WO2024077893A1 - 联合干细胞和水凝胶生物材料的制备及其在脊髓损伤中的应用 - Google Patents

联合干细胞和水凝胶生物材料的制备及其在脊髓损伤中的应用 Download PDF

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WO2024077893A1
WO2024077893A1 PCT/CN2023/086424 CN2023086424W WO2024077893A1 WO 2024077893 A1 WO2024077893 A1 WO 2024077893A1 CN 2023086424 W CN2023086424 W CN 2023086424W WO 2024077893 A1 WO2024077893 A1 WO 2024077893A1
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hydrogel
spinal cord
stem cells
cord injury
stem cell
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French (fr)
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李天晴
李鹏飞
张磊
陈衍颖
朱小庆
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昆明理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention belongs to the technical field of spinal cord injury repair, and specifically relates to a preparation method of a combined stem cell and hydrogel biomaterial and its application in spinal cord injury.
  • SCI Spinal cord injury
  • MSCs Mesenchymal stem cells
  • the nutritional factors secreted can promote cell survival in the injured area, improve the microenvironment of the injured area, and improve motor function after spinal cord injury. Therefore, mesenchymal stem cells have the characteristics of protecting neurons and promoting nerve regeneration.
  • Neuroepithelial stem cells are neural stem cells in the neural tube development stage, with strong proliferation and differentiation capabilities. After being transplanted to the site of spinal cord injury, neuroepithelial stem cells can provide cellular nutritional support to the injured site, protect nerve cells from apoptosis, and promote the growth of damaged axons; they can efficiently differentiate into neurons, integrate with host spinal cord neurons, promote nerve regeneration, re-establish neural circuits at the site of injury, reconstruct the corticospinal tract, and promote the recovery of spinal cord injury nerve function.
  • biomaterials As a stem cell carrier scaffold, biomaterials have good biocompatibility and degradability, can simulate the soft tissue environment, effectively support and guide axon regeneration and migration, inhibit the formation of glial scars, and are conducive to the delivery of nutrients and growth factors. Therefore, the combination of biomaterials and stem cells can avoid cell loss caused by cerebrospinal fluid flow and reduce immune cell attacks.
  • the combination of biomaterial hydrogel and stem cells can promote the recovery of motor function in spinal cord injury.
  • this paper proposes a combined stem cell and hydrogel biomaterial preparation and its application in spinal cord injury.
  • the present invention designs a combined stem cell and hydrogel biomaterial preparation and its application in spinal cord injury.
  • the present invention constructs hydrogel or hydrogel microspheres by methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA), which exhibits good cell compatibility.
  • mesenchymal stem cells and neuroepithelial stem cells are encapsulated by hydrogel or hydrogel microspheres to form a stable stem cell preparation, which is transplanted to the injured spinal cord to promote the regeneration of the injured spinal cord.
  • the present invention is implemented through the following technical scheme: an application of combined stem cells and hydrogel biomaterials in spinal cord injury, characterized in that the combined stem cells and hydrogel biomaterials are transplanted into the damaged spinal cord of patients with spinal cord injury as a cellular medicine for treating spinal cord injury to repair the damaged area.
  • Another object of the present invention is to provide a method for preparing a hydrogel or hydrogel microsphere carrying stem cells, characterized in that it comprises the following steps:
  • Step 1 Preparation of methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA);
  • Step 2 Dissolve GelMA and HAMA completely in a PBS solution containing a blue light initiator LAP, and crosslink and solidify to obtain a hydrogel or hydrogel microspheres;
  • Step 3 Encapsulate mesenchymal stem cells and/or neuroepithelial stem cells in the prepared hydrogel or hydrogel microspheres to obtain stem cell-loaded hydrogel or hydrogel microspheres.
  • methacrylated gelatin (GelMA) in the Step 1 is as follows: sodium carbonate is added to deionized water, gelatin is added to the sodium carbonate solution at a ratio of 50 to 200 g/L, and dissolved by stirring at 35 to 60° C., and then methacrylic anhydride is added, the volume mass ratio of methacrylic anhydride to gelatin is 1:1 to 3:1, and after the reaction is completed, dialyzed at room temperature, and freeze-dried to obtain methacrylated gelatin (GelMA).
  • the preparation method of methacrylated hyaluronic acid (HAMA) in the Step 1 is as follows: sodium hyaluronate is dissolved in deionized water at a ratio of 1 to 20 g/L, sodium carbonate is added to the obtained solution, and dissolved by stirring at 35 to 60° C., and then methacrylic anhydride is added, and the volume mass ratio of methacrylic anhydride to sodium hyaluronate is 1:1 to 3:1. After the reaction is completed, the hyaluronic acid is dialyzed with deionized water at room temperature, and freeze-dried to obtain methacrylated hyaluronic acid (HAMA).
  • the hydrogel or hydrogel microsphere preparation process is under light-proof conditions, and the concentrations of GelMA and HAMA are 1% to 10% wt% respectively; the concentration of the blue light initiator is less than 0.1 wt%; the volume ratio of the GelMA and HAMA aqueous solutions is 1:1; the cross-linking curing adopts 405 nm blue light, and the cross-linking time is 10 to 120 s.
  • the present invention provides a combined stem cell and hydrogel biomaterial for treating spinal cord injury.
  • the hydrogel raw material used is of biological origin, has low immunogenicity, has good biocompatibility, and has certain biological functions. After transplantation, it can reduce the excessive aggregation of astrocytes after spinal cord injury, reduce the formation of glial scars, promote the regeneration and repair of nerves after spinal cord injury, and further promote the behavioral recovery of animals with spinal cord injury.
  • FIG1 is a physical characteristic of the prepared hydrogel microspheres loaded with stem cells
  • FIG2 shows the preparation of a spinal cord injury model and the transplantation of hydrogel microspheres loaded with stem cells
  • FIG3 is a graph showing motor function scores of rats after spinal cord injury treatment with stem cell-loaded hydrogel microspheres
  • Figure 4 is a comparison of the spinal cord injury cavity area and glial scar formation in different spinal cord injury treatment groups after 4 weeks;
  • Figure 5 is a comparison of newly formed nerve fibers in the spinal cord injury in different spinal cord injury treatment groups 4 weeks later.
  • Proliferation and culture of stem cells using mesenchymal stem cells obtained from umbilical cord or other tissues, using neuroepithelial stem cells obtained by inducing differentiation from embryonic stem cells, induced pluripotent stem cells or somatic cells, etc., according to existing standard culture protocols, obtaining mesenchymal stem cells and neuroepithelial stem cells with good growth status; the incubator conditions are 37°C, 5% CO2 , and saturated humidity.
  • the combined stem cell hydrogels used were prepared by chemical methods, using bio-derived gelatin, methacrylic anhydride, bio-derived hyaluronic acid, blue light initiator LAP and other chemical reagents to prepare and synthesize hydrogel materials that can be cross-linked and cured by 405nm blue light: methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA);
  • GelMA methacrylated gelatin
  • HAMA methacrylated hyaluronic acid
  • the present invention provides a method for preparing a combined stem cell and hydrogel biomaterial for treating spinal cord injury, which specifically includes the following steps: culturing and amplifying stem cells, and preparing stem cell-carrying hydrogel or hydrogel microspheres.
  • the specific operation steps are as follows:
  • Step 1 Cultivation and expansion of stem cells.
  • the incubator conditions are 37°C, 5% CO2 , and saturated humidity.
  • the established umbilical cord mesenchymal stem cells (MSCs) and neuroepithelial stem cells (NESCs) are cultured to obtain stem cells with better growth status.
  • the obtained stem cells are digested and counted.
  • Step 2 Preparation of biomaterial hydrogel for carrying stem cells, the specific operation is as follows:
  • Sodium carbonate is added to the obtained deionized water, and the sodium carbonate aqueous solution is diluted and adjusted to a pH of 7 to 10.
  • Gelatin is added to the sodium carbonate solution at a ratio of 50 to 200 g/L, and stirred to dissolve at 35 to 60° C., and then methacrylic anhydride is added, and the volume mass ratio of methacrylic anhydride to gelatin is 1:1 to 3:1, and the reaction is stirred at room temperature for 24 hours; after the reaction is completed, it is dialyzed with deionized water at room temperature for one week, and the molecular weight cutoff of the dialysis bag is 8KDa to 14KDa.
  • methacrylic acid acylated gelatin (GelMA) is obtained after freeze-drying; sodium hyaluronate is dissolved in deionized water at a ratio of 1 to 20 g/L, sodium carbonate is added to the obtained solution, and the mixed aqueous solution is diluted and adjusted to The pH value is 7 to 10, and it is stirred and dissolved at 35 to 60° C., and then methacrylic anhydride is added, and the volume mass ratio of methacrylic anhydride to sodium hyaluronate is 1:1 to 3:1, and the reaction is stirred at room temperature for 24 hours; after the reaction is completed, it is dialyzed with deionized water at room temperature for one week, and the molecular weight cutoff of the dialysis bag is 8KDa to 14KDa.
  • methacrylic acid acylated hyaluronic acid (HAMA) is obtained after freeze-drying; under light-proof conditions, the GelMA and HAMA obtained after freeze-drying are completely dissolved in a PBS solution to prepare a hydrogel solution with a concentration of 1% to 10%wt%, respectively.
  • the hydrogel also contains a blue light initiator LAP, and the concentration of the blue light initiator is less than 0.1wt%.
  • Step 3 Preparation of hydrogel biomaterial microspheres for treating spinal cord injury, the specific operation is as follows:
  • the prepared GelMA and HAMA hydrogel solutions were evenly mixed in a volume ratio of 1:1 and kept away from light for later use.
  • 0.1-1 ⁇ l of the GelMA and HAMA hydrogel mixed liquid was aspirated with a micropipette and dropped into ice mineral oil to allow it to undergo physical low-temperature cross-linking to form hydrogel microspheres.
  • the hydrogel microspheres were irradiated with a 405 nm blue light lamp for 30 to 60 seconds to allow the hydrogel to undergo a cross-linking chemical reaction of photo-initiated free radical polymerization to prepare stable light-cured hydrogel microspheres.
  • Step 4 Preparation of combined stem cell hydrogel biomaterial microspheres for treating spinal cord injury, the specific operation is as follows:
  • the prepared GelMA and HAMA hydrogel solutions were evenly mixed in a volume ratio of 1:1 and kept away from light for later use.
  • the stem cells obtained after large-scale culture were digested into single cells and then centrifuged to precipitate. According to the ratio of 1 to 5 million cells per 1 ml of hydrogel, MSCs and NESCs were evenly resuspended in the hydrogel, and 0.1-1 ⁇ l of the resuspended cell-hydrogel mixture was aspirated with a micropipette and dropped into ice mineral oil to form hydrogel microspheres by physical low-temperature crosslinking.
  • the hydrogel microspheres formed were irradiated with a 405 nm blue light lamp for 30 to 60 seconds to make the hydrogel undergo a crosslinking chemical reaction of photo-initiated free radical polymerization to prepare stable light-cured stem cell-loaded hydrogel microspheres.
  • Step 5 Characterization of combined stem cell and hydrogel biomaterial microspheres for treatment of spinal cord injury:
  • FIG. 1a shows that the size and shape of the hydrogel microspheres are uniform and the roundness is good.
  • Figure 1b shows that the hydrogel microspheres have a porous structure.
  • the electron microscope photo shows that it has a rough and porous honeycomb structure.
  • the larger voids can form more specific surface area and space, providing space for cell adhesion, proliferation and extension for the encapsulated stem cells, which is conducive to cell adhesion.
  • Figure 1c shows that the stem cells are evenly distributed in the hydrogel microspheres in the form of dispersed single cells, and their particle size can meet the normal exchange of substances between cells and the external environment, which is 400-500 ⁇ m.
  • the prepared hydrogel microspheres were filtered and collected using a 100 ⁇ m cell sieve. After collection, the hydrogel microspheres were repeatedly rinsed with 250mL of sterile saline or sterile PBS solution to remove residual mineral oil on the surface.
  • the collected stem cell-carrying hydrogel microspheres were stored in a 4°C low-temperature refrigerator or transported in a low-temperature ice box for subsequent animal transplantation experiments.
  • a functional detection method of combined stem cells and hydrogel biomaterial microspheres for treating spinal cord injury of the present invention specifically comprises the following steps: preparation of a spinal cord injury model, transplantation of hydrogel microspheres carrying stem cells, behavioral evaluation of the treatment effect of spinal cord injury animals, acquisition of tissue samples, and observation of the spinal cord injury scar area and nerve regeneration.
  • the specific operation steps are as follows:
  • the method of full transection was adopted, and the animals were anesthetized with 5% isoflurane concentration for 3-5 minutes.
  • the skin of the experimental animals was prepared with electric small animal clippers in the skin preparation area.
  • the experimental animals were anesthetized with 2% isoflurane concentration for surgery, and 75% alcohol and iodine were used for disinfection in turn.
  • the skin and muscles of the T4 segment of the experimental animals were carefully peeled off, and the T4 segment bone lamina was carefully removed with a patterned forceps to fully expose the spinal cord of the experimental animals.
  • the spinal cord of the experimental animals was partially hooked out with an arc hook, and the spinal cord tissue was completely cut and exposed with microscissors.
  • a spinal cord cut gap of more than 2mm was formed.
  • a medical cotton swab was used to fully stop the bleeding, and then the wound was fully rinsed with saline. The next step was performed after the bleeding stopped.
  • Step 1 Transplantation of stem cell-loaded hydrogel microspheres. The specific steps are as follows;
  • the collected stem cell-loaded hydrogel microspheres were transplanted into the 2mm gap of the spinal cord injury of the experimental animal using a syringe, the exposed muscles and skin of the experimental animal were sutured layer by layer, the wound was rinsed with saline, and 20,000 units of penicillin sodium and 1 ml of saline were injected subcutaneously into the experimental animal after surgery.
  • the left side of Figure 2 shows the prepared spinal cord injury model
  • the right side of Figure 2 shows the transplantation of stem cell-loaded hydrogel microspheres into the spinal cord injury site.
  • Step 2 Postoperative care: After the experimental animals wake up, they are sent to a clean animal room for daily injections of sodium penicillin and postoperative care, which lasts for one to several weeks.
  • Step 1 Collection of behavioral scores of rats with spinal cord injury. The specific steps are as follows:
  • Step 2 Behavioral scores of rats with spinal cord injury were used to evaluate the therapeutic effect. The specific results are as follows;
  • the data were processed by GraphPad and the results are shown in Figure 3.
  • the results in Figure 3 show that there are significant differences between the groups of hydrogel microspheres carrying stem cells; after treatment with hydrogel microspheres carrying a combination of three types of stem cells, the scores of the treatment group gradually improved over time, proving that their motor function was continuing to recover, and the BBB score tended to stabilize 90 days after postoperative treatment.
  • the hind limb walking gait of the group treated with MSC+NESC hydrogel microspheres was significantly improved, the hind paws could flip over and walk on the ground, and they could frequently walk with weight, and coordinated movements of the forelimbs and hindlimbs were occasionally seen, and the BBB behavioral score was significantly better than that of other groups.
  • the rats with spinal cord injury were treated with stem cell-loaded hydrogel microspheres or hydrogel microspheres for 4 weeks, and then the animals were over-anesthetized with pentobarbital, and perfused with an infusion bag connected to a syringe.
  • Normal saline was injected from the left ventricle aorta and flowed out from the right atrium. After the outflowing liquid became transparent, it was perfused with 4% paraformaldehyde until the animal was immobile.
  • the intact spinal cord tissue with lamina was removed, and the spinal cord was peeled after being fixed with 4% paraformaldehyde overnight. After sucrose concentration gradient dehydration, the spinal cord tissue was frozen and sectioned with a thickness of 20 ⁇ m. The tissue was attached to an adhesive slide, dried at 37°C for one hour, and stored in a refrigerator at -20°C for later use.
  • Step 1 After the slide is taken out and rinsed with PBS solution (3 times), it is permeabilized overnight with 0.2% Triton X100 solution. After washing with PBS, add blocking solution (3% BSA) and place it in a wet box for 2 hours at room temperature. Add 100 ⁇ l of rabbit anti-rat GFAP antibody or mouse anti-rat NF (Neurofilament) antibody diluted with antibody diluent and place it in a wet box at 4°C overnight.
  • Step 2 Observation of the area of spinal cord injury scar. After spinal cord injury, astrocytes participate in the formation of glial scar tissue, which limits the regeneration of axons. Therefore, the size of glial scar tissue is one of the indicators for evaluating nerve regeneration and repair. Astrocytes were labeled with GFAP antibody. The results in Figure 4 show that there are a large number of activated GFAP+ astrocytes in the injury site in the control untreated group, accounting for the largest proportion, and the injury site has a large cavity area. The GFAP+ cells in the hydrogel microsphere group were reduced to a certain extent, which shows that the hydrogel microspheres have a certain function of inhibiting the formation of glial scars.
  • stem cells further induced the reduction of GFAP+ cells in the spinal cord injury site.
  • GFAP+ cells There was no significant difference in GFAP+ cells between the MSC microsphere treatment group, the NESC microsphere treatment group, and the MSC+NESC microsphere treatment group, but the cell distribution in the MSC+NESC microsphere treatment group was more regular and the cavity area was the smallest.
  • Implantation of stem cell-loaded hydrogel microspheres effectively prevented the excessive aggregation of astrocytes and inhibited the formation of glial scars.
  • the regeneration of new neurons is involved in the process of spinal cord regeneration.
  • Step 3 Observation of nerve regeneration, using NF (neuronal fiber filaments) to mark the fibers of regenerated nerves.
  • NF neurovascular fiber filaments
  • Preparation of combined stem cells and hydrogel biomaterials and their application in spinal cord injury The obtained mesenchymal stem cells and/or neuroepithelial stem cells are combined with the prepared hydrogel biomaterials to prepare stem cell-carrying hydrogels or hydrogel microspheres. And they can only take effect by being transplanted into the damaged spinal cord of patients with spinal cord injury, and are used as cell drugs for the treatment of spinal cord injury.
  • the mesenchymal stem cells used are not limited to those obtained from the umbilical cord, but also include those obtained from other tissues; the neuroepithelial stem cells are not limited to those obtained by differentiation from embryonic stem cells, but also include neuroepithelial stem cells differentiated from induced pluripotent stem cells or neuroepithelial stem cells obtained by direct transdifferentiation of somatic cells.

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Abstract

本发明公开了联合干细胞和水凝胶生物材料制备及其在脊髓损伤的应用，其特征在于，包括以下步骤：制备甲基丙烯酸酰化明胶（GelMA）和甲基丙烯酸酰化透明质酸（HAMA）；制备水凝胶溶液，联合标准培养的间充质干细胞或/和神经上皮干细胞包裹在制备的水凝胶中，得到载干细胞水凝胶或水凝胶微球；将载干细胞水凝胶或水凝胶微球移植到脊髓损伤处进行治疗并评估效果。本发明用于修复脊髓损伤，使用生物材料，免疫原性低，具备较好的细胞生物相容性，并具有一定生物学功能；移植后可以减少脊髓损伤后星形胶质细胞的过度聚集，减少神经胶质疤痕的形成，改善膀胱和运动功能，能够促进脊髓损伤后神经的再生和修复，进而促进脊髓损伤动物的行为恢复。

Description

联合干细胞和水凝胶生物材料的制备及其在脊髓损伤中的应用 技术领域
本发明属于脊髓损伤修复技术领域，具体涉及一种联合干细胞和水凝胶生物材料制备及其在脊髓损伤的应用。
背景技术
脊髓损伤(spinalcordinjury，SCI)是临床常见的一种致残性严重创伤，其后果是神经元死亡及轴突断裂、神经支配功能的丧失，导致患者发生严重的生理、心理和社会交往的障碍，同时给社会和家庭带来沉重负担，其治疗是医学界长期的难题和研究热点。传统观点认为脊髓损伤后星形胶质细胞(astrocyte，AS)增殖、胶质瘢痕和空洞形成是阻碍损伤脊髓轴突有效再生和生长的主要因素。而近年来随着神经生理学、神经细胞生物学、分子生物学飞速发展，脊髓损伤的病理生理、修复机制的研究得以不断深入，最新研究表明脊髓损伤后AS反应和胶质瘢痕微环境对神经元存活、轴突再生、连接，甚至内源性神经干激活、分化均有重要作用，从而是决定脊髓损伤后神经结构和功能修复的关键因素。
间充质干细胞(MesenchymalStemCells，MSCs)易于获取，无伦理限制，并具有多向分化潜能、具有强大的营养和免疫调节活性、低免疫原性、高度的安全性以及调控炎性反应的能力，分泌的营养因子可促进损伤区域细胞存活，改善损伤区域微环境，改善脊髓损伤后的运动功能。因此间充质干细胞具有神经元的保护和促进神经再生的特性。
神经上皮干细胞(NeuroepithelialStemCells,NESCs)是神经管发育阶段的神经干细胞，具有很强的增殖和分化能力。神经上皮干细胞移植到脊髓损伤部位后，可为损伤部位提供细胞营养支持，保护神经细胞免于凋亡，促进受损轴突的生长；可高效分化产生神经元，与宿主脊髓神经元整合，促进神经再生，在损伤处重新建立神经回路重建皮质脊髓束，促进脊髓损伤神经功能的恢复。
目前，国内已建立了一套无血清间充质干细胞的扩增方案，建立了一套高效稳定扩增神经上皮干细胞的培养体系，能够标准化生产NESCs和MSCs。
生物材料作为干细胞载体支架，具备良好的生物相容性、降解性，可以模拟软组织环境，有效支持和引导轴突再生、迁移、抑制胶质瘢痕的形成，有利于营养物质和生长因子传递，因此生物材料与干细胞的结合，能够避免脊髓液流动导致的细胞流失以及减少免疫细胞的攻击。生物材料水凝胶与干细胞联合可促进脊髓损伤运动功能的恢复。
当前的脊髓损伤缺乏有效的修复方法，干细胞的移植治疗为该病的治疗提供了可能，然而选择适合脊髓损伤的干细胞类型以及相关的干细胞递送系统仍然缺乏。
因此，为了解决上述问题，本文提出一种联合干细胞和水凝胶生物材料制备及其在脊髓损伤的应用。
发明内容
为了解决上述技术问题，本发明设计了一种联合干细胞和水凝胶生物材料制备及其在脊髓损伤的应用，本发明通过甲基丙烯酸酰化明胶(GelMA)和甲基丙烯酸酰化透明质酸(HAMA)构建水凝胶或水凝胶微球，表现出很好的细胞相容性，在此基础上利用水凝胶或水凝胶微球包裹间充质干细胞和神经上皮干细胞，形成稳定的干细胞制剂，通过移植到损伤的脊髓部位，促进损伤脊髓的再生。
为了达到上述技术效果，本发明是通过以下技术方案实现的：一种联合干细胞和水凝胶生物材料在脊髓损伤中的应用，其特征在于：将联合干细胞和水凝胶生物材料移植到脊髓损伤患者的损伤脊髓部位，作为治疗脊髓损伤的细胞药物，修复损伤部位。
本发明的另一目的在于提供一种载干细胞水凝胶或水凝胶微球的制备方法，其特征在于，包括以下步骤：
Step1：制备甲基丙烯酸酰化明胶(GelMA)和甲基丙烯酸酰化透明质酸(HAMA)；
Step2：将GelMA和HAMA完全溶解于含有蓝光引发剂LAP的PBS溶液中，交联固化得到水凝胶或水凝胶微球；
Step3：将间充质干细胞或/和神经上皮干细胞包裹在制备的水凝胶或水凝胶微球中，得到载干细胞水凝胶或水凝胶微球。
进一步的，所述的Step1中甲基丙烯酸酰化明胶(GelMA)的制备方法为：将碳酸钠加入到去离子水中，按50～200g/L的比例将明胶加入碳酸钠溶液中，于35～60℃下搅拌溶解，然后加入甲基丙烯酸酐，甲基丙烯酸酐与明胶体积质量比为1:1～3:1，反应完成后常温下透析，冷冻干燥后得到甲基丙烯酸酰化明胶(GelMA)。
进一步的，所述的Step1中甲基丙烯酸酰化透明质酸(HAMA)的制备方法为：按1～20g/L的比例将透明质酸钠溶解于去离子水中，使用碳酸钠加入得到的溶液中，于35～60℃下搅拌溶解，然后加入甲基丙烯酸酐，甲基丙烯酸酐与透明质酸钠体积质量比为1:1～3:1，反应完成后用去离子水在常温下透析，冷冻干燥后得到甲基丙烯酸酰化透明质酸(HAMA)。
进一步的，所述Step2中，水凝胶或水凝胶微球制备过程在避光条件下，GelMA和HAMA浓度分别为1％～10％wt％；所述蓝光引发剂的浓度小于0.1wt％；GelMA和HAMA水溶液体积比为1：1；所述的交联固化采用405nm蓝光，交联的时间为10～120s。
本发明的有益效果是：
本发明的一种联合干细胞和水凝胶生物材料治疗脊髓损伤，所使用的水凝胶原材料为生物来源，免疫原性低，具备较好生物相容性，具有一定生物功能；移植后可以减少脊髓损伤后星形胶质细胞的过度聚集，减少神经胶质疤痕的形成，能够促进脊髓损伤后神经的再生和修复，进而促进脊髓损伤动物的行为恢复；成本低、活性高、治疗效果明显的特征，并且不会产生症状波动的药物副作用，且能够持续在宿主体内长时间存活并产生活性，具有现有药物和(或)干细胞制剂无法比拟的优势，为建立脊髓损伤治疗策略和设计新型干细胞治疗药物提供实验依据和理论基础，拥有治疗脊损伤广阔应用前景和较高的商业价值。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是制备的载干细胞水凝胶微球的物理学特征；
图2是脊髓损伤模型的制备及载干细胞水凝胶微球的移植；
图3是载干细胞水凝胶微球治疗脊髓损伤后大鼠运动功能评分；
图4是不同脊髓损伤治疗组4周后，脊髓损伤空洞面积和神经胶质疤痕形成比较；
图5是不同脊髓损伤治疗组4周后，脊髓损伤新生神经纤维丝比较。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1
干细胞的扩增培养：使用脐带来源或其它组织获得的间充质干细胞，使用和胚胎干细胞或诱导多能干细胞或体细胞等诱导分化产生获得的神经上皮干细胞，按照已有的标准培养方案，获得生长状态较好的间充质干细胞和神经上皮干细胞；培养箱条件为37℃、5％CO2、饱和湿度。
载干细胞的水凝胶制备：所使用联合干细胞水凝胶的制备，利用化学的方法，使用生物来源的明胶、甲基丙烯酸酐、生物来源的透明质酸、蓝光引发剂LAP和其他化学试剂制备合成可405nm蓝光交联固化的水凝胶材料：甲基丙烯酸酰化明胶(GelMA)和甲基丙烯酸酰化透明质酸(HAMA)；
载干细胞和水凝胶生物材料用于治疗脊髓损伤的治疗效果评估：
1)脊髓损伤动物疾病模型的制备，利用手术的方法制备脊髓损伤动物全横断模型；
2)载干细胞水凝胶或水凝胶微球移植治疗脊髓损伤动物疾病模型；将制备的载干细胞水凝胶或水凝胶微球移植到动物脊髓损伤部位进行治疗；
3)脊髓损伤动物行为学评估治疗效果；使用载干细胞水凝胶或水凝胶微球对脊髓损伤动物进行治疗后，按照时间节点对实验动物运动状态进行评分，评估治疗效果；
4)脊髓损伤治疗后组织样本的获取：获取脊髓损伤动物治疗一段时间后脊髓组织并进行标本固定和组织切片获取；
5)脊髓损伤疤痕面积和神经再生情况观察评估，使用免疫荧光酶法进行染色进行观察，评估脊髓损伤治疗效果。
实施例2
本发明一种用于治疗脊髓损伤的联合干细胞和水凝胶生物材料制备方法，具体包括以下步骤：干细胞的培养扩增、载干细胞水凝胶或水凝胶微球的制备，具体操作步骤如下：
步骤1：干细胞的培养扩增，按照已有的标准培养方案，培养箱条件为37℃、5％CO2、饱和湿度，对建系的脐带间充质干细胞(MSCs)和神经上皮干细胞(NESCs)进行培养，获得生长状态较好的干细胞，将获取的干细胞进行消化并计数。
步骤2：用于载干细胞的生物材料水凝胶的制备，具体操作如下：
将碳酸钠加入得到去离子水中，把碳酸钠水溶液稀释调节至pH为7～10，按50～200g/L的比例将明胶加入碳酸钠溶液中，于35～60℃下搅拌溶解，然后加入甲基丙烯酸酐，甲基丙烯酸酐与明胶体积质量比为1:1～3:1，在常温下搅拌反应24小时；反应完成后用去离子水在常温下透析一周，透析袋截留分子量为8KDa～14KDa，透析结束后，冷冻干燥后得到甲基丙烯酸酰化明胶(GelMA)；按1～20g/L的比例将透明质酸钠溶解于去离子水中，使用碳酸钠加入得到的溶液中，把混合水溶液稀释调节至pH为7～10，于35～60℃下搅拌溶解，然后加入甲基丙烯酸酐，甲基丙烯酸酐与透明质酸钠体积质量比为1:1～3:1，在常温下搅拌反应24小时；反应完成后用去离子水在常温下透析一周，透析袋截留分子量为8KDa～14KDa，透析结束后，冷冻干燥后得到甲基丙烯酸酰化透明质酸(HAMA)；避光条件下，将冷冻干燥后得到的GelMA和HAMA完全溶解于PBS溶液中，配置成水凝胶溶液，浓度分别为1％～10％wt％，所述水凝胶中还含有蓝光引发剂LAP，所述蓝光引发剂的浓度小于0.1wt％。
步骤3：用于治疗脊髓损伤的水凝胶生物材料微球的制备，具体操作如下：
将配置好的GelMA和HAMA水凝胶溶液按照体积比为1：1的比例均匀混合后避光备用，用微量移液器吸取0.1-1μl的GelMA和HAMA水凝胶混合液体，滴加到冰的矿物油中，使其物理低温交联形成水凝胶微球，使用405nm蓝光灯进行照射形成的水凝胶微球，光照时间为30～60s，使水凝胶进行光引发自由基聚合的交联化学反应，制备成稳定的光固化的水凝胶微球。
步骤4：用于治疗脊髓损:联合干细胞水凝胶生物材料微球的制备，具体操作如下：
将配置好的GelMA和HAMA水凝胶溶液按照体积比为1：1的比例均匀混合后避光备用。将规模化培养后获取的干细胞消化为单细胞后，进行离心沉淀。按照每1ml水凝胶中100～500万cells的比率，将MSCs和NESCs均匀重悬在水凝胶中，用微量移液器吸取0.1-1μl的重悬的细胞-水凝胶混合液体，滴加到冰的矿物油中，使其物理低温交联形成水凝胶微球，使用405nm蓝光灯进行照射形成的水凝胶微球，光照时间为30～60s，使水凝胶进行光引发自由基聚合的交联化学反应，制备成稳定的光固化的载干细胞水凝胶微球。
步骤5：用于治疗脊髓损伤的联合干细胞和水凝胶生物材料微球的特征：
制备的水凝胶微球形态如图1所示，图1a表明，水凝胶微球大小形态均一，圆度较好。图1b表明，水凝胶微球具有多孔隙结构，其电镜照片显示了它具有粗糙多孔的蜂窝状结构，较大空隙可以形成更多比表面积和空间，为包裹的干细胞提供细胞黏附增殖和延伸的空间，有利于细胞的黏附。图1c表明干细胞呈分散单细胞形式均匀分布在水凝胶微球内，其粒径大小能满足细胞与外界环境的物质正常交换，为400～500μm。将制备好的水凝胶微球使用100μm细胞筛网进行过滤收集，收集后用250mL无菌生理盐水或无菌PBS溶液反复冲洗水凝胶微球，去除表面残留矿物油。收集好的载干细胞水凝胶微球使用4℃低温冰箱保存或使用低温冰盒运输，用于后续动物移植实验。
实施例3
参阅图2至图5所示，本发明一种用于治疗脊髓损伤的联合干细胞和水凝胶生物材料微球的功能检测方法，具体包括以下步骤：脊髓损伤模型的制作、载干细胞水凝胶微球的移植、脊髓损伤动物行为学评估治疗效果、组织样本的获取、脊髓损伤疤痕面积和神经再生情况观察，具体操作步骤如下：
1、脊髓损伤大鼠疾病模型的制备。选取200-250g清洁级成年雌性SD大鼠，并按照下面步骤进行模型制备：
采用全横断的方法，使用5％异氟烷浓度诱导麻醉动物3-5分钟，在备皮区域用电动小动物推剪对实验动物进行备皮。2％异氟烷浓度麻醉实验动物进行手术，依次使用75％酒精、碘伏涂抹消毒，小心剥离开实验动物T4段皮肤、肌肉，使用纹式钳小心去除T4段骨椎板，将实验动物脊髓充分暴露。使用弧型勾将实验动物脊髓部分勾出，使用显微剪完全剪断暴露脊髓组织，由于剪断脊髓组织的回缩，最后形成越2mm肉眼可见脊髓剪断空隙，使用医用棉签进行充分止血，再使用生理盐水对伤口充分进行冲洗，待出血止住后进行下一步操作。
2、载干细胞水凝胶或水凝胶微球移植治疗脊髓损伤大鼠模型，具体实施步骤如下；
步骤1：载干细胞水凝胶微球的移植治疗，具体操作如下；
使用注射器将收集好的载干细胞水凝胶微球移植到实验动物的脊髓损伤2mm空隙部位，逐层缝合实验动物暴露肌肉、皮肤，使用生理盐水冲洗伤口，术后对实验动物皮下注射20000单位的青霉素钠和生理盐水各1ml。如图2所示，图2左侧显示为制作的脊髓损伤模型，图2右侧为载干细胞水凝胶微球移植到脊髓损伤部位。
步骤2：术后护理：待实验动物苏醒后送至清洁级动物房每天注射青霉素钠和术后护理，护理持续一周至数周时间。
3、脊髓损伤动物行为学评估治疗效果，具体实施步骤如下；
步骤1：脊髓损伤大鼠行为学评分采集，具体操作如下：
动物手术一周后，将动物放置在空旷无障碍的场地使其自由活动，对动物的臀、膝、踝关节活动，以及前后肢协调性、躯干运动稳定性等情况进行观察。动物运动评分超过3分的剔除实验分组。实验动物术后一周，进行开放场地实验，应用脊髓损伤大鼠行为学评分(Basso-Beattie-Bresnahan,BBB)评分法对实验动物运动状态逐一进行评分，该评分主要对脊髓损伤大鼠的后置运动，前后肢协调性、驱赶稳定性等进行评价。评分持续到损伤治疗6个月后。本实验规定术后BBB评分不能超过3分，术后BBB评分超过3分的剔除实验组。
步骤2：脊髓损伤大鼠行为学评分评估治疗效果，具体结果如下；
数据经GraphPad进行处理显示，数据结果如图3所示，图3结果表明，各载干细胞水凝胶微球分组间差异显著；使用载三种类型干细胞组合的水凝胶微球治疗后，治疗组的评分随着时间的推移逐渐提高改善，证明其运动功能在持续恢复，术后治疗90天后BBB评分趋于稳定，载MSC+NESC水凝胶微球治疗组后肢行走步态明显的改善，后脚爪能翻转着地行走，后可以频繁负重步行,前后肢偶尔可见协调运动，BBB行为学评分显著优于其他组。
4、脊髓损伤治疗后组织样本的获取，具体实施步骤如下：
完成脊髓损伤动物行为学评估治疗效果后，使用载干细胞水凝胶微球或水凝胶微球治疗脊髓损伤大鼠4周后，使用戊巴比妥过度麻醉动物，并用输液袋连接注射器进行灌流，将生理盐水从左心室主动脉中注入，右心房流出，等到流出的液体呈现透明后，再用4％多聚甲醛进行灌流直至动物不动为止。取出完整带椎板脊髓组织，4％多聚甲醛固定过夜后剥出脊髓，蔗糖浓度梯度脱水后对脊髓组织进行冰冻切片，切片厚度20μm，将组织贴附于黏附载玻片上，37℃烘干一小时，-20℃冰箱保存备用。
5、脊髓损伤疤痕面积和神经再生情况观察评估，使用免疫荧光酶法进行染色进行观察，具体实施步骤如下：
步骤1：载玻片取出用PBS溶液冲洗后(3次)用0.2％Triton X100溶液进行过夜透膜，PBS洗涤后加封闭液(3％BSA)，室温下置于湿盒内封闭2小时。加抗体稀释液稀释后的兔抗大鼠GFAP抗体或小鼠抗大鼠NF(Neurofilament)抗体100μl，置于湿盒内4℃过夜， 第二天PBS洗涤后用抗体稀释液(同上)稀释过的驴抗兔IgG多抗或兔抗小鼠IgG多抗(1：800)和DAPI(1：1000)100μl，置于避光湿盒内室温下孵育1小时。PBS洗涤(3次)后，50％甘油-PBS溶液封片避光保存，置于激光共聚焦显微镜下观察切片染色情况并拍照。
步骤2：脊髓损伤疤痕面积观察，脊髓损伤后，星形胶质细胞参与形成神经胶质疤痕组织，限制了轴突的再生，因此神胶质疤痕组织的大小是评估神经再生和修复的指标之一，使用GFAP抗体标记星形胶质细胞。图4结果显示，对照未治疗组在损伤部位存在大量激活的GFAP+星形胶质细胞，占比最大，损伤部位具有较大的空腔面积。水凝胶微球组GFAP+细胞得到了一定层度的减少，这说明水凝胶微球具有一定抑制胶质疤痕形成的功能，干细胞的加入进一步诱导了脊髓损伤部位GFAP+细胞的减少。MSC微球治疗组、NESC微球治疗组以及MSC+NESC微球治疗组之间的GFAP+细胞未表现明显的差异，但MSC+NESC微球治疗组细胞分布更规则、空腔面积最少。植入载干细胞水凝胶微球有效的阻止了星形胶质细胞的过度聚集，抑制了神经胶质疤痕的产生。脊髓再生过程中包含着新生神经元的再生。
步骤3：神经再生情况观察，使用NF(神经纤维丝)来标记再生神经的纤维。图5结果表明对照未治疗组在损伤部位NF+神经纤维数目较少，形态稀疏，长度较短，无明显的神经纤维再生。水凝胶微球组在损伤部位可见少量的NF+神经纤维。干细胞治疗后的脊髓在损伤部位NF+的密度显著增加，神经纤维出现大量的再生，分布更加规则，MSC+NESC治疗组的NF+细胞比率高于MSC和NESC治疗组，神经纤维丝形态清晰呈线状连接脊髓损伤的前后段。
实施例4
联合干细胞和水凝胶生物材料的制备及其在脊髓损伤的应用，将获得的间充质干细胞或/和神经上皮干细胞，联合制备的水凝胶生物材料，制备载干细胞水凝胶或水凝胶微球。并通过移植到脊髓损伤疾病病人的损伤脊髓部位，才能发生作用，用于治疗脊髓损伤的细胞药物。所用的间充质干细胞不仅限于从脐带获得，还包括从其它组织获得的间充质干细胞；所属的神经上皮干细胞不仅限于从胚胎干细胞分化获得，还包括从诱导多能干细胞分化而来的神经上皮干细胞或通过体细胞直接转分化而获得的神经上皮干细胞。
在本说明书的描述中，参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节，也不限制该发明仅为所述的具体实施方式。显然，根据本说明书的内容，可作很多的修改和变化。本说明书选取并具体描述这些实施例，是为了更好地解释本发明的原理和实际应用，从而使所属技术领域技术人员能很好地理解和利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。

Claims (6)

  1. 一种联合干细胞和水凝胶生物材料在脊髓损伤中的应用，其特征在于：将联合干细胞和水凝胶生物材料移植到脊髓损伤患者的损伤脊髓部位，作为治疗脊髓损伤的细胞药物，修复损伤部位。
  2. 一种联合干细胞和水凝胶生物材料的制备方法，其特征在于，包括以下步骤：
    Step1：制备甲基丙烯酸酰化明胶(GelMA)和甲基丙烯酸酰化透明质酸(HAMA)；
    Step2：将GelMA和HAMA完全溶解于含有蓝光引发剂LAP的PBS溶液中，交联固化得到水凝胶或水凝胶微球；
    Step3：将间充质干细胞或/和神经上皮干细胞包裹在制备的水凝胶或水凝胶微球中，得到载干细胞水凝胶或水凝胶微球。
  3. 根据权利要求2所述的一种联合干细胞和水凝胶生物材料的制备方法，其特征在于，所述的Step1中甲基丙烯酸酰化明胶(GelMA)的制备方法为：将碳酸钠加入到去离子水中，按50～200g/L的比例将明胶加入碳酸钠溶液中，于35～60℃下搅拌溶解，然后加入甲基丙烯酸酐，甲基丙烯酸酐与明胶体积质量比为1:1～3:1，反应完成后常温下透析，冷冻干燥后得到甲基丙烯酸酰化明胶(GelMA)。
  4. 根据权利要求2所述的一种联合干细胞和水凝胶生物材料的制备方法，其特征在于，所述的Step1中甲基丙烯酸酰化透明质酸(HAMA)的制备方法为：按1～20g/L的比例将透明质酸钠溶解于去离子水中，使用碳酸钠加入得到的溶液中，于35～60℃下搅拌溶解，然后加入甲基丙烯酸酐，甲基丙烯酸酐与透明质酸钠体积质量比为1:1～3:1，反应完成后用去离子水在常温下透析，冷冻干燥后得到甲基丙烯酸酰化透明质酸(HAMA)。
  5. 根据权利要求2所述的一种联合干细胞和水凝胶生物材料的制备方法，其特征在于，所述Step2中，水凝胶或水凝胶微球制备过程在避光条件下，GelMA和HAMA浓度分别为1％～10％wt％；所述蓝光引发剂的浓度小于0.1wt％；GelMA和HAMA水溶液体积比为1：1；所述的交联固化采用405nm蓝光，交联的时间为10～120s。
  6. 根据权利要求2所述的一种联合干细胞和水凝胶生物材料的制备方法，其特征在于，所述的间充质干细胞从脐带获得，或从其它组织获得的间充质干细胞；所述的神经上皮干细胞从胚胎干细胞分化获得，或从诱导多能干细胞分化而来的神经上皮干细胞或通过体细胞直接转分化而获得的神经上皮干细胞。
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