Porous microcarrier capable of loading human umbilical cord mesenchymal stem cells as well as preparation method and application of porous microcarrier

Abstract

The invention discloses a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells and a preparation method and application of the porous microcarrier, and relates to the technical field of biomedical engineering. Comprising the following raw materials in parts by weight: 0.1-5 parts of methylacryloylated gelatin, 0.1-5 parts of methylacryloylated silk fibroin and 0.1-5 parts of methylacryloylated chondroitin sulfate. The preparation method of the porous microcarrier comprises the following steps: S1, constructing a microfluidic platform; s2, preparing microsphere liquid drops; s3, preparing a porous microcarrier containing gelatin, silk fibroin and chondroitin sulfate; according to the prepared gelatin-silk fibroin-chondroitin sulfate microcarrier capable of loading the hUMSCs, the silk fibroin and the chondroitin sulfate are doped in the gelatin, and compared with the gelatin microcarrier, by adjusting the physical performance of the gelatin-silk fibroin-chondroitin sulfate microcarrier capable of loading the hUMSCs, the anti-inflammatory capacity and the immune regulation capacity of the hUMSCs can be effectively improved, and the capacity of stem cells for treating related diseases is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a porous microcarrier capable of carrying human umbilical cord mesenchymal stem cells, and a preparation method and application thereof. Background Art

[0002] Human umbilical cord mesenchymal stem cells (hUMSCs) can be isolated from the Wharton's jelly of the human umbilical cord. They have the potential for self-renewal and multidirectional differentiation and can differentiate into osteoblasts, adipocytes, chondrocytes, etc. in vitro. Stem cells can replace damaged tissues and organs by differentiating into specific cells; they can also intervene in the cell functions of cells and immune cells in damaged tissues through secretion or paracrine effects (including various soluble factors, exosomes, and microcapsules, etc.), promote endogenous regeneration mechanisms, and achieve therapeutic effects such as new angiogenesis, anti-fibrosis, anti-apoptosis, anti-inflammation, and immunomodulation. MSCs transplantation has become a promising therapeutic strategy for damaged tissues. For example, a large number of studies have shown that the use of mesenchymal stem cells (MSCs) to treat chronic wounds in diabetes can regulate the inflammatory process, accelerate epidermal regeneration, significantly increase the number of microvessels and fibroblasts, make collagen arrangement more regular, reduce scar formation, and help repair skin appendages such as sweat glands and sebaceous glands.

[0003] In clinical studies, stem cell preparations directly applied to damaged tissues in the form of suspensions are difficult to colonize and survive at the lesion site, and stem cells will die by shearing during the injection process; the harsh microenvironment at the lesion site seriously affects the survival quality of cells after local administration, and usually within a few hours after transplantation, most cells are difficult to survive. Developing effective stem cell delivery vectors to build a good microenvironment for transplanted cells is crucial to achieving stem cell therapy.

[0004] Stem cell delivery vehicles are usually in the form of bulk hydrogels and microgels (microcarriers). Compared with bulk hydrogels, hydrogel microparticles (also known as microgels, or microcarriers, HMPs) have many unique properties: first, they are small in size and can be injected through small needles and catheters, which is conducive to minimally invasive delivery of cells and biologics; second, the HMP system itself is modular, and multiple HMP populations with different compositions, sizes and inclusions can be mixed to create different materials; third, particles can be combined and assembled through a variety of annealing methods such as covalent bonding, reversible bonding, electrostatic interaction, and hydrophobic interaction; fourth, granular hydrogels have significant porosity (or pore space), which not only facilitates the transport of nutrients and waste by providing a stable microporous connection network, but also facilitates the migration of host / transplanted cells and overall integration with surrounding tissues, thereby avoiding the need for material degradation before tissue growth. Injectable microporous annealed particle gel scaffolds have been studied and applied in many fields such as skin repair, bone repair, cartilage repair, osteoarthritis, stroke, and nerve repair. As a carrier for cell and drug delivery and a tissue engineering scaffold material, it has broad application prospects in the field of regenerative medicine. Common methods for preparing hydrogel microparticles include batch emulsion, microfluidic emulsion, photolithography, EHD spraying, and mechanical crushing. Hydrogel microspheres prepared by microfluidics have the advantages of better monodispersity, more controllable size adjustment, excellent encapsulation performance, and advanced controlled release performance.

[0005] At present, most commercial microcarriers or research microcarriers use gelatin as the main raw material, and are mainly used for stem cell expansion or stem cell exosome production. As a microtissue treatment, gelatin microcarriers have certain defects in specific indications, such as their degradation rate may not match the healing rate of damaged tissues (such as difficult-to-heal wounds), or the therapeutic ability of stem cells needs to be further improved. Silk fibroin, as a natural protein polymer macromolecule, has the characteristics of flexible molecular chains, good biocompatibility and degradability (generally degradation time is 8 to 12 weeks); its regular structure (crystallization mode) is diverse, and can achieve conversion and regulation between water-soluble structures and insoluble structures, which is more conducive to regulating the series of morphologies and properties of silk fibroin materials. In addition, silk fibroin has a good promoting effect on the proliferation of human keratinocytes and fibroblasts. A large number of studies have shown that mixed silk fibroin scaffolds may have better application prospects than pure silk fibroin in skin tissue regeneration and wound healing. Chondroitin sulfate (CS) is an anionic heteropolysaccharide that is widely present in the extracellular matrix in the form of proteoglycans. It is covalently linked to the core protein through O-glycosidic bonds and has anti-inflammatory, antioxidant, anti-tumor, anti-coagulant and anti-thrombotic activities. Chondroitin sulfate is biodegradable and biocompatible in nature and is used as a promising biomaterial for drug delivery and tissue engineering. Therefore, other active ingredients such as silk fibroin and chondroitin sulfate can be introduced for specific indications to develop corresponding microcarriers to achieve the purpose of microtissue therapy loaded with stem cells.

[0006] The technology of preparing microspheres through microfluidic platforms has been reported. Most of the work is to initiate polymerization through photocrosslinking. There are also reports that crosslinking is achieved through click chemistry reactions of specific groups. Its characteristics are that the microsphere droplets are solidified in the microfluidic channel, and the physical properties of the microspheres (size, stiffness, micro-nanotopological structure, pore structure, network size) can be adjusted to a limited extent. Therefore, this patent separates the generation of microsphere droplets and the solidification of microspheres into two independent steps, while introducing a gradient temperature control program and adjusting the ratio of dispersed phase mixed solvents to achieve freezing and pore formation, and then photocrosslinking and solidification while thawing to regulate the physical properties of the microspheres, thereby accurately regulating the cell fate of MSCs.

[0007] In summary, this patent intends to develop a new type of microcarrier with uniform particle size that can load MSCs by combining microfluidic technology and cryoporation technology, and utilize the chemical composition (gelatin, silk fibroin, chondroitin sulfate) and physical properties (size, stiffness, micro-nano topological structure, pore structure, network size) of the microcarrier to accurately regulate the cell fate of MSCs, such as improving its anti-inflammatory and immunomodulatory capabilities, thereby improving the effect of MSCs in treating specific lesions (such as burn wounds, diabetic wounds, osteoarthritis or pulpitis, etc.), and can also be used for the large-scale production of stem cells or their exosomes. Summary of the invention

[0008] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells. The microcarrier is prepared based on microfluidic technology, which is easy to operate and has controllable size. The prepared gelatin-silk fibroin-chondroitin sulfate microcarrier capable of loading hUMSCs is doped with silk fibroin and chondroitin sulfate in gelatin, and by adjusting its physical properties (size, stiffness, topological structure, pore structure, network size), compared with gelatin microcarriers, it can effectively upregulate the anti-inflammatory and immune regulatory abilities of hUMSCs, thereby improving the ability of stem cells to treat related diseases.

[0009] To achieve the above purpose, the present invention adopts the following technical solutions: A porous microcarrier capable of loading human umbilical cord mesenchymal stem cells comprises the following raw materials, calculated by mass concentration ratio: 0.1-5 parts of methacrylylated gelatin, 0.1-5 parts of methacrylylated silk fibroin and 0.1-5 parts of methacrylylated chondroitin sulfate.

[0010] The method for preparing the porous microcarrier capable of loading human umbilical cord mesenchymal stem cells comprises the following steps: S1, build a microfluidic platform; S2. Preparation of microsphere droplets: gelatin, silk fibroin and chondroitin sulfate are dissolved in a photoinitiator solution, and an integrated microfluidic device is used with a single-channel chip; the dispersed phase solution is connected to the water phase inlet of the chip, and the mobile phase containing a non-ionic emulsifier is connected to the oil phase inlet, and the pressure of the water phase and oil phase inlet channels is regulated to set the flow rate; the dispersed phase is a prepolymer solution containing gelatin, silk fibroin and chondroitin sulfate, which contains a photoinitiator and double bond groups with photo-induced responsive cross-linking; the mobile phase is an oil phase solution containing a specific concentration of a non-ionic emulsifier; uniform, stable and non-fusion-resistant microsphere droplets are prepared, and the diameter of the microsphere droplets is controlled between 20 μm and 500 μm; S3. Preparation of porous microcarriers containing gelatin-silk fibroin-chondroitin sulfate: The prepared microsphere droplets are placed in a gradient temperature freezer. Under the irradiation of ultraviolet light, the photoinitiator inside the microsphere droplets will trigger the photo-crosslinking reaction of the macromolecules of the cross-linked double bond groups. After the cross-linking is completed, the porous microcarrier is prepared. The excess toxic substances are removed with isopropanol and deionized water, and the porous microcarrier is then subjected to gradient freezing and freeze-dried for later use.

[0011] As a preferred embodiment: the dispersed phase in step S2 is water-soluble, the gelatin, silk fibroin and chondroitin sulfate form modified macromolecules, and the total concentration of the modified biomacromolecules contained in the dispersed phase is 0.5wt%~6wt%; the dispersed phase contains double bond groups that can be cross-linked by a photoinitiator, and the double bond groups include one or more of methacryloyl groups, methacrylic anhydride, maleic anhydride, glycidyl methacrylate, hydroxyethyl methacrylate, and N,N'-methylenebisacrylamide; the concentration of the double bond groups accounts for 1%~60% of the total mass.

[0012] As a preferred embodiment: the solvent of the dispersed phase in step S2 is water, and the dispersed phase can be doped with water-soluble organic solvents and inorganic salts, the organic solvent includes methanol, ethanol, isopropanol, glycerol, DMSO, and the inorganic salt includes sodium chloride, potassium chloride, calcium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate; wherein the PBS buffer solution containing 0.1% to 5% DMSO is a preferred embodiment.

[0013] As a preferred embodiment, the dispersed phase photoinitiator in step S2 is a soluble photoinitiator, which includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone; the concentration of the photoinitiator is 0.1% to 2%.

[0014] As a preferred embodiment, the mobile phase in step S2 includes HFE-7500 electronic fluorinated liquid containing 0.1% to 5% perfluoro surfactant, isopropyl palmitate containing 0.1% to 10% EM-180, and mineral oil or soybean oil containing 0.1% to 8% Span-80.

[0015] As a preferred solution: in step S2, the flow rate ratio of the dispersed phase to the mobile phase is 0.5:0.5~1, and the pipeline pressure ratio of the dispersed phase to the mobile phase is 1:0.5~1.

[0016] As a preferred solution: in step S3, the microsphere droplets are frozen overnight, and a gradient temperature freezing is selected, the temperature range is -20°C to -196°C, and the time is 30min to 12h to form pores inside the microspheres; the wavelength range of the cross-linking light is 350~450 nanometers, and the light intensity range is 10~100mw / cm2 The photo-crosslinking time is 30min~2h to achieve the curing and molding of microspheres.

[0017] The porous microcarrier capable of carrying human umbilical cord mesenchymal stem cells is used to carry human umbilical cord mesenchymal stem cells. The porous microcarrier is immersed in 75% alcohol for sterilization, replaced with DPBS several times, and excess alcohol is removed. Then, the human umbilical cord mesenchymal stem cells are inoculated on the microcarrier for culture; the microcarrier containing gelatin, silk fibroin, and chondroitin sulfate for inoculating human umbilical cord mesenchymal stem cells has a diameter range of 50 μm to 300 μm; the inoculation density of human umbilical cord mesenchymal stem cells is 10×10 4 / mg~1000×10 4 / mg.

[0018] As a preferred embodiment: the microcarrier containing gelatin, silk fibroin and chondroitin sulfate loaded with human umbilical cord mesenchymal stem cells is used for large-scale production of stem cells or their exosomes, and for preparing drugs for large-area wounds, difficult-to-heal wounds, osteoarthritis or pulpitis. The drug is used independently or mixed with other in situ formed hydrogels; the dosage form of the drug is selected from dressings, ointments, liniments or injectable dosage forms.

[0019] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical solution that: 1. The present invention prepares microcarriers based on microfluidic technology, which is easy to operate and has controllable size.

[0020] 2. The porous microcarriers prepared by the method of the present invention based on microfluidic technology and gradient freezing pore making technology can achieve size adjustment of the microcarriers by adjusting the microfluidic parameters; the hardness, pore size and topological structure of the microcarriers can be adjusted by adjusting the type, concentration and ratio of the prepolymer, or adjusting the composition of the solvent of the dispersed phase, or adjusting the gradient freezing program.

[0021] 3. The gelatin-silk fibroin-chondroitin sulfate microcarrier prepared by the present invention that can load hUMSCs is doped with silk fibroin and chondroitin sulfate in gelatin, and by adjusting its physical properties (size, stiffness, topological structure, pore structure, network size), it can effectively increase the anti-inflammatory ability (STC1) and immune regulation ability (PGE-2 and TSG-6) of hUMSCs compared to gelatin microcarriers, thereby improving the ability of stem cells to treat related diseases.

[0022] 4. The gelatin-silk fibroin-chondroitin sulfate microcarrier prepared by the present invention is a potential stem cell delivery carrier, which is expected to be used in tissue engineering, large-area wounds (such as burn wounds), difficult-to-heal wounds (such as diabetic wounds), osteoarthritis or pulpitis, and can also be used for large-scale production of stem cells or their exosomes.

[0023] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of the process for preparing the porous microcarrier of the present invention; Figure 2 The scanning electron microscope images of the porous microcarriers of different material components of the present invention; Figure 3 The optical image and live-dead staining image of the microcarrier carrying hUMSCs of the present invention; Figure 4 Schematic diagram of the cell proliferation ability of HUMSCs on microcarriers of different components of the present invention; Figure 5 This is a cytoskeleton staining image of the microcarrier carrying hUMSCs of the present invention; Figure 6 Schematic diagram of the expression of inflammation-related genes (STC1, IL-6), immune regulatory genes (PGE-2, TSG-6) and TGF-β of HUMSCs of the present invention on microcarriers of different components; Figure 7 Schematic diagram of wound closure of the effect of the microcarrier carrying hUMSCs on diabetic wound healing of the present invention; Figure 8 Schematic diagram of wound area statistics showing the effect of the hUMSCs-loaded microcarriers of the present invention on diabetic wound healing. DETAILED DESCRIPTION

[0025] The present invention Figure 1 As shown in FIG8 , a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells comprises the following raw materials, calculated by mass concentration ratio: 0.1 to 5 parts of methacrylylated gelatin, 0.1 to 5 parts of methacrylylated silk fibroin, and 0.1 to 5 parts of methacrylylated chondroitin sulfate.

[0026] The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells comprises the following steps: S1, build microfluidic platform; S2. Preparation of microsphere droplets: gelatin, silk fibroin and chondroitin sulfate are dissolved in a photoinitiator solution, and an integrated microfluidic device is used with a single-channel chip; the dispersed phase solution is connected to the water phase inlet of the chip, and the mobile phase containing a non-ionic emulsifier is connected to the oil phase inlet, and the pressure of the water phase and oil phase inlet channels is regulated to set the flow rate; the dispersed phase is a prepolymer solution containing gelatin, silk fibroin and chondroitin sulfate, which contains a photoinitiator and double bond groups with photo-induced responsive cross-linking; the mobile phase is an oil phase solution containing a specific concentration of a non-ionic emulsifier; uniform, stable and non-fusion-resistant microsphere droplets are prepared, and the diameter of the microsphere droplets is controlled between 20 μm and 500 μm; S3. Preparation of porous microcarriers containing gelatin-silk fibroin-chondroitin sulfate: The prepared microsphere droplets are placed in a gradient temperature freezer. Under the irradiation of ultraviolet light, the photoinitiator inside the microsphere droplets will trigger the photo-crosslinking reaction of the macromolecules of the cross-linked double bond groups. After the cross-linking is completed, the porous microcarrier is prepared. The excess toxic substances are removed with isopropanol and deionized water, and the porous microcarrier is then subjected to gradient freezing and freeze-dried for later use.

[0027] In step S2, the dispersed phase is water-soluble, the gelatin, silk fibroin and chondroitin sulfate form modified macromolecules, and the total concentration of the modified biomacromolecules contained in the dispersed phase is 0.5wt%~6wt%; the dispersed phase contains double bond groups that can be cross-linked by a photoinitiator, and the double bond groups include one or more of methacryloyl groups, methacrylic anhydride, maleic anhydride, glycidyl methacrylate, hydroxyethyl methacrylate, and N,N'-methylenebisacrylamide; the concentration of the double bond groups accounts for 1%~60% of the total mass.

[0028] The solvent of the dispersed phase in step S2 is water, and the dispersed phase can be doped with water-soluble organic solvents and inorganic salts, the organic solvents include methanol, ethanol, isopropanol, glycerol, DMSO, and the inorganic salts include sodium chloride, potassium chloride, calcium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate; wherein a PBS buffer solution containing 0.1% to 5% DMSO is a preferred embodiment.

[0029] The dispersed phase photoinitiator in step S2 is a soluble photoinitiator, which includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone; the concentration of the photoinitiator is 0.1% to 2%.

[0030] In step S2, the mobile phase includes HFE-7500 electronic fluorinated liquid containing 0.1% to 5% perfluoro surfactant, isopropyl palmitate containing 0.1% to 10% EM-180, and mineral oil or soybean oil containing 0.1% to 8% Span-80.

[0031] In step S2, the flow rate ratio of the dispersed phase to the mobile phase is 0.5:0.5~1, and the pipeline pressure ratio of the dispersed phase to the mobile phase is 1:0.5~1.

[0032] In step S3, the microsphere droplets are frozen overnight, using a gradient temperature freezing method, with a temperature range of -20°C to -196°C and a time of 30 min to 12 h to form pores inside the microspheres; the wavelength range of the cross-linking light used is 350 to 450 nanometers, and the light intensity range is 10 to 100 mw / cm 2 The photo-crosslinking time is 30min~2h.

[0033] The porous microcarrier can be used to load human umbilical cord mesenchymal stem cells. The porous microcarrier is immersed in 75% alcohol for sterilization, and DPBS is replaced several times to remove excess alcohol. Then, the human umbilical cord mesenchymal stem cells are inoculated on the microcarrier for culture. The diameter of the microcarrier containing gelatin, silk fibroin, and chondroitin sulfate used for inoculating human umbilical cord mesenchymal stem cells ranges from 50 μm to 300 μm. The inoculation density of human umbilical cord mesenchymal stem cells is 10×10 4 / mg~1000×10 4 / mg.

[0034] The microcarrier containing gelatin, silk fibroin and chondroitin sulfate loaded with human umbilical cord mesenchymal stem cells is used for large-scale production of stem cells or their exosomes, and is used to prepare drugs for large-area wounds, difficult-to-heal wounds, osteoarthritis diseases or pulpitis diseases. The drug is used independently or mixed with other in-situ formed hydrogels; the dosage form of the drug is selected from dressings, ointments, liniments or in vivo injectable dosage forms.

[0035] A method for preparing a porous microcarrier (gelatin-silk fibroin-chondroitin sulfate microcarrier) that can load and empower human umbilical cord mesenchymal stem cells (hUMSCs), first using microfluidic technology to prepare solid microsphere droplets of uniform size, wherein the mobile phase is an oil phase solution containing a non-ionic emulsifier of a specific concentration, and the dispersed phase is a prepolymer solution containing a photoinitiator and a modified biomacromolecule (such as gelatin, silk fibroin and chondroitin sulfate) with a double bond group having a photo-initiated responsive cross-linking. Then, it is placed in a refrigerator and frozen overnight at a gradient temperature, and then, under ultraviolet light, it is thawed and photocrosslinked and cured, and then isopropanol and deionized water are used to remove excess toxic substances, and finally, it is gradient frozen and freeze-dried for use. The morphological characteristics of the microcarrier are characterized and analyzed by microscopy and scanning electron microscopy (SEM). Subsequently, the microcarrier was used to carry human umbilical cord mesenchymal stem cells, and its biocompatibility and the cell function of hUMSCs after being carried were evaluated by live-dead staining (Calcein-AM / PI staining), CCK8, cytoskeleton staining, PCR, etc. Finally, the porous microcarrier loaded with and able to empower human umbilical cord mesenchymal stem cells (hUMSCs) was placed on the wound surface of diabetic rats to observe the healing of the wound.

[0036] The specific steps include: S1. Construction of microfluidic platform.

[0037] S2. Preparation of microsphere droplets: using a single-channel chip; the dispersed phase is a prepolymer solution containing a photoinitiator and modified biomacromolecules (such as gelatin, silk fibroin and chondroitin sulfate) with double bond groups having photoinitiator-responsive cross-linking, and the mobile phase is an oil phase solution containing a specific concentration of a non-ionic emulsifier; by adjusting the chip channel diameter, the flow rates of the dispersed phase and the mobile phase, etc., microsphere droplets of uniform size, stability and non-fusion resistance are prepared, and the diameter of the microsphere droplets is controlled between 20μm and 500μm.

[0038] S3. Preparation of microcarriers containing gelatin-silk fibroin-chondroitin sulfate: After preparing microsphere droplets of uniform size using microfluidic technology, we placed the microsphere droplets in a refrigerator at a gradient temperature and frozen overnight. Under the irradiation of ultraviolet light, the macromolecules containing double bond groups inside the microsphere droplets that contain photoinitiators that can trigger cross-linking reactions undergo cross-linking reactions. After the cross-linking is completed, isopropanol and deionized water are used to remove excess toxic substances, followed by gradient freezing and freeze-drying for later use.

[0039] S4. Preparation of porous microcarriers that can load and empower human umbilical cord mesenchymal stem cells (hUMSCs): The above microcarriers (50μm-300μm) were placed in a 75% alcohol solution for sterilization, and finally replaced with DPBS solution multiple times. Human umbilical cord mesenchymal stem cells were prepared into a cell suspension, mixed with an appropriate amount of microcarriers, inoculated in a 6-well plate for co-culture, and placed in a 37°C incubator for incubation. The medium was changed every other day, and samples were taken regularly for live-dead staining observation, CCK8 analysis, cytoskeleton staining, and PCR analysis.

[0040] The porous microcarriers used for inoculating stem cells need to be sterilized, and the methods include but are not limited to alcohol immersion, radiation sterilization, ethylene oxide sterilization, etc., as long as the topological structure of the microcarrier is not destroyed. Under laboratory conditions, 70-75% alcohol immersion sterilization is selected for 30 min to 2 h. Under sterile conditions, DPBS is replaced multiple times to remove residual alcohol.

[0041] S5. Treatment of diabetic rat wounds: 7-8 week-old male SD rats were purchased from an animal supply unit and fed normally in the laboratory environment for 2 days. Then, a certain amount of streptozotocin (STZ) was intraperitoneally injected into the rats and a high-fat diet was administered for 5-7 days. If the fasting blood glucose level exceeded 13 mmol / L for two consecutive times, the rats / mice were considered to have diabetes and were used for subsequent experiments. STZ-induced diabetic rats were intraperitoneally injected with sodium pentobarbital (50 mg / kg). After anesthesia, hair removal and full-thickness excision of skin wounds of a specific area (10 mm in diameter) were performed. After hemostasis, donut-shaped silicone sheets of corresponding sizes were sutured on the edges of the wounds to prevent the skin around the wounds from contracting. The wounds were set aside for use. This time point was recorded as day 0. Samples of different groups were injected into the wounds of rats for treatment, and the wounds were covered with 3M Tegaderm transparent wound dressings, and the dressings were tied and fixed with PE tape. The animals were housed individually to prevent further scratching of the wounds. At specific time points, the wound healing was photographed with a digital camera, and the wound closure rate was counted.

[0042] A method for preparing a porous microcarrier (gelatin-silk fibroin-chondroitin sulfate microcarrier) that can load and empower human umbilical cord mesenchymal stem cells (hUMSCs), first using microfluidic technology to prepare solid microsphere droplets of uniform size, wherein the mobile phase is an oil phase solution containing a non-ionic emulsifier of a specific concentration, and the dispersed phase is a prepolymer solution containing a photoinitiator and modified biomacromolecules (such as gelatin, silk fibroin and chondroitin sulfate) with double bond groups that can be cross-linked in response to photoinitiation. Then, the porous microcarrier is prepared by combining gradient temperature freezing pores and photocrosslinking technology; after disinfection and sterilization, the microcarrier can be inoculated with hUMSCs to achieve the microtissue treatment purpose of stem cell delivery.

[0043] Implementation Case 1: (1) Preparation of microcarriers First, prepare the dispersed phase solution: weigh 0.15g and 0.3g of methacrylated gelatin, silk fibroin, and chondroitin sulfate, respectively, and dissolve them in 10mL of photoinitiator solution. Then, use an integrated microfluidic device to generate a single-channel chip (such as Figure 1 ), the dispersed phase solution was connected to the water phase inlet of the chip, and the fluorinated oil containing non-ionic emulsifier was connected to the oil phase inlet. Then the pressure of the two inlet channels was adjusted and the flow rate was set. The water phase inlet was 100mbar and the oil phase inlet was 110mbar. The pipe was placed in a 50 ml centrifuge tube at the outlet. After the collection was completed, the fluorinated oil in the lower layer was moved to a new centrifuge tube, and the prepared microcarriers were placed in a 60mm culture dish. Then the culture dish containing the microcarriers was placed in a refrigerator for gradient freezing (-20℃, 1h; -80℃, 12h) overnight. The next day, the culture dish was placed under a 365nm ultraviolet lamp to irradiate the microcarriers therein to photocrosslink. After crosslinking, isopropanol was added to remove the excess fluorinated oil, and then deionized water was added to wash away the excess photoinitiator. Then, gradient freezing (-20℃, 1h; -80℃, 12h) was performed, and freeze-dried after freezing. The morphology of the samples was characterized by SEM (Quanta 400 FEG, FEI, USA).

[0044] (2) Result analysis: Figure 2 As shown in the figure, in addition to commercial microcarriers, five types of microcarriers were prepared, namely G, GC, GSC-1, GSC-2 and GSC-3. Among them, G is 1.5wt% GelMA, GC is 1.5wt% (GelMA: ChsMA=6.5:1), GSC-1 is 1.5wt% (GelMA: SilMA: ChsMA = 4.5:2:1), GSC-2 is 3wt% (GelMA: SilMA: ChsMA = 4.5:2:1) and GSC-3 is 3wt% (GelMA: SilMA: ChsMA=4.5:2:1) and 1% DMSO. Based on the SEM results, it was found that the diameters of the five prepared microcarriers were roughly distributed around 200μm, and they showed a porous structure similar to that of commercial gelatin microcarriers.

[0045] Implementation Case 2: (1) Culture and inoculation of human umbilical cord mesenchymal stem cells After obtaining informed consent from the volunteers, fresh umbilical cords of full-term newborns were obtained by cesarean section. After being rinsed with PBS, the arteries and veins were removed from the umbilical cords. The umbilical cord tissue was exposed and cut into small pieces (0.5-1.0 cm 3 ) and MSCs culture medium were used at 37°C and 5% CO 2hUMSCs migrate from the tissues after incubation under the conditions of . When the cell density reaches about 80%, the cells are digested with 0.25% trypsin (Gibco, USA), or passaged, or cryopreserved for breeding. Subsequently, the expression of surface markers CD105, CD90, CD73, CD45, CD34, CD14, CD19, and HLA-DR of MSCs in vitro was identified by flow cytometry; after the corresponding induction of differentiation, the osteogenic, adipogenic, and chondrogenic differentiation abilities were identified by alizarin red staining, oil red O staining, and alpha blue staining. hUMSCs samples were prepared, and the sample quality was strictly controlled through intermediate quality control and comprehensive quality inspection to reach the clinical application level, and long-term stability studies were conducted. This study used P3~P5 cells for further research. For the inoculation of hUMSCs, the microcarriers were pretreated before starting. The freeze-dried microcarriers were soaked in 75% alcohol for 1 hour, and then the alcohol was replaced with PBS for several times for standby use. Then, the sterilized microcarriers were placed in a low-adsorption well plate, and the digested cell suspension was diluted with 12.5×10 4 / mg and 25×10 4 / mg was added to 10mg of microcarriers, and after standing for 2 hours, the culture medium was added until the microcarriers were completely immersed. The medium was changed every other day.

[0046] (2) Live-dead staining Dilute 100 μL of 10× assay buffer in the Live / dead kit (Abbkine) with 900 μL of sterile water to 1 mL of 1× buffer and place it at 37°C. In the dark, take 1 μL LiveDye and 1 μL NucleDye from the Live / dead kit (Abbkine) and add them to 1 mL of 1× buffer, pipette evenly to prepare live / dead cell staining solution. After hUMSCs were cultured on microcarriers for 24 h and 72 h, the prepared staining reagent was added to the sample and placed in a constant temperature incubation at 37°C in the dark for 15 min for staining. After staining, wash the cells with 1 mL of PBS and observe cell viability using an inverted fluorescence microscope.

[0047] (3) Result analysis: hUMSCs were inoculated on microcarriers of different components and cultured for 24 h and 72 h. It was found that there was no obvious toxicity on the microcarriers, and the cell fluorescence was significantly enhanced on the third day (e.g. Figure 3 This indicates that the prepared microcarriers have good biocompatibility and greatly promote cell proliferation.

[0048] Implementation Case 3: (1) Cytoskeleton staining After hUMSCs were cultured on microcarriers for 7 days, the samples were fixed in 4% paraformaldehyde for 15 min, then permeabilized with 0.2% TritonX 100 permeabilization solution (Solarbio, USA) and allowed to stand for 10 min. PBS buffer containing 1% (v / v) PhalloidinTRITC dye was added to the samples and incubated in the dark for 30 min. Then 1 μg / mL DAPI dilution was added to the samples and incubated in the dark at room temperature for 15 min. The cell morphology was observed and imaged using an inverted fluorescence microscope.

[0049] (2) Proliferation capacity of hUMSCs on microcarriers with different components According to the manufacturer's instructions, the incubation solution was prepared at a ratio of cck-8: culture medium = 1:10. The microcarrier plate was removed from the incubator, the old culture medium was discarded, and it was washed once with DPBS. An equal volume of incubation solution was added and placed in a 37°C constant temperature incubator for 4 hours. Then the incubation solution was moved to a 96-well plate, 100 μL per well, and the cell proliferation ability was finally detected and analyzed using an ELISA reader.

[0050] (3) Anti-inflammatory and immunomodulatory abilities of hUMSCs on microcarriers with different components At specific time points, the microcarrier samples loaded with hUMSCs were collected and soaked with TRIzol reagent (Thermo, USA). RNA was extracted according to the conventional method, and cDNA reverse transcription was performed using PrimeScriptTM RT reagent Kit (TOYOBO). SYBR Green Realtime PCR Master Mix (TOYOBO) and real-time fluorescence quantitative PCR instrument (UltraSYBRMixture, CWBiotech, China) were used to quantitatively analyze genes such as stanniocalcin 1 (STC1), prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6), interleukin 6 (IL-6), and transforming growth factor-β (TGF-β). GAPDH was used as an internal reference, and the 2-ΔΔCt method was used for statistics. The expression of the target gene at each time point was marked as the multiple of the expression of the corresponding gene in hUMSCs loaded with commercial microcarriers. Each gene in each group of samples was measured three times.

[0051] (4) Result analysis: Combined with the live-dead staining results of the microcarrier, the cell proliferation experiment (CCK8 experiment) found that whether the cells were seeded at low density or high density, on the seventh day, G and GSC-1 showed a proliferation ability that was better than that of commercial cells. Figure 4This also proves that the change of material concentration greatly affects the hardness of the material itself. The microcarrier with low concentration has a soft material, which is conducive to cell attachment. Then the cells on the microcarrier were stained with cytoskeleton, such as Figure 5 As shown, the two groups with slower proliferation (GSC-2 and GSC-3) only had a small number of cells on the surface, but the overall morphology of the cells did not change much. Finally, the expression of inflammation-related genes (STC1, IL-6), immune regulation-related genes (TSG-6 and PGE2), and repair-related genes (TGF-β) of hUMSCs inoculated with these five microcarriers was tested. Figure 6 As shown in the figure, compared with commercial gelatin microcarriers, GSC microcarriers, especially GSC-3 microcarriers, greatly increased the expression of TSG-6, PGE2 and TGF-β related genes of hUMSC; in addition, the expression of pro-inflammatory related genes (IL-6) of hUMSC on GSC-3 microcarriers was minimal. This result shows that the chemical composition (gelatin, silk protein, chondroitin sulfate) and physical properties (size, stiffness, micro-nano topological structure, pore structure, network size) of the microcarrier can be used to precisely control the cell fate of hUMSC, such as improving its anti-inflammatory and immunomodulatory abilities.

[0052] Implementation Case 4: (1) Effects of different microcarriers on the healing of diabetic wounds All mice were approved for all safety procedures according to the institutionally approved animal care guidelines. Male diabetic mice aged 12-14 weeks were randomly divided into two treatment groups: "untreated control group", "hUMSC-treated group" and "microcarriers with different components + hUMSC". An excisional wound healing model with scaffold was used. Briefly, the dorsal surface of the mice was shaved and depilated under anesthesia. Two full-thickness wounds with a diameter of 10 mm were made in the midline of the back of the mice. To prevent wound contraction, a ring-shaped silicone was used to limit the healing of the wound. Microcarriers with different components were mixed with an in situ formable hyaluronic acid solution and placed on the excisional wounds. Pictures were taken regularly to observe the wound healing of diabetic mice.

[0053] (2) Result analysis: In order to verify the effects of microcarriers with different components on wound healing, hUMSCs were mixed with hyaluronic acid solution on microcarriers and placed on a diabetic mouse model. By taking pictures of the wounds and calculating the wound area at different days, it was found that ( Figure 7 and Figure 8 ), GSC-3 microcarriers effectively promoted wound healing compared with PBS and other groups.

[0054] The design emphasis of the present invention is: 1. The present invention prepares microcarriers based on microfluidic technology, which is easy to operate and has controllable size.

[0055] 2. The porous microcarriers prepared by the method of the present invention based on microfluidic technology and gradient freezing pore making technology can achieve size adjustment of the microcarriers by adjusting the microfluidic parameters; the hardness, pore size and topological structure of the microcarriers can be adjusted by adjusting the type, concentration and ratio of the prepolymer, or adjusting the composition of the solvent of the dispersed phase, or adjusting the gradient freezing program.

[0056] 3. The gelatin-silk fibroin-chondroitin sulfate microcarrier prepared by the present invention that can load hUMSCs is doped with silk fibroin and chondroitin sulfate in gelatin, and by adjusting its physical properties (size, stiffness, topological structure, pore structure, network size), it can effectively increase the anti-inflammatory ability (STC1) and immune regulation ability (PGE-2 and TSG-6) of hUMSCs compared to gelatin microcarriers, thereby improving the ability of stem cells to treat related diseases.

[0057] 4. The gelatin-silk fibroin-chondroitin sulfate microcarrier prepared by the present invention is a potential stem cell delivery carrier, which is expected to be used in tissue engineering, large-area wounds (such as burn wounds), difficult-to-heal wounds (such as diabetic wounds), osteoarthritis or pulpitis, and can also be used for large-scale production of stem cells or their exosomes.

[0058] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A porous microcarrier capable of loading human umbilical cord mesenchymal stem cells, characterized in that: Calculated by mass concentration ratio, the raw materials include: 0.1-5 parts of methacrylylated gelatin, 0.1-5 parts of methacrylylated silk fibroin and 0.1-5 parts of methacrylylated chondroitin sulfate.

2. A method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells as claimed in claim 1, characterized in that: The steps include: S1, build microfluidic platform; S2. Preparation of microsphere droplets: gelatin, silk fibroin and chondroitin sulfate are dissolved in a photoinitiator solution, and an integrated microfluidic device is used with a single-channel chip; the dispersed phase solution is connected to the water phase inlet of the chip, and the mobile phase containing a non-ionic emulsifier is connected to the oil phase inlet, and the pressure of the water phase and oil phase inlet channels is regulated to set the flow rate; the dispersed phase is a prepolymer solution containing gelatin, silk fibroin and chondroitin sulfate, which contains a photoinitiator and double bond groups with photo-induced responsive cross-linking; the mobile phase is an oil phase solution containing a specific concentration of a non-ionic emulsifier; uniform, stable and non-fusion-resistant microsphere droplets are prepared, and the diameter of the microsphere droplets is controlled between 20 μm and 500 μm; S3. Preparation of porous microcarriers containing gelatin-silk fibroin-chondroitin sulfate: The prepared microsphere droplets are placed in a gradient temperature freezer. Under the irradiation of ultraviolet light, the photoinitiator inside the microsphere droplets will trigger the photo-crosslinking reaction of the macromolecules of the cross-linked double bond groups. After the cross-linking is completed, the porous microcarrier is prepared. The excess toxic substances are removed with isopropanol and deionized water, and the porous microcarrier is then subjected to gradient freezing and freeze-dried for later use.

3. The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 2, characterized in that: In step S2, the dispersed phase is water-soluble, the gelatin, silk fibroin and chondroitin sulfate form modified macromolecules, and the total concentration of the modified biomacromolecules contained in the dispersed phase is 0.5wt%~6wt%; the dispersed phase contains double bond groups that can be cross-linked by a photoinitiator, and the double bond groups include one or more of methacryloyl groups, methacrylic anhydride, maleic anhydride, glycidyl methacrylate, hydroxyethyl methacrylate, and N,N'-methylenebisacrylamide; the concentration of the double bond groups accounts for 1%~60% of the total mass.

4. The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 2, characterized in that: The solvent of the dispersed phase in step S2 is water, and the dispersed phase can be mixed with water-soluble organic solvents and inorganic salts. The organic solvent includes methanol, ethanol, isopropanol, glycerol, and DMSO, and the inorganic salt includes sodium chloride, potassium chloride, calcium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate.

5. The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 2, characterized in that: The dispersed phase photoinitiator in step S2 is a soluble photoinitiator, which includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone; the concentration of the photoinitiator is 0.1% to 2%.

6. The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 2, characterized in that: The mobile phase in step S2 includes HFE-7500 electronic fluorinated liquid containing 0.1% to 5% perfluoro surfactant, isopropyl palmitate containing 0.1% to 10% EM-180, and mineral oil or soybean oil containing 0.1% to 8% Span-80.

7. The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 2, characterized in that: In step S2, the flow rate ratio of the dispersed phase to the mobile phase is 0.5:0.5~1, and the pipeline pressure ratio of the dispersed phase to the mobile phase is 1:0.5~1.

8. The method for preparing a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 2, characterized in that: In step S3, the microsphere droplets are frozen overnight, using a gradient temperature freezing method, with a temperature range of -20°C to -196°C and a time of 30 min to 12 h to form pores inside the microspheres; the wavelength range of the cross-linking light is 350 to 450 nanometers, and the light intensity range is 10 to 100 mw / cm 2 The photo-crosslinking time is 30min~2h to achieve the curing and molding of microspheres.

9. A use of a porous microcarrier capable of carrying human umbilical cord mesenchymal stem cells as claimed in claim 1, characterized in that: The porous microcarrier is used to load human umbilical cord mesenchymal stem cells. The porous microcarrier is immersed in 75% alcohol for sterilization, and DPBS is replaced several times to remove excess alcohol. Then, the human umbilical cord mesenchymal stem cells are inoculated on the microcarrier for culture. The diameter of the microcarrier containing gelatin, silk fibroin, and chondroitin sulfate used for inoculating human umbilical cord mesenchymal stem cells ranges from 50 μm to 300 μm. The inoculation density of human umbilical cord mesenchymal stem cells is 10×10 4 / mg~1000×10 4 / mg.

10. The use of a porous microcarrier capable of loading human umbilical cord mesenchymal stem cells according to claim 9, characterized in that: The microcarrier containing gelatin, silk fibroin and chondroitin sulfate loaded with human umbilical cord mesenchymal stem cells is used for large-scale production of stem cells or their exosomes, and is used to prepare drugs for large-area wounds, difficult-to-heal wounds, osteoarthritis or pulpitis. The drug is used independently or mixed with other in situ formed hydrogels; the dosage form of the drug is selected from dressings, ointments, liniments or in vivo injectable dosage forms.

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