CN118001455A - Nerve construct capable of promoting regeneration of various tissues and preparation method and application thereof - Google Patents

Abstract

The invention relates to a nerve construct capable of promoting regeneration of various tissues, a preparation method and application thereof. The nerve construct capable of promoting the regeneration of various tissues is a three-dimensional composite scaffold prepared from an organic hydrogel matrix material, an inorganic bioactive material and nerve-related cells.

Description

Nerve construct capable of promoting regeneration of various tissues and preparation method and application thereof

Technical Field

The invention relates to a nerve construct capable of promoting regeneration of various tissues, a preparation method and application thereof, belonging to the field of biological materials.

Background

The human body is a unified and complex organic whole and is assembled by various tissues and organs. And the systems are tightly coordinated, and the interaction completes various life activities. Wherein the nervous system plays a dominant role in the process. The central nerve is responsible for maintaining homeostasis of the body and controlling motor behavior of the body. Peripheral nerves are distributed throughout each tissue organ and participate in the development of the tissue and the repair of the injury. For example, sensory nerves in bone determine the formation of primary/secondary ossification centers and vascularization during skeletal development. Nerves also regulate skeletal muscle maturation and contraction behavior by forming neuromuscular junctions with muscle cells. In summary, the nervous system plays a role as a commander to maintain homeostasis and physiological functions of various tissues and organs.

Tissue damage repair is a complex physiological process that requires guidance of the nervous system and synergy with other systems. The peripheral nerve first senses a "tissue repair signal" from the damaged area and transmits the signal to the central nervous system, further initiating the tissue repair process. In addition, the nervous system is involved in the repair and regeneration of injury to tissues by secreting various neurotrophic factors and neurotransmitters. Therefore, based on the common characteristic of nerve regulation and control tissue regeneration, the development of a tissue engineering construct with nerve activity is expected to realize the repair and regeneration of injuries of various nerve innervation tissues.

Disclosure of Invention

In view of the above, the present invention provides a neural construct having neural activity and promoting regeneration of various tissues, and a preparation method and application thereof. The nerve construct has excellent nerve activity and can effectively promote the damage repair of nerve tissues. In addition, the nerve construct has the characteristic of enhancing the functions of other tissue cells, including but not limited to osteogenic differentiation of bone marrow mesenchymal stem cells, vascularization of endothelial cells, formation of neuromuscular junctions with muscle cells and the like, and can effectively realize the repair and regeneration of injuries of various tissues.

In one aspect, the present invention provides a neural construct that promotes regeneration of a plurality of tissues, the neural construct being a three-dimensional composite scaffold prepared from an organic hydrogel matrix material, an inorganic bioactive material, and neural-related cells.

Preferably, the inorganic bioactive material contains at least one of lithium, calcium, silicon, magnesium, manganese, molybdenum, zinc, copper, strontium and iron; preferably, the inorganic bioactive material is microsphere with a particle size distribution of 0.1-100 μm.

Preferably, the inorganic bioactive material is an inorganic material containing lithium, calcium and silicon elements.

Preferably, the inorganic bioactive material is a calcium lithium silicate biological ceramic material, preferably calcium lithium silicate microspheres with particle size distribution of 0.1-100 μm.

Preferably, the inorganic bioactive material is present in an amount of 0.01 to 20wt%, preferably 3 to 10wt%, based on the weight of the organic hydrogel matrix material.

Preferably, the organic hydrogel matrix material is at least one of gelatin, methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan and polyethylene glycol.

Preferably, the organic hydrogel matrix material is at least one of methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan, and polyethylene glycol.

Preferably, the neural-related cells include: at least one of neural stem cells, glial cells, neurons, PC12 cells, preferably neural stem cells;

The cell density of the nerve cells in the nerve construct capable of promoting the regeneration of various tissues is 10-1000 ten thousand/mL.

In another aspect, the present invention provides a method of preparing a neural construct that promotes regeneration of a plurality of tissues, comprising:

(1) Mixing an inorganic bioactive material, an organic hydrogel matrix material, a photoinitiator, water and nerve cells to obtain inorganic bioactive ink;

(2) And (3) carrying out layer-by-layer deposition printing on the obtained inorganic bioactive ink after the pre-curing treatment, then carrying out cross-linking curing molding by using blue light, and finally culturing to obtain the nerve construct capable of promoting the regeneration of various tissues.

Preferably, when the raw materials are selected, the organic hydrogel matrix material is a compound of gelatin and at least one of methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan and polyethylene glycol, preferably a compound of methacryloylated gelatin and gelatin; more preferably, the mass ratio of at least one of the methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan and polyethylene glycol to gelatin in the organic hydrogel matrix material is 10:1-1:10, preferably 1:5-5:1, more preferably 1:3-3:1;

the photoinitiator comprises: at least one of phenyl 2,4, 6-trimethylbenzoyl lithium phosphinate and an ultraviolet initiator I2959; the addition amount of the photoinitiator is 0.1-0.5 wt% of the mass of water;

The parameters of the pre-curing treatment include: the temperature of the pre-curing treatment is-20 to 15 ℃, preferably 4 ℃; the time of the pre-curing treatment is 5-60 min, preferably 20min;

The parameters of the layer-by-layer deposition printing include: setting the temperature of the charging barrel to be 10-25 ℃, the temperature of the deposition platform to be 0-10 ℃, the printing needle head to be 12-27G, the moving speed of the needle head to be 1-12 mm/s, and the extrusion pressure to be 10-200 kPa;

The parameters of the culture include: the composition of the culture solution used comprises: 90 to 99.5vol% of DMEM/F12 basal medium, 1 to 10vol% of fetal calf serum and 0.5 to 2vol% of antibiotics; the temperature of the culture is 33-40 ℃; the culture solution is replaced every 2-3 days. Preferably, the antibiotic may be penicillin streptomycin or the like.

In yet another aspect, the present invention provides the use of a neural construct for the preparation of a plurality of tissue regeneration materials, including neural tissue regeneration materials, bone tissue regeneration materials, and muscle tissue regeneration materials;

Preferably, the nerve tissue regeneration material comprises spinal cord regeneration material and peripheral nerve regeneration material;

Preferably, the bone tissue regeneration material comprises skull regeneration material and femur regeneration material;

Preferably, the muscle tissue regeneration material comprises a volumetric injury repair material of the tibialis anterior.

The invention has the beneficial effects that:

The nerve construct provided by the invention not only has excellent nerve activity, but also can effectively regulate and control the differentiation behaviors of various tissue cells, so that the nerve construct can be used for repairing and regenerating various complex tissues, including but not limited to spinal cord, bones, muscles and other tissues.

Drawings

In FIG. 1, (a-b) are scanning electron microscope pictures of lithium calcium silicate microspheres (LCS), (c) are particle size distribution curves of the LCS microspheres, and (d) are XRD patterns of the LCS microspheres;

FIG. 2 (a) is a scanning electron microscope image of a methacryloylated gelatin/gelatin hydrogel (GG) compounded with LCS microspheres of different contents, wherein the mass fraction of methacryloylated gelatin is 4%, the mass fraction of gelatin is 2%, and the mass percentages of LCS microspheres compared with the mass percentages of methacryloylated gelatin are respectively 0, 2%, 5% and 10%, corresponding to the names GG, GG-2LCS, GG-5LCS and GG-10LCS, (b) is a shear thinning curve of each group of bio-inks, and (c) is a curve of the modulus of each group of bio-inks with the change of frequency;

FIG. 3 is a photograph of staining live/dead cells of neural constructs containing neural stem cells prepared with different concentrations of LCS microsphere bio-ink when cultured for 1, 7 and 14 days, wherein the live cells are marked green and the dead cells are marked red;

FIG. 4 shows gene expression of neural constructs with different LCS microsphere content during 7 days of culture, (a) Nestin, (b) Tuj1, (c) GFAP;

FIG. 5 shows immunofluorescent protein staining analysis of biological 3D printed GG and GG-5LCS neural constructs, wherein (a) is a GFAP and Tuj1 dual immunofluorescent staining picture of neural constructs cultured for 7 days, (b-c) is a statistical result of the proportion of GFAP and Tuj1 positive cells, (D) is a MAP2 immunofluorescent staining picture of neural constructs cultured for 10 days, and (e) is a statistical result of MAP2 positive cells;

FIG. 6 shows results of nerve constructs at 7 and 14 days of subcutaneous implantation in rats, where (a) is a macroscopic picture of the nerve construct and (b) is the H & E staining result;

FIG. 7 is an in vivo neuronal differentiation characterization of the neural construct, wherein (a-b) is immunofluorescent-stained picture and statistical analysis of the neuronal marker Tuj1 protein and (c-d) is immunofluorescent-stained picture and statistical analysis of the neuronal maturation marker MAP2 protein;

FIG. 8 is a representation of the multicellular regulatory behavior of a neural construct, wherein (a) is ALP staining pictures and statistical results for 7 days of co-culture of the neural construct with rat bone marrow mesenchymal stem cells (BMSCs), and Alizarin Red (ARS) staining pictures and statistical results for 10 days of co-culture, (b) is statistics of matrigel angiogenesis experimental pictures and number of vascular connections and number of vascular meshes after 6h co-culture of the neural construct with Human Umbilical Vein Endothelial Cells (HUVECs), and (c) is statistics of Tuj1/AchRs double immunofluorescent protein staining pictures and number of neuromuscular junctions after 3 days of co-culture of the neural construct with rat myoblasts (L6);

FIG. 9 is a BBB score of 8 weeks after implantation of the neural construct prepared according to the present invention into a rat full-cross spinal cord injury site;

FIG. 10 is a representation of the promotion of spinal cord injury and functional recovery after implantation of the neural construct prepared according to the present invention into a rat at a full-cross spinal cord injury site, wherein (a) is the measured action potential of each spinal cord group and (b) is the result of statistical analysis of the action potential;

FIG. 11 is a macroscopic image of spinal cord tissue and H & E staining results for each group after 8 weeks of neural construct implantation;

FIG. 12 is an evaluation of nerve repair in rat spinal cord, wherein (a-b) is immunofluorescent staining of neuronal marker Tuj1 protein and statistical result, (c-d) is immunofluorescent staining of glial cell marker GFAP protein and (e-f) is immunofluorescent staining of angiogenic marker CD31 protein and statistical result;

FIG. 13 is an evaluation of the ability of a nerve construct prepared according to the present invention to repair bone regeneration after implantation into the skull of a rat, wherein (a) is a Micro-CT scan of bone regeneration, (b) is an H & E stained picture of bone tissue, (c) is the bone volume fraction at the bone defect, and (d) is the bone density at the bone defect;

FIG. 14 is a representation of the performance of new bone growth, innervation, vascularization at a bone defect, wherein (a) is immunofluorescent-stained with bone-related protein OPN, (b) is immunofluorescent-stained with neurofilament protein NF, (c) is immunofluorescent-stained with the vascularization marker CD31, (d) is statistical for OPN protein, (e) is statistical for NF protein, and (f) is statistical for CD31 protein;

FIG. 15 is an assessment of the ability of a nerve construct prepared according to the present invention to regenerate muscle after implantation into a rat tibialis anterior volumetric defect, wherein (a) is a picture of the muscle of each group after 8 weeks of implantation, (b) is the weight percent of the muscle of each group (as compared to the contralateral side) 8 weeks after surgery, (c) is the results of the H & E staining and Masson staining pictures of each group of muscle;

FIG. 16 is a graph showing muscle fiber regeneration, vascularization and innervation performance at muscle injury, wherein (a) is an immunofluorescent staining picture of the MHC of muscle fibers, (b) is a statistical result of MHC, (c) is an immunofluorescent staining picture of angiogenin CD31, (d) is a statistical result of CD31, (e) is an immunofluorescent staining picture of neuromuscular junction, (f) is a statistical result of NF, and (g) is a statistical result of AchRs.

Detailed Description

The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof.

The invention provides a nerve construct capable of promoting regeneration of various tissues, which consists of an organic hydrogel matrix material, an inorganic bioactive material and nerve cells. The above construct has excellent neural activity.

In an alternative embodiment, the cells contained in the construct are neural related cells, can exhibit excellent neural activity under the induction of inorganic bioactive materials, can be used for regenerating neural tissues, and can regulate the specific differentiation and functions of other tissue cells through intercellular actions, such as osteogenic differentiation of bone marrow mesenchymal stem cells, vascularization of endothelial cells, maturation of muscle cells and the like. The nerve related cell is at least one of neural stem cell, neuron and glial cell. The density of cells in the bio-ink is 10-1000 tens of thousands/mL because lower density cells will be difficult to survive in a three-dimensional hydrogel scaffold, while higher density cells reduce their activity due to nutritional competition, limited adhesion sites, etc. The scaffold containing nerve cells is a nerve stem cell, and the nerve stem cell is a stem cell with heterogeneous differentiation potential, and can be differentiated into neurons, glial cells, oligodendrocytes and the like. Thus, the neural constructs described herein can promote regeneration of a variety of tissues including the spinal cord of central nervous tissue, as well as innervating tissue bone and muscle, and the like.

In alternative embodiments, the organic hydrogel, as a matrix material for the neural construct, may provide three-dimensional support and adhesion sites for cells, facilitating long-term survival and functionalization of the cells. In some technical schemes, the organic hydrogel is at least one of collagen, gelatin, methacryloylated gelatin, chitosan, sodium alginate and hyaluronic acid. The mass fraction of the hydrogel is 1-20%, preferably 3-10%. If the concentration of the hydrogel is too high, the survival of internal cells is not facilitated, and if the concentration is too low, the matrix network is sparse, and the printability is poor. In the invention, the organic hydrogel material is preferably a mixture of methacryloylated gelatin and gelatin, wherein the mass fraction of the methacryloylated gelatin is 4 percent, the mass fraction of the gelatin is 2 percent, and the gelatin can be used as a sacrificial phase after crosslinking to form a macroporous network structure, thereby being more beneficial to adhesion and survival of nerve cells.

In alternative embodiments, the inorganic bioactive material acts as a "bioactive component" in the construct, acting to enhance cell viability, regulating cell-specific differentiation. Further, the inorganic bioactive material contains elements with nerve regulating and controlling effects such as lithium, calcium, silicon, magnesium, manganese, molybdenum, copper, zinc and the like. The inorganic bioactive material selected in the invention is a biological ceramic material containing lithium, calcium and silicon, the morphology is uniform sphere, and the particle size is between 1 and 100 mu m. The mass of the inorganic bioactive material in the nerve construct accounts for 0.001-20% of the mass of the methacryloylated gelatin, and is preferably 1-10%. On the one hand, if the inorganic content is too high, potential cytotoxicity may occur due to the burst effect of the polyions. If the inorganic content is too low, it may be difficult to exert its biological activity. The inorganic bioactive material used in the invention is a biological ceramic material containing three elements of Li, ca and Si. Among them, li plays an important role, which has been widely demonstrated to have good neuroprotection and neurotrophic effects, and also promotes survival of neural stem cells and neuronal differentiation through GSK-3β pathway.

In the invention, the neural construct can effectively regulate and control the specific differentiation behaviors of other tissue cells. For example, the neural construct induces osteogenic differentiation of bone-related cells, which are one or more of bone marrow mesenchymal stem cells, osteoblasts, osteoprogenitor cells, dental pulp stem cells. The neural construct induces vascularization of blood vessel-related cells, which are one or more of endothelial cells, endothelial progenitor cells, vascular smooth muscle cells. The neural construct induces maturation of muscle-related cells, which are one or more of myoblasts, muscle stem cells.

The following illustrates a method of preparing a neural construct that can promote regeneration of various tissues.

Preparing calcium lithium silicate microsphere (LCS) with particle size of 0.1-100 μm by spray granulation.

Preparation of the biological ink: a small amount of photoinitiator LAP was weighed and fully dissolved in deionized water, and a proper amount of methacryloylated gelatin and gelatin particles were added and fully dissolved in a 50 ℃ water bath. After treatment with a 0.22 μm bacterial filter membrane. The LCS microspheres were weighed and sterilized by UV light and added to the sterile hydrogel solution. The cells are treated and then added into the organic-inorganic composite solution, and then the cells can be put into a charging barrel for low-temperature pre-crosslinking.

Biological 3D printing process: the invention adopts an extrusion type biological 3D printer to prepare the nerve construct. The printing parameters were as follows: the temperature of the barrel was set at 18 ℃, the deposition platform temperature at 4 ℃, the print needle at 27G, the needle travel speed at 8mm/s, and the extrusion pressure at 30kPa. After printing, the hydrogel is crosslinked by blue light, and the nerve construct can be obtained.

The neural construct is placed in a 24-well plate, and a neural-related culture medium is added for culture, wherein the behaviors of cell survival, proliferation and the like are characterized by using the neural proliferation culture medium, and the behaviors of cell differentiation and the like are characterized by using the neural differentiation culture medium. The medium was changed every other day. Because nerve cells tend to survive and proliferate on soft matrices, hard matrix materials can hinder nerve cell activity, but too soft matrix materials are detrimental to print formation and have poor structural integrity. In order to solve the contradictory dilemma, the invention provides that the used organic hydrogel matrix material is a compound of methacrylic acid acylated Gelatin (GelMA) and Gelatin (Gelatin), the GelMA and Gelatin compound biological ink has the characteristic of 'complementary network', wherein GelMA is taken as a framework material, gelatin is taken as a sacrificial phase material to only assist in printing the ink, blue light crosslinking after printing is finished leads to permanent solidification of the GelMA network, and Gelatin can be slowly dissolved away from the inside of a bracket, leaving a macroporous network which is mutually communicated. The combination not only can support printing of a bracket material with good structural accuracy, but also can reduce the overall strength of the bracket after printing, and is beneficial to survival, attachment and proliferation of neural stem cells. Preferably, the combination of 4% GelMA and 2% Gelatin is used in the present invention, and only 4% GelMA framework material is left after Gelatin is removed, it should be noted that the pure 4% GelMA hydrogel material cannot complete the printing process due to its lower strength. It should be noted that at least one of gellan gum, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol may be substituted for the methacryloylated gelatin in the present invention.

In the invention, the nerve construct capable of promoting the regeneration of various tissues is an inorganic material functionalized nerve construct and can be used for the injury repair and regeneration of various tissues. Specifically, the neural stem cells exhibit good survival, proliferation and neuron differentiation ability under the stimulation of inorganic materials, and the neural activity of the neural construct is effectively improved. (2) The nerve construct shows multicellular regulatory activity, namely, the in vitro co-culture experimental result shows that the nerve construct can effectively promote the osteogenic differentiation of bone marrow mesenchymal stem cells, the formation of calcium nodules, the vascularization of endothelial cells and the in vitro formation of neuromuscular junctions with muscle cells. (3) The nerve construct prepared by the invention has the capability of promoting the regeneration of various tissues, and the in vivo animal experiment results show that the nerve construct can effectively promote the injury repair and functional recovery of spinal cord, promote the bone regeneration of neurovascularization, and repair and nerve innervation of volumetric muscle injury.

The present invention will be further illustrated by the following examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below. Unless otherwise specified, each percentage refers to a mass percent.

Example 1 preparation of organic-inorganic composite bio-ink:

Lithium Calcium Silicate (LCS) microspheres were prepared using a spray granulation process, as follows: firstly, according to the mol ratio of TEOS: HNO ₃：H₂ o=1: 0.16:8, respectively weighing TEOS, nitric acid and deionized water according to the proportion, mixing and stirring until the solution is completely clarified; the molar ratio of TEOS: ca (NO ₃)₂·4H₂O：LiNO₃ =1:1:0.5) respectively weighing calcium nitrate and lithium nitrate, adding calcium nitrate into the solution, stirring until the calcium nitrate is completely dissolved, adding lithium nitrate, continuously stirring for 3 hours, adding deionized water with twice volume to dilute the solution, continuously stirring for 1 hour, placing the mixed solution into a spray granulator for spray granulation, setting parameters such as the temperature of 250 ℃ and the rotating speed of a centrifugal fan of 300Hz, the feeding speed of a peristaltic pump of 40r/min and the rotating speed of a draught fan of 30Hz, sintering the obtained green particles in a high-temperature furnace after spraying, setting the temperature of 940 ℃ and the sintering time of 3 hours, and the heating speed of 2 ℃/min, dispersing the particles in absolute ethyl alcohol after sintering, and filtering with the aid of vacuum suction filtration by a 1000-mesh screen, and drying in an oven after the completion of filtration, thus obtaining the lithium calcium silicate microspheres (LCS).

Weighing a proper amount of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate (LAP), fully dissolving in deionized water, wherein the LAP content is 0.25%, and then dissolving methacryloylated gelatin (GelMA) and gelatin particles in the LAP solution to obtain a mixed hydrogel solution (GG) with the GelMA mass fraction of 4% and the gelatin mass fraction of 2%. The hydrogel solution was then sterilized by filtration using a 0.22 μm filter. Weighing a proper amount of LCS microspheres, and fully irradiating and sterilizing under UV. Sterile LCS microspheres were added to the sterile GG hydrogel solution and designated GG, GG-2LCS, GG-5LCS, GG-10LCS, based on the mass percent of LCS microspheres and GelMA. Finally, neural stem cells are added into the mixed organic-inorganic hydrogel, and the cell density is 300 ten thousand/mL.

In fig. 1, a-b are LCS microspheres prepared using spray granulation, and it can be seen that LCS is uniformly spherical with no significant agglomeration between particles. The particle size of the LCS microspheres was counted to show that the LCS microspheres had a particle size ranging from 4 to 15 μm and an average particle size of 9.23. Mu.m. The XRD results in FIG. 1 d show that the LCS microspheres have Li ₂Ca₄Si₄0₁₃ phase, which demonstrates successful preparation of LCS microspheres.

In fig. 2a shows the microscopic morphology of hydrogels with different LCS microsphere contents, it can be seen that the composite hydrogel is internally of a macroporous structure which is mutually communicated, and LCS microspheres are uniformly distributed on the walls of the hydrogel network. In fig. 2 b it is shown that the introduction of LCS microspheres does not affect the shear thinning properties of the hydrogels and can still be used for extruded bio-3D printing.

EXAMPLE 2 preparation of the neural construct according to the invention and in vitro biological Activity

Lithium Calcium Silicate (LCS) microspheres were prepared using a spray granulation process, as follows: firstly, according to the mol ratio of TEOS: HNO ₃：H₂ o=1: 0.16:8, respectively weighing TEOS, nitric acid and deionized water according to the proportion, mixing and stirring until the solution is completely clarified; the molar ratio of TEOS: ca (NO ₃)₂.4H2O: liNO3=1:1:0.5) respectively weighing calcium nitrate and lithium nitrate, adding calcium nitrate into the solution, stirring until the calcium nitrate is completely dissolved, adding lithium nitrate, continuously stirring for 3 hours, adding deionized water with twice volume to dilute the solution, continuously stirring for 1 hour, placing the mixed solution into a spray granulator for spray granulation, setting parameters such as the temperature of 250 ℃, the rotating speed of a centrifugal fan of 300Hz, the feeding speed of a peristaltic pump of 40r/min and the rotating speed of a draught fan of 30Hz, sintering the obtained green particles in a high-temperature furnace after spraying, setting the temperature of 940 ℃, the sintering time of 3 hours and the heating speed of 2 ℃/min, dispersing the particles in absolute ethyl alcohol after sintering, and carrying out 1000-mesh vacuum suction filtration assistance, and drying in an oven after screen filtration completion, thus obtaining the lithium calcium silicate microspheres (LCS).

Firstly, weighing a proper amount of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate (LAP), fully dissolving in deionized water, wherein the LAP content is 0.25%, and then dissolving methacryloylated gelatin (GelMA) and gelatin particles in the LAP solution to obtain a mixed hydrogel solution (GG) with the GelMA mass fraction of 4% and the gelatin mass fraction of 2%. The hydrogel solution was then sterilized by filtration using a 0.22 μm filter. Weighing a proper amount of LCS microspheres, and fully irradiating and sterilizing under UV. Sterile LCS microspheres were added to the sterile GG hydrogel solution and designated GG, GG-2LCS, GG-5LCS, GG-10LCS, based on the mass percent of LCS microspheres and GelMA. Finally, neural stem cells are added into the mixed organic-inorganic hydrogel, and the cell density is 300 ten thousand/mL.

The biological ink is filled into a charging barrel of a biological 3D printer and cooled for 20min at 4 ℃, and then is filled into a machine for printing, wherein the printing parameters are as follows: the temperature of the cylinder is set to 18 ℃, the temperature of the deposition platform is set to 4 ℃, the printing needle head is set to 27G, the moving speed of the needle head is set to 8mm/s, the extrusion pressure is set to 30kPa, and the number of layers is set to 6. After printing, the hydrogel is crosslinked for 15s by using blue light, and the bracket can be obtained. Finally, placing the bracket in an incubator (37 ℃,5% CO ₂) for culturing, wherein the composition of the culture solution comprises DMEM/F12 basal medium (90-99.5%), fetal bovine serum (1-10%), antibiotics (penicillin streptomycin, 0.5-2%); the fluid was changed every 2 days to obtain a neural construct that promoted regeneration of various tissues. In the resulting neural construct, gelatin was not present, and gelatin had dissolved away from the scaffold as early as the initial stage of culture (1-2 days).

The neural constructs were cultured in vitro for 1, 7 and 14 days for cell viability using the Calcein-AM/PI live dead cell staining kit. Staining working solution was prepared in a ratio of PBS: AM: pi=1000:2:3, and the scaffold was immersed in the staining working solution and incubated in an incubator at 37 ℃ for 20min. Followed by washing with PBS and photographing with a fluorescence microscope. Wherein living cells are marked green and dead cells are marked red.

The expression of the neural differentiation genes of NSCs was detected using RT-qPCR experiments, respectively. After 10 days of culture, the scaffolds were lysed using GelMA lysate to isolate cells. Then 1mL of Trizol reagent was added to lyse the cells, and RNA of the cells was extracted. After completion of the cleavage, transfer to an EP tube and add 200. Mu.L of chloroform, mix well and centrifuge at 12000rpm/min for 10min using a cryogenic centrifuge at 4 ℃. And then sucking the supernatant to mix with the isopropanol solution, shaking and uniformly mixing, centrifuging for 10min at the temperature of 4 ℃ at the rotation speed of 12000rpm/min by using a low-temperature centrifuge, and obtaining the white precipitate at the bottom of the EP tube, namely the RNA. After washing with ethanol, the RNA precipitate was dissolved in DEPC water and the concentration of RNA was measured using a spectrophotometer (Nano Drop, thermo, usa). RNA was reverse transcribed into cDNA using PRIMESCRIPT A1 st Strand cDNA synthesis kit, and then subjected to a PCR reaction StepOnePlus REAL TIME SYSTEMS. Wherein GAPDH is housekeeping gene and pure GG scaffold is control group. After completion of the reaction, data were processed using the method 2 ^-ΔΔCt to analyze the gene expression level of each group.

The effect of LCS microspheres on the neural differentiation of NSCs and expression of maturation-related proteins in scaffolds was characterized by immunofluorescent protein staining. The method comprises the following specific steps: (1) After the bracket is cultured for 10 days, the culture medium in the pore plate is sucked off, 4% paraformaldehyde solution is added into each pore for fixing for 30min, and PBS buffer solution is used for cleaning for 3 times, and each time lasts for 5min; (2) Adding 0.1% (v/v) Triton-X solution for cell permeation treatment for 5min, and washing with PBS buffer solution for 3 times each for 5min; (3) Adding 5% bovine serum albumin (BSA, beyotime, china) and blocking at room temperature for 1h to avoid nonspecific staining; (4) Adding a proper amount of primary antibody, and after incubating overnight at 4 ℃ in a refrigerator, washing with PBS buffer solution for 3 times, each time for 5min; (5) Adding the corresponding specific secondary antibody, incubating for 1h in a 37 ℃ oven, and washing with PBS buffer solution for 3 times, each time for 5min; (7) Cytoskeleton and nuclei were labeled with phalloidin and DAPI, respectively. Finally, specific protein expression was photographed using a laser scanning confocal microscope (CLSM, TCS SP8, leica, germany). It is worth noting that the expression of the mature neuronal marker MAP2 was stained when the scaffolds were cultured in vitro for 14 days.

FIG. 3 shows the survival of the neural constructs with different LCS microsphere contents in the in vitro culture process, and it can be seen that the scaffolds of each group have obvious green fluorescence and almost no red fluorescence, which indicates that the cells in the scaffolds have high survival rate and GG-LCS bio-ink has excellent biocompatibility.

In FIG. 4a, it is shown that the Nestin gene is significantly down-regulated in the scaffolds containing LCS microspheres compared to the pure GG scaffolds, indicating that LCS microspheres can reduce the dryness of NSCs and promote neural differentiation. In addition, GG-5LCS scaffold significantly promoted the high expression of the neuron-specific gene Tuj1, indicating that it can induce differentiation of NSCs toward neurons (b in FIG. 4). Whereas LCS microspheres had no significant effect on expression of the glial marker GFAP (c in fig. 4). In general, the introduction of LCS microspheres into the bio-ink can reduce the dryness of NSCs in the scaffold and promote the differentiation of the NSCs towards neurons, wherein GG-5LCS group has the most excellent neural differentiation regulating effect. Thus, the scaffold group containing 5% lcs microspheres is preferred for subsequent experiments.

It can be seen in fig. 5 that there is a higher density of GFAP green fluorescence in the GG group, but Tuj1 is less expressed, indicating that neural stem cells are more prone to differentiate into glial cells, and only a small number of neural stem cells differentiate into neurons. In contrast, a significantly increased Tuj1 red fluorescence high expression was seen in the GG-5LCS group, indicating that LCS microspheres significantly increased the cell fraction of neural stem cells differentiated into neurons. The statistical results of b-c in FIG. 5 show that there is no significant difference in the proportion of GFAP positive cells in the two groups, but that the proportion of Tuj1 positive cells in the GG-5LCS stent group (21.89.+ -. 2.45%) is significantly greater than the proportion of Tuj1 positive cells in the GG stent group (11.12.+ -. 2.2%). After 14 days of in vitro culture, the expression of the mature neuronal marker MAP2 protein in the scaffold was analyzed. As shown in FIG. 5 d-e, the proportion of MAP2 positive cells in the GG-5LCS scaffold was significantly greater than that in the GG scaffold, indicating that LCS microspheres can effectively promote differentiation of neural stem cells in the scaffold into neurons and promote neuronal maturation.

EXAMPLE 3 in vivo biological Activity of the neural construct according to the present invention

To explore the in vivo bioactivity and neural differentiation capacity of the neural constructs, 2 sets of scaffolds were prepared: GG-NSC (containing NSCs, no LCS microspheres, cell density of 300 ten thousand/mL) and GG-LCS-NSC (containing LCS microspheres and NSCs, cell density of 300 ten thousand/mL, content of LCS microspheres of 5wt% of GelMA dry weight) were prepared as described in example 2. After printing, the neural construct is obtained by in vitro pre-culture for 7 days, and is implanted subcutaneously after stabilization. After anesthetizing the mice, the back was shaved, the back skin was cut, and the expansion was performed using forceps to form a subcutaneous cavity. The wound is then sutured after placement of the stent in the cavity. And finally, placing the mice in a proper environment for raising, taking out the subcutaneous support at the back after 7 days and 14 days of implantation, photographing, and then soaking in 4% paraformaldehyde for fixing.

The taken scaffold samples were dehydrated using 10% and 30% concentration sucrose solutions after fixation with 4% paraformaldehyde. Following embedding using OCT, the tissue was cut into pieces of 20 μm thickness using a frozen microtome (Thermo, germany).

Hematoxylin & eosin (H & E) staining: (1) After the sections were dried at room temperature, the residual OCT was removed by washing twice with PBS for 10min each. (2) The sections were stained in hematoxylin stain (Beyotime, china) for 5min, then washed thoroughly with running water for 5min to remove excess hematoxylin stain. (3) The sample is soaked in 1% hydrochloric acid-ethanol (1 mL hydrochloric acid+99 mL absolute ethanol) solution for differentiation for 30s, and is manually pulled for several times until the solution is colorless. (4) Dyeing with eosin dye (Beyotime, china) for 5min, washing with tap water until the water is colorless. (5) The sections were dehydrated in gradient ethanol and the xylene solution was transparent and then sealed with neutral gum.

Immunofluorescent protein staining: (1) After the sections were dried at room temperature, the residual OCT was removed by washing twice with PBS for 10min each. (2) Antigen retrieval using PK enzyme (P78893, abcone, china), incubation for 15min at 37 ℃ followed by blocking for 1h with 10% horse serum (Biosharp, china) mixed with 0.3% triton-PBS; (3) Dropwise adding the diluted primary antibody solution to the surface of a sample, and finally, washing the sample at 4 ℃ overnight with PBS for 3 times every 10min the next day; (4) After PBS is washed, specific fluorescent secondary anti-staining solution is added, and the mixture is incubated for 1h at room temperature; (5) After PBS washing, DAPI dye was added to stain the nuclei. After incubation for 10min at room temperature, washing with PBS, drying the surface water, and dripping immunofluorescence anti-attenuation sealing tablet, so as to observe under a fluorescence microscope. The primary antibodies used in this section of the experiment were as follows: beta-III Tubulin (1:300, ab18207, abcam, U.S.) and MAP2 (1:500, ab5392, abcam, U.S.).

As shown in fig. 6 a, the presence of blood scab was seen around the stent after 7 days of implantation, indicating the presence of a slight inflammatory response. However, after 14 days of implantation, it can be seen that host blood vessels infiltrate into the inside of the stent, indicating that the stent is well integrated with the host and has excellent biocompatibility. The H & E staining results of fig. 6 b indicate that fibrous tissue can penetrate into and integrate well with the interior of the neural construct.

FIG. 7a shows that there is a greater area of expression of Tuj1 positive protein in the GG-LCS-NSC group compared to the GG-NSC group. Statistical results showed that Tuj1 positive area of GG-LCS-NSC group was 4.94 times that of GG-NSC group (b in fig. 7). In addition, there was also more expression of MAP2 positive protein in GG-LCS-NSC group, 2.35 times the area of GG-NSC group (c-d in FIG. 7). The above results indicate that GG-LCS-NSC neural constructs have good neuronal differentiation and neuronal maturation properties in vivo.

Example 4 multicellular regulatory capability of neural constructs according to the invention:

In example 4, to investigate the regulation of the neural construct on other tissue cells, the neural construct was co-cultured directly/indirectly with various tissue cells. 4 sets of scaffolds were prepared: GG (pure hydrogel scaffold), GG-LCS (hydrogel scaffold containing LCS microspheres, content of LCS microspheres being 5wt% of dry weight of GelMA), GG-NSC (containing NSCs, no LCS microspheres, cell density of 300 ten thousand/mL) and GG-LCS-NSC (containing LCS microspheres and NSCs, cell density of 300 ten thousand/mL, content of LCS microspheres being 5wt% of dry weight of GelMA), were prepared as described in example 2.

First, neural constructs were explored to contribute to bone effects. The neural constructs and bone marrow mesenchymal stem cells (BMSCs) were Transwell co-cultured, with BMSCs seeded in the lower chamber and each set of scaffolds placed in the upper chamber. After 7 days of co-culture, ALP activity of BMSCs was evaluated using an ALP staining kit, and after 10 days of co-culture, mineralization and deposition of calcium nodules was evaluated using an alizarin red staining kit.

Next, the vascular-contributing ability of the neural constructs was explored, and the neural constructs and Human Umbilical Vein Endothelial Cells (HUVECs) were Transwell co-cultured, wherein the lower chamber was pre-plated with matrigel followed by seeding with HUVECs, and each set of scaffolds was placed in the upper chamber. After 6h co-culture, the lower cells were fixed, the angiogenesis of HUVECs was photographed using an optical microscope and statistically analyzed using Image J.

The nerve can form a neuromuscular junction (NMJs) with the muscle cells to effectively control the maturation and contraction behaviour of the muscle cells. To explore the ability of neural constructs to form NMJs with muscle cells in vitro, 2 neural constructs were prepared: GG-NSCs (containing NSCs, without LCS microspheres) and GG-LCS-NSCs (containing LCS microspheres and NSCs) were prepared as described in example 2. Rat myoblasts (L6) were inoculated directly onto the neural construct and co-cultured for 3 days. A 4% paraformaldehyde fixing scaffold was then used. The formation of NMJs was assessed using a double immunofluorescent protein staining experiment, in which red fluorescence Tuj1 represents neurons and green fluorescence AchRs represents acetylcholine. The co-localization phenomenon of Tuj1 and AchRs represents the formation of NMJs.

As shown in FIG. 8a, higher levels of ALP activity and mineralization nodules were observed in the GG-LCS and GG-LCS-NSC groups compared to the GG and GG-NSC groups, and quantitative analysis confirmed that GG-LCS-NSC had the best bone-promoting properties. It is worth mentioning that GG-LCS has better ability to promote osteogenic differentiation than GG group for GG-LCS group and GG group without neural stem cells, which is caused by the supported LCS microsphere. The inorganic material LCS microspheres themselves also have excellent ability to promote osteogenic differentiation by sustained release of biologically active Li, ca and Si ions. According to the above results, the biological 3D printed GG-LCS-NSC neural construct has the most excellent osteogenic ability.

As shown in fig. 8 b, the most dense vascular network was observed in the GG-LCS-NSC group compared to the other groups, and the quantitative result showed that the number of vascular connections and the number of meshes in the GG-LCS-NSC group were the greatest, indicating that the inorganic LCS microsphere functionalized neural construct had the most excellent vascularization ability. Furthermore, better angiogenesis may also be observed in the GG-NSC group, possibly due to beneficial cellular interactions between NSCs and HUVECs. Indicating a tight interaction between the nerve and the blood vessel.

As shown in fig. 8 c, co-localization of AchRs and Tuj1 was observed in both GG-NSC-L6 and GG-LCS-NSC-L6 groups, indicating that neural constructs were able to form NMJs in vitro and muscle cells, with more NMJs observed in GG-LCS-L6 co-cultured groups, demonstrating that formation of NMJs is favored under regulatory action of LCS microspheres of inorganic material, again demonstrating the superior biological effects of inorganic material.

Example 5 spinal cord injury repair effects of the neural construct of the present invention:

The full-transection spinal cord injury model was used in this example 5 to explore the potential of neural constructs for use in repair of central nervous system injury. SD rats (female, weighing 220-250 g) were used to build the model. A total of 5 groups were set: (1) blank (without any treatment); (2) GG (pure hydrogel scaffold), (3) GG-LCS (hydrogel scaffold containing LCS microspheres, content of LCS microspheres being 5wt% of dry weight of GelMA), (4) GG-NSC (containing NSCs, no LCS microspheres, cell density of 300 ten thousand/mL) and (5) GG-LCS-NSC (containing LCS microspheres and NSCs, cell density of 300 ten thousand/mL, content of LCS microspheres being 5wt% of dry weight of GelMA), were prepared as described in example 2. Rats were first anesthetized and the dorsal skin was incised to expose the T9-T11 vertebrae. After the vertebrae at the corresponding positions are resected using scissors, the spinal cord is exposed and fully transected, producing a full transection lesion of about 3mm in length. Commercial gelatin sponges were used for hemostasis. Each set of nerve constructs was then implanted into a 3mm defect and finally the skin was sutured and the wound was swabbed with iodophor. Manual assistance in urination is performed twice a day in the morning and evening after operation until mice recover from spontaneous urination.

Exercise function assessment: the recovery of hind limb motor function of mice was observed weekly after surgery and scored according to the Basso Beattie-Bresnahan (BBB) scoring principle. Wherein the total paralysis state is 0 point, and the total free movement state is 21 points. In addition, electrophysiological tests were performed on recovery of nerve conduction function in mice 8 weeks after surgery. After anesthetizing the mice, a defect is created in the skull to expose the brain. The stimulation electrodes are placed on the brain surface corresponding to the movement area of the cortex. Recording electrodes were then inserted at the tibialis anterior muscle of the contralateral hind limb to record the value of the Motor Evoked Potential (MEP) under current stimulation.

As shown in fig. 9, the BBB scores of all groups increased over time, indicating that rats with different degrees of motor function were still severely paralyzed after 8 weeks of recovery implantation in the SCI group, with BBB scores of 3±0.63, with only slight ankle movement, but only hind limbs being dragged. In contrast, BBB scores of the GG-LCS-NSC group were 8.67.+ -. 0.82, indicating better recovery of hindlimb motor function.

Electrophysiological analysis was further performed to quantitatively characterize the motor function recovery of mice. As shown in FIGS. 10 a-b, the mean amplitudes of the Motor Evoked Potentials (MEPs) of SCI, GG, GG-LCS, GG-NSC and GG-LCS-NSC groups were 4.37.+ -. 1.15, 5.16.+ -. 0.83, 7.37.+ -. 1.20, 8.86.+ -. 1.66 and 11.30.+ -. 1.10. Mu.V, respectively, and it was found that the signal transduction function of mice of GG-LCS-NSC group was best. Taken together, the above results indicate that the GG-LCS-NSC group has the best ability to restore motor function in rats.

As shown in fig. 11, the untreated SCI group formed a large void in the defect, which is typical of traumatic spinal cord injury. The cavity structure interrupts the nerve signal transmission, and the lower limb movement function is difficult to recover. However, the group into which the neural construct is implanted may significantly reduce the lesion cavity, with the GG-NSC and GG-LCS-NSC groups being more effective. In particular, GG-LCS-NSC group, continuous neo-nerve tissue can be seen, and no obvious cavity structure exists.

Further, immunofluorescent staining was used to evaluate nerve regeneration at the lesion. As shown in FIGS. 12 a-d, only few Tuj1 positive neurons were observed in the SCI group, but the GFAP expression level was more, indicating that the SCI group was unfavorable for the regeneration of neurons due to the presence of a large-area cavity structure in the damaged area, and the formation of glial scar at the cavity hindered the connection and regeneration of neurons. In contrast, more Tujl positive neurons were observed in the GG-LCS-NSC group, while the GFAP expression level was lower, indicating that the GG-LCS-NSC group had the highest neuron density involved in spinal cord injury repair and the formation of glial scar was the weakest. In addition, immunofluorescent staining of CD31 was performed on the spinal cord injury area to assess vascularization of the injury area. As shown in FIG. 12 e-f, the GG-LCS-NSC construct has the ability to induce angiogenesis as shown by the increased area of CD 31-positive fluorescence in the GG-LCS-NSC group compared to the other groups. In summary, the LCS microsphere-based neural constructs can significantly promote nerve regeneration and vascularization, thereby repairing damaged spinal cord and restoring its physiological function, and exhibit great potential for use in promoting central nerve regeneration.

Example 6 bone repair effects of neural constructs according to the invention:

The ability of neural constructs to repair bone defects was investigated in this example 6 using the rat skull critical damage model. The following 5 groups were set: (1) blank (without any treatment); (2) GG (pure hydrogel scaffold), (3) GG-LCS (hydrogel scaffold containing LCS microspheres, content of LCS microspheres being 5wt% of dry weight of GelMA), (4) GG-NSC (containing NSCs, no LCS microspheres, cell density of 300 ten thousand/mL) and (5) GG-LCS-NSC (containing LCS microspheres and NSCs, cell density of 300 ten thousand/mL, content of LCS microspheres being 5wt% of dry weight of GelMA), were prepared as described in example 2. Firstly, injecting a proper dosage of chloral hydrate solution into the abdominal cavity to anesthetize the rat, then using a shaver to remove the hair of the head of the rat, wiping the rat clean by using normal saline and carrying out iodophor disinfection; gently swiping the head skin using a scalpel to expose the skull, incising the periosteum along the sagittal suture of the skull and separating the periosteum using forceps; then using a drilling hole with the diameter of 5mm to establish skull defects at two sides of the sagittal suture; the printed stent material is then implanted. 8 weeks after surgery, the skull was removed and immersed in 4% paraformaldehyde solution. The bone regeneration, the nerve innervation and the vascularization are analyzed by using methods such as Micro-CT, histological staining and the like.

In fig. 13a, the bone repair of the coronal and sagittal interfaces at each set of bone defects is shown (green: new bone). From this, it can be seen that the Blank group had only a small amount of bone formation at the edge of the defect, and a large void remained in the middle of the bone defect, indicating that it was difficult to self-heal. There was a different degree of bone regeneration in each group implanted with scaffolds, with the GG-LCS-NSC exhibiting the best bone repair characteristics, the newly generated bone densely distributed at the defect, indicating a higher quality of bone regeneration. Statistical analysis of bone regeneration in each group revealed that GG-LCS-NSC scaffolds had the highest fresh bone volume/total volume (BV/TV) and bone density (BMD) (c-d in FIG. 13). From the H & E staining results of fig. 13b, it can be seen that the Blank group only sees a small amount of new bone at the edges and the middle bone defect is filled with fibrous tissue. In each group of stent implants, different degrees of new bone formation can be observed. In particular, the area of new bone growth in the GG-LCS-NSC group is the largest. In addition, the new bone of the stent-implanted group can be tightly combined with surrounding host bone tissue, thus proving good osseointegration property.

The immunofluorescent staining results of FIG. 14 show that GG-LCS-NSC group has the highest density of OPN, NF, CD protein expression, demonstrating that GG-LCS-NSC group has the most excellent osteogenic, innervating and vascularizing capacity. The nerve construct based on LCS microspheres well achieved neurovascularized bone regeneration.

EXAMPLE 7 muscle repair Effect of the neural construct of the present invention

A volumetric muscle injury model (tibialis anterior (TA) defect) was developed in this example 7 to assess the ability of the neural construct to promote skeletal muscle regeneration and innervation. Male SD rats (body weight range 220-250 g) were selected for the preparation of tibial anterior muscle defects. The following 6 groups were set: (1) Sham group (intact group); (2) blank (without any treatment); (3) GG (pure hydrogel scaffold), (4) GG-LCS (hydrogel scaffold containing LCS microspheres, content of LCS microspheres being 5wt% of dry weight of GelMA), (5) GG-NSC (containing NSCs, no LCS microspheres, cell density of 300 ten thousand/mL) and (6) GG-LCS-NSC (containing LCS microspheres and NSCs, cell density of 300 ten thousand/mL, content of LCS microspheres being 5wt% of dry weight of GelMA), were prepared as described in example 2. The rats were first anesthetized and then the skin of the left hind limb was incised to expose the tibialis anterior. According to the formula: w (g) =0.0017×body weight (g) -0.0716 to resect the tibial anterior muscle, the weight of the resected muscle being about 40% of the total tibial anterior muscle. Each set of stents is then implanted into the defect and covered by skin.

Muscle regeneration rate analysis: after 8 weeks of implantation, the tibial muscle of the left hind limb of the rat was removed and weighed (M1), while the contralateral tibial muscle was removed and weighed (M2). The weight of the tibial muscle is calculated by the following company: TA mux weight=m1/m2×100%.

Histological analysis: muscle tissue was immersed in 4% paraformaldehyde solution and then sectioned for histological analysis. The muscle regeneration effect was analyzed by H & E staining, masson staining, immunofluorescence staining, and the like.

As shown in fig. 15 a, the Non-treated group showed significant muscle atrophy, while the group implanted with the scaffold showed some degree of muscle recovery. Statistical results showed that the TA muscle weights (contralateral percentages) of Sham, non-treated, GG-LCS, GG-NSC and GG-LCS-NSC groups were 99.41.+ -. 8.07%, 54.69.+ -. 7.78%, 67.03.+ -. 2.45%, 75.83.+ -. 3.59%, 83.01.+ -. 6.82% and 89.52.+ -. 5.29%, respectively (b in FIG. 15). It can be seen that the tibialis anterior muscle of the GG-NSC and GG-LCS-NSC groups has a volume close to that of the Sham group and that muscle repair is better. Overall, GG-NSC and GG-LCS-NSC groups showed the best muscle repair effect.

H & E staining and Masson trichromatic staining were used to further evaluate the effect and quality of muscle repair. As shown in fig. 15 c, sham group showed that the natural muscle consisted of directional arranged muscle fibers. The muscle repair condition of the Non-treated group and the GG group is poor, and the defect part still has obvious cavity structure and infiltrates fibrous tissues. In contrast, the GG-LCS-NSC group can observe that newly generated muscle fibers are filled in the defect part of the muscle, the muscle fibers are well arranged, and the GG-LCS-NSC group is integrated with surrounding host muscle tissues to a certain extent, so that better muscle repair effect is shown.

Longer and directionally distributed myofiber structures can be seen for the Sham group shown in fig. 16 a-b, while the Non-treated and GG groups have fewer and disordered amounts of neo-myofibers. In contrast, a large number of neomyofibers were observed in the GG-LCS-NSC group and distributed in a directional arrangement, indicating high quality muscle repair. In addition, GG-LCS-NSC group had high expression of CD31 protein (red fluorescence), indicating good degree of vascularization in neo-muscle tissue (c-d in FIG. 16). Finally, NF/AchR double immunofluorescent staining was used to assess neuromuscular junction formation in vivo. Higher densities of NF and AchR were observed in the GG-LCS-NSC group compared to the remaining groups, and co-localization of NF with AchR also confirmed the presence of neuromuscular junctions (e-g in fig. 16).

The above results indicate that the LCS microsphere based neural construct can effectively promote the repair of volumetric muscle injuries and restore vascularization and innervation effects at the site of the injury, and that the reformation of neuromuscular junctions contributes to the restoration of muscle contraction function, which is crucial for achieving functional muscle regeneration.

In summary, the scaffold of the present invention only contains neural stem cells, which confers the scaffold with multi-functional applicability. Firstly, it can be seen from fig. 6-7 that the neural construct has good in vivo neuron differentiation capability, so that the neural construct not only can promote the damage repair of nerve tissues, but also has a certain repair effect on the damage repair (such as bones, muscles and the like) of innervating tissues. From fig. 9-12, it can be seen that the neural construct is capable of well promoting repair of full-transection spinal cord injury, connecting damaged spinal cord tissue, restoring transmission of electrical signals, restoring motor functions of lower limbs of rats, and the like. In addition, the neural construct is effective in promoting osteogenic differentiation of bone marrow mesenchymal stem cells, vascularization of endothelial cells, and in vitro formation of neuromuscular junctions with muscle cells based on the multicellular regulatory ability of the nerve (fig. 8). On the basis, we further verify the in vivo repair effect of the nerve construct on the nerve innervation tissue injury on the animal experiment level. From fig. 13-14, it can be seen that the nerve construct can effectively promote the damage repair of the rat skull, promote the innervation and vascularization of the new bone tissue, and prove the application potential in hard tissue repair. In addition, as can be seen from fig. 15-16, the nerve construct can also effectively promote the regeneration of volumetric muscle injury of tibialis anterior, effectively restore the weight of muscle, promote the regeneration and maturation of muscle fibers, vascularization and formation of neuromuscular junctions, realize functional repair of muscle injury, and prove the application potential of the nerve construct in soft tissue repair.

Claims (12)

1. A nerve construct capable of promoting regeneration of various tissues, which is a three-dimensional composite scaffold prepared from an organic hydrogel matrix material, an inorganic bioactive material and nerve-related cells.

2. The nerve construct of claim 1, wherein the inorganic bioactive material comprises at least one of lithium, calcium, silicon, magnesium, manganese, molybdenum, zinc, copper, strontium, and iron; preferably, the inorganic bioactive material is microsphere with a particle size distribution of 0.1-100 μm.

3. The nerve construct of claim 2, wherein the inorganic bioactive material is an inorganic material comprising elements of lithium, calcium, and silicon.

4. A neural construct according to claim 3, wherein the inorganic bioactive material is a lithium calcium silicate bioceramic material, preferably lithium calcium silicate microspheres with a particle size distribution of 0.1-100 μm.

5. The neural construct of any of claims 1-4, wherein the inorganic bioactive material is present in an amount of 0.01 to 20wt%, preferably 3 to 10wt%, based on the weight of the organic hydrogel matrix material.

6. The nerve construct of any one of claims 1-5, wherein the organic hydrogel matrix material is at least one of gelatin, methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol.

7. The nerve construct of claim 6, wherein the organic hydrogel matrix material is at least one of methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol.

8. The nerve construct of any one of claims 1-7, wherein the nerve-related cell comprises: at least one of neural stem cells, glial cells, neurons, PC12 cells, preferably neural stem cells;

The cell density of the nerve cells in the nerve construct capable of promoting the regeneration of various tissues is 10-1000 ten thousand/mL.

9. A method of preparing a neural construct according to any one of claims 1 to 8, which promotes regeneration of a plurality of tissues, comprising:

(1) Mixing an inorganic bioactive material, an organic hydrogel matrix material, a photoinitiator, water and nerve cells to obtain inorganic bioactive ink;

(2) And (3) carrying out layer-by-layer deposition printing on the obtained inorganic bioactive ink after the pre-curing treatment, then carrying out cross-linking curing molding by using blue light, and finally culturing to obtain the nerve construct capable of promoting the regeneration of various tissues.

10. The preparation method according to claim 9, wherein when selecting the raw materials, the organic hydrogel matrix material is a complex of gelatin and at least one of methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol, preferably a complex of methacryloylated gelatin and gelatin; more preferably, the mass ratio of at least one of the methacryloylated gelatin, gellan gum, sodium alginate, hyaluronic acid, chitosan and polyethylene glycol to gelatin in the organic hydrogel matrix material is 10:1-1:10;

The photoinitiator comprises: at least one of phenyl 2,4, 6-trimethylbenzoyl lithium phosphinate and an ultraviolet initiator I2959; the addition amount of the photoinitiator is 0.1-0.5 wt% of the mass of water.

11. The method according to claim 9 or 10, wherein the parameters of the pre-curing treatment include: the temperature of the pre-curing treatment is-20 to 15 ℃, preferably 4 ℃; the time of the pre-curing treatment is 5-60 min, preferably 20min;

The parameters of the layer-by-layer deposition printing include: setting the temperature of the charging barrel to be 10-25 ℃, the temperature of the deposition platform to be 0-10 ℃, the printing needle head to be 12-27G, the moving speed of the needle head to be 1-12 mm/s, and the extrusion pressure to be 10-200 kPa;

the parameters of the culture include: the composition of the culture solution used comprises: 90 to 99.5vol% of DMEM/F12 basal medium, 1 to 10vol% of fetal calf serum and 0.5 to 2vol% of antibiotics; the temperature of the culture is 33-40 ℃; preferably, the culture medium is replaced every 2-3 days, and the antibiotic is penicillin streptomycin.

12. Use of the neural construct of any one of claims 1-8, for the preparation of a plurality of tissue regeneration materials, wherein the plurality of tissue regeneration materials comprises neural tissue regeneration material, bone tissue regeneration material and muscle tissue regeneration material;

Preferably, the nerve tissue regeneration material comprises spinal cord regeneration material and peripheral nerve regeneration material;

Preferably, the bone tissue regeneration material comprises skull regeneration material and femur regeneration material;

Preferably, the muscle tissue regeneration material comprises a volumetric injury repair material of the tibialis anterior.