implants

Abstract

The present invention relates to an implant (10) comprising a support structure (12) manufactured from the form of a porous material for receiving and supporting stem cells (14) in a supported state, wherein the implant is for receiving a culture medium in the form of a mixture of basal media and neuroinduction media (20), and the shape and size of the support structure (12) are determined to fit into and complementarily receive an implant zone (16). The implant zone (16) is a space defined by the resected portion of the target spinal cord (18). The mixture of basal media and neuroinduction media (20) provides nutrients for the development of stem cells (14), and growth factors in the form of neurotrophin 3, neurotrophin 4 and brain-derived neurotrophic factor (BDNF) promote and regulate the development of stem cells (14), these growth factors are typically components of the culture medium mixture (20).

Description

Technical Field

[0001] The present invention relates to implants. In particular, the present invention relates to implants for use in regenerative medical procedures.

Background Art

[0002] Tissue damage in mammals can cause a temporary, impaired or permanent loss of tissue structure and / or function. The natural repair of damaged tissue involves two processes, namely regeneration and / or replacement, depending on the type of tissue. Regeneration relates to new growth, whereby at least the tissue structure and function in part of the damaged or destroyed tissue are restored. Replacement relates to the construction of connective tissue in the damaged area, which results in scarring and the loss of tissue structure and function. Examples of tissues that can be regenerated are hematopoietic tissue, epithelium, liver, bone and muscle. Tissues such as myocardium, pancreas and spinal cord respond to injury by forming scars.

[0003] Despite muscle and bone being listed as regenerative tissues, regeneration can be hindered by the presence of large gaps resulting from severe damage. Also, tissue degeneration due to aging or disease can prevent regeneration, regardless of the type of injury or tissue.

[0004] The nervous system is composed of the central nervous system and the peripheral nervous system. Damage to the central nervous system often occurs instantaneously and typically does not regenerate. However, due to neuroplasticity, the nervous system can reorganize to recover at least partial function. Surgical intervention can reduce or prevent secondary damage by relieving pressure on the spinal cord or brain. However, the damage caused by primary injury can result in long-term effects such as partial or complete paralysis.

[0005] Damage to the peripheral nervous system can occur instantaneously or over time. The potential for regeneration in the peripheral nervous system is greater and can occur over a considerable period. Surgical options for peripheral nerve damage include, but are not limited to, scar tissue removal, direct nerve repair, and grafting.

[0006] Regenerative medicine is a medical field that focuses on the replacement, engineering, or regeneration of cells, tissues, or organs. Stem cell therapy is a key aspect of regenerative medicine, offering a promising option for enhancing endogenous repair through the engineering or regeneration of new tissues. Stem cell therapy typically involves harvesting stem cells from the subject's own body (autologous) or a donor (allogeneic). After harvesting, the stem cells are processed and prepared for injection or transplantation into the subject's body at the site of injury or disease. As with any emerging technology, there are many challenges to overcome in order to achieve effective and efficient stem cell therapy.

[0007] These challenges include the limited ability of stem cells to survive and effectively differentiate after transplantation. This can be due to a lack of endogenous stimuli to induce effective differentiation and / or a lack of nutrient supply for development and maturation. Furthermore, difficulties in handling or handling stem cells can lead to cell death during transplantation. In addition, without adequate support and guidance, stem cells may be unable to integrate into surrounding tissues or differentiate into the desired cell type. This is particularly true in areas of the body where fluid flow can displace the implanted stem cells.

[0008] In light of these challenges, there is a clear need for technologies that enable easy handling of stem cells during transplantation, support for stem cells during and after transplantation, adequate nutrient supply for development and maturation, and stimulation and support for differentiation or continued differentiation of stem cells.

[0009] The present invention addresses at least some of the above-mentioned needs. [Overview of the project]

[0010] According to a first aspect of the present invention, To receive and support stem cells in a supported state, To accept a culture medium containing nutrients, and We provide implants equipped with a support structure for receiving growth factors to promote and control stem cell development. Here, the support structure can be sized and shaped to fit the implant zone in the target and thereby be complementarily accepted.

[0011] For the purposes of this specification, biodegradability should be understood as the ability of a substance to be broken down and absorbed by the body. Bioabsorption should be understood as the ability to be absorbed by living tissues.

[0012] The support structure may be manufactured or synthesized using biological materials, biochemical materials, biocompatible materials, hemostatic materials, degradable and absorbable materials, and / or bioabsorbable materials. The materials may be porous and / or fibrous. The support structure may have an internal lattice structure. The support structure may be constructed by 3D printing or additive manufacturing. The support structure may be manufactured or synthesized from hygroscopic and / or absorbent materials. The support structure may define a receptive zone, preferably in the form of gaps or pores, within which stem cells can be received and supported in a supported state. The receptive zone may have a size in the range of 150 μm to 1000 μm in width. During hydration and / or absorption, the receptive zone may have a size in the range of 150 μm to 500 μm in width. Reducing the size of the receptive zone during hydration and / or absorption can facilitate the support of stem cells in a supported state. The support structure may have pores defined to allow insertion of stem cells into its inner region. It should be understood that the pores help to facilitate the filling, saturation, and / or distribution of stem cells substantially throughout the support structure. The pores may extend substantially coaxially with the support structure. The pores may be formed by using a thin, elongated member such as a needle. Multiple pores may be defined in the support structure to further facilitate the filling, saturation, and / or distribution of stem cells throughout the support structure. Each of the multiple pores may extend over various depths within the support structure, preferably over two-thirds of the length of the support structure. The support structure may have a substantially cylindrical shape. The support structure may have a length in the range of 1 to 4 cm, preferably 2 cm. The support structure may have a diameter in the range of 0.8 to 1.2 cm, preferably 1 cm.

[0013] The support structure may be synthesized or manufactured from a natural polymer. The natural polymer may be one or more forms of any of the group including proteoglycans, gelatinous substrates, graphene, oxidized cellulose, dextrose, hyaluronic acid, starch, graphene oxide, fibrin, alginate substrates, chitosan, collagen, and any combination thereof. The natural polymer may be in the form of collagen, preferably in the form of degradable collagen. In particular, degradable collagen may include purified cross-linked type I collagen. The collagen may be derived from bovine Achilles tendon. The collagen can be degraded and absorbed within 20 to 40 days after implantation, preferably within 30 days. The support structure may be in the form of an RCP Resorbable Collagen Plug (RCP Resorbable Collagen PLUG®).

[0014] The support structure may be synthesized or manufactured from a synthetic polymer. The synthetic polymer may be one or more forms from the group including polystyrene, poly-L-lactic acid, polyester, polyethylene glycol, polycyanoacrylate, polyurethane, polystat, polyglycolic acid, and poly-dl-lactic acid-co-glycolic acid.

[0015] The support structure may be synthesized or manufactured from a ceramic material. The ceramic material may be any one or more forms from the group including hydroxyapatite and tricalcium phosphate.

[0016] The support structure may be synthesized or manufactured from one or more combinations of natural polymers, synthetic polymers, and / or ceramic materials.

[0017] The support structure may be sized and shaped by molding and / or cutting processes. Multiple support structures may be provided to facilitate their adaptation to implant zones of various sizes and / or shapes. The multiple support structures may be arranged in contact with each other to form a substantially continuous structure, which allows the implant to fit into the implant zone and be complementarily received by the implant zone.

[0018] The implant zone may be the site of damage, destruction, or deterioration of tissue within the subject's body. The implant zone may be the space defined by any one of the following: damaged, deteriorated, severed, torn, or excised tissue within the subject's body. More specifically, the implant zone may be the space defined by the excision of a portion of the subject's spinal cord. Alternatively, the implant zone may be the space defined by severed or excised portions of the subject's peripheral nerves. The subject may be a mammal.

[0019] For the purposes of this specification, stem cell development should be understood to include one or more of the following: differentiation, continued differentiation, proliferation, migration, and multicellular morphogenesis. Furthermore, determined stem cells should be understood as stem cells determined to form a particular tissue type or cell type, and partially differentiated stem cells should be understood as stem cells that exhibit morphologically identifiable features in the early stages of a particular tissue type or cell type, or cells that express molecular markers indicating that they will become a particular tissue type or cell type.

[0020] Stem cells can be any one form of the group, including totipotent stem cells, pluripotent stem cells, compound pluripotent stem cells, and unipotent stem cells. Preferably, stem cells may be compound pluripotent stem cells. Stem cells may be at any stage of differentiation toward the specific cell type of interest. The differentiation stage may be one or more of the group, including undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells. A mixture of stem cells at different differentiation stages may be provided. The mixture may contain one or more of undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells. Preferably, the mixture may contain partially differentiated stem cells and fully differentiated stem cells. It should be understood that including fully differentiated stem cells in the mixture can ensure that the specific cell type or tissue type of interest is present in sufficient quantities within the implant.

[0021] Partially differentiated stem cells may exhibit early morphologically distinct features of nerve cells. This feature may be the formation of axonal crests. Partially differentiated stem cells may express one or more neuronal-specific molecular markers. These molecular markers may be one or more of the following: NeuN, Tuji, synapsin, PAX6, SOX1, SOX2, bActin, nestin, MAP2, and / or GFAP.

[0022] Fully differentiated stem cells may take the form of oligodendrocytes or Schwann cells. Oligodendrocytes may take the form of oligodendrocyte progenitor cells (OPCs) and / or premyelinate oligodendrocytes. It should be understood that fully differentiated oligodendrocytes or Schwann cells may promote myelination of one or more other stem cells, either during development or / or when fully developed. Furthermore, it should be understood that fully differentiated oligodendrocytes may promote myelination or remyelination of endogenous neurons in the target central nervous system after transplantation, while fully differentiated Schwann cells may promote myelination or remyelination of endogenous neurons in the target peripheral nervous system after transplantation. Endogenous neurons may be damaged or injured neurons within the implant zone. Oligodendrocytes, or more specifically, OPCs, may express one or more molecular markers from the group including SOX10, NKX2.2, OLIG1, and OLIG2. Oligodendrocytes, or more specifically, premyelinate oligodendrocytes, may express one or more molecular markers from the group including OLIG1, OLIG2, SOX10, NKX2.2, ZFP191, ZFP488, ZFP536, SOX17, and NKX6.2.

[0023] Stem cells are made from 1 cm of material. 3 It may have a concentration in the range of 150,000 to 1,500,000 per unit. Excess stem cells may be provided to differentiate stem cells into the target cell type or any other cell type that may be formed as a result of intercellular signaling or endogenous signaling after transplantation. In excess, stem cells are added to 1 cm³ of material. 3 The concentration can range from 2,000,000 to 3,000,000 per unit. Fully differentiated stem cells can constitute 2-4% of the total number of stem cells, preferably in the 3% range. Stem cells can be obtained during any passage, preferably any of passages 3-9. More specifically, fully differentiated stem cells can be obtained during passages 5 and / or 6. Stem cells can be at any stage of their cell cycle.

[0024] Stem cells may be in one or more forms from the group including embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs), dental pulp stem cells (DPSCs), olfactory nerve sheath cells (OECs), cancer stem cells (CSCs), and / or human fetal brain neural stem cells (hNSCs). MSCs may include bone marrow mesenchymal stem cells (BM-MSCs), human umbilical cord mesenchymal stem cells (HUC-MSCs), and adipose-derived mesenchymal stem cells (AD-MSCs). NSCs may include neural progenitor cells, neutral crest cells, and / or Schwann cell precursors. Preferably, stem cells may be in the form of MSCs, and more preferably in the form of AD-MSCs. MSCs or AD-MSCs may be identifiable by the expression of positive and / or negative molecular markers. MSCs or AD-MSCs may be identifiable in any of the passages discussed above. Positive molecular markers may be one or more from the group including CD73, CD90, and CD105. Negative molecular markers may be one or more from the group including CD34, CD116, CD19, and CD45. Stem cells may be derived from the subject to promote stem cell compatibility by the subject and / or inhibit rejection. Therefore, stem cells may be autologous, or they may be allogeneic.

[0025] The medium can be in the form of a growth medium or a culture medium. The medium can be a solidifiable medium. In its non-solidified form, the medium can have a viscosity that allows it to penetrate at least a portion of the support structure. It should be understood that when the medium is introduced into the support structure, typically in its non-solidified form, the medium has a viscosity that facilitates absorption by the support structure, preferably via capillary action, due to its porous nature. Further, the medium can be introduced into the support structure in an amount sufficient to allow the medium to surround the support structure. The medium can be protein-free. The medium can be in any one or more forms of a group including a neurobasal medium, a neural induction medium, a saline medium, a phosphate-buffered saline medium, a wash medium, a holding medium, a stem cell-based medium, a Roswell Park Memorial Institute medium, a Dulbecco's Modified Eagle Medium, a Dulbecco's Modified Eagle Medium / F12, a HAM / F12 medium, and a HAM / F10 medium. The medium can contain additives. The additives can be in any one or more forms of a group including essential amino acids, vitamins, and minerals. A second medium or subsequent media may be provided, and each of the media is arranged in layers within or around the support structure. Also, each medium may differ in terms of its form and / or the additives contained therein.

[0026] Growth factors, preferably multiple thereof, when in contact with stem cells, may promote and regulate the development of stem cells in the implant and / or any other endogenous stem cells in the implant zone. Growth factors may have reduced or no effect on fully differentiated stem cells in the implant, more specifically, fully differentiated oligodendrocytes or Schwann cells. Endogenous stem cells may be in the form of neural progenitor cells. When in contact with stem cells and / or endogenous stem cells, growth factors may promote and regulate the differentiation of one or more cells from the group including neurons, astrocytes, oligodendrocytes, interneurons, microglia, and Schwann cells. Growth factors may be components of the culture medium. Growth factors may be in the form of one or more of the group including enzymes having kinase or phosphatase activity, enzymes having tyrosine kinase activity, cytokines, lipid-soluble steroid hormones, nitric oxide, oligodendrocyte growth factor, and reactive oxygen species (ROS). The growth factors may be in the form of brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin 4, and / or glial cell line-derived neurotrophic factor (GDNF). Brain-derived neurotrophic factor may have a concentration in the range of 0.2 to 0.8 μl per 50 ml of medium, preferably 0.5 μl per 50 ml of medium. Neurotrophin 3 or 4 may have a concentration in the range of 5 to 15 μl per 50 ml of medium, preferably 10 μl per 50 ml of medium, and GDNF may have a concentration in the range of 5 to 15 μg / ml, preferably 10 μg / ml. The second medium or subsequent medium may contain different growth factors to differently promote and control the development of stem cells and / or endogenous stem cells.

[0027] A cover may be provided for covering at least a part of the support structure that has received stem cells, a medium, and a growth factor. The cover can facilitate the delay of absorption of the support structure after transplantation. The cover can prevent the shedding of any one or more of the stem cells, the medium, and the growth factor after transplantation, where the shedding can be caused by the flow of the subject's body fluid. The cover can facilitate the induction of the subject's body fluid away from the support structure. The body fluid can be in the form of the subject's cerebrospinal fluid or blood. The cover can be synthesized or manufactured from a porous and / or fibrous material to allow at least some absorption of the subject's body fluid and then allow the exudation of the body fluid towards the support structure. It should be understood that the exudation of the body fluid can supply additional nutrients to the stem cells within the implant and can result in the migration of endogenous stem cells within the area of the implant. The cover can be synthesized or manufactured from the aforementioned types of materials. The cover can be in the form of a sheet, and more specifically, it can be in the form of a rectangular sheet. The sheet can be sized to have a length in the range of 6 to 10 mm, preferably a length of 7.5 mm. The sheet can be sized to have a thickness in the range of 1 to 2 mm, preferably a thickness of 1 mm. The sheet can be sized to have a width in the range of 2 to 4 mm, preferably a width of 2.5 mm. The cover can be in the form of RCT Resorbable Collagen Tape (registered trademark).

[0028] Alternatively, the cover can be in the form of a coating medium that coats at least a part of the support structure that has received stem cells, a medium, and a growth factor. The coating medium can be a medium that can be solidified. The solidified form of the coating medium can be more rigid than the medium received by the support structure. The coating medium can be permeable to allow the subject's body fluid to permeate towards the support structure.

[0029] According to a second aspect of the present invention, a support structure, a receiving zone defined within the support structure for receiving stem cells in a supported state, An implant is provided which includes a culture medium contained in at least a portion of a support structure that has received supported stem cells.

[0030] The supporting structure may receive one or more growth factors to control stem cell development.

[0031] The support structure can be sized and shaped to fit the implant zone in the target area and to be complementarily received by the implant zone.

[0032] The supporting structure, receptive zone, stem cells, culture medium, growth factors, and implantation zone may be of the types described above.

[0033] A cover may be provided to cover at least a portion of the support structure receiving the stem cells, culture medium, and growth factors. The cover may be of the type described above.

[0034] The target could be a mammal.

[0035] According to a third aspect of the present invention, An implant is provided having a support structure for supporting partially differentiated stem cells in a supported state.

[0036] The supporting structure can receive a culture medium containing nutrients and one or more growth factors to promote and control stem cell development.

[0037] The support structure can be sized and shaped to fit the implant zone in the target area and to be complementarily received by the implant zone.

[0038] The supporting structure, stem cells, culture medium, growth factors, and implant zone may be of the types described above.

[0039] A cover may be provided to cover at least a portion of the support structure receiving the stem cells, culture medium, and growth factors. The cover may be of the type described above.

[0040] The target could be a mammal.

[0041] According to a fourth aspect of the present invention, the aforementioned implant for use in regenerative medicine procedures is provided.

[0042] Regenerative medicine treatments may include one or more of the following: spinal cord regeneration, nerve regeneration, cartilage regeneration, bone regeneration, soft tissue regeneration, regenerative treatment for Alzheimer's disease, regenerative treatment for dementia, regenerative treatment for diabetes, treatment for macular degeneration, treatment for glaucoma, nephron regeneration, regenerative treatment for multiple sclerosis, treatment for cancer, and any autoimmune disease.

[0043] Regenerative medicine procedures may be suitable for mammals.

[0044] According to a fifth aspect of the present invention, a method for regenerative treatment of injury or disease, wherein the method is This includes transplanting the implant described above into the implant zone defined in the subject.

[0045] The implant zone may be the site of damage, destruction, or deterioration of tissue within the subject's body. The implant zone may be the space defined by any one of the following: damaged, deteriorated, severed, torn, or excised tissue within the subject's body. More specifically, the implant zone may be the space defined by the excision of a portion of the subject's spinal cord. Alternatively, the implant zone may be the space defined by severed, ruptured, destroyed, or excised portions of the subject's peripheral nerves. The subject may be a mammal.

[0046] According to a sixth aspect of the present invention, a method for preparing an implant, Obtaining stem cells, This involves introducing stem cells into a supporting structure, where the stem cells are supported by the supporting structure in a supported state. This involves introducing the culture medium into the support structure, where the culture medium provides nutrients to the stem cells while the medium is supported. A method comprising introducing growth factors into a supporting structure, wherein the growth factors control and promote the development of stem cells in a state supported by the growth factors.

[0047] Stem cells can be obtained from tissue taken from a subject requiring implantation. Stem cells can be autologous or allogeneic. Stem cells can be obtained from tissue using any known technique. Stem cells can be of the types described above. To determine whether stem cells have been obtained, or to identify the type or morphology of the obtained stem cells, this method may include performing a quantitative polymerase chain reaction (qPCR) test to confirm whether the stem cells or potential stem cells express molecular marks indicating that they are stem cells or stem cells of interest. To identify the presence of MSCs, more specifically AD-MSCs, qPCR is performed to detect positive and / or negative molecular markers. Positive molecular markers may be one or more from the group including CD73, CD90, and CD105. Negative molecular markers may be one or more from the group including CD34, CD116, CD19, and CD45.

[0048] This method may involve completely differentiating stem cells into a desired cell or tissue type using any known technique. The desired cell type may be in the form of oligodendrocytes or Schwann cells. More specifically, oligodendrocytes may be in the form of oligodendrocyte progenitor cells (OPCs) and / or premyelinate oligodendrocytes. Completely differentiating stem cells into oligodendrocytes is 1) A step of plating undifferentiated stem cells onto a culture plate using culture medium, 2) The seeded stem cells are incubated at a temperature in the range of 37°C to 37.5°C for 2 to 5 days, typically 3 days. 3) A step of priming stem cells using priming medium, 4) A step of initiating stem cell differentiation using a differentiation medium, 5) A process of completely differentiating stem cells using terminal differentiation medium, 6) The process may include a step of determining whether the stem cells have differentiated or whether they have differentiated into oligodendrocytes.

[0049] In step (1) above, the culture medium may be in the form of DMEM / F12. DMEM / F12 may contain FBS and / or gentamicin. FBS and gentamicin may have concentrations ranging from 10-15% and 1-2%, respectively. Preferably, FBS and gentamicin may have concentrations of 10% and 1%, respectively.

[0050] In step (3) above, the priming medium may be any preferred form of priming medium. The priming medium may contain trypsin and ethylenediaminetetraacetic acid (EDTA) at concentrations ranging from 0.2 to 0.3% [v / v] and 0.01 to 0.03% [v / v], respectively. Preferably, the concentrations of trypsin and EDTA may be 0.25% [v / v] and 0.02% [v / v], respectively.

[0051] In step (4) above, the differentiation medium may be in any suitable medium form. The differentiation medium may contain retinoic acid and / or sonic hedgehog protein (SHH). Retinoic acid may be added to the differentiation medium from a stock solution having a concentration in the range of 18 to 22 mM, preferably 20 mM. The retinoic acid stock solution may be added to the differentiation medium in a ratio of 0.01:0.1 ml. SHH may be added to the differentiation medium from a stock solution having a concentration in the range of 23 μg / L to 27 μg / L, preferably 25 μg / L. The SHH stock solution may be added to the differentiation medium in a ratio of 1:100 ml.

[0052] In step (5) above, the terminal differentiation medium may be any suitable form of terminal differentiation medium. The terminal differentiation medium may contain neurotrophin 3 and / or platelet-derived growth factor (PDGF). PDGF may be added to the terminal differentiation medium from a stock solution having a concentration of 1 μg / mL. The PDGF stock solution may be added to the terminal differentiation medium in a ratio of 1:50 mL. PDGF may be in the form of PDGFα. Neurotrophin 3 may be added to the terminal differentiation medium from a stock solution having a concentration of 1 μg / mL. The neurotrophin 3 stock solution may be added to the terminal differentiation medium in a ratio of 1:33 mL.

[0053] In step (6) above, a qPCR test may be used to determine whether the stem cells express molecular markers characteristic of oligodendrocytes. Molecular markers may include one or more from the group including SOX10, OLIG1, OLIG2, NKX2.2, ZFP191, ZFP488, ZFP536, SOX17, and NKX6.2.

[0054] The support structure may be of the types described above. The support structure may be prepared by puncturing it to form one or more pores to allow the insertion of stem cells into the inner region of the support structure. It should be understood that the pores work to facilitate the filling, saturation, and / or distribution of stem cells throughout the support structure. The pores may extend substantially coaxially with respect to the support structure. The pores may extend at various depths within the support structure, preferably over two-thirds of the length of the support structure. The support structure may be punctured using a thin, elongated member such as a needle.

[0055] Stem cells can be introduced into a support structure using a stem cell suspension. The stem cell suspension may contain a mixture of stem cells at different stages of differentiation. The mixture may contain one or more of undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells. Preferably, the mixture may contain undifferentiated stem cells and fully differentiated stem cells. Fully differentiated stem cells may be in the form of oligodendrocytes or Schwann cells. Oligodendrocytes may be in the form of oligodendrocyte progenitor cells (OPCs) and / or premyelinate oligodendrocytes. The stem cell suspension can be introduced into the support structure via one or more pores, either dropwise or by pouring.

[0056] The culture medium may be of the type described above. The culture medium may be introduced into the support structure by immersing at least a portion of the support structure in the unsolidified culture medium. Preferably, the culture medium may be introduced into the support structure by immersing the entire support structure in the unsolidified culture medium. Alternatively, the culture medium may be introduced into the support structure in its unsolidified form, preferably through pores, dropwise or by pouring. The culture medium can then be solidified. In its solidified form, the culture medium may allow the stem cells to be held in place relative to the support structure in a supported state.

[0057] The growth factors, preferably a plurality thereof, may be of the types described above. The growth factors may be introduced into the support structure, preferably by dropping or pouring, through one or more pores. Preferably, the growth factors may be components of the culture medium.

[0058] This method may involve enabling undifferentiated stem cells, determined stem cells, and / or partially differentiated stem cells to develop in a supported state before transplantation into a subject. The stem cells may be allowed to develop to any stage of differentiation toward a specific cell type of interest. The differentiation stage may be one or more of the group including undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells, preferably partially differentiated stem cells. Partially differentiated stem cells may exhibit early morphologically distinct features of nerve cells. These features may include the formation of axonal crests. Partially differentiated stem cells may express molecular markers indicating the desired tissue or cell type.

[0059] This method may include monitoring the development of stem cells that have been prepared to develop under support before transplantation into a target. By monitoring stem cells, a desired stage of differentiation toward a specific cell type of interest can be identified. The differentiation stage may be one or more of the group including undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells. Preferably, the differentiation stage may be partially differentiated stem cells. Monitoring may include culturing a portion of the stem cells separately from the stem cells contained in the implant. Culturing the stem cells to be monitored may be initiated simultaneously with the stem cells introduced into the implant, using the culture medium and growth factors used in the implant. Furthermore, monitoring may include periodically observing the cultured stem cells using a light microscope to identify early characteristic features of the desired cell type, and / or performing a qPCR test to confirm whether the stem cells express molecular marks indicating differentiation toward the desired cell type. Early characteristic features may include axonal hilllets. Molecular markers may include one or more of the following: NeuN, Tuji, Synapsin, PAX6, SOX1, SOX2, bActin, Nestin, MAP2, and / or GFAP.

[0060] This method involves wrapping or covering at least a portion of a support structure receiving stem cells and / or culture medium and / or growth factors with a cover of the type described above.

[0061] An implant according to the present invention will be described by the following non-limiting examples with reference to the attached drawings. [Brief explanation of the drawing]

[0062] [Figure 1] This is a cross-sectional view of a first embodiment of the implant according to the present invention. [Figure 2] Figure 1 is a three-dimensional schematic diagram showing the implant. [Figure 3] This is a three-dimensional schematic diagram showing the morphological support structure of a porous material, with an insert of a scanning electron microscope image of the material. [Figure 4] This is a three-dimensional schematic diagram showing a portion of the spinal cord with a resected area. [Figure 5] This is a three-dimensional schematic diagram showing different implant placements within the implant zone. [Figure 6] This is a three-dimensional schematic diagram showing different implant placements within the implant zone. [Figure 7] This is a three-dimensional schematic diagram showing the development of stem cells within the implant during use. [Figure 9] This is a three-dimensional schematic diagram showing the development of stem cells within the implant during use. [Figure 8] This is a three-dimensional schematic diagram showing the implant of Figure 1 having a cover according to the present invention. [Figure 10] This is a schematic diagram showing partially differentiated stem cells, including axonal hilllets. [Modes for carrying out the invention]

[0063] In the first embodiment of the present invention shown in Figures 1 to 8, reference numeral 10 generally refers to an implant.

[0064] In this embodiment, the implant 10 includes a support structure 12 made of porous material for receiving a culture medium in the form of a basal and neuroinduction medium mixture 20 for receiving and supporting stem cells 14 in a supported state, as clearly shown in Figure 3, the support structure 12 is shaped and sized to fit into and thereby complementarily receive an implant zone 16, which is a space defined by the resected portion of the target spinal cord 18, as shown in the figure. The basal and neuroinduction medium mixture 20 provides nutrients for the development of stem cells 14, and growth factors in the form of neurotrophins 3, neurotrophins 4, and brain-derived neurotrophic factor (BDNF) for promoting and controlling the development of stem cells 14, these growth factors are components of the culture medium mixture 20.

[0065] The support structure 12 is a natural polymer in the form of collagen. More specifically, the support structure is in the form of degradable collagen, and even more specifically, in the form of degradable purified cross-linked type I collagen. Typically, degradable purified cross-linked type I collagen is degraded and absorbed by the subject within approximately 30 days after implantation. Degradation and absorption of collagen allows for the unhindered and continuous development of stem cells 14, while simultaneously supporting the developing stem cells during the fragile early stages of development. The collagen is derived from bovine Achilles tendon and is in the form of RCP Resorbable Collagen Plug (RCP Resorbable Collagen PLUG®). The RCP Resorbable Collagen Plug (RCP Resorbable Collagen PLUG®) has dimensions of 1 cm × 2 cm. If the length of the implant zone 16 exceeds the length of the support structure 12, multiple support structures 12 may be provided in an end-to-end configuration, as clearly shown in Figure 5. Alternatively, a portion of the second support structure 22 may be cut to a desired length and positioned end-to-end with the first support structure 12 (as clearly shown in Figure 6). The support structure 12 is defined with holes 24 to allow insertion of stem cells 14 into its inner region. It should be understood that the holes 24 serve to facilitate the filling, saturation, and distribution of stem cells 14 substantially throughout the entire support structure 12. The holes 24 are formed by using an elongated member such as a needle. Although not shown in the figure, multiple holes can be defined in the support structure 12 to further facilitate the filling, saturation, and distribution of stem cells substantially throughout the entire support structure 12. The multiple holes may be of different depths over two-thirds of the length of the support structure 12.

[0066] The support structure 12 defines the morphological receptive zone of the pores 26, which is most clearly shown in Figure 3. Stem cells 14 are recepted within the pores 26 and supported by the support structure 12. The pores 26 have a typical width of 300–600 μm. The support structure 12 is hygroscopic and adsorbent, absorbing moisture when exposed to air and / or the culture medium mixture 20. During hydration and / or absorption, the pores 26 have a typical width of 150–300 μm. The reduction in pore size that occurs during hydration and / or absorption facilitates the support of stem cells 14 in the supported state.

[0067] Stem cells 14 are morphologically distinct stem cells, specifically mesenchymal stem cells (MSCs), and more specifically, adipose-derived mesenchymal stem cells (AD-MSCs). MSCs or AD-MSCs can be identified by the expression of positive and negative molecular markers. Positive molecular markers are CD73, CD90, and CD105. Negative molecular markers are CD34, CD116, CD19, and CD45. Stem cells 14 are primarily a mixture of partially differentiated and fully differentiated stem cells 14. Partially differentiated stem cells 14 exhibit morphologically distinct features of nerve cells. This feature is the formation of axonal hilllets 50. Partially differentiated stem cells express one or more molecular markers specific to nerve cells. Molecular markers include one or more of the following: NeuN, Tuji, synapsin, PAX6, SOX1, SOX2, bActin, nestin, MAP2, and / or GFAP. Fully differentiated stem cells 14 are in the form of oligodendrocytes 34, more specifically, oligodendrocyte progenitor cells (OPCs) and premyelinated oligodendrocytes. Oligodendrocytes 34 express one or more molecular markers from the group including SOX10, OLIG1, OLIG2, NKX2.2, ZFP191, ZFP488, ZFP536, SOX17, and NKX6.2. Including fully differentiated stem cells 14 in the mixture ensures that a sufficient amount of specific cell types are present within the implant 10 and implant zone 16 after transplantation. The concentration of stem cells 14 is measured in 1 cm³ of the support structure 12.3 Each cell contains 2.5 million cells, thus supplying an excess of stem cells 14 so that they can differentiate into a cell type determined by growth factors, or any other cell type determined by intercellular or endogenous signaling after transplantation. Fully differentiated stem cells 14 constitute approximately 3% of the total number of stem cells 14. Stem cells 14 are obtained during any of passages 3 to 9. More specifically, fully differentiated stem cells 14 are obtained during passages 5 to 6.

[0068] The culture medium mixture 20 is a culture medium that can be solidified. In its unsolidified form, the culture medium mixture 20 has a viscosity that allows it to penetrate at least a portion of the support structure 12. When the unsolidified culture medium mixture 20 is introduced into the support structure 12, it should be understood that it has a viscosity that promotes absorption by the support structure 12, typically due to its porous properties resulting from capillary action. Furthermore, the culture medium mixture 20 is introduced into the support structure 12 in an amount sufficient to allow the culture medium mixture 20 to surround the support structure 12. When the culture medium mixture 20 is received by the support structure 12 and in a solidified form, it facilitates the support of the stem cells 14 in their position relative to the support structure 12 in the supported state.

[0069] When growth factors are in contact with stem cells 14, they promote and regulate the development of stem cells 14 within the implant 10, and may have little or no effect on fully differentiated stem cells 14, more specifically fully differentiated oligodendrocytes 34. When growth factors are in contact with endogenous stem cells 28 within the region of the implant zone 16, they promote and regulate the development of endogenous stem cells 28. Endogenous stem cells 28 are typically in the form of neural progenitor cells released from the central canal during spinal cord resection 18. The development of stem cells 14 and / or endogenous stem cells 28 includes one or more of the following: differentiation, continued differentiation and proliferation, and may further include cell migration or multicellular morphogenesis. When differentiated, stem cells 14 and / or endogenous stem cells 28 are one or more of the following: neurons 30, astrocytes 32, oligodendrocytes 34, interneurons 36 and microglia 38. By including partially differentiated stem cells 14 in the implant 10, post-transplant survival and successful differentiation are promoted, and the release of neurotoxins in synapses that occur in fully developed neurons 30 during handling is suppressed. The culture medium mixture 20, along with growth factors, may promote the regeneration of the target axon 40. The implant 10 may provide favorable conditions for myelination 42 of one or more of the stem cells 14 that are differentiating into neurons 30 or have already been differentiated, endogenous stem cells 28 that are in the process of differentiating into neurons 30 or have already been differentiated, and the regenerated axon 40. Oligodendrocytes 34 may promote myelination 42 of the regenerated axon 40 and / or differentiated neurons 30, and may also promote the remyelination of damaged or damaged endogenous neurons 40.

[0070] The implant 10 includes a cover in the form of a rectangular sheet 44 to cover a portion of the support structure 12 that has received stem cells 14, a culture medium mixture 20, and growth factors. The sheet 44 facilitates the delay of degradation and absorption of the support structure 12 after implantation, thereby extending the support time provided by the support structure, and suppresses the shedding of one or more of the stem cells 14, culture medium mixture 20, and growth factors after implantation, where shedding may be caused by the flow of the target cerebrospinal fluid. The sheet 44 is in the form of a porous material that allows it to absorb the target cerebrospinal fluid and thereby extrude toward the support structure 12. The sheet 44 is sized to have a length of 7.5 mm, a thickness of 1 mm, and a width of 2.5 mm. The sheet 44 is in the form of RCT Degradable Absorbable Collagen Tape®.

[0071] In a second embodiment of the present invention shown in Figure 9, the implant 10 can be fitted into an implant zone 16 which is in the form of a space defined by a severed peripheral nerve 46. Growth factors control and promote the regeneration of the axon 40 of the peripheral nerve 46. The stem cells 14 are a mixture mainly of partially differentiated stem cells 14 and fully differentiated stem cells 14, the fully differentiated stem cells being Schwann cells 48. Furthermore, growth factors control and promote the continued differentiation of partially differentiated stem cells 14 into Schwann cells 48. The Schwann cells 48 can facilitate myelination 42 of the regenerated axon 40.

[0072] A third embodiment of the present invention relates to a method for preparing an implant 10, the method being: To obtain stem cell 14, The process involves introducing stem cells 14 into a support structure 12 made from a porous material, wherein the stem cells 14 are supported by the support structure 12 in a supported state. The method involves introducing a culture medium in the form of a mixture 20 of neural basal medium and neural induction medium into a support structure 12, thereby supplying nutrients to the stem cells 14 in a state in which the culture medium mixture 20 is supported. The method involves introducing growth factors in the form of neurotrophin 3, neurotrophin 4, and BDNF into a supporting structure 12, thereby controlling, promoting, and introducing the development of stem cells 14 in a state supported by the growth factors.

[0073] Stem cells 14 are obtained from tissue acquired from subjects requiring implants. Therefore, stem cells 14 are autologous. Stem cells 14 are obtained using known techniques. Stem cells 14 are MSCs, and more specifically, AD-MSCs. To determine whether stem cells 14 are MSCs or more specifically AD-MSCs, quantitative polymerase chain reaction (qPCR) assays are performed to detect the expression of positive and negative molecular markers specific to MSCs or AD-MSCs. The positive molecular markers are CD73, CD90, and CD105. The negative molecular markers are CD34, CD116, CD19, and CD45.

[0074] This method involves completely differentiating several stem cells 14 into a desired cell type or tissue type. The desired cell types are oligodendrocytes 34, more specifically, oligodendrocyte progenitor cells (OPCs) and premyelinated oligodendrocytes. Completely differentiating stem cells 14 into oligodendrocytes 34 involves the following steps: 1) Seeding undifferentiated stem cells 14 onto a culture plate using culture medium, 2) Incubate the seeded stem cells 14 at a temperature of 37°C for 3 days, 3) Priming stem cells 14 using priming medium, 4) Initiating the differentiation of stem cells 14 using differentiation medium, 5) Completely differentiate stem cells 14 using terminal differentiation medium, 6) This includes determining whether the stem cells 14 are in the process of differentiating into oligodendrocytes 34 or have already been differentiated.

[0075] In step (1) above, the culture medium is in the form of DMEM / F12 containing FBS and / or gentamicin. FBS and gentamicin are present at concentrations of 10% and 1%, respectively.

[0076] In step (3) above, the priming medium is any preferred form of priming medium. The priming medium contains trypsin and ethylenediaminetetraacetic acid (EDTA) at concentrations of 0.25% [v / v] and 0.02% [v / v], respectively. The priming medium is kept at a temperature in the range of 37°C to 37.5°C, preferably 37°C. Stem cells 14 are added to the priming medium to form a cell suspension. The cell suspension is incubated for a period of 3 to 6 minutes, preferably 5 minutes. The cell suspension is then centrifuged to form a pellet of stem cells 14. The pellet is resuspended in fresh priming medium. The resuspended stem cells 14 are seeded onto a culture plate and incubated at a temperature in the range of 37 to 37.5°C, preferably 37°C. The stem cells 14 are incubated for 18 days. During incubation, the priming medium is at least partially removed and replaced with fresh priming medium periodically, preferably every 7 days.

[0077] In step (4) above, the differentiation medium is any preferred form of differentiation medium. The differentiation medium contains retinoic acid and / or sonic hedgehog protein (SHH). Retinoic acid is added to the differentiation medium from a stock solution having a concentration of 20 mM. The retinoic acid stock solution is added to the differentiation medium in a ratio of 0.01:0.1 ml. SHH is added to the differentiation medium from a stock solution having a concentration of 25 μg / L. The SHH stock solution is added to the differentiation medium in a ratio of 1:100 ml. Stem cells 14 are seeded on a culture plate using the differentiation medium. The seeded stem cells 14 are incubated for 10 days at a temperature in the range of 37°C to 37.5°C, preferably 37°C. During the incubation period, the differentiation medium is at least partially removed and replaced periodically, preferably every 7 days, with fresh differentiation medium.

[0078] In step (5) above, terminal differentiation is in the form of any preferred terminal differentiation medium. The terminal differentiation medium contains neurotrophin 3 and platelet-derived growth factor (PDGF). PDGF is in the form of PDGFα. PDGFα is added to the terminal differentiation medium from a stock solution having a concentration of 1 μg / mL. The PDGFα stock solution is added to the terminal differentiation medium in a ratio of 1:50 ml. Neurotrophin 3 is added to the terminal differentiation medium from a stock solution having a concentration of 1 μg / mL. The neurotrophin 3 stock solution is added to the terminal differentiation medium in a ratio of 1:33 ml. Stem cells 14 are seeded on a culture plate using the terminal differentiation medium. The seeded stem cells 14 are incubated for 14 days at a temperature in the range of 37°C to 37.5°C, preferably 37°C. During the incubation period, the terminal differentiation medium is at least partially removed and replaced periodically, preferably every 7 days, with fresh terminal differentiation medium.

[0079] In step (6) above, a qPCR test is used to determine whether the stem cells 14 express molecular markers characteristic of oligodendrocytes 34. The molecular markers include one or more of SOX10, OLIG1, OLIG2, NKX2.2, ZFP191, ZFP488, ZFP536, SOX17, and NKX6.2. More specifically, for OPC34, the molecular markers include one or more of SOX10, NKX2.2, OLIG1, and OLIG2, and for premyelinate oligodendrocytes 34, the molecular markers include one or more of OLIG1, OLIG2, SOX10, NKX2.2, ZFP191, ZFP488, ZFP536, SOX17, and NKX6.2.

[0080] This method involves using a subcutaneous injection needle (not shown) to puncture the support structure 12 to form a hole 24, thereby enabling the insertion of stem cells 14 into the inner region of the support structure 12. The hole 24 is defined substantially coaxially with the support structure 12.

[0081] Stem cells 14 are introduced into the support structure 12 using a stem cell suspension (not shown). The stem cell suspension is introduced into the support structure 12 in a dropper manner through the pores 24.

[0082] The culture medium mixture 20 is introduced into the support structure 12 by immersing it in the culture medium mixture 20 while it is still in an unsolidified state. Subsequently, the culture medium mixture 20 solidifies, thereby supporting the stem cells 14 in their position relative to the supported support structure 12.

[0083] The growth factors are introduced into the support structure 12 along with the culture medium mixture 20. The growth factors are components of the culture medium mixture 20.

[0084] This method involves developing fully undifferentiated stem cells 14 in a supported solidified culture medium mixture 20. These stem cells may be undifferentiated cells and / or determined cells. The stem cells 14 can be developed until partially differentiated. The stem cells 14 partially differentiate to become one or more of the following: neurons 30, astrocytes 32, oligodendrocytes 34, interneurons 36, and Schwann cells 48. Including partially differentiated stem cells 14 in the implant 10 promotes successful post-transplant survival and development, and suppresses the release of neurotoxins in synapses that occur in fully developed neurons 30 during handling.

[0085] This method involves monitoring the development of stem cells 14 that are allowed to develop until partially developed. Monitoring involves culturing a portion of the stem cells 14 separately from the stem cells 14 contained in the implant 10. Culturing the monitored stem cells 14 is initiated simultaneously with the introduction of the stem cells 14 into the implant 10. Monitoring involves periodically observing the cultured stem cells 14 using a light microscope to identify early characteristic features of the desired cell type, and / or performing a qPCR test to confirm whether the stem cells 14 express molecular markers indicating differentiation into the desired cell type, which is a neuron. The early characteristic feature is the formation of axonal hilllets 50, as shown in Figure 10. Molecular markers include one or more of the following: nestin, PAX6, SOX1, SOX2, bActin, NeuN, Tujl, synapsin, MAP2, and / or GFAP.

[0086] While this specification describes only specific embodiments of the present invention, it will be understood by those skilled in the art that other modifications, variations, and possibilities of the present invention may exist. Such modifications, variations, and possibilities should therefore be considered to fall within the spirit and scope of the present invention and thus form part of the present invention as described and / or illustrated herein. It should be further understood that the examples are provided to further illustrate the present invention and to help those skilled in the art to understand it, and are not intended to be construed as unduly limiting the reasonable scope of the present invention.

[0087] The inventors believe that the implant according to the present invention is advantageous in that it brings about improvement of damaged, devastated, and / or deteriorated tissue through regeneration. The implant according to the present invention may facilitate the handling of stem cells during transplantation. By using partially differentiated stem cells, the survival and successful development of transplanted stem cells are facilitated, and the release of neurotoxins in synapses that often occurs in fully developed neurons during handling is suppressed. Furthermore, the implant supports developing stem cells, and as a result, it is easier to integrate the implant into the environment in which it is implanted.

Claims

1. It is an implant, The implant includes a support structure, The aforementioned support structure, In order to receive and support stem cells in a supported state, To accept a culture medium containing nutrients, and This is for receiving growth factors that promote and control the development of the aforementioned stem cells. The support structure is an implant that can be sized and shaped to fit the target implant zone and thereby be complementarily accepted.

2. The implant according to claim 1, wherein the support structure is manufactured or synthesized from one or more materials selected from the group including biological, biochemical, biocompatible, hemostatic, degradable, and bioabsorbable materials.

3. The implant according to claim 1 or 2, wherein the support structure is manufactured or synthesized from a hygroscopic and / or absorbent material.

4. The implant according to any one or more of claims 1 to 3, wherein the support structure is constructed by 3D printing or additive manufacturing.

5. The implant according to any one or more of claims 1 to 4, wherein the support structure has an internal lattice structure.

6. The implant according to any one or more of claims 1 to 5, wherein the support structure defines a receptive zone for receiving and supporting the stem cells in the supported state.

7. The implant according to claim 6, wherein the receiving zone is in the form of a gap or pore.

8. The implant according to claim 6 or 7, wherein the size of one or more receptor zones is reduced during hydration and / or absorption by the support structure, thereby facilitating the support of the stem cells in the supported state.

9. The implant according to claim 8, wherein the receiving zone has a width in the range of 150 to 1000 μm, and during hydration and / or absorption by the support structure, the width of the receiving zone is reduced to a range of 150 to 500 μm.

10. The implant according to any one or more of claims 1 to 9, wherein a hole for enabling the insertion of the stem cells into the inner region is defined within the support structure.

11. The implant according to claim 10, wherein the hole extends substantially coaxially with the support structure and typically spans two-thirds of the length of the support structure.

12. The implant according to claim 10 or 11, wherein a plurality of holes are defined within the support structure.

13. The implant according to claim 12, wherein each of the holes extends to a different length within the support structure.

14. The implant according to any one or more of claims 1 to 13, wherein the support structure has a substantially cylindrical shape.

15. The aforementioned support structure has the following dimensions Lengths ranging from 1 to 4 cm; and The implant according to claim 14, having a diameter in the range of 0.8 to 1.2 cm.

16. The implant according to any one or more of claims 1 to 15, wherein the support structure is manufactured or synthesized from a natural polymer.

17. The implant according to claim 16, wherein the natural polymer is one selected from the group comprising proteoglycans, gelatinous substrates, graphene, oxidized cellulose, dextrose, hyaluronic acid, starch, graphene oxide, fibrin, alginate substrates, chitosan, collagen, and any combination thereof.

18. The implant according to claim 17, wherein the natural polymer is in the form of collagen.

19. The implant according to claim 18, wherein the collagen is in the form of biodegradable and absorbable collagen.

20. The implant according to claim 18 or 19, wherein the collagen includes cross-linked type I collagen.

21. The implant according to any one or more of claims 18 to 20, wherein the collagen is derived from the bovine Achilles tendon.

22. The implant according to any one or more of claims 19 to 21, wherein the collagen is degradable and absorbable within 20 to 40 days after transplantation.

23. The implant according to any one or more of claims 18 to 22, wherein the support structure is in the form of an RCP-resorbable collagen plug (RCP-Resorbable Collagen Plug®).

24. The implant according to any one or more of claims 1 to 15, wherein the support structure is manufactured or synthesized from a synthetic polymer.

25. The implant according to claim 24, wherein the synthetic polymer is one or more selected from the group consisting of polystyrene, poly-L-lactic acid, polyester, polyethylene glycol, polycyanoacrylate, polyurethane, polystat, polyglycolic acid, and poly-dl-lactic acid-coglycolic acid.

26. The implant according to any one or more of claims 1 to 15, wherein the support structure is manufactured or synthesized from a ceramic material.

27. The implant according to claim 26, wherein the ceramic material is one or more selected from the group comprising hydroxyapatite and tricalcium phosphate.

28. The implant according to any one or more of claims 16 to 27, wherein the support structure is manufactured or synthesized from a combination of the natural polymer, the synthetic polymer and / or the ceramic material.

29. The implant according to any one or more of claims 1 to 28, wherein the support structure is sized and shaped by a molding and / or cutting process.

30. The implant according to any one or more of claims 1 to 29, wherein a plurality of support structures are provided to enable their adaptation to the implant zone such that the support structures are complementarily received by the implant zone.

31. The implant according to claim 30, wherein the plurality of support structures are arranged in contact with each other to form a substantially continuous structure, and the structure allows the support structures to fit into the implant zone and be complementarily received by the implant zone.

32. The implant according to claim 31, wherein the plurality of support structures are arranged end-to-end to form the substantially continuous structure.

33. The implant according to any one or more of claims 1 to 32, wherein the implant zone is a site of damage, destruction and / or deterioration of tissue within the body of the target.

34. The implant according to any one or more of claims 1 to 33, wherein the implant zone is a space defined by one or more of the group including damaged, broken, deteriorated, severed, torn, and excised tissues within the body of the subject.

35. The implant according to claim 34, wherein the implant zone is a space defined by the resection of a portion of the target spinal cord.

36. The implant according to claim 34, wherein the implant zone is a space defined by the resection of a portion of the peripheral nerve of the target.

37. The implant according to any one or more of claims 1 to 36, wherein the subject is a mammal.

38. The implant according to any one or more of claims 1 to 37, wherein the stem cells are in any one form from the group including totipotent stem cells, pluripotent stem cells, compound pluripotent stem cells, and unipotent stem cells.

39. The implant according to claim 38, wherein the stem cells are in the form of pluripotent stem cells.

40. The implant according to any one or more of claims 1 to 39, wherein the stem cells are in any differentiation stage toward a specific cell type of interest.

41. The implant according to claim 40, wherein the differentiation stage is selected from one or more of the group including undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells.

42. The implant according to claim 41, wherein the stem cells are partially differentiated.

43. The implant according to claim 41, wherein the stem cells are fully differentiated.

44. The implant according to claim 40 or 41, wherein a mixture of stem cells at different stages of differentiation is provided.

45. The implant according to claim 44, wherein the mixture comprises undifferentiated stem cells, determined stem cells, partially differentiated stem cells and / or fully differentiated stem cells.

46. The implant according to claim 45, wherein the mixture comprises partially differentiated stem cells and fully differentiated stem cells.

47. The implant according to any one or more of claims 41, 42, 45, or 46, wherein the partially differentiated stem cells are stem cells that exhibit the early morphologically distinct characteristics of being nerve cells.

48. The implant according to claim 47, wherein the morphologically distinct feature is the formation of an axonal hillette.

49. The implant according to any one or more of claims 41, 42, 45, or 46, wherein the partially differentiated stem cells can be identified by the expression of one or more molecular markers specific to nerve cells.

50. The implant according to claim 49, wherein the neuronal cell-specific molecular marker is one or more of the group consisting of NeuN, Tuji, Synapsin, PAX6, SOX1, SOX2, bActin, NESTIN, MAP2, and / or GFAP.

51. The implant according to any one or more of claims 41, 43, 45, or 46, wherein the fully differentiated stem cells are in the form of oligodendrocytes or Schwann cells.

52. The implant according to claim 51, wherein the oligodendrocyte is in the form of an oligodendrocyte progenitor cell (OPC) and / or a premyelinating oligodendrocyte.

53. The implant according to claim 52, wherein the OPC can be identified by the expression of one or more molecular markers from the group including SOX10, NKX2.2, OLIG1, and OLIG2.

54. The implant according to claim 52 or 53, wherein the premyelinate oligodendrocyte can be identified by the expression of one or more molecular markers from the group comprising OLIG1, OLIG2, SOX10, NKX2.2, ZFP191, ZFP488, ZFP536, SOX17, and NKX6.

2.

55. The aforementioned stem cells are in the material of the support structure 1 cm ３ An implant according to any one or more of claims 1 to 54, having a concentration in the range of 150,000 to 1,500,000 per unit.

56. The implant according to claim 55, wherein an excess number of stem cells are provided to enable the differentiation of the stem cells into a target cell type and / or any other cell type formed after transplantation as a result of intercellular signaling and / or endogenous signaling.

57. The aforementioned stem cells, in excess, make up 1 cm of the material of the support structure. ３ The implant according to claim 56, having a concentration in the range of 2,000,000 to 3,000,000 per unit.

58. The implant according to claim 55 or 57, wherein the concentration of the fully differentiated stem cells is 2 to 4% of the total number of stem cells.

59. The implant according to any one or more of claims 1 to 58, wherein the stem cells are obtained during any of the passages.

60. The implant according to claim 59, wherein the stem cells are obtained during any of passages 3 to 9.

61. The implant according to any one or more of claims 1 to 60, wherein the stem cells are one or more forms from the group including embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs), dental pulp stem cells (DPSCs), olfactory nerve sheath cells (OECs), cancer stem cells (CSCs), and human fetal brain neural stem cells (hNSCs).

62. The implant according to claim 61, wherein the MSC is one form selected from the group including bone marrow mesenchymal stem cells (BM-MSC), human umbilical cord mesenchymal stem cells (HUC-MSC), and adipose-derived mesenchymal stem cells (AD-MSC).

63. The implant according to claim 61, wherein the NSC is in the form of a neural progenitor cell, a neutral crest cell, and / or a Schwann cell precursor.

64. The implant according to claim 61, wherein the stem cells are in the form of MSCs.

65. The implant according to claim 62, wherein the MSC is in the form of an AD-MSC.

66. The implant according to claim 65, wherein the AD-MSC can be identified by the expression of a positive molecular marker selected from any one or more of the group including CD73, CD90, and CD105.

67. The implant according to claim 65, wherein the AD-MSC can be identified by the expression of a negative molecular marker selected from any one or more of the group including CD34, CD116, CD19, and CD45.

68. The implant according to any one or more of claims 1 to 67, wherein the stem cells are of autologous origin.

69. The implant according to any one or more of claims 1 to 68, wherein the stem cells are allogeneic.

70. The implant according to any one or more of claims 1 to 69, wherein the culture medium is in the form of a growth medium or a culture medium.

71. The implant according to any one or more of claims 1 to 70, wherein the culture medium is a culture medium that can be solidified.

72. The implant according to claim 71, wherein the culture medium has a viscosity in an unsolidified state that allows the penetration of at least a portion of the material of the support structure.

73. The implant according to claim 71 or 72, wherein the culture medium is in an unsolidified state and has a viscosity that facilitates absorption by the material of the support structure.

74. The implant according to claim 72 or 73, wherein a sufficient amount of the culture medium is provided to allow the culture medium to penetrate into and surround the support structure.

75. The implant according to any one or more of claims 1 to 74, wherein the culture medium is protein-free.

76. The implant according to any one or more of claims 1 to 75, wherein the culture medium is selected to provide nutrients to the stem cells within the implant and / or any other endogenous stem cells within the region of the implant zone, and to promote their development when in contact with them.

77. The implant according to any one or more of claims 1 to 76, wherein the culture medium is one or more forms from the group comprising basal neural medium, neural induction medium, physiological saline medium, phosphate-buffered physiological saline medium, washing medium, retention medium, stem cell-based medium, Roswell Park Memorial Laboratory medium, Dulbecco's Modified Eagle medium, Dulbecco's Modified Eagle medium / F12, HAM / F12 medium, and HAM / F10 medium.

78. The implant according to any one or more of claims 1 to 77, wherein the culture medium contains an additive.

79. The implant according to claim 78, wherein the additive is in the form of essential amino acids, vitamins and / or minerals.

80. The implant according to any one or more of claims 1 to 79, comprising a second culture medium or a subsequent culture medium, each of which is arranged in layers within or around the support structure.

81. The implant according to claim 80, wherein each of the culture media comprises different morphologies and / or different additives.

82. The implant according to any one or more of claims 1 to 81, wherein the growth factor is selected to promote and control the development of the stem cells within the implant and / or any other endogenous stem cells within the region of the implant zone.

83. The implant according to claim 76 or 82, wherein the endogenous stem cells are in the form of neural progenitor cells.

84. The implant according to claim 82 or 83, wherein, when the growth factor is in contact with the stem cells and / or endogenous stem cells within the implant, it promotes and controls the differentiation of the stem cells into one or more of the group including neurons, astrocytes, oligodendrocytes, interneurons, microglia, and Schwann cells.

85. The implant according to any one or more of claims 82 to 84, wherein the growth factor is one or more forms selected from the group consisting of an enzyme having kinase activity or phosphatase activity, an enzyme having tyrosine kinase activity, a cytokine, a lipid-soluble steroid hormone, nitric oxide, oligodendrocyte growth factor, and reactive oxygen species (ROS).

86. The implant according to any one or more of claims 82 to 85, wherein the growth factor is one or more forms selected from the group comprising brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin 4, and glial cell line-derived neurotrophic factor (GDNF).

87. An implant according to any one or more of claims 1 to 86, comprising multiple growth factors.

88. The implant according to claim 87, wherein one or more growth factors are components of the culture medium.

89. The implant according to claim 88, wherein the BDNF has a concentration in the range of 0.2 to 0.8 μL per 50 mL of culture medium.

90. The implant according to claim 88 or 89, wherein the neurotrophin 3 or 4 has a concentration in the range of 5 to 15 μL per 50 mL of culture medium.

91. The implant according to any one or more of claims 88 to 90, wherein the GDNF has a concentration in the range of 5 to 15 μg per 1 mL of culture medium.

92. The implant according to claim 88, wherein the second culture medium or subsequent culture medium contains different growth factors for differently promoting and controlling the development of the stem cells and / or endogenous stem cells.

93. The implant according to any one or more of claims 1 to 92, comprising a cover for covering at least a portion of the support structure that has received the stem cells, the culture medium, and one or more growth factors.

94. The implant according to claim 93, wherein the cover is manufactured or synthesized from the same material used in the manufacture or synthesis of the support structure, or from one or more of the materials described in any one of claims 2 to 4, 7, 16 to 22, or 24 to 28.

95. The implant according to claim 94, wherein the material is absorbent to allow absorption of one or more of the target's bodily fluids after implantation, and to allow the bodily fluids to seep into the support structure.

96. The implant according to claim 95, wherein the bodily fluid comprises one or more of cerebrospinal fluid and blood.

97. The implant according to claim 95, wherein the bodily fluid is cerebrospinal fluid.

98. The implant according to any one or more of claims 94 to 97, wherein the cover is manufactured or synthesized from a porous and / or fibrous material to allow the migration of endogenous cells and / or endogenous stem cells to the support structure.

99. The implant according to any one or more of claims 93 to 98, wherein the cover is in the form of a sheet.

100. The implant according to claim 99, wherein the sheet is substantially rectangular.

101. The cover has the following dimensions: Lengths ranging from 6 to 10 mm. A thickness in the range of 1 to 2 mm, An implant according to claim 99 or 100, having a width in the range of 2 to 4 mm.

102. The implant according to any one or more of claims 94 to 101, wherein the cover is in the form of RCT Resorbable Collagen Tape (registered trademark).

103. The implant according to claim 93, wherein the cover is in the form of a coating medium that coats at least a portion of the support structure receiving stem cells, culture medium, and growth factors.

104. The implant according to claim 103, wherein the coating medium is a culture medium that can be solidified.

105. The implant according to claim 104, wherein the coated culture medium, in its solidified form, is more rigid than the culture medium received by the support structure.

106. The implant according to any one or more of claims 103 to 105, wherein the coating culture medium is permeable in order to allow the bodily fluids of the target to permeate toward the support structure.

107. It is an implant, Support structure and, A receptive zone defined within the support structure for receiving stem cells in a supported state, A culture medium contained in at least a portion of the support structure that has received the stem cells in the supported state, Implants, including those mentioned above.

108. Support structure for supporting partially differentiated stem cells in a supported state Implants, including those mentioned above.

109. An implant according to any one or more of claims 1 to 108, for use in the target regenerative medicine procedure.

110. The implant for use according to claim 109, wherein the regenerative medicine treatment includes one or more of the group including spinal cord regeneration treatment, nerve regeneration treatment, cartilage regeneration treatment, bone regeneration treatment, soft tissue regeneration treatment, Alzheimer's disease regeneration treatment, dementia regeneration treatment, diabetes regeneration treatment, macular degeneration treatment, glaucoma treatment, nephron regeneration treatment, multiple sclerosis regeneration treatment, cancer treatment, and any autoimmune disease.

111. The implant according to claim 109 or 110, wherein the subject is a mammal.

112. A method for regenerative treatment of tissue damage or disease, A method comprising implanting an implant according to any one or more of claims 1 to 108 into an implant zone defined within a subject.

113. The method according to claim 112, wherein the implant zone is a site of damage, destruction or deterioration of the tissue within the body of the target.

114. The method according to claim 112 or 113, wherein the implant zone is a space defined by any one of the group including damaged tissue, deteriorated tissue, severed tissue, torn tissue, and excised tissue within the body of the subject.

115. The method according to claim 114, wherein the implant zone is a space defined by the resection of a portion of the target spinal cord.

116. The method according to claim 114, wherein the implant zone is a space defined by the portion of the target peripheral nerve that has been severed, cut, destroyed, or excised.

117. The method according to any one or more of claims 112 to 116, wherein the subject is a mammal.

118. A method for preparing an implant according to any one or more of claims 1 to 108, wherein the method is Obtaining stem cells, The method involves introducing stem cells into a support structure, wherein the stem cells are supported by the support structure in a supported state. Introducing a culture medium into the support structure, wherein the culture medium provides nutrients to the stem cells in the supported state. A method comprising introducing a growth factor into the support structure, wherein the growth factor controls and promotes the development of the stem cells in the state in which the growth factor is supported.

119. The method according to claim 118, wherein the stem cells are obtained from tissue, and the tissue is obtained from a subject requiring the implant.

120. The method according to claim 118, wherein the stem cells are obtained from a tissue, and the tissue is any tissue suitable for the subject.

121. The method according to claim 119, wherein the stem cells are of autologous origin.

122. The method according to claim 120, wherein the stem cells are allogeneic.

123. The method according to any one or more of claims 119 to 122, wherein the stem cells are obtained from the tissue using any known technique.

124. The method according to any one or more of claims 118 to 123, comprising determining whether or not stem cells were obtained, or identifying the type or morphology of the obtained stem cells.

125. The method according to claim 124, comprising performing a quantitative polymerase chain reaction (qPCR) test to determine whether or not stem cells were obtained, or to identify the type or morphology of the obtained stem cells.

126. The method according to claim 125, wherein the qPCR test is performed to confirm whether MSCs or AD-MSCs have been obtained by confirming whether the stem cells express any one or more molecular markers selected from the group including CD73, CD90, CD105, CD34, CD116, CD19, and CD45.

127. The method according to any one or more of claims 118 to 126, comprising completely differentiating the stem cells into a target cell type or tissue type.

128. The method according to claim 127, wherein the target cell type is in the form of an oligodendrocyte.

129. The method according to claim 128, wherein the oligodendrocyte is an OPC and / or a premyelinating oligodendrocyte.

130. The method according to claim 127, wherein the target cell type is in the form of a Schwann cell.

131. The method according to any one or more of claims 118 to 130, comprising preparing the support structure by puncturing the support structure to form one or more holes for enabling the introduction of the stem cells into the inner region of the support structure.

132. The method according to claim 131, wherein the hole extends substantially coaxially with the support structure.

133. The method according to claim 131 or 132, wherein the support structure is punctured using a thin, elongated member.

134. The method according to claim 133, wherein the thin, elongated member is in the form of a needle.

135. The method according to any one or more of claims 118 to 134, wherein the introduction of the stem cells into the support structure is achieved using a stem cell suspension.

136. The method according to claim 135, wherein the stem cell suspension comprises a mixture of stem cells at different differentiation stages.

137. The method according to claim 136, wherein the mixture comprises one or more of undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells.

138. The method according to claim 137, wherein the mixture comprises undifferentiated stem cells and fully differentiated stem cells.

139. The method according to claim 137 or 138, wherein the fully differentiated stem cells are in the form of oligodendrocytes.

140. The method according to claim 139, wherein the oligodendrocyte is in the form of an OPC and / or a premyelinating oligodendrocyte.

141. The method according to claim 137 or 138, wherein the fully differentiated stem cells are in the form of Schwann cells.

142. The method according to any one or more of claims 135 to 141, wherein the stem cell suspension is introduced into the support structure through one or more pores.

143. The method according to any one or more of claims 118 to 142, wherein the culture medium is introduced into the support structure by immersing at least a portion of the support structure in the unsolidified culture medium.

144. The method according to any one or more of claims 118 to 142, wherein the culture medium in an unsolidified form is introduced into the support structure through one or more pores.

145. The method according to claim 143 or 144, comprising solidifying the culture medium introduced into the support structure, thereby enabling the stem cells to be held in a predetermined position relative to the support structure in a supported state.

146. The method according to any one or more of claims 137 to 145, comprising enabling the undifferentiated stem cells, determined stem cells, or partially differentiated stem cells to develop in the supported state before transplanting the implant into the target.

147. The method according to claim 146, wherein the undifferentiated stem cells, determined stem cells, or partially differentiated stem cells are able to develop to any stage during differentiation toward a specific cell type of interest.

148. The method according to claim 147, wherein the differentiation stage includes one or more of the group consisting of undifferentiated stem cells, determined stem cells, partially differentiated stem cells, and fully differentiated stem cells.

149. The method according to claim 147 or 148, wherein the differentiation step includes partially differentiated stem cells.

150. The method according to claim 148 or 149, wherein the partially differentiated stem cells exhibit early morphologically distinct characteristics of nerve cells.

151. The method according to claim 150, wherein the aforementioned early morphologically distinct feature is the formation of an axonal hill.

152. The method according to any one or more of claims 146 to 151, comprising monitoring the development of the undifferentiated stem cells, determined stem cells, or partially differentiated stem cells in the supported state before implanting the implant into the subject.

153. The method according to claim 152, wherein the monitoring includes culturing a portion of the stem cells separately from the stem cells contained in the implant, and the culturing is initiated simultaneously with the stem cells introduced into the support structure using a culture medium and growth factors introduced into the support structure.

154. The method according to claim 153, wherein the monitoring includes periodically observing the cultured stem cells using an optical microscope to determine whether the stem cells have formed axonal hills, which are characteristic of partially differentiated stem cells.

155. The method according to claim 153, wherein the monitoring of the cultured stem cells includes periodically performing a qPCR test to confirm whether the stem cells express a molecular marker indicating a desired differentiation stage.

156. The method according to claim 155, wherein the molecular marker comprises one or more of the group comprising NeuN, Tuji, Synapsin, PAX6, SOX1, SOX2, bActin, NESTIN, MAP2, and / or GFAP.

157. The method according to any one or more of claims 118 to 156, wherein the method includes wrapping or covering at least a portion of the implant with a cover.

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