easier for patients to accept.
    [0017] [0017] (2) The support cells used in manipulated tissue nerve grafts include Schwann cells and various stem cells. Most of these cells are allogeneic cells, which can cause immunogenicity during implantation. The ECM contained in the manipulated tissue nerve grafts developed in the present invention is obtained after decellularizing cultured cells, thus leading to less or no immunogenicity. The manipulated tissue nerve grafts are suitable for a large population of users.
    [0018] [0018] (3) The manipulated tissue nerve grafts developed by the present invention are prepared by a three-dimensional cell culture method in microgravity, which allows the supporting cells to be uniformly adhered to the inner and outer surfaces of lumen filaments and ECM obtained after decellularization is also uniformly distributed over the inner and outer surfaces of luminal filaments, thus facilitating nerve regeneration.
    [0019] [0019] (4) The manipulated tissue nerve grafts developed by the present invention do not contain exogenous toxic substances, which can probably be introduced during the preparation process and also have excellent biocompatibility and biodegradability, as well as good mechanical property. The nerve conduit wall exhibits a microporous 3D structure, which facilitates the transport of nutrients required by nerve development. The silk fibroin filaments included in the conduit light are prepared by electrostatic wiring, having a wide specific surface area to serve as a space required for the development of nerve cells.
    [0020] [0020] The details of the present invention are shown below by the use of the Figures and working examples. PICTURE'S DESCRIPTION
    [0021] [0021] Fig. 1 - (A) Light photomicrograph of Schwann cells. (B) Microphotography of the immunohistochemistry of Schwann cells, which are immunopositive for S100 (similar to a gray spindle) with the nuclei marked by Hoechst33342 (gray dot).
    [0022] [0022] Fig. 2 - (A) Light microphotography of fibroblasts (100x). (B) Microphotography of the immunohistochemistry of fibroblasts, which are immunopositive for their marker (gray) with the nuclei marked by Hoechst33342 (white).
    [0023] [0023] Fig. 3 - (A) Light microphotography of cutaneous stem cells (100x). (B) Microphotography of immunohistochemistry of cutaneous stem cell spheres (100x), which are immunopositive for Versican (a) and Nestin (b) with nuclei marked by Hoechst33342 (c) and (d) is the fusion of (a ), (b) and (c). (C) Microphotography of the immunohistochemistry of cutaneous stem cell spheres (100x), which are immunopositive for Vimentin (a) and Nestin (b) with nuclei marked by Hoechst33342 (c) and (d) is the fusion of (a ), (b) and (c).
    [0024] [0024] Fig. 4 - is the induced differentiation of stem cells into Schwann cells. Light microphotography (100x) (A) and local magnification (B) showing Schwann cell colonies that are formed by induced differentiation of cutaneous stem cells.
    [0025] [0025] Fig. 5 - Microphotography of the immunohistochemistry of Schwann cells formed by the induced differentiation of cutaneous stem cells (100x). Differentiated Schwann cells are immunopositive for S100 (A) with nuclei marked by Hoechst33342 (B). (C) is the fusion of (A) and (B).
    [0026] [0026] Fig. 6 - Flow cytometry of bone marrow mesenchymal stem cells and immunohistochemistry with CD molecules.
    [0027] [0027] Fig. 7 - Micrograph of ECM immunohistochemistry. (A) ECM light micrograph on the material surface after decellularization. (B) ECM is immunopositive for anti-FN. (C) ECM is immunopositive for anti-LN. (D) is the fusion of (B) and (C).
    [0028] [0028] Fig. 8 - Effects of ECM on neuron growth. (A) Micrograph of the immunohistochemistry of neurons cultured in ECM, where the ECM group refers to neurons cultured on cultured ECM and an N-ECM group refers to neurons cultured on ECM obtained after the decellularization of support cells. (B) cellular vitality of neurons. (C) Western blot images showing the expression of the neural cell adhesion molecule (NCAM) and the protein associated with axon growth (GAP43).
    [0029] [0029] Fig. 9 - Scanning electron micrograph (SEM) of a tissue nerve graft manipulated with modified ECM. (A) SEM of the inner surface of a nerve conduit containing support cells. The support cells are evenly distributed over the inner surface. (B) SEM of the inner surface of a nervous conduit treated by decellularization. ECM is generated and visible. (C) SEM of fibroin filaments of the thirst within the light of the nerve conduit that contain support cells. (D) SEM of fibroin filaments from the seat within the light of the nervous conduit treated by decellularization.
    [0030] [0030] Fig. 10 - Micrograph of the immunohistochemistry of ECM on the surface of fibroin filaments within the light of a nervous conduit treated by decellularization. (A) Immunopositive for anti-FN. (B) Immunopositive for anti-FN. (C) filaments of silk fibroin. (D) is the fusion of (A), (B) and (C).
    [0031] [0031] Fig. 11 - Regrowth of axons marked with NF (graphic scale, 200 μm) for a group with material in one week (A) or 2 weeks (C) after grafting and for a group with ECM in one week ( B) or 2 weeks (D) after grafting.
    [0032] [0032] Fig. 12 - Histogram for statistical comparisons of regenerated nerve fibers between the indicated groups.
    [0033] [0033] Fig. 13 - SEM micrograph of the cross section of a regenerated nerve in the middle portion (graphic scale, 5 μm) for a group with material (A) and a group with ECM (B). SCHEME DETAILS
    [0034] [0034] Unless otherwise defined, all expressions used in the present invention have the same meaning as commonly used by researchers in the field of tissue manipulation.
    [0035] [0035] The present invention will be described further with reference to working examples. The descriptions of these working examples are used only to illustrate the present invention, but are not intended to limit the scope of the present invention in any way.
    [0036] [0036] In the following examples, various processes and methods that are not described in detail are conventional methods known in the field of tissue manipulation. Working example 1: Culture and purification of Schwann cells
    [0037] [0037] Newborn SD rats (1 day old) are killed and disinfected with alcohol. The sciatic nerves on both sides are removed and placed on an ice-bath plate to remove epineuros and adhesions. After collagenase / pancreatin digestion, centrifugation is performed and the supernatant is discarded. Then, the cells are resuspended in complete medium and seeded on a PDL-coated culture disc for 24 h culture. Subsequently, a complete medium containing cytarabine (10 μΜ) is replaced for one culture for 48 h and then a complete medium containing HRG (50 ng / ml) and Forsklin (2 μΜ) is replaced for continuous culture with the medium that is changed every 3 days until cell fusion, followed by pancreatin digestion and centrifugation to obtain cell pellets, which are resuspended using 1 ml of complete medium containing Thyl.1 (1: 1000) and to allow incubation on ice for 2 h. Centrifugation is performed and the supernatant is discarded. The cells are resuspended using a mixture of DMEM and complement (3: 1), incubated for 1 h at 37 ° C, cleaned twice using the complete medium after centrifugation and re-inoculated on the culture disc. The medium is changed every other day and the cells can be used after growth to coat the container (cell culture and identification are shown in Fig. 1). Working example 2: Culture of fibroblasts derived from the skin
    [0038] [0038] Newborn SD rats (1 day old) are killed and disinfected with alcohol. The dorsal skins are removed and placed in a pre-cooled dissection solution to remove the subcutaneous tissue (fat, hypodermic fascia layer and blood vessels). After washing three times with PBS, the skins are cut into pieces (<1mm x 1mm), using a surgical blade and completely digested using type I collagenase (1 mg / ml). The supernatant is discarded. The cells are resuspended in complete medium and seeded on a culture disc. After 90% of the cells are fused, the cells are subcultured. Contamination by the epithelium can occur during the primary culture process and most cells of the parenchyma (epithelial and endothelial cells) will gradually die after several subcultures. Therefore, the cells used in the present invention are cells that have been subcultured for more than three generations (cell culture and identification are as shown in Fig.2). Working example 3: Cutaneous stem cell culture and differentiation directed to Schwann cells.
    [0039] [0039] This is conducted as previously described (Jeffrey A Biernaskie, Ian A McKenzie, Jean G Toma & Freda D Miller, Skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny NATURE PROTOCOLS, 2006 (1): 2803-2812). In summary, newborn SD rats (1 day old) are killed and disinfected with alcohol. The dorsal skins are removed and placed in a pre-cooled dissection solution to remove the subcutaneous tissue (fat, hypodermic fascia layer and blood vessels). After washing three times with PBS, the skins are cut into pieces (<1mm x 1mm), using a surgical blade and completely digested using pancreatin 0.1% or collagenase type XI (1 mg / ml) for 45 to 60 min at 37 ° C and then digestion is completed using a complete medium. Centrifugation is carried out and the supernatant discarded. The suspension culture is performed in DMEMJ / F12 (3: 1) containing a secondary antibody 0.1%, fungizone 40 μg / ml, FGF2 40 ng / ml and supplement B27 2%. The resulting cell spheres are subcultured to obtain a sufficient amount of cutaneous stem cells. Cell culture and identification are shown in Fig. 3).
    [0040] [0040] Cutaneous stem cells are differentiated into Schwann cells through the following steps. The cutaneous stem cells are grown in a differentiation medium I (DMEM / F12 (3: 1) containing a secondary antibody 0.1%, fungizone 40 μg / ml, FGF2 40 ng / ml, supplement B27 2% and FBS 10 %) for 3 days and then grown in a differentiation medium II (DMEM / F 12 (3: 1) containing a 0.1% secondary antibody, forskolin 5 μm, heregulin-1β 50 ng / ml, supplement N2 2% and FBS 1%) for two or three weeks, thus forming Schwann cell colonies (Fig. 4.A). Schwann cells (SKP-SCs) differentiated from cutaneous stem cells proliferate after colony amplification (Fig. 4.B). The results of immunohistochemistry show that the cells are immunopositive for S100 (Fig. 5). Working example 4: Culture of bone marrow mesenchymal stem cells
    [0041] [0041] Adult SD rats are killed by disarticulation, immersed for 5 min in 75% alcohol. The femur and fetal bone are removed under aseptic conditions and the medullary cavities are exposed and washed with basal IMDM culture solution to collect the bone marrow. The bone marrow obtained is repeatedly pumped with an injector and prepared in a single cell suspension, which is filtered through a 200 mesh sieve, placed in a horizontal centrifuge for centrifugation (1000 rpm x 5 min.). Then the supernatant is discarded. The cells are seeded at a density of 4 x 105 / cm2 in a complete IMDM culture solution (containing 10% fetal bovine serum) for culture. The entire solution is changed after 24 h and the cells not adhered to the wall are removed. Then, half the dose of the solution is changed every 3 days. The cell modality and the growth situation are observed daily under an inverted microscope. When the cells cover the bottom of the culture disc and are 90% fused, the cells are subcultured (cell culture and identification are shown in Fig. 6). Working example 5: Decellularization of support cells.
    [0042] [0042] Support cells (for example, Schwann cells) are grown on a culture disc. When the cells cover the bottom of the culture disk and are fused at 90%, the cells are cultured for two weeks in a differentiation medium (H-DMEM + FBS 15% + HRG 50 ng / ml + forskolin 2 μm + Vc 50 μg / ml) to stimulate ECM secretion. After washing with PBS, the cells are decellularized and subjected to hypotonic treatment for 10 min in sterile deionized water at 37 ° C. Cell extracts (PBS containing 0.5% TritonX-100 and 20 mM aqueous ammonia) are added to break the cells for 10 to 15 min at 37 ° C and Dnase I (4 mg / ml) is added for digestion for 30 min at 37 ° C to remove the DNA. The ECM obtained is as shown in Fig. 7. The partial ECM components are identified by LN and FN immunohistochemistry. ECM is fixed for 30 min at room temperature with 4% paraformaldehyde and to allow the reaction with primary antibodies, that is, anti-mouse laminin (LN) and rabbit anti-fibronectin (FN), respectively, for one night at 4 ° C, followed by incubation with secondary antibodies, i.e., goat anti-rabbit IgG antibody conjugated with FTIC (1: 200) and goat anti-mouse IgG antibody conjugated with TRITC (1: 200), respectively, for 2 h at room temperature and detection under confocal fluorescent microscopy (DMR, Leica) (Figs. 7 BD). Working example 6: Natural ECM is beneficial for axon growth
    [0043] [0043] Ganglion neurons of the dorsal root are seeded on a culture disc coated with ECM derived from acellular support cells, as obtained in Working Example 5 or with commercially available ECM (SIGMA, product Mo. E0282) for a culture of 48 h, the cell vitality of neurons is detected by the MTT method, while axon growth is observed by immunohistochemistry and the expression of GAP43 and CAM is detected by Western Blotting. The results are shown in Fig. 8. There is no statistical difference in the cell viability of neurons over the two types of ECM. The immunohistochemistry results, however, show that the neuron axon is obviously longer grown on acellular ECM derived from support cells than on acquired ECM and Western Blotting shows that the expression of GAP43 or NCAM in neurons grown on the acellular ECM derived from support cells is greater than that of neurons grown on the acquired ECM, respectively (Fig. 8). Work example 7: Preparation of manipulated tissue nerve grafts containing support cells.
    [0044] [0044] Chitosan nerve conduits and silk fibroin filaments are treated aseptically, 120 pieces of silk fibroin monofilaments are inserted into the light of a chitosan nerve conduit as luminous charges to give a composite to the nervous conduit. 100 ml of a complete medium (DMEM + 2% FBS + 50 ng / ml HRG + 2 μM forskolin) are slowly placed in a culture vessel using a peristaltic pump and then 2.5 x 107 cells and a sterile nerve conduit are added, followed by processing with a peristaltic pump to ensure the final cell density of 1 x 105 / ml. After the air is exhausted from the culture vessel, a microgravity circulation perfusion culture is started with the bioreactor placed in an incubator at 37 ° C, CO2 to ensure full contact and adhesion of the cells over the nervous conduit by adjusting of the rotation speed of 10 rpm in the first 24 h to make the nervous conduit suspended in a culture solution by adjusting the rotation speed after 24 h. After cultivating for 2 days, the medium is replaced by a differentiation medium (H-DMEM + FBS 15% + HRG 50 ng / ml + forskolin 2 μΜ + Vc 50 μg / ml) every 3 days to promote the secretion of ECM and then allowed to grow for another two weeks before the nerve conduit lined by the support cells is removed, resulting in the manipulated tissue nerve graft.
    [0045] [0045] SEM shows that the support cells are evenly distributed on both surfaces of the nervous conduit and silk fibroin filaments (Figs. 9A and 9C). To perform SEM observation, the manipulated tissue nerve graft is fixed with 4% glutaric acid, washed three times with PBS, fixed with 1% osmic acid at room temperature for 2 h, washed twice with PBS, dehydrated in graduated concentrations (30, 50, 70, 80, 95 and 100%) of ethanol for 10 min, respectively, and then incubated by changing the medium with a mixture of ethyl alcohol and tertiary butyl alcohol (1: 1) and pure tertiary butyl alcohol for 10 min at a time. After lyophilization and a platinum spray, the sample is observed with scanning electron microscopy (TCS SP2, Leica). Working example 8: Construction of tissue nerve grafts manipulated with modified ECM derived from support cells
    [0046] [0046] The manipulated tissue nerve grafts containing support cells, as prepared in Working Example 7, are subjected to decellularization. They are washed with PBS, placed in sterile deionized water at 37 ° C for hypotonic treatment, followed by lysis of the cell in a non-ionic detergent (consisting of 0.5% TritonX-100 and 20 mM aqueous ammonia) for 10 to 15 min. at 37 ° C and in a non-ionic detergent plus Dnase I (4 mg / ml) for digestion for 30 in at 37 ° C to remove DNA.
    [0047] [0047] SEM shows that ECM components derived from support cells are uniformly distributed over both surfaces of the nervous conduit and silk fibroin filaments (Figs. 9B and 9D). The prepared modified ECM manipulated tissue nerve graft is frozen at -80 ° C prior to use. The reserved ECM components after decellularization are observed, respectively, under SEM and detected by immunohistochemistry, where SEM detection is the same as that of example 7 and an immunohistochemical method is the same as that of example 5. results are shown in Fig. 10. Working example 9: Repair of sciatic nerve defects in rats with tissue nerve grafts manipulated with modified cell-derived ECM.
    [0048] [0048] Rat sciatic nerve defects are repaired with tissue nerve grafts manipulated with modified cell-derived ECM and function restoration is detected using a number of methods, such as immunohistochemistry, electron transmission microscopy (TEM), electrophysiology, morphological evaluation of target muscles.
    [0049] [0049] The rat sciatic nerve injury model with a 10 mm defect is established. The animals are randomly divided into two groups: one group is using nerve grafts from tissue manipulated with modified cell-derived ECM to repair sciatic nerve defects in rats, called the ECM group, and the other group is using nerve grafts from common manipulated tissue (without ECM modification) for the repair of sciatic nerve defects in rats, called the material group. One or two weeks after surgery, the animals are perfused transcardiacally and the regenerated nerve segments are then collected and cut into sections. Immunohistochemistry with NF shows that nerve growth in the ECM group is faster than the material group.
    [0050] [0050] NF-positive nerve fibers in the ECM group are relatively dense and more evenly distributed (Fig. 11A-D) and statistical comparisons are shown in Fig. 12. The sciatic nerves of all animals are exposed under appropriate anesthesia three months after surgery and electrophysiological detection is performed. The amplitude of the average compound muscle action potential (CMAP) is 4.63 ± 0.13 mN and 6.89 ± 2.85 mV in the material group and in the ECM group, respectively, and there is a significant difference (P <0 , 05) between the two groups. TEM shows that three months after surgery, some of the regenerated myelinated nerve fibers are dispersed in the middle portion with less demyelinated nerve fibers in both groups (Figs. 13A and B). The thicknesses of the myelin sheaths of the nerves are 0.63 ± 0.27 μm and 0.82 ± 0.39 μm in the material group and in the ECM group, respectively, and there is a significant difference (P <0.05 ) between the two groups.
    权利要求:
    Claims (4)
    [0001]
    Manipulated tissue nerve graft to repair peripheral nerve defect, characterized by the fact that it consists of a nervous conduit and an ECM and that it has the characteristics that the ECM is secreted by autologous or allogeneic cells and obtained through decellularization, where it involves the use of support cells, which are alone or in combination with skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells or induced pluripotent stem cells, and wherein the nervous conduit is made of biodegradable materials, which are alone or as a combination of biodegradable materials which are one or more of silk fibroin, chitosan, collagen, polylactic acid or polyglycolic acid.
    [0002]
    Method of preparing a manipulated tissue nerve graft, as defined in claim 1, characterized by the fact that the manipulated tissue nerve graft is prepared by decellularizing the nerve graft containing support cells to generate ECM secreted by the cells of support and then the nerve graft with modified ECM, where support cells are alone or in combination with skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells or induced pluripotent stem cells, and wherein the nervous conduit is made of biodegradable materials, which are alone or as a combination of biodegradable materials which are one or more of silk fibroin, chitosan, collagen, polylactic acid or polyglycolic acid.
    [0003]
    Method for preparing the manipulated tissue nerve graft according to claim 2, characterized by the fact that the nerve graft containing support cells is formed through a three-dimensional rotational culture in microgravity.
    [0004]
    Method for preparing the manipulated tissue nerve graft according to claim 3, characterized in that the preparation of the nerve graft containing support cells and the construction of the decellularized manipulated tissue nerve graft: (1) Preparation of manipulated tissue nerve grafts that contain support cells; the complete medium is slowly placed in a culture flask and then 2.5 x 107 cells and a sterile nerve conduit is added, followed by processing with a peristaltic pump to ensure the final cell density of 1 x 105 / ml; then, the air is evacuated from the culture flask, a perfusion circulation culture in rotational microgravity is started with a rotary bioreactor placed in an incubator with CO2 at 37 ° C to ensure full contact and adhesion of the cells over the nervous conduit suspended in a culture solution by adjusting the rotation speed to 10 rpm in the first 24 h and then to make the nerve conduit suspended in a culture solution by adjusting the rotation speed after 24 h; after cultivating for 2 days, the medium is switched to a differentiation medium to promote ECM secretion and left to cultivate for another two weeks before the nerve conduit coated with the supporting cells is removed; (2) Construction of decellularized manipulated tissue nerve grafts; the manipulated tissue nerve graft that contains a support cell is subjected to decellularization; it is washed with phosphate-buffered saline (PBS), placed in sterile deionized water at 37 ° C for hypotonic treatment for 10 minutes, followed by lysis of the cell in a non-ionic detergent for 10 to 15 min at 37 ° C and then added in a non-ionic detergent with the addition of DNase I (4 mg / ml) for digestion for 30 min at 37 ° C to remove DNA; the acellular product is kept at -80 ° C for use.
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    同族专利:
    公开号 | 公开日
    BR112015017174A2|2017-07-11|
    WO2014114043A1|2014-07-31|
    EP2949349B1|2018-10-31|
    EA201591373A1|2016-01-29|
    EP2949349A4|2016-11-02|
    US20150352255A1|2015-12-10|
    AU2013375655B2|2016-11-17|
    US9492589B2|2016-11-15|
    EA038957B1|2021-11-15|
    AU2013375655A1|2015-07-23|
    CN103041450B|2015-06-10|
    CN103041450A|2013-04-17|
    EP2949349A1|2015-12-02|
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    法律状态:
    2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |
    2019-05-21| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2019-09-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-03-17| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-06-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-09-24| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/05/2013, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    CN201310028903.6A|CN103041450B|2013-01-25|2013-01-25|Cell matrix modified tissue engineering nerve graft for repairing peripheral nerve injury and preparation method thereof|
    CN201310028903.6|2013-01-25|
    PCT/CN2013/076239|WO2014114043A1|2013-01-25|2013-05-27|Cell matrix modified tissue engineering nerve graft for repairing peripheral nerve injury and preparation method thereof|
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