Microspheres for guiding microvascular network reconstruction and a preparation method thereof

Abstract

This invention provides microspheres for guiding microvascular network reconstruction and a method for preparing the same. The microspheres comprise a gel matrix formed by crosslinking TGF-β-grafted oxidized bacterial cellulose and methacrylamide gelatin under the action of a photoinitiator, and endothelial cells loaded on the surface of the gel matrix; the gel matrix has a porous structure. Through minimally invasive multi-point injection, the vascular branches grown from adjacent microspheres can rapidly anastomose with each other, seamlessly connecting with the host's existing blood vessels to form a dense and widely interconnected microvascular network, ultimately achieving efficient blood perfusion and functional recovery of ischemic tissues.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and tissue engineering technology, and relates to a microsphere for guiding the reconstruction of microvascular networks and its preparation method, specifically to a microsphere for in situ reconstruction of functional vascular microcirculation networks in ischemic diseases and its preparation method. Background Technology

[0002] Chronic ischemic diseases (such as myocardial infarction and severe lower limb ischemia) have extremely high rates of disability and mortality. Their fundamental pathology lies not only in the blockage of large blood vessels, but also in the large-area occlusion and functional failure of the microvascular network in the peripheral microcirculation. Among existing clinical treatments, traditional revascularization strategies (such as vascular bypass surgery, endovascular balloon angioplasty, and stent implantation) primarily target the opening of macroscopic large arteries. However, because the microvascular bed in deep ischemic tissues is already damaged, simply opening large blood vessels often fails to effectively improve blood perfusion in peripheral tissues, ultimately leading to tissue necrosis or amputation.

[0003] To address the challenge of microcirculatory revascularization, directly transplanting exogenous endothelial cells into ischemic areas to generate new microvessels in situ has become a promising therapeutic approach. However, simply injecting free cells can lead to massive cell death (apoptosis) due to the harsh microenvironment (hypoxia, inflammation) in the ischemic area, resulting in extremely low early colonization rates. To protect cells, current interventions often employ biomaterials (such as various hydrogel carriers) for cell delivery. However, in-depth research has revealed that existing biomaterials exhibit serious structural and functional defects in practical applications, leading to poor angiogenesis outcomes, specifically in the following aspects: Traditional natural soft matrix hydrogels (such as pure alginate, pure collagen, or low-concentration gelatin) cause cells to become "trapped": Although these materials have good biocompatibility, their physical texture is usually too soft, with extremely low mechanical strength (modulus). When endothelial cells are encased in these soft matrices, they cannot obtain sufficient mechanical support to activate cellular skeletal remodeling. This causes the cells to become trapped, like being stuck in quagmire, in a "migration-blind state." The cells can only maintain a shrunken state and cannot actively extend pseudopodia to break through the physical barrier of the material, ultimately becoming trapped inside and unable to take root and grow into ischemic tissue.

[0004] Macroscopic in-situ gelation systems or bulk patch materials create "transplant islands": Current delivery strategies often involve injecting liquid precursors locally into the lesion and cross-linking them to form a monolithic macroscopic hydrogel, or directly implanting large hydrogel patches. The dense cross-linked network within these large, encapsulated carriers creates significant spatial steric hindrance. This results in the transplanted endothelial cells being completely physically isolated from the host's existing microcirculation by the material "wall." Even if new blood vessels can germinate within the material, they can only form isolated "transplant islands," unable to cross the material boundary and establish effective blood connectivity and circulation with the host's blood vessels.

[0005] Single-factor delivery or burst-release materials lead to the "prone decline" of newly formed blood vessels: True blood vessels require not only endothelial cells forming the lumen, but also surrounding supporting cells (such as pericytes and smooth muscle cells) for long-term stability. Many existing bioactive materials typically only load a single early pro-angiogenic factor (such as VEGF or bFGF), or suffer from burst-release defects (i.e., the drug is rapidly released and exhausted in the initial implantation stage). This results in the initial stimulation of numerous disorganized endothelial lumens, but after blood vessel growth, the material fails to provide the necessary biological signals for vascular maturation during critical periods. Such exposed new blood vessels, lacking supporting cell encapsulation, are extremely fragile and immature, easily leaking and rapidly degenerating in the complex inflammatory microenvironment of the body, leading to complete loss of long-term perfusion function. Summary of the Invention

[0006] To address the technical problems of low survival, restricted migration, spatial physical hindrance, and easy degeneration of newly formed blood vessels in existing endothelial cell transplantation technologies, the present invention aims to provide a microsphere for guiding the reconstruction of microvascular networks and its preparation method. The microspheres provided by the present invention can be injected at multiple points through minimally invasive procedures, and the vascular branches grown from adjacent microspheres can quickly anastomose with each other and seamlessly connect with the host's original blood vessels to form a dense and widely interconnected microvascular network, ultimately achieving efficient blood perfusion and functional recovery of ischemic tissues.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a microsphere for guiding the reconstruction of a microvascular network, the microsphere comprising a gel matrix formed by crosslinking oxidized bacterial cellulose grafted with TGF-β factor with methacrylamide gelatin under the action of a photoinitiator, and endothelial cells loaded on the surface of the gel matrix; the gel matrix having a porous structure.

[0008] In this invention, the dual-network structure formed by oxidizing bacterial cellulose and methacrylamide gelatin significantly enhances the Young's modulus of the carrier, providing solid physical support for endothelial cells, triggering intracellular mechanosensory responses, activating cytoskeleton reorganization and the ability to actively extend and migrate outwards, and the porous structure of the gel matrix provides more adhesion sites for endothelial cells; TGF-β factor grafted onto the oxidizing bacterial cellulose backbone enables the sustained release of cytokines, continuously promoting endothelial cell migration and budding.

[0009] The microspheres provided by this invention, through minimally invasive multi-point injection, become independent budding nodes, eliminating the physical steric hindrance of macroscopic materials. This allows the vascular branches grown from adjacent microspheres to rapidly anastomose with each other and seamlessly connect with the host's original blood vessels, forming a dense and widely interconnected microvascular network. Ultimately, this achieves in-situ reconstruction of the vascular microcirculation network in ischemic diseases, restoring tissue blood perfusion, and saving ischemic tissue and motor function.

[0010] Preferably, the ratio of the dry weight of the gel matrix to the number of endothelial cells is (20~30) mg: 1×(10) mg. 6 ~10 10 )indivual.

[0011] The specific point values ​​in (20~30) can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.

[0012] 1×(10 6 ~10 10 The specific point value in ) can be 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 1×10 8 1×10 9 Or 1×10 10 wait.

[0013] Preferably, the mass ratio of the oxidized bacterial cellulose grafted with TGF-β factor to the methacrylamide gelatin is (0.1~0.5):(5~15).

[0014] The specific point values ​​in (0.1~0.5) can be 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, etc.

[0015] The specific point values ​​in (5~15) can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, etc.

[0016] Preferably, the photoinitiator comprises any one or a combination of at least two of the following: lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (abbreviated as photoinitiator LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (abbreviated as photoinitiator 2959), or phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide (abbreviated as photoinitiator BAPO).

[0017] Preferably, the mass ratio of the photoinitiator to methacrylamide gelatin is 1:(10~30).

[0018] The specific point values ​​in (10~30) can be 10, 12, 15, 17, 20, 23, 25, 28 or 30, etc.

[0019] Preferably, the oxidized bacterial cellulose grafted with TGF-β factor is prepared by a method comprising the following steps: (1) Bacterial cellulose, catalyst and oxidant are mixed in buffer solution and subjected to oxidation reaction to obtain oxidized bacterial cellulose; (2) Osmotic pressure regulator, carboxyl activator and TGF-β factor are added sequentially to the buffer containing oxidized bacterial cellulose to carry out the grafting reaction and obtain the oxidized bacterial cellulose grafted with TGF-β factor (abbreviated as TGF-β-oxidized bacterial cellulose).

[0020] Preferably, in step (1), the buffer solution is PBS buffer.

[0021] Preferably, the catalyst is 2,2,6,6-tetramethylpiperidine oxide (TEMPO).

[0022] Preferably, the ratio of bacterial cellulose to catalyst is 1 g:(0.05~0.5) mmol.

[0023] The specific point values ​​in (0.05~0.5) can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, etc.

[0024] Preferably, the oxidant includes any one or a combination of at least two of sodium chlorite, potassium chlorite, sodium hypochlorite, or potassium hypochlorite.

[0025] Preferably, the ratio of bacterial cellulose to oxidant is 1 g:(10~30) mmol.

[0026] The specific point values ​​in (10~30) can be 10, 12, 15, 17, 20, 23, 25, 28 or 30, etc.

[0027] Preferably, the temperature of the oxidation reaction is 45~55℃, for example, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃; the time is 36~72 h, for example, 36 h, 38 h, 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h or 72 h, etc.

[0028] Preferably, in step (2), the buffer solution is a MES buffer solution.

[0029] Preferably, the osmotic pressure regulator comprises sodium chloride and / or potassium chloride.

[0030] Preferably, the ratio of the oxidizing bacterial cellulose to the osmotic pressure regulator is 60 mg:(20~30) mmol.

[0031] The specific point values ​​in (20~30) can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.

[0032] Preferably, the carboxyl activator includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (abbreviated as EDC) and N-hydroxysuccinimide (abbreviated as NHS).

[0033] Preferably, the ratio of the oxidizing bacterial cellulose to N-hydroxysuccinimide is 60 mg:(2~5) mmol.

[0034] The specific point values ​​in (2~5) can be 2, 2.2, 2.5, 2.7, 3, 3.3, 3.5, 3.8, 4, 4.5 or 5, etc.

[0035] Preferably, the ratio of the oxidizing bacterial cellulose to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 60 mg:(5~10) mmol.

[0036] The specific point values ​​in (5~10) can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10, etc.

[0037] Preferably, the ratio of oxidizing bacterial cellulose to TGF-β factor is 60 mg:(20~30) μg.

[0038] The specific point values ​​in (20~30) can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.

[0039] Preferably, the grafting reaction temperature is 25~35℃, for example, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃ or 35℃, etc.; the time is 10~14 h, for example, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, 12.5 h, 13 h, 13.5 h or 14 h, etc.

[0040] Preferably, the particle size of the gel matrix is ​​50~500 μm, for example, it can be 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm or 500 μm.

[0041] Preferably, the porous structure is prepared by a method comprising the following steps: After oxidizing bacterial cellulose grafted with TGF-β factor and methacrylamide gelatin crosslinked to form a gel, the mixture was subjected to pre-freezing, sublimation drying, desorption drying and collagenase treatment in sequence to obtain a gel matrix with a porous structure.

[0042] The specific method of this invention can prepare appropriate pores on the gel matrix, which not only provides adhesion sites for endothelial cells, but also facilitates the confluence of vascular branches and improves the microvascular network reconstruction effect.

[0043] Preferably, the pre-freezing temperature is -70 to -90°C, for example, -70°C, -72°C, -75°C, -77°C, -80°C, -83°C, -85°C, -88°C, or -90°C; and the time is 4 to 6 hours, for example, 4 hours, 4.2 hours, 4.5 hours, 4.7 hours, 5 hours, 5.3 hours, 5.5 hours, 5.8 hours, or 6 hours.

[0044] Preferably, the temperature for the analytical drying is 15~25℃, for example, it can be 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃, etc.; the time is 10~15 h, for example, it can be 10 h, 10.5 h, 11 h, 11.5 h, 12 h, 12.5 h, 13 h, 13.5 h, 14 h, 14.5 h or 15 h, etc.

[0045] Preferably, the sublimation drying temperature is -30 to -40°C, for example, -30°C, -31°C, -32°C, -33°C, -34°C, -35°C, -36°C, -37°C, -38°C, -39°C, or -40°C; and the time is 8 to 28 hours, for example, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 17 hours, 20 hours, 23 hours, 25 hours, or 28 hours.

[0046] Preferably, the sublimation drying process includes: first treating at -30 to -35℃ (e.g., -30℃, -30.5℃, -31℃, -31.5℃, -32℃, -32.5℃, -33℃, -33.5℃, -34℃, -34.5℃, or -35℃, etc.) for 8 to 12 hours (e.g., 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, or 12 hours, etc.), then treating at -35 to -40℃ (e.g., -35℃, -35.5℃, -36℃, -36.5℃, -37℃, -37.5℃, -38℃, -38.5℃, -39℃, -39.5℃, or -40℃, etc.) for 12 to 16 hours (e.g., 12 hours, 12.5 hours, 13 hours, 13.5 hours, etc.). h, 14 h, 14.5 h, 15 h, 15.5 h or 16 h, etc.).

[0047] In this invention, a specific two-stage sublimation drying process can prepare appropriate pores on the gel matrix, which not only provides adhesion sites for endothelial cells, but also facilitates the confluence of vascular branches and improves the microvascular network reconstruction effect.

[0048] Preferably, the temperature of the collagenase treatment is 33~38℃, for example, 33℃, 33.5℃, 34℃, 34.5℃, 35℃, 35.5℃, 36℃, 36.5℃, 37℃, 37.5℃ or 38℃; and the time is 3~14 min, for example, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min or 14 min.

[0049] Preferably, the collagenase treatment process includes: first treating at 33~35℃ (e.g., 33℃, 33.2℃, 33.5℃, 33.7℃, 34℃, 34.5℃, 34.8℃, or 35℃, etc.) for 3~5 min (e.g., 3 min, 3.2 min, 3.5 min, 3.7 min, 4 min, 4.3 min, 4.5 min, 4.8 min, or 5 min, etc.), and then treating at 35~38℃ (e.g., 35℃, 35.2℃, 35.5℃, 35.7℃, 36℃, 36.3℃, 36.5℃, 36.8℃, 37℃, 37.5℃, or 38℃, etc.) for 7~9 min (e.g., 7 min, 7.2 min, 7.5 min, 7.7 min, 8 min, 8.3 min, 8.5 min, 8.8 min, or 9 min, etc.).

[0050] In this invention, a specific two-stage collagenase treatment can prepare appropriate pores on the gel matrix, which not only provides adhesion sites for endothelial cells, but also facilitates the confluence of vascular branches and improves the microvascular network reconstruction effect.

[0051] Preferably, the concentration of the collagenase used is 0.1~0.3 mg / mL, for example, it can be 0.1 mg / mL, 0.12 mg / mL, 0.15 mg / mL, 0.17 mg / mL, 0.2 mg / mL, 0.23 mg / mL, 0.25 mg / mL, 0.28 mg / mL or 0.3 mg / mL, etc.

[0052] Preferably, the terminating agent for the collagenase treatment is an EDTA solution.

[0053] In a second aspect, the present invention provides a method for preparing microspheres as described in the first aspect, the method comprising: An aqueous phase was prepared by mixing oxidized bacterial cellulose grafted with TGF-β factor, methacrylamide gelatin, a photoinitiator, and water; the aqueous phase was sheared with an oil phase to form microdroplets, which were then cross-linked and cured to prepare a porous structure. Endothelial cells were then inoculated to obtain the microspheres.

[0054] Preferably, the flow rate ratio of the oil phase to the water phase is (40~60):1.

[0055] The specific point values ​​in (40~60) can be 40, 42, 45, 47, 50, 53, 55, 58 or 60, etc.

[0056] Preferably, the light wavelength for cross-linking curing is 360~410 nm, for example, it can be 360 ​​nm, 370 nm, 380 nm, 390 nm, 400 nm or 410 nm.

[0057] Preferably, the cross-linking curing time is 30~90 s, for example, it can be 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s, 65 s, 70 s, 75 s, 80 s, 85 s or 90 s, etc.

[0058] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0059] Preferably, the oil phase includes any one or a combination of at least two of paraffin oil, Span 80, Span 60, castor oil, or soybean oil.

[0060] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a dual-network structure formed by oxidized bacterial cellulose and methacrylamide gelatin to provide robust physical support for endothelial cells, activating cytoskeleton remodeling and their ability to actively extend and migrate outwards. The porous structure of the gel matrix provides endothelial cells with more adhesion sites. TGF-β factor is grafted onto the oxidized bacterial cellulose backbone, continuously promoting endothelial cell migration and budding. Microspheres are injected at multiple points via minimally invasive techniques to form independent budding nodes, eliminating the physical steric hindrance of macroscopic materials. This allows vascular branches growing from adjacent microspheres to rapidly anastomose with each other, seamlessly connecting with the host's existing blood vessels to form a dense and widely interconnected microvascular network. Ultimately, this achieves in-situ reconstruction of the vascular microcirculation network in ischemic diseases, restoring tissue blood perfusion, rescuing ischemic tissue, and restoring motor function. Attached Figure Description

[0061] Figure 1 Infrared spectra of bacterial cellulose, oxidized bacterial cellulose, and TGF-β-oxidized bacterial cellulose in Preparation Example 1; Figure 2 These are scanning electron microscope images of the porous microgels in Example 1, Comparative Example 1, and Comparative Example 2. Figure 3 This is a laser confocal image of the microspheres used in Example 1 to guide microvascular network reconstruction after 14 days of culture in DMEM medium; Figure 4 This is a small animal imaging image of a BALB / c-Nude athymic nude mouse lower limb ischemia model treated with microsphere injection to guide microvascular network reconstruction, as described in Example 1. Detailed Implementation

[0062] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0063] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0064] The sources of materials used in the following specific embodiments are as follows: Dissolve 5 g peptone, 5 g yeast extract, 100 g D-fructose, and 1 g magnesium sulfate heptahydrate in 1 L of deionized water. After sterilization, adjust the pH to 5.5 and inoculate with 10% (v / v) activated Acetobacter xylinum (ATCC 23767) culture (1×10⁻⁶). 6 The bacterial cellulose (CFU / mL) was fermented at 30℃ with shaking at 120 rpm for 120 h. The bacterial cellulose generated on the surface of the culture medium was collected, washed three times with deionized water, then three times with 0.1wt% NaOH aqueous solution, and finally repeatedly washed with deionized water until the pH reached 7.0. The mixture was homogenized at 15000 rpm for 15 min and then freeze-dried to obtain bacterial cellulose.

[0065] TGF-β factor was purchased from Abclonal, model RP01458; methacrylamide gelatin was purchased from EFL, model EFL-GM-30 / 60 / 90; collagenase (type II) was purchased from MCE, model HY-E70005B; HUVEC cells were purchased from Shanghai Anwei Biotechnology Co., Ltd., model AW-CH0165.

[0066] Preparation Example 1 This preparation example provides an oxidizing bacterial cellulose grafted with TGF-β factor, which is prepared by a method comprising the following steps: (1) 1 g of bacterial cellulose, 0.1 mmol TEMPO and 20 mmol sodium chlorite were mixed in 300 mL PBS buffer and reacted at 50 °C for 48 h. After the reaction was completed, the mixture was centrifuged at 7000 rpm for 10 min, the precipitate was collected, washed and lyophilized to obtain oxidized bacterial cellulose.

[0067] (2) Take 60 mg of oxidizing bacterial cellulose and disperse it in 100 mL of MES buffer. Then add 25 mmol sodium chloride, 3 mmol NHS, 6 mmol EDC and 25 μg TGF-β factor in sequence. React at 30℃ for 12 h to obtain TGF-β-oxidizing bacterial cellulose.

[0068] Preparation Example 2 This preparation example provides an oxidizing bacterial cellulose grafted with TGF-β factor, which is prepared by a method comprising the following steps: (1) 1 g of bacterial cellulose, 0.2 mmol TEMPO and 10 mmol potassium chlorite were mixed in 300 mL PBS buffer and reacted at 45 °C for 72 h. After the reaction was completed, the mixture was centrifuged at 7000 rpm for 10 min, the precipitate was collected, washed and lyophilized to obtain oxidized bacterial cellulose.

[0069] (2) Take 60 mg of oxidizing bacterial cellulose and disperse it in 100 mL of MES buffer. Then add 30 mmol sodium chloride, 2 mmol NHS, 5 mmol EDC and 30 μg TGF-β factor in sequence. React at 35℃ for 10 h to obtain TGF-β-oxidizing bacterial cellulose.

[0070] Preparation Example 3 This preparation example provides an oxidizing bacterial cellulose grafted with TGF-β factor, which is prepared by a method comprising the following steps: (1) 1 g of bacterial cellulose, 0.3 mmol TEMPO and 30 mmol sodium hypochlorite were mixed in 300 mL PBS buffer and reacted at 55 °C for 36 h. After the reaction was completed, the mixture was centrifuged at 7000 rpm for 10 min, the precipitate was collected, washed and lyophilized to obtain oxidized bacterial cellulose.

[0071] (2) Take 60 mg of oxidizing bacterial cellulose and disperse it in 100 mL of MES buffer. Then add 20 mmol sodium chloride, 5 mmol NHS, 10 mmol EDC and 20 μg TGF-β factor in sequence. React at 25℃ for 14 h to obtain TGF-β-oxidizing bacterial cellulose.

[0072] Example 1 This embodiment provides a microsphere for guiding microvascular network reconstruction, the preparation method of which includes: (1) Take 0.3 g of TGF-β-oxidative bacterial cellulose provided in Preparation Example 1, 10 g of methacrylamide gelatin, and 0.5 g of photoinitiator LAP and dissolve them in 100 mL of water to prepare an aqueous phase. Use paraffin oil containing 5% (v / v) Span 80 as the oil phase, set the flow rate ratio of the oil phase to the aqueous phase to be 50:1, and use a coaxial microfluidic device to shear the aqueous phase to form microdroplets. Crosslink and cure under 405 nm blue light for 60 s, clean and remove the surface oil phase to obtain microgels.

[0073] (2) Take the microgel obtained in step (1), pre-freeze it at -80℃ for 5 h, and then perform sublimation drying: first treat at -33℃ for 10 h, then at -37℃ for 14 h. Next, perform desorption drying at 20℃ for 12 h, and then treat it with 0.2 mg / mL collagenase: first treat at 34℃ for 4 min, then at 36℃ for 8 min. Finally, wash with PBS solution containing 0.1% (w / v) EDTA to obtain porous microgel.

[0074] (3) On a dry weight basis, take 25 mg of the porous microgel prepared in step (2) and mix it with 10 mg of water. 6 HUVEC cells were suspended in 2 mL of DMEM medium and co-cultured in a 6-well plate with ultra-low adsorption. During the first 8 hours, the cells were gently pipetted once every 2 hours to ensure uniform adhesion of the cells to the porous microgel surface. After 24 hours of co-culture, the microspheres that guide the reconstruction of the microvascular network were obtained.

[0075] Example 2 This embodiment provides a microsphere for guiding microvascular network reconstruction, the preparation method of which includes: (1) Take 0.5 g of TGF-β-oxidative bacterial cellulose provided in Preparation Example 2, 15 g of methacrylamide gelatin, and 0.5 g of photoinitiator 2959 and dissolve them in 100 mL of water to prepare an aqueous phase. Use paraffin oil containing 5% (v / v) Span 80 as the oil phase, set the flow rate ratio of the oil phase to the aqueous phase to be 60:1, and use a coaxial microfluidic device to shear the aqueous phase to form microdroplets. Crosslink and cure under 365 nm ultraviolet light for 60 s, wash to remove the surface oil phase, and obtain microgels.

[0076] (2) Take the microgel obtained in step (1), pre-freeze it at -70℃ for 6 h, and then perform sublimation drying: first treat at -30℃ for 12 h, then at -35℃ for 16 h. Next, perform desorption drying at 25℃ for 10 h, and then treat it with 0.3 mg / mL collagenase: first treat at 33℃ for 5 min, then at 35℃ for 9 min. Finally, wash with PBS solution containing 0.1% (w / v) EDTA to obtain porous microgel.

[0077] (3) On a dry weight basis, take 20 mg of the porous microgel prepared in step (2) and mix it with 10 mg of the microgel. 6 HUVEC cells were suspended in 2 mL of DMEM medium and co-cultured in a 6-well plate with ultra-low adsorption. During the first 8 hours, the cells were gently pipetted once every 2 hours to ensure uniform adhesion of the cells to the porous microgel surface. After 24 hours of co-culture, the microspheres that guide the reconstruction of the microvascular network were obtained.

[0078] Example 3 This embodiment provides a microsphere for guiding microvascular network reconstruction, the preparation method of which includes: (1) Take 0.1 g of TGF-β-oxidative bacterial cellulose provided in Preparation Example 3, 5 g of methacrylamide gelatin, and 0.5 g of photoinitiator LAP and dissolve them in 100 mL of water to prepare an aqueous phase. Use paraffin oil containing 5% (v / v) Span 80 as the oil phase, set the flow rate ratio of the oil phase to the aqueous phase to be 40:1, and use a coaxial microfluidic device to shear the aqueous phase to form microdroplets. Crosslink and cure under 405 nm blue light for 60 s, clean and remove the surface oil phase to obtain microgels.

[0079] (2) Take the microgel obtained in step (1), pre-freeze it at -90℃ for 4 h, and then perform sublimation drying: first treat at -35℃ for 8 h, then at -40℃ for 12 h. Next, perform desorption drying at 15℃ for 15 h, and then treat it with 0.1 mg / mL collagenase: first treat at 35℃ for 3 min, then at 38℃ for 7 min. Finally, wash with PBS solution containing 0.1% (w / v) EDTA to obtain porous microgel.

[0080] (3) On a dry weight basis, take 30 mg of the porous microgel prepared in step (2) and mix it with 10 mg of water. 6 HUVEC cells were suspended in 2 mL of DMEM medium and co-cultured in a 6-well plate with ultra-low adsorption. During the first 8 hours, the cells were gently pipetted once every 2 hours to ensure uniform adhesion of the cells to the porous microgel surface. After 24 hours of co-culture, the microspheres that guide the reconstruction of the microvascular network were obtained.

[0081] Example 4 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Embodiment 1 is that in step (2), “sublimation drying: first treat at -33℃ for 10 h, then treat at -37℃ for 14 h” is replaced with “sublimation drying: first treat at -37℃ for 14 h, then treat at -33℃ for 10 h”, while the other steps remain unchanged.

[0082] Example 5 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Embodiment 1 is that in step (2), “sublimation drying: first treat at -33℃ for 10 h, then treat at -37℃ for 14 h” is replaced with “sublimation drying: treat at -33℃ for 24 h”, while the rest of the steps remain unchanged.

[0083] Example 6 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Embodiment 1 is that in step (2), “sublimation drying: first treat at -33℃ for 10 h, then treat at -37℃ for 14 h” is replaced with “sublimation drying: treat at -37℃ for 24 h”, while the other steps remain unchanged.

[0084] Example 7 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Embodiment 1 is that in step (2), the "sublimation drying: first at -33℃ for 10 h, then at -37℃ for 14 h" is not performed. All other steps remain unchanged.

[0085] Example 8 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Example 1 is that in step (2), “treat with 0.2 mg / mL collagenase: first treat at 34℃ for 4 min, then treat at 36℃ for 8 min” is replaced with “treat with 0.2 mg / mL collagenase: first treat at 36℃ for 8 min, then treat at 34℃ for 4 min”. All other steps remain unchanged.

[0086] Example 9 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Example 1 is that in step (2), “treat with 0.2 mg / mL collagenase: first treat at 34℃ for 4 min, then treat at 36℃ for 8 min” is replaced with “treat with 0.2 mg / mL collagenase: treat at 34℃ for 12 min”. All other steps remain unchanged.

[0087] Example 10 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Embodiment 1 is that in step (2), “treat with 0.2 mg / mL collagenase: first treat at 34℃ for 4 min, then treat at 36℃ for 8 min” is replaced with “treat with 0.2 mg / mL collagenase: treat at 36℃ for 12 min”. All other steps remain unchanged.

[0088] Example 11 This embodiment provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this embodiment and Example 1 is that in step (2), the treatment with 0.2 mg / mL collagenase is not performed: first at 34°C for 4 min, then at 36°C for 8 min. All other steps remain unchanged.

[0089] Comparative Example 1 This comparative example provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this example and Example 1 is that in step (1), “0.3 g of TGF-β-oxidative bacterial cellulose provided in Preparation Example 1” is replaced with “0.3 g of oxidative bacterial cellulose involved in Preparation Example 1”, while the rest of the steps remain unchanged.

[0090] Comparative Example 2 This comparative example provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this example and Example 1 is that in step (1), "0.3 g of TGF-β-oxidative bacterial cellulose provided in Example 1" was not added, while the rest of the steps remained unchanged.

[0091] Comparative Example 3 This comparative example provides a microsphere for guiding the reconstruction of microvascular networks. The only difference between this example and Example 1 is that in step (1), “0.3 g of TGF-β-oxidized bacterial cellulose provided in Preparation Example 1” is replaced with “0.3 g of oxidized bacterial cellulose involved in Preparation Example 1 and 125 μg of TGF-β factor”, while the rest of the steps remain unchanged.

[0092] Comparative Example 4 This comparative example provides a microsphere for guiding microvascular network reconstruction, which differs from Example 1 only in that step (2) was not performed. Instead, 25 mg of the microgel prepared in step (1) was taken by dry weight and mixed with 10... 6 HUVEC cells were co-cultured for 24 h, with all other steps remaining unchanged.

[0093] Comparative Example 5 This comparative example provides a microsphere for guiding microvascular network reconstruction, the preparation method of which includes: Take 0.3 g of the TGF-β-oxidative bacterial cellulose provided in Preparation Example 1, 10 g of methacrylamide gelatin, and 0.5 g of photoinitiator LAP, dissolve them in 100 mL of DMEM medium, and mix with 4.32 × 10⁻⁶ ppm of the solution. 8 HUVEC cells were thoroughly mixed to prepare an aqueous phase. Paraffin oil containing 5% (v / v) Span 80 was used as the oil phase, with an oil-to-aqueous phase flow rate ratio of 50:1. Using a coaxial microfluidic device, the oil phase sheared the aqueous phase to form microdroplets. The microgels were cross-linked and cured under 405 nm blue light for 60 s, and the surface oil phase was removed by washing to obtain a microgel. The microgels were cultured in DMEM medium for 24 h to obtain the microspheres that guide microvascular network reconstruction.

[0094] Test Example 1 Infrared spectroscopy analysis was performed on the bacterial cellulose (BNC), oxidative bacterial cellulose (OBNC), and TGF-β-oxidative bacterial cellulose (OBNC@TGF) involved in Preparation Example 1. Figure 1 As shown, TGF-β successfully modified oxidative bacterial cellulose.

[0095] Test Example 2 The porous microgels prepared in Example 1, Comparative Example 1, and Comparative Example 2 were analyzed by scanning electron microscopy. Figure 2As shown, the present invention creates a porous structure in the microspheres through a pore-forming process, which provides more adhesion sites for endothelial cells.

[0096] Test Example 3 The microspheres prepared in Example 1 for guiding microvascular network reconstruction were cultured in DMEM medium in a 6-well plate with ultra-low adsorption for 14 days, and the attachment and growth status of HUVEC cells on the gel matrix was recorded. Figure 3 As shown, endothelial cells can survive and proliferate continuously on the surface of the microspheres provided by this invention.

[0097] Test Example 4 Eight-week-old BALB / c-Nude athymic nude mice were anesthetized with sevoflurane and placed in a supine position. A 1 cm incision was made in the left hind limb groin, and the femoral artery was fully accessed, then ligated and cut proximally and distally. The incision was sutured after the operation. Microspheres for guiding microvascular network reconstruction prepared in Examples 1-11 and Comparative Examples 1-5 were resuspended in PBS buffer (50-80 microspheres / mL). Using a standard 23G needle syringe, the microsphere suspension was injected evenly into the muscle tissue of the ischemic hind limb of the mice at three points (medial, lateral, and anterior thigh). Small animal imaging analysis was performed at fixed time points (sample size n=8), and the blood perfusion ratio (ischemic / non-ischemic hind limb) was calculated. The control group was treated with an equal volume of PBS solution.

[0098] like Figure 4 As shown, after implantation, the microspheres prepared in Example 1 provide multiple budding nodes in a distributed spatial manner. The endothelial cells loaded on the surface are pre-activated by matrix mechanics and actively migrate to the ischemic area to bud, connect with each other and integrate with the host. Subsequently, under the action of delayed-release TGF-β, supporting cells are generated in situ, and finally a long-term stable microvascular network is constructed in the depth of ischemia to achieve blood flow restoration.

[0099] As shown in Table 1, and as can be seen from Examples 1-3, the microspheres provided by the present invention, through minimally invasive multi-point injection, become independent budding nodes, eliminating the physical steric hindrance of macroscopic materials, so that the vascular branches grown from adjacent microspheres can quickly anastomose with each other and seamlessly connect with the host's original blood vessels, forming a dense and widely interconnected microvascular network, ultimately realizing the in-situ reconstruction of the vascular microcirculation network in ischemic diseases, restoring tissue blood perfusion, and saving ischemic tissue and motor function.

[0100] As shown in Comparative Example 5, endothelial cells, encased in the gel matrix, are unable to actively extend pseudopodia to overcome the physical barrier of the material, ultimately becoming trapped inside and unable to take root and grow into ischemic tissue. As shown in Comparative Example 3, TGF-β factor, physically mixed into the gel matrix, is rapidly released and exhausted in the early stages of implantation, failing to provide pro-angiogenic biological signals during critical periods.

[0101] Comparative Example 4 shows that the porous structure facilitates endothelial cell adhesion and the convergence of vascular branches. Examples 4-7 show that specific sublimation drying treatment can prepare appropriate pores in the gel matrix; Examples 8-11 show that specific collagenase treatment can prepare appropriate pores in the gel matrix, which not only provides adhesion sites for endothelial cells but also facilitates the convergence of vascular branches, thereby improving the microvascular network reconstruction effect.

[0102] Table 1 This invention illustrates a microsphere for guiding microvascular network reconstruction and its preparation method through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0103] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0104] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A microsphere for guiding the reconstruction of a microvascular network, characterized in that, The microspheres comprise a gel matrix formed by crosslinking TGF-β-grafted oxidized bacterial cellulose and methacrylamide gelatin under the action of a photoinitiator, and endothelial cells loaded on the surface of the gel matrix; the gel matrix has a porous structure.

2. The microspheres according to claim 1, characterized in that, The ratio of the dry weight of the gel matrix to the endothelial cells is (20-30) mg: 1 x (10 6 ~10 10 ) cells; Preferably, the mass ratio of the oxidized bacterial cellulose grafted with TGF-β factor to the methacrylamide gelatin is (0.1~0.5):(5~15); Preferably, the photoinitiator comprises any one or a combination of at least two of the following: lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, or phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide; Preferably, the mass ratio of the photoinitiator to methacrylamide gelatin is 1:(10~30).

3. The microspheres according to claim 1 or 2, characterized in that, The TGF-β-grafted oxidized bacterial cellulose was prepared by a method comprising the following steps: (1) Bacterial cellulose, catalyst and oxidant are mixed in buffer solution and subjected to oxidation reaction to obtain oxidized bacterial cellulose; (2) Osmotic pressure regulator, carboxyl activator and TGF-β factor are added sequentially to the buffer containing oxidized bacterial cellulose to carry out the grafting reaction and obtain the oxidized bacterial cellulose grafted with TGF-β factor.

4. The microspheres according to claim 3, characterized in that, In step (1), the buffer solution is PBS buffer; Preferably, the catalyst is 2,2,6,6-tetramethylpiperidine oxide; Preferably, the ratio of bacterial cellulose to catalyst is 1 g:(0.05~0.5) mmol; Preferably, the oxidant includes any one or a combination of at least two of sodium chlorite, potassium chlorite, sodium hypochlorite, or potassium hypochlorite; Preferably, the ratio of bacterial cellulose to oxidant is 1 g:(10~30) mmol; Preferably, the oxidation reaction is carried out at a temperature of 45-55°C for a time of 36-72 h.

5. The microspheres according to claim 3 or 4, characterized in that, In step (2), the buffer solution is MES buffer solution; Preferably, the osmotic pressure regulator comprises sodium chloride and / or potassium chloride; Preferably, the ratio of the oxidizing bacterial cellulose to the osmotic pressure regulator is 60 mg:(20~30) mmol; Preferably, the carboxyl activator comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; Preferably, the ratio of the oxidizing bacterial cellulose to N-hydroxysuccinimide is 60 mg:(2~5) mmol; Preferably, the ratio of the oxidizing bacterial cellulose to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 60 mg:(5~10) mmol; Preferably, the ratio of oxidizing bacterial cellulose to TGF-β factor is 60 mg:(20~30) μg; Preferably, the grafting reaction is carried out at a temperature of 25-35°C for 10-14 hours.

6. The microspheres according to any one of claims 1 to 5, characterized in that, The porous structure is prepared by a method comprising the following steps: After oxidizing bacterial cellulose grafted with TGF-β factor and methacrylamide gelatin crosslinked to form a gel, the mixture was subjected to pre-freezing, sublimation drying, desorption drying and collagenase treatment in sequence to obtain a gel matrix with a porous structure.

7. The microspheres according to claim 6, characterized in that, The pre-freezing temperature is -70~-90℃, and the time is 4~6 hours; Preferably, the temperature for the analytical drying is 15~25℃, and the time is 10~15 h; Preferably, the sublimation drying temperature is -30~-40℃, and the time is 8~28 h; Preferably, the sublimation drying process includes: first treating at -30~-35℃ for 8~12 h, and then treating at -35~-40℃ for 12~16 h.

8. The microspheres according to claim 6 or 7, characterized in that, The collagenase treatment was performed at a temperature of 33-38°C for 3-14 minutes. Preferably, the collagenase treatment process includes: first treating at 33~35℃ for 3~5 min, and then treating at 35~38℃ for 7~9 min; Preferably, the concentration of the collagenase used is 0.1~0.3 mg / mL; Preferably, the terminating agent for the collagenase treatment is an EDTA solution.

9. The method for preparing microspheres according to any one of claims 1 to 8, characterized in that, The preparation method includes: An aqueous phase was prepared by mixing oxidized bacterial cellulose grafted with TGF-β factor, methacrylamide gelatin, a photoinitiator, and water; the aqueous phase was sheared with an oil phase to form microdroplets, which were then cross-linked and cured to prepare a porous structure. Endothelial cells were then inoculated to obtain the microspheres.

10. The preparation method according to claim 9, characterized in that, The flow rate ratio of the oil phase to the water phase is (40~60):1; Preferably, the wavelength of light used for cross-linking and curing is 360~410 nm; Preferably, the cross-linking curing time is 30~90 s; Preferably, the oil phase includes any one or a combination of at least two of paraffin oil, Span 80, Span 60, castor oil, or soybean oil.

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