CN115501253A - Preparation of combined stem cell and hydrogel biomaterial and application of combined stem cell and hydrogel biomaterial in spinal cord injury - Google Patents
Preparation of combined stem cell and hydrogel biomaterial and application of combined stem cell and hydrogel biomaterial in spinal cord injury Download PDFInfo
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- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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Abstract
The invention discloses a preparation method of a combined stem cell and hydrogel biomaterial and application thereof in spinal cord injury, which is characterized by comprising the following steps: preparing methacrylic acylated gelatin (GelMA) and methacrylic acylated hyaluronic acid (HAMA); preparing a hydrogel solution, and wrapping the mesenchymal stem cells or/and the neuroepithelial stem cells which are subjected to combined standard culture in the prepared hydrogel to obtain stem cell-loaded hydrogel or hydrogel microspheres; the stem cell-loaded hydrogel or hydrogel microspheres are transplanted to the spinal cord injury to treat the spinal cord injury and evaluate the effect. The invention is used for repairing spinal cord injury, uses biological materials, has low immunogenicity, better cell biocompatibility and certain biological function; after transplantation, excessive accumulation of astrocytes after spinal cord injury can be reduced, formation of glial scars is reduced, bladder and motor functions are improved, regeneration and repair of nerves after spinal cord injury can be promoted, and behavior recovery of animals with spinal cord injury is promoted.
Description
Technical Field
The invention belongs to the technical field of spinal cord injury repair, and particularly relates to preparation of a combined stem cell and hydrogel biomaterial and application of the combined stem cell and hydrogel biomaterial in spinal cord injury.
Background
Spinal Cord Injury (SCI) is a common disabling severe trauma in clinic, with the consequences of neuronal death and axon rupture, loss of innervation function, resulting in severe physical, psychological and social interaction impairment for patients, as well as a heavy burden on society and families, and its treatment is a long-term problem and research focus in the medical community. Conventional wisdom holds that Astrocyte (AS) proliferation, glial scarring and cavity formation following spinal cord injury are major factors that hinder efficient regeneration and growth of injured spinal axons. In recent years, with the rapid development of neurophysiology, neurocytobiology and molecular biology, the research on pathophysiology and repair mechanism of spinal cord injury is continuously and deeply carried out, and the latest research shows that AS reaction and glial scar microenvironment after spinal cord injury have important effects on neuron survival, axon regeneration and connection, and even activation and differentiation of endogenous nerve stem, so that the AS reaction and the glial scar microenvironment after spinal cord injury are key factors for determining the repair of nerve structure and function after spinal cord injury.
Mesenchymal Stem Cells (MSCs) are easily obtained, are not restricted by ethics, have multidirectional differentiation potential, have strong nutrition and immunoregulatory activity, low immunogenicity, high safety and the ability of regulating and controlling inflammatory reaction, and the secreted trophic factors can promote the survival of cells in damaged areas, improve the microenvironment in the damaged areas and improve the motor function after spinal cord injury. Therefore, the mesenchymal stem cells have the characteristics of protecting neurons and promoting nerve regeneration.
Neuroepithelial stem cells (NESCs) are neural stem cells in the neural tube development stage, and have strong proliferation and differentiation abilities. After the neuroepithelial stem cells are transplanted to the injured part of the spinal cord, the neuroepithelial stem cells can provide cell nutrition support for the injured part, protect nerve cells from apoptosis and promote the growth of injured axons; can efficiently differentiate to generate neurons, integrate with host spinal cord neurons, promote nerve regeneration, reestablish a nerve circuit at a damaged part to rebuild corticospinal tracts, and promote the recovery of the nerve function of spinal cord injury.
At present, a set of serum-free mesenchymal stem cell amplification schemes is established domestically, a set of culture systems for efficiently and stably amplifying neuroepithelial stem cells is established, and NESCs and MSCs can be produced in a standardized manner.
The biomaterial is used as a stem cell carrier bracket, has good biocompatibility and degradability, can simulate soft tissue environment, effectively support and guide axon regeneration and migration, inhibit the formation of colloid scars, and is beneficial to the transfer of nutrient substances and growth factors, so that the combination of the biomaterial and the stem cells can avoid cell loss caused by spinal fluid flow and reduce the attack of immune cells. The combination of the biomaterial hydrogel and the stem cells can promote the recovery of the motor function of spinal cord injury.
Current spinal cord injuries lack effective repair methods and transplantation therapy of stem cells offers the possibility for treatment of the disease, however selection of stem cell types appropriate for spinal cord injury and associated stem cell delivery systems are still lacking.
Therefore, in order to solve the above problems, proposed herein is a combined stem cell and hydrogel biomaterial preparation and its application in spinal cord injury.
Disclosure of Invention
In order to solve the technical problems, the invention designs preparation of a combined stem cell and hydrogel biomaterial and application of the combined stem cell and hydrogel biomaterial in spinal cord injury.
In order to achieve the technical effects, the invention is realized by the following technical scheme: use of a combined stem cell and hydrogel biomaterial in spinal cord injury, characterized in that: the combined stem cell and hydrogel biomaterial is transplanted to the injured spinal cord part of a spinal cord injury patient to be used as a cell medicament for treating spinal cord injury to repair the injured part.
Another object of the present invention is to provide a method for preparing a stem cell-loaded hydrogel or hydrogel microsphere, which comprises the following steps:
step1: preparing methacrylic acylated gelatin (GelMA) and methacrylic acylated hyaluronic acid (HAMA);
step2: completely dissolving GelMA and HAMA in a PBS solution containing a blue light initiator LAP, and crosslinking and curing to obtain hydrogel or hydrogel microspheres;
step3: and wrapping the mesenchymal stem cells or/and the neuroepithelial stem cells in the prepared hydrogel or hydrogel microspheres to obtain the stem cell-loaded hydrogel or hydrogel microspheres.
Further, the preparation method of the methacrylic acid acylated gelatin (GelMA) in Step1 comprises the following steps: adding sodium carbonate into deionized water, adding gelatin into a sodium carbonate solution according to the proportion of 50-200 g/L, stirring and dissolving at 35-60 ℃, then adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the gelatin is 1-3.
Further, the preparation method of the methacrylic acid acylated hyaluronic acid (HAMA) in Step1 comprises the following steps: dissolving sodium hyaluronate in deionized water according to the proportion of 1-20 g/L, adding sodium carbonate into the obtained solution, stirring and dissolving at 35-60 ℃, then adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the sodium hyaluronate is 1-1.
Further, in Step2, the concentrations of GelMA and HAMA are respectively 1-10 percent by weight percent in the preparation process of the hydrogel or the hydrogel microspheres under the condition of keeping out of the sun; the concentration of the blue light initiator is less than 0.1wt%; the volume ratio of GelMA to HAMA aqueous solution is 1:1; the crosslinking curing adopts 405nm blue light, and the crosslinking time is 10-120 s.
The invention has the beneficial effects that:
the stem cell and hydrogel biomaterial combined for treating spinal cord injury, disclosed by the invention, adopts the hydrogel raw material as a biological source, is low in immunogenicity, has better biocompatibility and has a certain biological function; after the transplantation, the excessive accumulation of astrocytes after spinal cord injury can be reduced, the formation of glial scars can be reduced, the regeneration and repair of nerves after spinal cord injury can be promoted, and the behavior recovery of animals with spinal cord injury can be further promoted; the composition has the characteristics of low cost, high activity and obvious treatment effect, does not generate the side effect of a medicine with fluctuating symptoms, can continuously survive in a host body for a long time and generate activity, has the advantages which cannot be compared with the existing medicine and (or) stem cell preparation, provides experimental basis and theoretical basis for establishing a spinal cord injury treatment strategy and designing a novel stem cell treatment medicine, and has wide application prospect in treating spinal cord injury and higher commercial value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a physical characteristic of the prepared stem cell-loaded hydrogel microspheres;
FIG. 2 is the preparation of a spinal cord injury model and the transplantation of hydrogel microspheres carrying stem cells;
FIG. 3 is a rat motor function score after treatment of spinal cord injury with stem cell-loaded hydrogel microspheres;
FIG. 4 is a comparison of the void area of spinal cord injury and glial scar formation after 4 weeks in different spinal cord injury treatment groups;
FIG. 5 is a comparison of the regenerated neurofibrillary filaments from spinal cord injury after 4 weeks in different spinal cord injury treatment groups.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
And (3) expanding and culturing stem cells: mesenchymal stem cells obtained from umbilical cord sources or other tissues are used, embryonic stem cells or induced pluripotent stem cells or somatic cells and the like are used for inducing differentiation to generate obtained neuroepithelial stem cells, and the mesenchymal stem cells and the neuroepithelial stem cells with better growth state are obtained according to the existing standard culture scheme; the conditions of the incubator were 37 ℃ and 5% CO 2 And saturated humidity.
Preparation of hydrogel carrying stem cells: preparing the combined stem cell hydrogel, and chemically preparing and synthesizing the hydrogel material capable of crosslinking and curing by using biological gelatin, methacrylic anhydride, biological hyaluronic acid, a blue light initiator LAP and other chemical reagents, wherein the biological gelatin, the biological hyaluronic acid, the blue light initiator LAP and the other chemical reagents are used for: acylated methacrylate gelatin (GelMA) and acylated methacrylate hyaluronic acid (HAMA);
evaluation of therapeutic effect of stem cell-loaded and hydrogel biomaterials for treatment of spinal cord injury:
1) Preparing a spinal cord injury animal disease model, namely preparing a spinal cord injury animal full-transection model by using an operation method;
2) Transplanting stem cell-loaded hydrogel or hydrogel microsphere to treat a spinal cord injury animal disease model; transplanting the prepared stem cell-loaded hydrogel or hydrogel microsphere to the spinal cord injury part of the animal for treatment;
3) Behavioral assessment of spinal cord injury animals for treatment efficacy; after the stem cell-loaded hydrogel or hydrogel microspheres are used for treating the spinal cord injury animal, scoring is carried out on the motion state of the experimental animal according to time points, and the treatment effect is evaluated;
4) Obtaining a tissue sample after spinal cord injury treatment: obtaining spinal cord tissues after a spinal cord injury animal treats for a period of time, and fixing a specimen and obtaining a tissue section;
5) And (3) observing and evaluating the scar area and nerve regeneration condition of spinal cord injury, and observing by using an immunofluorescence enzyme method to dye so as to evaluate the treatment effect of spinal cord injury.
Example 2
The invention relates to a preparation method of a combined stem cell and hydrogel biomaterial for treating spinal cord injury, which specifically comprises the following steps: the method comprises the following specific operation steps of culture and amplification of stem cells and preparation of stem cell-loaded hydrogel or hydrogel microspheres:
step1: the stem cells were cultured and expanded according to the standard culture protocol, the incubator conditions were 37 ℃ and 5% CO 2 And (3) culturing umbilical cord Mesenchymal Stem Cells (MSCs) and neuroepithelial stem cells (NESCs) to obtain stem cells with a good growth state, digesting the obtained stem cells and counting the stem cells under the saturated humidity.
Step2: the preparation of the biomaterial hydrogel for carrying the stem cells comprises the following specific operations:
adding sodium carbonate into the obtained deionized water, diluting and adjusting the pH value of a sodium carbonate aqueous solution to 7-10, adding gelatin into the sodium carbonate solution according to the proportion of 50-200 g/L, stirring and dissolving at 35-60 ℃, then adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the gelatin is 1; dialyzing for one week with deionized water at normal temperature after the reaction is finished, wherein the cut-off molecular weight of a dialysis bag is 8 KDa-14 KDa, and after the dialysis is finished, freezing and drying to obtain methacrylic acidylated gelatin (GelMA); dissolving sodium hyaluronate in deionized water according to the proportion of 1-20 g/L, adding sodium carbonate into the obtained solution, diluting and adjusting the pH of the mixed aqueous solution to 7-10, stirring and dissolving at 35-60 ℃, then adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the sodium hyaluronate is 1-3; dialyzing for one week with deionized water at normal temperature after the reaction is finished, wherein the cut-off molecular weight of a dialysis bag is 8 KDa-14 KDa, and after the dialysis is finished, freezing and drying to obtain methacrylic acid acylated hyaluronic acid (HAMA); under the condition of keeping out of the sun, completely dissolving GelMA and HAMA obtained after freeze drying in PBS solution to prepare hydrogel solution, wherein the concentration of the hydrogel is 1-10 percent by weight respectively, the hydrogel also contains blue light initiator LAP, and the concentration of the blue light initiator is less than 0.1 percent by weight.
And 3, step3: the preparation of the hydrogel biomaterial microspheres for treating spinal cord injury comprises the following specific operations:
and (3) preparing the prepared GelMA and HAMA hydrogel solutions according to a volume ratio of 1:1, sucking 0.1-1 mu l of GelMA and HAMA hydrogel mixed liquid by a micropipette, dripping the GelMA and HAMA hydrogel mixed liquid into ice mineral oil, performing physical low-temperature crosslinking to form hydrogel microspheres, irradiating the formed hydrogel microspheres by using a 405nm blue-light lamp for 30-60 s so that the hydrogel performs crosslinking chemical reaction of photoinitiation free radical polymerization, and preparing the stable photocuring hydrogel microspheres.
And 4, step 4: the preparation method of the stem cell hydrogel biomaterial microspheres for treating spinal cord injury comprises the following specific operations:
preparing the prepared GelMA and HAMA hydrogel solutions according to the volume ratio of 1:1, and keeping out of the sun for later use. And digesting the stem cells obtained after large-scale culture into single cells, and then carrying out centrifugal precipitation. Uniformly suspending MSCs and NESCs in hydrogel according to the ratio of 100-500 ten thousand cells in 1ml of hydrogel, sucking 0.1-1 mul of the suspended cell-hydrogel mixed liquid by a micropipette, dripping the suspended cell-hydrogel mixed liquid into ice mineral oil to form hydrogel microspheres through physical low-temperature crosslinking, irradiating the hydrogel microspheres by using a 405nm blue-light lamp for 30-60 s to perform crosslinking chemical reaction of photo-initiated free radical polymerization on the hydrogel, and preparing the stable photo-cured stem cell-loaded hydrogel microspheres.
And 5: characteristics of combined stem cells and hydrogel biomaterial microspheres for the treatment of spinal cord injury:
the shape of the prepared hydrogel microspheres is shown in figure 1, and figure 1a shows that the hydrogel microspheres are uniform in size and shape and good in roundness. FIG. 1b shows that the hydrogel microsphere has a porous structure, and an electron micrograph shows that the hydrogel microsphere has a rough and porous honeycomb structure, and larger gaps can form more specific surface areas and spaces, so that the spaces for cell adhesion proliferation and cell extension are provided for the encapsulated stem cells, and cell adhesion is facilitated. FIG. 1c shows that stem cells are uniformly distributed in hydrogel microspheres in the form of dispersed single cells, and the particle size of the stem cells can meet the requirement of normal exchange of substances between the cells and the external environment and is 400-500 μm. And filtering and collecting the prepared hydrogel microspheres by using a 100-micron cell screen, repeatedly washing the hydrogel microspheres with 250mL of sterile normal saline or sterile PBS (phosphate buffer solution) after collection, and removing residual mineral oil on the surfaces. The collected stem cell-loaded hydrogel microspheres are stored in a low-temperature refrigerator at 4 ℃ or transported by using a low-temperature ice box for subsequent animal transplantation experiments.
Example 3
Referring to fig. 2 to 5, the method for detecting the function of the combined stem cell and hydrogel biomaterial microsphere for treating spinal cord injury according to the present invention specifically includes the following steps: the method comprises the following specific operation steps of making a spinal cord injury model, transplanting hydrogel microspheres carrying stem cells, evaluating the treatment effect of the spinal cord injury animal behavioural, obtaining a tissue sample, and observing the scar area and nerve regeneration condition of the spinal cord injury, wherein the specific operation steps are as follows:
1. preparing a disease model of a spinal cord injury rat. 200-250g of clean grade adult female SD rats were selected and model preparation was performed according to the following procedure:
the full transection method is adopted, 5% isoflurane concentration is used for inducing the anesthetized animal for 3-5 minutes, and the experimental animal is preserved by an electric small animal clipper in a skin preparation area. The experimental animal is anesthetized with the isoflurane concentration of 2% for operation, 75% alcohol and iodophor are sequentially used for smearing and disinfecting, the T4 section of skin and muscle of the experimental animal are carefully stripped, the T4 section of bone vertebral plate is carefully removed by using a schlieren clamp, and the spinal cord of the experimental animal is fully exposed. The method comprises the following steps of hooking out the spinal cord part of an experimental animal by using an arc hook, completely cutting exposed spinal cord tissues by using a micro-scissors, forming a macroscopic spinal cord cutting gap with the distance of 2mm due to retraction of the cut spinal cord tissues, fully stopping bleeding by using a medical cotton swab, fully washing a wound by using normal saline, and performing the next operation after the bleeding is stopped.
2. The stem cell-loaded hydrogel or hydrogel microsphere is transplanted to a rat model for treating spinal cord injury, and the specific implementation steps are as follows;
step1: the stem cell-loaded hydrogel microsphere is used for transplantation treatment, and the specific operation is as follows;
transplanting the collected stem cell-loaded hydrogel microspheres to a gap position of 2mm of spinal cord injury of the experimental animal by using an injector, suturing the exposed muscle and skin of the experimental animal layer by layer, flushing the wound by using normal saline, and injecting 20000 units of penicillin sodium and normal saline respectively into the experimental animal subcutaneously after the operation. As shown in fig. 2, the left side of fig. 2 shows the prepared spinal cord injury model, and the right side of fig. 2 shows the stem cell-loaded hydrogel microspheres transplanted to the spinal cord injury site.
Step2: and (3) postoperative care: after the experimental animal revives, the experimental animal is sent to a clean animal room for injection of penicillin sodium every day and postoperative care lasts for one week to several weeks.
3. Evaluating the treatment effect of the animal with spinal cord injury by ethology, and specifically implementing the steps as follows;
step1: collecting behavioral scores of rats with spinal cord injuries, and specifically operating as follows:
after one week of animal operation, the animals are placed in an open barrier-free field to move freely, and the hip, knee and ankle joint activities, coordination of front and rear limbs, body motion stability and other conditions of the animals are observed. Culling test groups with animal movement scores exceeding 3 points. One week after the operation of the experimental animal, an open field experiment is carried out, the exercise states of the experimental animal are graded one by using a behavioral grading (Basso-Beattie-Bresnahan, BBB) method of the spinal cord injury rat, and the grading is mainly used for evaluating the postposition exercise, the coordination of limbs, the driving stability and the like of the spinal cord injury rat. Scoring was continued until 6 months after injury treatment. The experiment stipulates that the postoperative BBB score cannot exceed 3 points, and the postoperative BBB score exceeds 3 points of rejection experiment groups.
Step2: evaluating the treatment effect of the spinal cord injury rat by behavioral scoring, wherein the specific result is as follows;
the data is processed by GraphPad to show that the data result is shown in figure 3, and the figure 3 result shows that the difference among the hydrogel microsphere groups loaded with the stem cells is obvious; after the treatment by using the hydrogel microspheres loaded with the combination of the three types of stem cells, the score of a treatment group is gradually improved along with the passage of time, and the motor function of the treatment group is proved to be continuously recovered, the BBB score tends to be stable after 90 days of postoperative treatment, the walking gait of hind limbs of the treatment group loaded with the MSC + NESC hydrogel microspheres is obviously improved, hind paws can walk on the ground in a turnover mode, the hind paws can walk frequently with a load, the fore limbs and the aft limbs occasionally can move coordinately, and the BBB ethological score is obviously superior to that of other groups.
4. The method comprises the following specific implementation steps of obtaining a tissue sample after spinal cord injury treatment:
after the behavioral assessment of the spinal cord injury animal is completed, after the stem cell-loaded hydrogel microspheres or hydrogel microspheres are used for treating the spinal cord injury rat for 4 weeks, the animal is overanesthetized by pentobarbital, an infusion bag is connected with an injector for perfusion, physiological saline is injected from the left ventricle aorta, the right atrium flows out, and after the flowing liquid is transparent, 4% paraformaldehyde is used for perfusion until the animal is immobile. Taking out the intact spinal cord tissue with vertebral plate, fixing with 4% paraformaldehyde overnight, stripping off spinal cord, dehydrating with sucrose concentration gradient, freezing and slicing the spinal cord tissue to slice thickness of 20 μm, attaching the tissue on an adhesive slide, drying at 37 deg.C for one hour, and storing in refrigerator at-20 deg.C for use.
5. The method comprises the following specific steps of (1) observing and evaluating the scar area of spinal cord injury and the nerve regeneration condition, and observing by dyeing with an immunofluorescence enzyme method:
step1: after the slide was taken out and washed with PBS solution (3 times), overnight permeation was carried out with 0.2% TritonX100 solution, and after washing with PBS, blocking solution (3% BSA) was added and the slide was blocked in a wet box at room temperature for 2 hours. Mu.l of rabbit anti-rat GFAP antibody or mouse anti-rat NF (Neurofament) antibody diluted with the antibody diluent was placed in a wet box overnight at 4 ℃, and the next day of PBS washing was followed by 100. Mu.l of donkey anti-rabbit IgG polyclonal antibody or rabbit anti-mouse IgG polyclonal antibody (1. After PBS washing (3 times), the sections were sealed and stored in dark with 50% glycerol-PBS solution, and then observed under a confocal laser microscope for staining and photographed.
Step2: the area of the scar of the spinal cord injury is observed, after the spinal cord injury, astrocytes participate in forming glial scar tissues, and axon regeneration is limited, so that the size of the glial scar tissues is one of indexes for evaluating nerve regeneration and repair, and the astrocytes are marked by using a GFAP antibody. The results in fig. 4 show that the control untreated group had a large number of activated GFAP + astrocytes at the site of injury, with the largest proportion, and the site of injury had a larger luminal area. The GFAP + cells of the hydrogel microsphere group are reduced to a certain degree, which shows that the hydrogel microspheres have a certain function of inhibiting the formation of colloid scars, and the addition of the stem cells further induces the reduction of the GFAP + cells at the spinal cord injury part. GFAP + cells did not show significant differences between the MSC microsphere treatment group, NESC microsphere treatment group, and MSC + NESC microsphere treatment group, but the MSC + NESC microsphere treatment group had more regular cell distribution and minimal cavity area. The stem cell-loaded hydrogel microspheres are implanted to effectively prevent the excessive accumulation of astrocytes and inhibit the generation of glial scars. Regeneration of new neurons is involved in spinal cord regeneration.
And step3: nerve regeneration was observed by using NF (nerve fiber filament) to label the fibers of the regenerated nerve. The results in fig. 5 show that the control untreated group had fewer NF + nerve fibers at the injury site, sparse morphology, shorter length, and no significant nerve fiber regeneration. The hydrogel microsphere group showed a small number of NF + nerve fibers at the site of injury. The density of NF + at the injured part of the spinal cord after stem cell treatment is obviously increased, the nerve fibers are regenerated in a large amount and are distributed more regularly, the ratio of NF + cells in the MSC + NESC treatment group is higher than that in the MSC and NESC treatment group, and the nerve fibers are in clear and linear shapes and are connected with the front and rear sections of the spinal cord injury.
Example 4
The preparation of the combined stem cell and hydrogel biomaterial and the application thereof in spinal cord injury are implemented by combining the obtained mesenchymal stem cells or/and neuroepithelial stem cells with the prepared hydrogel biomaterial to prepare stem cell-loaded hydrogel or hydrogel microspheres. And can only act by being transplanted to the damaged spinal cord part of a patient with spinal cord injury disease, and is used as a cell medicine for treating spinal cord injury. The mesenchymal stem cells used are not limited to those obtained from umbilical cord but include those obtained from other tissues; the neural epithelial stem cells include not only the neural epithelial stem cells differentiated from embryonic stem cells, but also the neural epithelial stem cells differentiated from induced pluripotent stem cells or obtained by direct transdifferentiation of somatic cells.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (6)
1. Use of a combined stem cell and hydrogel biomaterial in spinal cord injury, characterized in that: the combined stem cell and hydrogel biomaterial is transplanted to the damaged spinal cord part of a spinal cord injury patient to be used as a cell medicament for treating spinal cord injury to repair the damaged part.
2. A preparation method of combined stem cells and hydrogel biomaterials is characterized by comprising the following steps:
step1: preparing methacrylic acylated gelatin (GelMA) and methacrylic acylated hyaluronic acid (HAMA);
step2: completely dissolving GelMA and HAMA in a PBS solution containing a blue light initiator LAP, and crosslinking and curing to obtain hydrogel or hydrogel microspheres;
step3: and wrapping the mesenchymal stem cells or/and the neuroepithelial stem cells in the prepared hydrogel or hydrogel microspheres to obtain the stem cell-loaded hydrogel or hydrogel microspheres.
3. The method for preparing a combined stem cell and hydrogel biomaterial as claimed in claim 2, wherein the method for preparing methacrylated gelatin (GelMA) in Step1 comprises the following steps: adding sodium carbonate into deionized water, adding gelatin into a sodium carbonate solution according to the proportion of 50-200 g/L, stirring and dissolving at 35-60 ℃, then adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the gelatin is 1-3.
4. The method for preparing a combined stem cell and hydrogel biomaterial as claimed in claim 2, wherein the method for preparing methacrylated hyaluronic acid (HAMA) in Step1 comprises the following steps: dissolving sodium hyaluronate in deionized water according to the proportion of 1-20 g/L, adding sodium carbonate into the obtained solution, stirring and dissolving at 35-60 ℃, then adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the sodium hyaluronate is 1-1.
5. The method for preparing a combined stem cell and hydrogel biomaterial according to claim 2, wherein in Step2, the hydrogel or hydrogel microsphere is prepared under the condition of keeping out light, the GelMA and HAMA concentrations are respectively 1-10% by weight; the concentration of the blue light initiator is less than 0.1wt%; the volume ratio of GelMA to HAMA aqueous solution is 1:1; the crosslinking curing adopts 405nm blue light, and the crosslinking time is 10-120 s.
6. The method of claim 2, wherein the mesenchymal stem cells are obtained from umbilical cord or from other tissues; the neuroepithelial stem cell is obtained by differentiation from an embryonic stem cell, or a neuroepithelial stem cell obtained by differentiation from an induced pluripotent stem cell or a neuroepithelial stem cell obtained by direct transdifferentiation from a somatic cell.
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| WO2024077893A1 (en) * | 2022-10-14 | 2024-04-18 | 昆明理工大学 | Preparation of combination of stem cells and hydrogel as biomaterial and use of combination in spinal cord injuries |
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| CN113713179A (en) * | 2021-09-06 | 2021-11-30 | 山东大学 | High-comprehensive-performance photocuring biological 3D printing composite hydrogel and preparation method and application thereof |
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