CN114773692B - Active biogel capable of controlling NGF release for long term and application thereof - Google Patents
Active biogel capable of controlling NGF release for long term and application thereof Download PDFInfo
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- CN114773692B CN114773692B CN202210695751.4A CN202210695751A CN114773692B CN 114773692 B CN114773692 B CN 114773692B CN 202210695751 A CN202210695751 A CN 202210695751A CN 114773692 B CN114773692 B CN 114773692B
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Abstract
The invention belongs to the field of biomedical tissue engineering, and discloses an active biogel capable of controlling NGF release for a long time and application thereof. The active biological gel consists of NGF-gelatin-polyisopropylacrylamide hydrogel and NGF-chitosan hydrogel according to the volume ratio of 1:1.05-1:1.5, wherein the load capacity of NGF in the NGF-gelatin-polyisopropylacrylamide hydrogel is 100-200ng/ml, and the load capacity of NGF in the NGF-chitosan hydrogel is 100-200 ng/ml; the NGF-gelatin-polyisopropylacrylamide hydrogel and the NGF-chitosan hydrogel. The gel can be filled in a liquid sac cavity of an injury part, promotes angiogenesis, inhibits scar infiltration, inhibits inflammation, improves a microenvironment of the injury part, activates endogenous neural stem cells, induces the endogenous neural stem cells to be differentiated into mature neurons in a high proportion, and finally leads to improvement of dysfunction.
Description
Technical Field
The invention belongs to the field of biomedical tissue engineering, and particularly relates to an active biogel capable of controlling NGF release for a long time and application thereof.
Background
The central nervous system includes the brain and spinal cord, and brain and spinal cord injuries are the leading causes of death and disability in all ages of the population in all countries, with annual treatment costs of brain injuries amounting to about $ 4000 billion worldwide. This places a significant economic burden on the patient and society. Due to the diversity of brain injuries, pathophysiological changes and clinical treatment after the injury are complicated. The results of the last thirty years of research have shown that multipotent neural stem/progenitor cells (NSCs/NPCs) persist throughout the life cycle of mammals in specific regions of the brain and spinal cord, namely the central tubular regions of the annual large hippocampal Dentate Gyrus (DG), the subventricular zone (SVZ) and the spinal cord. These endogenous NSC/NPC may play regenerative and reparative roles after brain or spinal cord injury. However, after the central nervous system is damaged, the microenvironment of the damaged area becomes very severe due to rupture of blood vessels, edema, ischemia, hypoxia, infiltration and accumulation of inflammatory factors and the like, and the microenvironment is like saline-alkali soil, so that the proliferation of neural stem progenitor cells and the differentiation towards the direction of neurons are inhibited, the neural stem cells are driven to differentiate towards astrocytes, and finally, glial scars are formed. Further preventing the generation and functional recovery of neurons in the brain or spinal cord. Therefore, how to regulate and control the microenvironment in the damaged area and change the severe microenvironment (saline-alkali soil) into a good environment (black land) with anti-inflammation and pro-neuroprotection (neurotrophic and pro-neurogenesis) is one of the scientific problems to be solved at present. Many growth factors and neurotrophic factors (such as NGF \ IGF-1, bFGF, BDNF, etc.), and some peptides (such as Erythropoietin, Thymosin beta 4, etc.) can promote the development and regeneration of the central nervous system. However, most of the protein factors and peptides have extremely short half-life (seconds to minutes) in physiological environment, are not easy to be retained in the damaged part and hardly pass through the blood-cerebrospinal fluid barrier, so that the clinical utilization efficiency of the protein factors and peptides is extremely low.
Spinal cord injury in adult mammals not only destroys the original spinal cord anatomy, leading to cell death. Secondary injury (i.e., secondary injury) is also triggered by inflammation, demyelination and glial cell proliferation (scarring), among others, all of which ultimately result in loss of function below the level of the injury. The number of newly added spinal cord injury patients in China is as high as 12-14 ten thousand every year, the economic consumption of the treatment of the spinal cord injury is also remarkable, the cost of each spinal cord injury patient is 30-40 ten thousand dollars according to the age and the anatomical segment of the injury, and the clinical effective treatment means is still lacked.
The key reason for the inability to repair following brain and spinal cord injury is the inability of neurons to regenerate. Currently, filling of damaged areas of the brain or spinal cord with biomaterial scaffold-gels is a widely accepted method of treating central nervous system injury. The biomaterial scaffolds commonly used are various hydrogels. For example, chinese patent CN 112933293 a discloses an in situ injectable hydrogel for treating central nervous system injury and a preparation method thereof, wherein the hydrogel is formed by chemically coupling multi-arm polyethylene glycol-X and multi-arm polyethylene glycol-Y modified by arginine-glycine-aspartic acid via functional groups to X-Y electrodes, and nano/micro particles of sustained-release immunomodulatory and/or antioxidant drugs and growth factors loaded on the hydrogel through chemical coupling reaction are coated in the hydrogel, but no corresponding convention and explanation are made for the optimal action concentration and controlled release time course of the growth factors, nor are comparisons made between biological actions of various growth factors. Chinese patent CN101301494A discloses a hydrogel material for repairing central nerve and a preparation method thereof, wherein 1-5wt% of polyethylene glycol aqueous solution, 0.1-2wt% of polylysine or staphylococcal protein A and nano molten iron oxide aqueous solution are mixed and stirred in a shaking way; adding glutaraldehyde solution until the concentration reaches 1-4wt%, and reacting at 4 deg.C for 2-24 hr; and then freeze-dried. The patent does not provide any convention and explanation as to whether the gel can promote the generation of neurons, and secondly the hydrogel does not provide biochemical clues such as neurotrophic factors required for the regeneration, migration and directional growth of processes of the neurons, does not promote the regeneration of blood vessels and inhibit the infiltration of scars, and the initiator-glutaraldehyde can have adverse effects in vivo. Chinese patent CN 107205809 a discloses a biological compound for nerve bundle regeneration comprising a degradable and biocompatible implantable tubular hybrid structure, a method of said biological compound and its use for regenerating nerve bundles in diseases affecting the central nervous system, preferably parkinson's disease. In the tubular inner cavity thereofContaining growth factors (neurotrophic factors NGF, BDNF or GDNF) and/or drugs (dopaminergic agents) and using initiators including glutaraldehyde. The patent lacks the convention and explanation on the optimal acting concentration, proportion and controlled release time course of various growth factors. The biological mixture takes fibrin, collagen or agarose as main components, has the defects of poor mechanical property, high degradation speed and the like, and has the key function of being incapable of activating endogenous neural stem cells and inducing the differentiation of the cells into neurons. Chinese patent CN 104910391 a discloses a preparation method of biodegradable hydrogel with bioactivity. The invention takes poly (lactide-co-ethylene oxide-co-fumaric acid as a hydrogel framework and a polypeptide cross-linking agent (Ac) 2 KYIGSRG is crosslinked, and GIKAVAK-Ac polypeptide and thiol-substituted Nogo-66 polypeptide are grafted to form degradable hydrogel which can promote the adhesion and growth of nerve cells. The patent does not make an appointment and explanation on whether the hydrogel can activate endogenous NSC, induce the endogenous NSC to differentiate into neurons, promote the regeneration of neurites and angiogenesis, inhibit scar infiltration and the like, and does not identify and make an appointment on the release time course and the biological activity of the Nogo-66 polypeptide combined with the hydrogel.
Disclosure of Invention
The invention aims to provide an active biological gel which is used for treating central nervous system injury and diseases and can control NGF release for a long time and a preparation method thereof. The gel can be filled in a liquid sac cavity of an injured part, promote angiogenesis, inhibit scar infiltration, inhibit inflammation, improve microenvironment of the injured part, activate endogenous neural stem cells, induce high-proportion differentiation of the endogenous neural stem cells into mature neurons, and finally cause improvement of dysfunction.
The preparation method of the active biological gel which is provided by the invention and can control NGF release for a long time and is used for treating central nervous system injury and diseases comprises the following steps:
(1) modifying the type I collagen to obtain gelatin dry powder;
(2) preparing the gelatin-polyisopropylacrylamide hydrogel by using the gelatin dry powder in the step (1);
(3) preparing chitosan hydrogel;
(4) under the action of an initiator, reacting the gelatin-polyisopropylacrylamide hydrogel prepared in the step (2) with Nerve Growth Factor (NGF), neurotrophin 3 (NT-3) and basic fibroblast growth factor (bFGF) respectively to obtain Nerve Growth Factor (NGF) -gelatin-polyisopropylacrylamide hydrogel, neurotrophin 3 (NT-3) -gelatin-polyisopropylacrylamide hydrogel and basic fibroblast growth factor (bFGF) -gelatin-polyisopropylacrylamide hydrogel respectively;
(5) under the action of an initiator, reacting the chitosan hydrogel prepared in the step (3) with Nerve Growth Factor (NGF), neurotrophin 3 (NT-3) and basic fibroblast growth factor (bFGF) respectively to obtain Nerve Growth Factor (NGF) -chitosan gel, neurotrophin 3 (NT-3) -chitosan gel and basic fibroblast growth factor (bFGF) -chitosan gel;
in the step (1), the collagen I is modified by modifying the collagen I with hydrogen peroxide; the specific modification method comprises the following steps: weighing a certain amount of type I collagen at room temperature, dissolving in hydrogen peroxide solution to prepare gelatin solution, and freeze-drying to obtain gelatin dry powder.
Preferably, the concentration of the hydrogen peroxide may be 0.02 to 1mmol, further 0.1 to 0.8mmol, and particularly 0.5 mmol.
The proportion of the type I collagen to the hydrogen peroxide solution is 1 g: 0.01L.
In order to fully dissolve the type I collagen, the dissolving temperature is 48-85 ℃, and the dissolving time can be 4-12 hours, particularly 8 hours.
The freeze-drying time may be 3 to 6 hours, specifically 5 hours.
In the step (2), the method for preparing the gelatin-polyisopropylacrylamide hydrogel specifically comprises the following steps: dissolving the gelatin dry powder, N-isopropylacrylamide (NIPAM), Tetramethylethylenediamine (TEMED) and N, N-subunit Bisacrylamide (BIS) in deionized water, introducing nitrogen, stirring, adding genipin after complete dissolution, rapidly and uniformly stirring, and standing at room temperature for 2-4 hours to obtain hydrogel; soaking the hydrogel in deionized water for 48-60 hours, wherein the deionized water is thoroughly replaced every 30 minutes in the soaking process; then taking out the hydrogel and soaking the hydrogel into deionized water at the temperature of 40-46 ℃, soaking for 48-60 hours, and completely replacing the deionized water every 30 minutes to remove residual monomers; and finally, placing the obtained gelatin-polyisopropylacrylamide hydrogel in deionized water for storage at 4 ℃ for later use.
In the above method, the mass ratio of the gelatin dry powder to N-isopropylacrylamide (NIPAM), Tetramethylethylenediamine (TEMED) and N, N-subunit Bisacrylamide (BIS) can be (1: 0.02), (1: 0.001) and (1: 0.05).
The ratio of genipin to gelatin is 8 mmol: 1g of the total weight of the composition.
In the step (3), the method for preparing the chitosan hydrogel comprises the following steps: and (3) carrying out crosslinking reaction on the chitosan solution under the condition that the pH value is 4.0-5.5 by using genipin as a crosslinking agent to obtain the chitosan hydrogel.
The dosage ratio of the chitosan to the genipin is (3 g:1.6 mol).
The crosslinking reaction is carried out at room temperature, and the time of the crosslinking reaction can be 60-72 h.
According to one embodiment of the present invention, a method of preparing a chitosan hydrogel is as follows: dissolving chitosan in 1% acetic acid solution to prepare 3% chitosan solution, and eluting residual acetic acid; dissolving genipin in sterile deionized water to prepare 8mmol/L genipin solution; mixing 3% chitosan solution and 8mmol/L genipin solution according to a volume ratio of 1:0.002, slowly stirring the mixed solution at 4 ℃ for 60-72h to obtain chitosan hydrogel, and storing in a refrigerator at 4 ℃ for later use.
In the step (4), the initiator is genipin or a mixture of genipin and genipin in a mass ratio of 1: (0.1-1) a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;
the concentration of the nerve growth factor solution is 20-300ng/ml, the mass concentration of the initiator is 0.1-1%,
the reaction conditions of the reaction are 4 ℃ environment.
The gelatin-polyisopropylacrylamide hydrogel acts with Nerve Growth Factor (NGF) with different doses (20-300 ng/ml), and the optimum acting dose is (100-200 ng/ml);
the gelatin-polyisopropylacrylamide hydrogel acts with the neurotrophin 3 (NT-3) with different doses (20-300 ng/ml), and the most suitable acting dose is NT-3-gelatin-polyisopropylacrylamide hydrogel with the dose of (100-;
the gelatin-polyisopropylacrylamide hydrogel acts with different dosages (20-300 ng/ml) of basic fibroblast growth factor (bFGF), and the optimal acting dosage is 50-100ng/ml of bFGF-gelatin-polyisopropylacrylamide hydrogel.
In the above process step (5), the initiator is selected from genipin.
The reaction conditions of the reaction are 4 ℃ environment.
The chitosan hydrogel acts with Nerve Growth Factor (NGF) with different doses (20-300 ng/ml), and NGF-chitosan hydrogel with the acting dose of (100-200ng/ml) is selected;
the chitosan hydrogel acts with neurotrophin 3 (NT-3) with different doses (20-300 ng/ml), and NT-3-chitosan hydrogel with the acting dose of (100-;
the chitosan hydrogel is acted with different dosages (20-300 ng/ml) of basic fibroblast growth factor (bFGF), and bFGF-chitosan hydrogel with the acting dosage (50-100ng/ml) is selected.
In the above step (4) and step (5), the optimum dose is: 1) the method is characterized in that the in vitro and brain-derived neural stem cells are co-cultured for 14 days, and the gels carrying different dosages of NGF, NT-3 and bFGF respectively promote NSC to mature neurons (MAP 2) + ) Number of differentiation, length of mature neuronal processAnd quantitatively analyzing and comparing the number of the proliferated NSCs to determine an optimal action range, namely the NGF/NT-3/bFGF-gel which embodies the optimal action range can respectively induce the NSCs to differentiate to neurons at a high proportion to the maximum extent, promote the growth and extension of neuron processes and promote the NSCs to proliferate and renew continuously. 2) Meanwhile, through an in-vivo implantation (implantation in a spinal cord injury area or a brain injury area) experiment, the effect of activating endogenous Neural Stem Cells (NSCs) and recruiting the migration of the cells to the injury area to be mature neurons by quantitative analysis and comparison of NGF/NT-3/bFGF-gel loaded with different doses is evaluated within half a year of implantation; quantitatively analyzing the effects of the NGF/NT-3/bFGF-gels loaded with different doses on promoting angiogenesis, inhibiting scar infiltration and inhibiting inflammatory infiltration respectively, and finally screening the NGF/NT-3-gel with the optimal dose (100-200ng/ml) and the bFGF-gel with the optimal dose (50-100 ng/ml).
The invention also protects an active biogel.
The invention provides a certain active biological gel, which consists of a gel A and a gel B,
wherein the gel A is selected from any one of the following: an optimal dosage (100 and 200ng/ml) of NGF-polyisopropylacrylamide hydrogel, an optimal dosage (100 and 200ng/ml) of NT-3-polyisopropylacrylamide hydrogel and an optimal dosage (50 to 100ng/ml) of bFGF-polyisopropylacrylamide hydrogel;
the gel B is selected from any one of the following: NGF-chitosan hydrogel with the selected dose (100-;
the growth factors contained in the gel A and the gel B are the same, namely both contain NGF or both contain NT-3 or both contain bFGF;
the ratio (volume ratio) of the gel A to the gel B is 1:1.05-1:1.5, and the ratio of the gel A to the gel B is 1: 1.05.
Specifically, the active biogel is selected from any one of the following:
1) NGF active biological gel consists of NGF gel A and NGF gel B,
wherein the NGF gel A is selected from NGF-gelatin-polyisopropylacrylamide hydrogel with optimal dose (100-200ng/ml), and the NGF gel B is selected from NGF-chitosan hydrogel with dose (100-200 ng/ml); the ratio (volume ratio) of the NGF gel A to the NGF gel B is 1: 1.05.
2) The NT-3 active biogel consists of NT-3 gel A and NT-3 gel B,
wherein the NT-3 gel A is selected from NT-3-gelatin-polyisopropylacrylamide hydrogel with optimal dose (100-; the ratio (volume ratio) of the NT-3 gel A to the NT-3 gel B is 1: 1.05.
3) bFGF active biological gel, which consists of bFGF gel A and bFGF gel B,
wherein, the bFGF gel A is selected from bFGF-gelatin-polyisopropylacrylamide hydrogel with optimal dose (50-100ng/ml), and the bFGF gel B is selected from bFGF-chitosan hydrogel with dose (50-100 ng/ml); the ratio (volume ratio) of the gel A to the gel B is 1: 1.05.
The active biogel can be used for reconstructing or repairing tissue or organ defects. Including but not limited to neural tissue.
Two NGF active biogels (polyisopropylacrylamide hydrogel and chitosan gel) with optimal acting dosage (100-200ng/ml) are mixed and used according to an optimal ratio (1: 1.05), namely the NGF active biogel (100-200ng/ml) is co-cultured with the brain-derived neural stem cells in vitro and detected by an enzyme-linked immunosorbent assay (Elisa): NGF (100-200ng/ml) active biogel can control long-term and stable NGF release for up to 16 weeks under physiological environment (FIG. 4);
two NT-3-active biogels (poly isopropyl acrylamide hydrogel and chitosan gel) with the optimal dosage (100-200ng/ml) are mixed and used according to the optimal proportion (1: 1.05), namely the NT-3(100-200ng/ml) active biogel is co-cultured with spinal cord-derived or brain-derived neural stem cells in vitro and detected by enzyme linked immunosorbent assay (Elisa): it can control the long-term and stable release of NT-3 for up to 14 weeks in physiological environment (figure 5);
two bFGF-active biogels with the optimal dosage (50-100ng/ml) are mixed according to the optimal proportion (1: 1.05) and used, namely the bFGF- (50-100ng/ml) active biogel is co-cultured with spinal cord-derived or brain-derived neural stem cells in vitro and detected by an enzyme-linked immunosorbent assay (Elisa): it can control the long-term and stable release of bFGF for up to 5 weeks in physiological environment (FIG. 6);
the optimal action active biological gel is the optimal action active biological gel which is screened out by co-culture and/or in-vivo implantation experiments of in vitro and neural stem cells respectively, can obviously promote the brain-derived neural stem cells to differentiate to neurons in high proportion, promote the growth of neuron processes, activate endogenous neural stem cells after adult spinal cords or brain injuries, recruit the endogenous neural stem cells to injury areas, differentiate to neurons, inhibit inflammatory reaction and inhibit scar infiltration, namely NGF (100-200ng/ml) -active biogel, which comprises two parts, namely 1) NGF (100-200ng/ml) -gelatin-polyisopropylacrylamide hydrogel has a biological guide effect, can simulate the development and growth environment of neurons, activate endogenous neural stem cells for a long time, induce the endogenous neural stem cells to differentiate into mature neurons in a high proportion; simultaneously, biochemical clues can be provided for the directional migration of the neurons and the directional growth of the neurites; 2) NGF (100-200ng/ml) -chitosan gel provides physical and chemical support for recruitment, adhesion and migration of nerve cells, and can inhibit infiltration of inflammatory cells and promote angiogenesis. Mixing the two NGF-active biological gels (polyisopropylacrylamide hydrogel and chitosan gel) in the 1) and the 2) according to the proportion (1: 1.05-1: 1.5) to obtain the NGF (100-200ng/ml) active biological gel, wherein the NGF can be controllably released for 16 weeks in a physiological environment, and the NGF active biological gel has optimal effects on the aspects of promoting the high-proportion differentiation of brain-derived or spinal cord-derived neural stem cells to neurons, promoting the growth of neuron processes, activating endogenous nerve generation after adult spinal cords or brain injury, inhibiting inflammatory reaction, inhibiting scar infiltration and the like.
The invention also protects the application of the active biological gel.
The application is the application in the following 1) and/or 2):
1) the application in preparing products for reconstructing or repairing tissue or organ defects;
2) the application in preparing products for treating central nervous system injury and diseases;
in the above applications, the tissue includes, but is not limited to, neural tissue.
Such central nervous system lesions include, but are not limited to, different types of brain or spinal cord injuries.
Such products include, but are not limited to, biomaterial scaffolds.
The reconstruction or repair of a tissue or organ defect is embodied in at least one of the following aspects:
1) activating endogenous neural stem cells, and recruiting the cells to migrate to the damaged area of the brain or spinal cord;
2) a high proportion of the cells differentiate into mature neurons;
3) promoting the regeneration of blood vessels;
4) inhibiting inflammatory response;
5) inhibiting scar infiltration.
The active biogel for treating central nervous system injury and disease disclosed by the invention, which can control NGF release for a long time, has a simple preparation method, can be repeatedly suitable for different types of brain or spinal cord injuries, can control NGF release in a physiological environment for 16 weeks, greatly activates endogenous neural stem cells, recruits the endogenous neural stem cells to a brain or spinal cord injury area, differentiates into mature neurons in a high proportion, promotes blood vessel regeneration, inhibits inflammatory reaction, inhibits scar infiltration, and finally improves dysfunction.
Drawings
FIG. 1 is a scanning electron micrograph of NGF-gelatin-polyisopropylacrylamide hydrogel in example 1.
FIG. 2 is a scanning electron micrograph of NGF-chitosan gel in example 4.
FIG. 3 is a photograph showing the identification of the activity of the biogel in example 11, which can control NGF release over a long period of time.
FIG. 4 is a controlled release profile of NGF-active biogel loaded at 100 ng/ml; wherein, S NGF represents NGF freeze-dried powder solution; NGF-gel representative: NGF (100ng/ml) -gelatin-polyisopropylacrylamide hydrogel and NGF (100ng/ml) -chitosan gel are mixed into NGF-active biological gel in the optimal action ratio of 1: 1.05; note: the graph shows the S NGF curve superimposed on the abscissa.
FIG. 5 is a controlled release profile of NT-3-active biogel loaded at 100 ng/ml; wherein, S NT-3 represents NT-3 freeze-dried powder solution; NT-3-gel represents: NT-3-active biogel which is formed by mixing NT-3(100ng/ml) -gelatin-polyisopropylacrylamide hydrogel and NT-3(100ng/ml) -chitosan gel according to the optimal action ratio of 1: 1.05; note: the graph of NT-3 coincides with the abscissa.
FIG. 6 is a controlled release profile of a bFGF-active biogel loaded at 100 ng/ml; wherein, S bFGF represents bFGF freeze-dried powder solution; bFGF-gel representation: the best action proportion of the bFGF (100ng/ml) -gelatin-polyisopropylacrylamide hydrogel to the bFGF (100ng/ml) -chitosan gel is 1: 1.05; note: the curve of sbfgf coincides with the abscissa in the figure.
FIG. 7 is a quantitative analysis of the high rate of differentiation of neural stem cells into neurons promoted by gelatin-polyisopropylacrylamide hydrogels loaded with different doses (20-300 ng/ml) of NGF.
FIG. 8 is a quantitative analysis of the growth of neurites promoted by gelatin-polyisopropylacrylamide hydrogels loaded with different doses (20-300 ng/ml) of NGF.
FIG. 9 is a quantitative analysis of the differentiation of the bioactive gel loaded with different doses of NGF into mature neurons at half a year after surgery, respectively, in activating endogenous Neural Stem Cells (NSCs) and recruiting them to migrate to the lesion area.
FIG. 10 is a quantitative analysis of the effect of bioactive gels loaded with different doses of NGF in promoting revascularization half a year after surgery.
FIG. 11 is a quantitative analysis of the effect of bioactive gels loaded with different doses of NGF in inhibiting inflammatory infiltration half a year after surgery.
Figure 12 is a quantitative analysis of the effect of bioactive gels loaded with different doses of NGF in inhibiting scar infiltration half a year after surgery.
FIG. 13 is a quantitative analysis of the ability of gelatin-polyisopropylacrylamide hydrogels of different dosages of NT-3 to promote neurite outgrowth.
FIG. 14 is a quantitative analysis of the high rate of differentiation of neural stem cells into neurons promoted by gelatin-polyisopropylacrylamide hydrogels with different dosages of NT-3.
FIG. 15 is a quantitative analysis of the differentiation of NT-3-gelatin-polyisopropylacrylamide hydrogels loaded with different doses into mature neurons upon activation of endogenous Neural Stem Cells (NSCs) and recruitment of migration to the lesion field, respectively, half a year after surgery.
FIG. 16 is a quantitative analysis of the high rate of differentiation of neural stem cells into neurons promoted by gelatin-polyisopropylacrylamide hydrogels loaded with different doses of bFGF.
FIG. 17 is a graph of the quantitative analysis of the neurite outgrowth promoting ability of gelatin-polyisopropylacrylamide hydrogels loaded with different doses of bFGF.
FIG. 18 is a quantitative analysis of the differentiation of the gelatin bioactive gel loaded with different doses of bFGF into mature neurons at half a year post-surgery, activating endogenous Neural Stem Cells (NSCs) and recruiting them to migrate to the lesion area.
FIG. 19 is a quantitative analysis of differentiation of NGF/NT-3/bFGF-bioactive gels into mature neurons at half a year post-surgery, respectively, upon activation of endogenous Neural Stem Cells (NSCs) for recruitment to lesion differentiation.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
The Nerve Growth factor used in the following examples is commonly known as Mouse Nerve Growth factor for Injection (Mouse Nerve Growth for Injection) under the trade name of Jinlujie, produced by Wuhanhai Biopharmaceutical Co., Ltd., available from national drug group chemical Co., Ltd., specification of 20ug (9000 AU)/vial.
The generic name of Neurotrophin 3 (NT-3) used in the following examples is Recombinant Human Neurotrophin 3(Recombinant Human neurotropin-3, manufactured by PROSPEC Inc., USA, with a specification of 2 ug/bottle.
The basic fibroblast growth factor (bFGF) used in the following examples is commonly known as Recombinant Human bFGF Protein (Recombinant Human bFGF Protein) and manufactured by Hitachi Hayazai BioProvisions Inc. at a specification of 50 ug/vial.
The devices and the like used in the following examples are commercially available or commonly used in the industry.
Example 1: preparation of NGF-gelatin-polyisopropylacrylamide hydrogel
The active gelatin-polyisopropylacrylamide hydrogel is prepared by the following steps
(1) Weighing a certain amount (1 g) of type I collagen at room temperature, dissolving in 10mL of 0.5mmol hydrogen peroxide solution, fully dissolving at 60 deg.C for 8 hr to obtain gelatin solution, and freeze drying for 5 hr to obtain gelatin solid powder.
(2) Dissolving 1g of gelatin solid powder, 0.02g of N-isopropylacrylamide (NIPAM), 0.001g of Tetramethylethylenediamine (TEMED) and 0.05g of N, N-subunit Bisacrylamide (BIS) in 10mL of deionized water, introducing nitrogen, stirring, adding 0.002mL of 8mmol of genipin after complete dissolution, quickly stirring uniformly, standing at room temperature for 2-4 hours to prepare hydrogel, soaking the hydrogel in deionized water at normal temperature for 48-60 hours, and completely replacing the deionized water once every 30 minutes in the soaking process. Then taking out the hydrogel and soaking the hydrogel in deionized water at the temperature of 40-46 ℃ for 48-60 hours, and completely replacing the deionized water every 30 minutes to remove residual monomers. Finally, the hydrogel is placed in deionized water and stored at 4 ℃ for standby.
(3) And (3) under the action of an initiator-8 mmol genipin, reacting the gelatin-polyisopropylacrylamide hydrogel prepared in the step (2) with Nerve Growth Factors (NGF) with different dosages (20-300 ng/ml, namely 1ml of hydrogel corresponds to 20-300ng of NGF), so as to obtain the NGF-gelatin-polyisopropylacrylamide hydrogel loaded with different dosages (20-300 ng/ml). The scanning electron micrograph is shown in FIG. 1 (the dose of NGF is 100 ng/ml).
Through in vitro and spinal cord-derived or brain-derived neural stem cell co-culture and in vivo implantation (implantation in spinal cord injury area or brain injury area) experiments, the NGF-gelatin-polyisopropylacrylamide hydrogel with the optimal acting dose (100-200ng/ml) is screened out.
The specific experimental method is as follows:
1) through in vitro co-culture with brain-derived neural stem cells for 14 days, NGF-gelatin-polyisopropylacrylamide hydrogel loaded with different doses of NGF is respectively promoted to mature neurons from NSC (MAP 2) + ) The differentiation quantity, the length of the mature neuron processes and the quantity of the proliferated NSCs are quantitatively analyzed and compared to determine the optimal action range, namely the NGF-gel embodying the optimal action range can maximally induce the NSCs to differentiate to the neurons in a high proportion, promote the growth and extension of the neuron processes and promote the NSCs to proliferate and renew continuously.
Specific experimental methods reference published articles: yang Z, Duan H, Mo L, et al, The effect of The dock of NT-3/chitosan carriers on The promotion and differentiation of neural stem cells [ J ]. Biomaterials, 2010, 31(18):4846.
The results are shown below:
FIG. 7 is a quantitative analysis of the high rate of differentiation of neural stem cells into neurons promoted by gelatin-polyisopropylacrylamide hydrogel loaded with different doses of NGF; as can be seen from FIG. 7, the NGF-gelatin-polyisopropylacrylamide hydrogel applied at a dose (100-200ng/ml) maximally induced the NSC differentiation to neurons at a high rate.
FIG. 8 is a graph of the quantitative analysis of the growth of neurites promoted by gelatin-polyisopropylacrylamide hydrogels loaded with different doses of NGF; as can be seen from FIG. 8, the NGF-gelatin-polyisopropylacrylamide hydrogel at the dose of 100-200ng/ml maximally promoted neurite outgrowth.
2) Meanwhile, the effect of gelatin-polyisopropylacrylamide hydrogel loaded with different doses of NGF on activating endogenous Neural Stem Cells (NSCs) and recruiting the NGF to the injury region to differentiate into mature neurons is evaluated within half a year through an in vivo implantation (implantation in a spinal cord injury region) experiment; quantitatively analyzing the effects of gelatin-polyisopropylacrylamide hydrogel loaded with different doses of NGF on promoting angiogenesis, inhibiting inflammatory infiltration and inhibiting scar infiltration respectively, and finally screening the NGF biogel with the optimal dose (100-200ng/ml) (figure 10-figure 12).
Specific experimental methods can be referred to as follows: yang Z, Zhang A, Duan H, et al NT3-chitosan elastomers to enable functional recovery after screw core in J. Proceedings of the National Academy of Sciences of the United States of America (PNAS), 2015, 112(43) 13354 and 13359.
2.Hao P, Duan H, Hao F, et al. Neural repair by NT3-chitosan via enhancement of endogenous neurogenesis after adult focal aspiration brain injury[J]. Biomaterials, 2017, 140:88-102.
Briefly described as follows: immunofluorescent staining of BrdU/Tuj1 and BrdU/NeuN neonatal neurons
Samples were taken at a specific time after surgery and immunofluorescent staining was performed, and all stained samples were subjected to sequential scanning by confocal laser microscopy in order to quantify double positive cells.
Each BrdU + cell was detected under high power microscopy on the full z-axis manually, and only cells co-labeled with the specific target protein marker were scored as double positive cells. The cell number is expressed as: pieces/mm 2. All data are expressed as means ± standard deviation,
SPSS is used for statistical analysis, the Shapiro-Wilk method detects the data normal distribution condition, Leven detects the homogeneity of variance, the one-factor variance analysis compares the difference among multiple groups, and the p is less than 0.05 and is considered to have statistical difference.
The results are shown below:
FIG. 9 shows the quantitative analysis of the differentiation of gelatin-polyisopropylacrylamide hydrogel loaded with different doses of NGF into mature neurons during half a year after operation, respectively, after the activation of endogenous Neural Stem Cells (NSCs) and the recruitment of the cells to the lesion, and the results suggest that gelatin-polyisopropylacrylamide hydrogel with 100-200ng/ml of NGF has the best effect of promoting the differentiation of endogenous neural stem cells into neurons.
FIGS. 10-12 are quantitative analyses of the effects of gelatin-polyisopropylacrylamide hydrogels loaded with different doses of NGF on promoting revascularization, inhibiting inflammatory infiltration, and inhibiting scar infiltration, respectively, at half a year after surgery, suggesting that: 100-200ng/ml NGF-gel has the best effects in promoting angiogenesis, inhibiting inflammatory infiltration and inhibiting scar infiltration.
Example 2: preparation of NT-3-gelatin-polyisopropylacrylamide hydrogel
The active gelatin-polyisopropylacrylamide hydrogel is prepared by the following steps
Steps (1) to (2) were the same as in example 1.
(3) And (3) under the action of an initiator-8 mmol genipin, reacting the gelatin-polyisopropylacrylamide hydrogel prepared in the step (2) with different doses (20-300 ng/ml) of neurotrophin 3 (NT-3) respectively to obtain the active gelatin-polyisopropylacrylamide hydrogel loaded with different doses (20-300 ng/ml) of NGF.
The optimum dose (100-200ng/ml) of NT-3-gelatin-polyisopropylacrylamide hydrogel (FIG. 13-FIG. 14) was selected by in vitro co-culture with brain-derived neural stem cells (the specific experimental method is the same as that in example 1). In half a year after operation, the effect of the gelatin-polyisopropylacrylamide hydrogel carrying NT-3 with different doses on the activation of endogenous neurogenesis to generate new neurons is quantitatively analyzed, and finally the NT-3-gelatin-polyisopropylacrylamide hydrogel with the optimal acting dose (100-200ng/ml) is screened out (FIG. 15), and the specific experimental method is the same as that in example 1.
The results are shown below:
FIGS. 13 to 14 are quantitative analyses of the ability of gelatin-polyisopropylacrylamide hydrogels loaded with different dosages of NT-3 to promote the differentiation of neural stem cells into neurons at a high ratio and promote the growth of processes; as can be seen from FIG. 13, the NT-3-gelatin-polyisopropylacrylamide hydrogel with the acting dose (100-200ng/ml) can maximally induce the outgrowth of NSC and neurons; as shown in FIG. 14, the NT-3-gelatin-polyisopropylacrylamide hydrogel applied at a dose of 100-200ng/ml was able to maximally induce NSC differentiation toward neurons at a high rate.
Fig. 15 is a quantitative analysis of the differentiation of NT-3-gelatin-polyisopropylacrylamide hydrogel loaded with different doses into mature neurons at half a year after surgery, respectively, in the activation of endogenous Neural Stem Cells (NSCs) to recruit their migration to the lesion field, and the results suggest: the NT-3-gelatin-polyisopropylacrylamide hydrogel with the concentration of 100-200ng/ml has the best effect of promoting the differentiation of endogenous neural stem cells into neurons.
Example 3: preparation of bFGF-gelatin-polyisopropylacrylamide hydrogel
The active gelatin-polyisopropylacrylamide hydrogel is prepared by the following steps
Steps (1) to (2) were the same as in example 1.
(3) And (3) under the action of an initiator-8 mmol genipin, reacting the gelatin-polyisopropylacrylamide hydrogel prepared in the step (2) with different doses (20-300 ng/ml) of basic fibroblast growth factor (bFGF) respectively to obtain the active gelatin-polyisopropylacrylamide hydrogel loaded with different doses (20-300 ng/ml) of bFGF.
Through in vitro and spinal cord-derived or brain-derived neural stem cell co-culture experiments (the specific experimental method is the same as that in example 1), the bFGF-gelatin-polyisopropylacrylamide hydrogel with the optimal acting dose (50-100ng/ml) is screened out.
The results are shown below:
FIGS. 16 to 17 are graphs showing the quantitative analysis of the ability of gelatin-polyisopropylacrylamide hydrogel loaded with different doses of bFGF to promote the differentiation of neural stem cells into neurons at a high ratio and to promote outgrowth of processes; as can be seen from FIG. 16, the bFGF-gelatin-polyisopropylacrylamide hydrogel applied at a dose (50-100ng/ml) maximally induced the NSC to differentiate toward neurons at a high rate; as can be seen from FIG. 17, the bFGF-gelatin-polyisopropylacrylamide hydrogel applied at a dose (50-100ng/ml) maximally induced the outgrowth of NSCs and neurons.
Fig. 18 is a quantitative analysis of the differentiation of gelatin-polyisopropylacrylamide hydrogels loaded with different doses of bFGF into mature neurons upon activation of endogenous Neural Stem Cells (NSCs) recruiting their migration to the lesion differentiation at half a year post-surgery, with the results suggesting: the gelatin-polyisopropylacrylamide hydrogel of 50-100ng/ml bFGF has the best effect of promoting the differentiation of endogenous neural stem cells into neurons.
Example 4: preparation of NGF-chitosan gel
NGF-chitosan gel was prepared as follows
(4) Dissolving chitosan in acetic acid solution with volume fraction of 1%, preparing chitosan solution with mass fraction of 3%, and eluting residual acetic acid. And dissolving genipin in sterile deionized water to prepare 8mmol/L genipin solution. Mixing a 3% chitosan solution and a 8mmol/L genipin solution according to the volume ratio of 1:0.002, and slowly stirring the mixed solution at 4 ℃ for 60-72h to obtain the chitosan hydrogel.
(5) And (3) respectively reacting the chitosan gel prepared in the step (4) with different dosages (20-300 ng/ml) of Nerve Growth Factor (NGF) under the action of an initiator-8 mmol of genipin to obtain NGF-chitosan gel loaded with different dosages (20-300 ng/ml). The scanning electron micrograph thereof is shown in FIG. 2.
Example 5: preparation of NT-3-Chitosan gel
NT-3-chitosan gel was prepared as follows
Step (4) was the same as in example 4.
(5) And (3) reacting the chitosan gel prepared in the step (4) with different dosages (20-300 ng/ml) of neurotrophin 3 (NT-3) under the action of an initiator-8 mmol of genipin to obtain NT-3-chitosan gel loaded with different dosages (20-300 ng/ml).
Example 6: preparation of bFGF-Chitosan gel
The bFGF-chitosan gel was prepared as follows
Step (4) was the same as in example 4.
(5) And (3) reacting the chitosan gel prepared in the step (4) with different dosages (20-300 ng/ml) of basic fibroblast growth factor (bFGF) under the action of an initiator-8 mmol of genipin to obtain the bFGF-chitosan gel loaded with different dosages (20-300 ng/ml).
Example 7: preparation of biological gel capable of controlling NGF release for long time for treating central nervous system injury and disease
The optimum dose (100-200ng/ml) of NGF-gelatin-polyisopropylacrylamide hydrogel prepared in example 1 was mixed with the optimum dose (100-200ng/ml) of NGF-chitosan gel prepared in example 4 in a ratio (volume ratio) of 1: 1.05.
Co-culturing the mixed gel with spinal cord-derived or brain-derived neural stem cells in vitro, and detecting by an enzyme-linked immunosorbent assay (Elisa): NGF (100-200ng/ml) -active biogel which can control the long-term and stable release of NGF for at least 16 weeks under physiological conditions, is obtained as a biogel for the long-term controlled release of NGF for the treatment of central nervous system injuries and diseases.
Example 8: preparation of biological gel capable of controlling NT-3 release for long time for treating central nervous system injury and disease
The NT-3-gelatin-polyisopropylacrylamide hydrogel at the optimum dose (100-200ng/ml) prepared in example 2 was mixed with the NT-3-chitosan hydrogel at the optimum dose (100-200ng/ml) prepared in example 5 at a ratio of 1: 1.05.
Co-culturing the mixed gel with spinal cord-derived or brain-derived neural stem cells in vitro, and detecting by an enzyme-linked immunosorbent assay (Elisa): NT-3 (100) -200ng/ml) -active biological gel which can control the long-term and stable release of NT-3 for at least 14 weeks under physiological environment, namely, biological gel which can control the release of NT-3 for a long time and is used for treating central nervous system injury and diseases is obtained. Of these, 100ng/ml,150ng/ml,200ng/ml, three doses were released up to 14 weeks with no statistical difference between groups.
Example 9: preparation of biological gel capable of controlling bFGF release for long time for treating central nervous system injury and disease
The optimum dose (50-100ng/ml) of bFGF-gelatin-polyisopropylacrylamide hydrogel prepared in example 3 was mixed with the optimum dose (50-100ng/ml) of bFGF-chitosan gel prepared in example 6 at a ratio of 1: 1.05.
Co-culturing the mixed gel with spinal cord-derived or brain-derived neural stem cells in vitro, and detecting by an enzyme-linked immunosorbent assay (Elisa): bFGF (50-100ng/ml) -active biological gel which can control the long-term and stable bFGF release for at least 5 weeks under physiological environment, namely the biological gel which can control the bFGF release for a long time and is used for treating central nervous system injury and diseases. Of these, 50ng/ml,1000ng/ml, both doses were released for up to 5 weeks with no statistical difference between groups.
Example 10 NGF active biogel can significantly promote the production of new neurons
The optimal biological effects of the three NGF/NT-3/bFGF-active biogels obtained by examples 7,8,9 were compared.
The effect of the three NGF/NT-3/bFGF-active biogels on the activation of endogenous Neural Stem Cells (NSCs) and the recruitment of the cells to the lesion area to mature neurons is evaluated within half a year of implantation in vivo (implantation in spinal cord injury area) experiments and respectively compared quantitatively (the specific experimental method refers to example 1).
The results are shown in FIG. 19:
the quantitative analysis of the differentiation of NGF/NT-3/bFGF-bioactive gel into mature neurons at half a year after operation, with the results shown that: 100-200ng/ml NGF-gel was most effective in promoting differentiation of endogenous neural stem cells into neurons.
Example 11 biological Activity Performance test
The biological activity of the bio-gel prepared in example 7 and capable of controlling NGF release over a long period of time for the treatment of central nervous system injury and disease was verified by chick embryo Dorsal Root Ganglion (DRG) gel co-culture with chick embryo Dorsal Root Ganglion (DRG) as the study object on day 8 of the embryo.
The specific process is as follows: chick embryo dorsal root ganglia dissected from chick embryos of day 8 of the embryo were co-cultured with the above biogel for 48 hours, and then the growth state of the dorsal root ganglia prominence was observed under a scanning electron microscope.
The results show (see fig. 3) that chick embryo dorsal root ganglion cells adhere to the biogel and outgrow processes that form tight junctions with the material and that the processes of the DRGs exhibit radial growth. This result demonstrates that the bio-gel prepared in example 7, which can controllably release NGF over a long period of time, has excellent biocompatibility, and can promote neuron survival, growth, adhesion and migration.
Controlled release kinetics of NGF-active biogels
Soluble NGF lyophilized powder (S NGF) was used as a control, and the NGF-biogel prepared in example 7 (100ng/ml) was co-cultured with brain-derived NSC and detected by enzyme-linked immunosorbent assay (Elisa): the two active gels were mixed at a volume ratio (1: 1.05) to control the long-term and stable release of NGF for up to 16 weeks in a physiological environment (FIG. 4).
Claims (10)
1. An active biological gel capable of controlling NGF release for a long time, which consists of NGF-gelatin-polyisopropylacrylamide hydrogel and NGF-chitosan hydrogel, wherein the loading amount of NGF in the NGF-gelatin-polyisopropylacrylamide hydrogel is 100-200ng/ml, and the loading amount of NGF in the NGF-chitosan hydrogel is 100-200 ng/ml; the volume ratio of the NGF-gelatin-polyisopropylacrylamide hydrogel to the NGF-chitosan hydrogel is 1:1.05-1: 1.5; the NGF represents nerve growth factor.
2. The active biogel of claim 1, wherein: the volume ratio of the NGF-gelatin-polyisopropylacrylamide hydrogel to the NGF-chitosan hydrogel is 1: 1.05.
3. Active biogel according to claim 1 or 2, characterised in that: the preparation method of the NGF-gelatin-polyisopropylacrylamide hydrogel comprises the following steps:
(a1) modifying the type I collagen to obtain dry gelatin powder;
(b1) preparing the gelatin-polyisopropylacrylamide hydrogel by using the gelatin dry powder in the step (a 1);
(c1) reacting the gelatin-polyisopropylacrylamide hydrogel prepared in the step (b 1) with NGF under the action of an initiator to obtain NGF-gelatin-polyisopropylacrylamide hydrogel; the initiator is selected from genipin.
4. The active biogel of claim 3, wherein:
in the step (a 1), the method for modifying type I collagen comprises modifying type I collagen with hydrogen peroxide; the specific modification method comprises the following steps: weighing I type collagen at room temperature, dissolving in hydrogen peroxide solution to obtain gelatin solution, and freeze drying to obtain gelatin dry powder;
the concentration of the hydrogen peroxide is 0.02-1 mmol/mL;
the proportion of the type I collagen to the hydrogen peroxide solution is 1 g: 0.01L;
the dissolving temperature of the type I collagen is 48-85 ℃, and the dissolving time is 4-12 hours;
the freeze-drying time is 3-6 hours.
5. The active biogel of claim 3, wherein:
in the step (b 1), the method for preparing the gelatin-polyisopropylacrylamide hydrogel specifically comprises the following steps: dissolving the gelatin dry powder, N-isopropylacrylamide, tetramethylethylenediamine and N, N-subunit bisacrylamide in deionized water, introducing nitrogen, stirring, adding genipin after completely dissolving, stirring uniformly, and standing at room temperature for 2-4 hours to prepare hydrogel; soaking the hydrogel in deionized water for 48-60 hours, wherein the deionized water is thoroughly replaced every 30 minutes in the soaking process; then taking out the hydrogel and soaking the hydrogel into deionized water at the temperature of 40-46 ℃, soaking for 48-60 hours, and completely replacing the deionized water every 30 minutes to remove residual monomers; and finally, placing the obtained gelatin-polyisopropylacrylamide hydrogel in deionized water for storage at 4 ℃ for later use.
6. The active biogel of claim 3, wherein:
in the step (c 1), the concentration of the NGF solution is 20-300ng/ml, and the mass concentration of the initiator is 0.1-1%.
7. Active biogel according to claim 1 or 2, characterised in that: the preparation method of the NGF-chitosan hydrogel comprises the following steps:
(a2) preparing chitosan hydrogel;
(b2) reacting the chitosan hydrogel prepared in the step (a 2) with NGF under the action of an initiator to obtain NGF-chitosan gel; the initiator is genipin.
8. The active biogel of claim 7, wherein: the method for preparing the chitosan hydrogel comprises the following steps: and (3) carrying out crosslinking reaction on the chitosan solution under the condition that the pH value is 4.0-5.5 by using genipin as a crosslinking agent to obtain the chitosan hydrogel.
9. Use of an active biogel as claimed in any of claims 1 to 8, comprising 1) and/or 2) of:
1) the application in preparing products for reconstructing or repairing tissue or organ defects;
2) the application in preparing products for treating central nervous system injury and diseases.
10. Use according to claim 9, characterized in that: the tissue includes, but is not limited to, neural tissue;
the central nervous system injury comprises different types of brain or spinal cord injury;
the product comprises a biomaterial scaffold;
the reconstruction or repair of a tissue or organ defect is embodied in at least one of the following aspects:
1) activating endogenous neural stem cells, and recruiting the endogenous neural stem cells to migrate to a brain or spinal cord injury area;
2) a high proportion of the cells differentiate into mature neurons;
3) promoting the regeneration of blood vessels;
4) inhibiting inflammatory response;
5) inhibiting scar infiltration.
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