A microgel-assembled bilayer scaffold for integrated osteochondral repair and its preparation method

Abstract

This invention discloses a microgel-assembled bilayer scaffold for integrated osteochondral repair and its preparation method. The preparation method includes the following steps: (1) synthesizing gelatin modified with carbon-carbon double bonds and dopamine; (2) preparing modified gelatin composite microgels loaded with bioactive glass and chondroitin sulfate respectively; (3) preparing hyaluronic acid modified with carbon-carbon double bonds and phenylboronic acid; (4) suspending the modified gelatin composite microgels loaded with bioactive glass and the modified gelatin composite microgels loaded with chondroitin sulfate in modified hyaluronic acid solutions respectively, and loading them into two different syringes; (5) injecting the two microgel suspensions sequentially into a mold, and constructing the microgel-assembled bilayer scaffold in situ under ultraviolet light. The scaffold of this invention has an open porous structure and good cell compatibility, which can promote the bidirectional differentiation of bone marrow mesenchymal stem cells into chondrogenic and osteogenic cells, and can be used for integrated regeneration and repair of osteochondral tissue defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a microgel-assembled bilayer scaffold for integrated osteochondral repair and its preparation method. Background Technology

[0002] Due to the high complexity and unique biological characteristics of native osteochondral tissue, reconstructing the intricate microstructure of the osteochondral tissue interface remains a challenging problem. Currently, research in osteochondral tissue engineering primarily focuses on the design of biomimetic scaffolds. The traditional manufacturing strategy for tissue engineering scaffolds employs a "top-down" approach, seeding cells onto biodegradable three-dimensional biomimetic scaffolds, such as sponges, fibers, and hydrogels. With the help of growth factors or biochemical cues, the cells filling the scaffold generate an appropriate extracellular matrix. However, scaffolds designed using the "top-down" strategy often fail to reproduce the complex microstructural features of the osteochondral tissue interface. Therefore, designing a scaffold that can mimic the different biochemical properties of osteochondral tissue is crucial.

[0003] The recently adopted "bottom-up" modular assembly approach offers an alternative strategy for reconstructing the complex microstructures of natural tissues by improving tissue and extracellular matrix formation. This strategy assembles smaller, microscale modules into a larger macrostructure, providing greater control over the layered tissue composition and structure compared to homogeneous bulk structures. Microgels are an emerging material for tissue engineering applications and represent excellent modular components for constructing macroscopic scaffolds, showing great potential in osteochondral tissue engineering.

[0004] Bone marrow mesenchymal stem cells are pluripotent stem cells that can differentiate into chondrocytes or osteoblasts under appropriate biological, chemical, and physical signals. However, conventional hydrogel scaffolds have limited ability to guide bidirectional cell differentiation, lack the inherent physical structure and properties required for repairing osteochondral tissue, cannot well mimic the biological environment, and are insufficient to replace defective osteochondral tissue.

[0005] Currently, many studies are dedicated to utilizing various growth factors to improve osteochondral tissue engineering scaffolds, thereby enhancing the bidirectional differentiation induction of bone marrow mesenchymal stem cells. These growth factors include TGF-β and BMPs, which provide crucial support for cartilage and bone tissue repair by promoting cell proliferation, matrix synthesis, and differentiation. However, it is worth noting that excessive release of growth factors may lead to adverse effects such as overgrowth and heterotopic ossification, which may limit their application in clinical treatment. Furthermore, the high cost of growth factor preparation and treatment remains a challenge. Summary of the Invention

[0006] The present invention aims to provide a microgel-assembled bilayer scaffold for integrated osteochondral repair and its preparation method. This bilayer scaffold utilizes hyaluronic acid modified with carbon-carbon double bonds and phenylboronic acid as an assembly cross-linking agent, achieving spatial assembly of modified gelatin / bioactive glass composite microgels and modified gelatin / chondroitin sulfate composite microgels through a bottom-up strategy. It possesses an open porous structure and good cell compatibility, promoting bidirectional differentiation of bone marrow mesenchymal stem cells into chondrocytes and osteoblasts, and can be used for integrated regenerative repair of osteochondral tissue defects.

[0007] The objective of this invention is achieved through one of the following technical solutions.

[0008] A method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair includes the following steps:

[0009] (1) In a buffer solution, a photoinitiator, carbon-carbon double bond and dopamine-modified gelatin and bioactive glass are added to obtain a modified gelatin / bioactive glass suspension aqueous solution; In a buffer solution, a photoinitiator, carbon-carbon double bond and dopamine-modified gelatin and chondroitin sulfate are added to obtain a modified gelatin / chondroitin sulfate composite aqueous solution.

[0010] (2) The modified gelatin / bioactive glass suspension aqueous solution and the modified gelatin / chondroitin sulfate composite aqueous solution are respectively added to liquid paraffin containing Span 80, and stirred to form modified gelatin / bioactive glass droplets and modified gelatin / chondroitin sulfate droplets. The resulting droplets are then subjected to free radical polymerization under ultraviolet light to achieve internal cross-linking of the microgel. After washing and filtration, the modified gelatin / bioactive glass composite microgel and the modified gelatin / chondroitin sulfate composite microgel are obtained.

[0011] (3) The modified gelatin / bioactive glass composite microgel is suspended in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing photoinitiator, and loaded into syringe A; the modified gelatin / chondroitin sulfate composite microgel is suspended in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing photoinitiator, and loaded into syringe B.

[0012] (4) Two microgel suspensions were injected into the mold in sequence using a syringe, and the microgel assembly double scaffold was constructed in situ under ultraviolet light.

[0013] Preferably, the chondroitin sulfate in step (1) is derived from bovine cartilage, has a molecular weight of 10-20 kDa, and a mass concentration of 10 mg / mL to 60 mg / mL in the buffer solution, more preferably 40 mg / mL; the bioactive glass has a particle size of 10-200 nm and a mass concentration of 10 mg / mL to 50 mg / mL in the buffer solution.

[0014] Preferably, the diameter of the microgel obtained in step (2) is 100 μm to 400 μm.

[0015] Preferably, the molecular weight of the hyaluronic acid used in step (3) is 1000KDa to 1500KDa; the mass concentration of the carbon-carbon double bond and phenylboronic acid double-modified hyaluronic acid in the buffer solution is 5mg / mL to 30mg / mL.

[0016] Preferably, the mass concentration of the modified gelatin / bioactive glass composite microgel suspended in step (3) in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing a photoinitiator is 50 mg / mL to 200 mg / mL; the mass concentration of the modified gelatin / chondroitin sulfate composite microgel suspended in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing a photoinitiator is 50 mg / mL to 200 mg / mL.

[0017] Preferably, in step (4), the syringe injects the two microgel suspensions into the mold sequentially by first injecting the modified gelatin / bioactive glass composite microgel suspension with syringe A, and then injecting the modified gelatin / chondroitin sulfate composite microgel suspension with syringe B.

[0018] Preferably, the photoinitiator in steps (1) and (3) is photoinitiator I 2959, with a mass concentration of 5 mg / mL in the buffer solution; the mass concentration of the carbon-carbon double bond and dopamine-modified gelatin in step (1) is 200 mg / mL in the buffer solution; the volume ratio of the composite aqueous solution or suspension aqueous solution to liquid paraffin in step (2) is 1:15; and the volume ratio of Span 80 to liquid paraffin is 1:30.

[0019] Preferably, the ultraviolet irradiation time in step (2) is 10 min, and the ultraviolet irradiation time in step (4) is 1 min to 5 min.

[0020] Preferably, the stirring speed in step (2) is 300 rpm; the stirring time is 30 min.

[0021] Preferably, the organic solvent used for washing the microgel in step (2) can be petroleum ether or acetone.

[0022] Preferred,

[0023] The preparation of the carbon-carbon double bond and dopamine-modified gelatin in step (1) includes the following steps:

[0024] Gelatin was dissolved in phosphate buffer solution, and methacrylic anhydride was added dropwise. After reacting for 3-5 hours, the reaction was terminated with 5 times the amount of phosphate buffer solution and stirred until homogeneous. The reactants were dialyzed and then freeze-dried to obtain carbon-carbon double-bond modified gelatin. The carbon-carbon double-bond modified gelatin was then dissolved in morpholine ethanesulfonic acid buffer solution, and the carboxyl groups were activated by adding a carboxylic acid activator. Under a nitrogen atmosphere, dopamine hydrochloride was added, and the reaction was carried out in the dark for 24 hours. The reactants were dialyzed and then freeze-dried to obtain carbon-carbon double-bond and dopamine-modified gelatin. The gelatin was derived from pig skin (gel strength ~ 240g Bloom).

[0025] The preparation of the carbon-carbon double bond and phenylboronic acid-modified hyaluronic acid in step (3) includes the following steps:

[0026] Hyaluronic acid was dissolved in morpholine ethanesulfonic acid buffer, and 3-aminophenylboronic acid was added. After reacting in the dark for 24 hours, the reactants were dialyzed and then lyophilized to obtain phenylboronic acid-modified hyaluronic acid. Then, phenylboronic acid-modified hyaluronic acid was dissolved in deionized water, and methacrylic anhydride was added dropwise to adjust the pH to 8-9. After reacting for 12 hours, the reactants were dialyzed and then lyophilized to obtain carbon-carbon double bond and phenylboronic acid-double modified hyaluronic acid.

[0027] A microgel-assembled bilayer scaffold for integrated osteochondral repair, prepared by any of the above preparation methods.

[0028] Based on the different physiological characteristics of osteocartilage tissue, this invention introduces chondroitin sulfate and bioactive glass into a double-layer scaffold to specifically guide bidirectional cell differentiation and achieve complex microstructural features at the osteocartilage tissue interface.

[0029] Compared to traditional hydrogels, the microgels used in this invention not only exhibit microstructural features closer to those of natural tissues, but also can load biomaterial components with different biochemical cues. Furthermore, the microgels are spatially assembled using a bottom-up strategy to construct macroscopic structural scaffolds.

[0030] This invention utilizes ultraviolet light to initiate free radical polymerization of double bonds on modified hyaluronic acid and dynamic bonding between phenylboronic acid on modified hyaluronic acid and dopamine on modified gelatin composite microgels to construct microgels in situ to assemble bilayer scaffolds.

[0031] Compared with the prior art, the present invention has the following advantages and effects:

[0032] (1) The microgels prepared in this invention have a size of 100μm to 400μm, and the smaller size has excellent injection performance.

[0033] (2) The microgel-assembled bilayer scaffold prepared in this invention has an open porous structure and good biocompatibility, which is beneficial to cell growth and nutrient transport inside.

[0034] (3) The microgel assembly double-layer scaffold prepared in this invention can promote the chondrogenic differentiation of BMSCs in the upper layer and facilitate the osteogenic differentiation of BMSCs in the lower layer, and can be used for integrated regeneration and repair of osteochondral tissue defects. Attached Figure Description

[0035] Figure 1 Bright field plot, diameter size distribution, and injectability verification plot of the microgel prepared in Example 3.

[0036] Figure 2 The image shows the qRT-PCR test results of the microgel prepared in Example 1.

[0037] The Control group consisted of pure differentiation culture medium, the G Ms group consisted of modified gelatin microgels prepared in Example 1, GC1Ms, GC4Ms and GC6Ms consisted of modified gelatin / chondroitin sulfate microgels with added 0.02, 0.08 and 0.12 g of chondroitin sulfate, respectively, and GB1Ms, GB2Ms and GB5Ms consisted of modified gelatin / bioactive glass microgels with added 0.02, 0.04 and 0.1 g of bioactive glass, respectively.

[0038] Figure 3 The images show the physical specimen and SEM image of the microgel-assembled bilayer scaffold prepared in Example 5.

[0039] Figure 4 Cell compatibility evaluation of the microgel-assembled bilayer scaffold prepared in Example 5.

[0040] Figure 5 This is an immunofluorescence staining image of the microgel-assembled bilayer scaffold prepared in Example 5. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0042] The preparation of the carbon-carbon double bond and dopamine-modified gelatin in step (1) includes the following steps:

[0043] 10g of gelatin was weighed and added to 100mL of phosphate buffer solution. The solution was stirred and dissolved at 45℃. 12mL of methacrylic anhydride was added dropwise. After reacting for 5 hours, the reaction was terminated with 500mL of phosphate buffer solution and stirred until homogeneous. The reactants were dialyzed and then lyophilized to obtain carbon-carbon double-bond modified gelatin. 3g of the carbon-carbon double-bond modified gelatin was then weighed and dissolved in 300mL of morpholine ethanesulfonic acid buffer (100mM, pH 5.5). 0.5751g of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and 0.3453g of N-hydroxysuccinimide were added and stirred for 30 minutes to activate the carboxyl groups. Under a nitrogen atmosphere, 0.5689g of dopamine hydrochloride was added, and the reaction was carried out in the dark for 24 hours. The reactants were dialyzed and then lyophilized to obtain carbon-carbon double-bond and dopamine-modified gelatin.

[0044] The preparation of the carbon-carbon double bond and phenylboronic acid-modified hyaluronic acid in step (3) includes the following steps:

[0045] 1 g of hyaluronic acid was dissolved in 300 mL of morpholine ethanesulfonic acid buffer (100 mM, pH 5.5). Then, 0.692 g of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added to the hyaluronic acid solution and stirred for 30 min to activate the carboxyl group of the hyaluronic acid. 0.129 g of 3-aminophenylboronic acid was added, and the reaction was carried out in the dark for 24 h. The reaction mixture was dialyzed and lyophilized to obtain phenylboronic acid-modified hyaluronic acid. Next, 2 g of phenylboronic acid-modified hyaluronic acid was dissolved in 200 mL of deionized water, and 7.594 mL of methacrylic anhydride was added dropwise to adjust the pH to 8-9. The reaction was carried out at 4 °C for 12 h. The reaction mixture was then dialyzed and lyophilized to obtain carbon-carbon double-bonded and phenylboronic acid-modified hyaluronic acid.

[0046] Example 1

[0047] (1) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0, 0.02, 0.04 or 0.1g of 45S5 bioactive glass and add it to 2mL of phosphate buffer solution to obtain modified gelatin / bioactive glass suspension aqueous solution;

[0048] (3) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0, 0.02, 0.08 or 0.12g of chondroitin sulfate and dissolve them in 2mL of phosphate buffer solution to obtain modified gelatin / chondroitin sulfate composite aqueous solution.

[0049] (4) Modified gelatin / bioactive glass suspension aqueous solution and modified gelatin / chondroitin sulfate composite aqueous solution were added to a continuous oil phase containing 1 mL Span 80 and 30 mL liquid paraffin, respectively, and mechanically stirred at 300 rpm for 30 min to form modified gelatin droplets; the droplets were then placed under 365 nm ultraviolet light for 10 min to initiate free radical polymerization and achieve internal cross-linking of the droplets. The liquid paraffin and Span 80 on the surface of the microgel were then washed with petroleum ether and filtered through a stainless steel mesh screen to obtain modified gelatin / bioactive glass microgels and modified gelatin / chondroitin sulfate microgels with a certain diameter.

[0050] Example 2

[0051] (1) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0.1g of 45S5 bioactive glass and add them to 2mL of phosphate buffer solution to obtain modified gelatin / bioactive glass suspension aqueous solution;

[0052] (2) Add the modified gelatin / bioactive glass suspension aqueous solution to a continuous oil phase containing 1 mL Span 80 and 30 mL liquid paraffin, and mechanically stir at 300 rpm for 30 min to form modified gelatin / bioactive glass droplets; then place the droplets under 365 nm ultraviolet light for 10 min to initiate free radical polymerization to achieve internal cross-linking of the droplets; then wash the liquid paraffin and Span 80 on the surface of the microgel with petroleum ether, and filter through a stainless steel mesh screen to obtain a modified gelatin / bioactive glass composite microgel with a certain diameter.

[0053] (3) Weigh 0.005g of carbon-carbon double bond and phenylboronic acid double-modified hyaluronic acid and 0.005g of I 2959 photoinitiator and dissolve them in 1mL of phosphate buffer solution to obtain modified hyaluronic acid aqueous solution. Then weigh 50mg of lyophilized modified gelatin / bioactive glass composite microgel, rehydrate it and suspend it in modified hyaluronic acid solution, and put it into a 5mL syringe.

[0054] (4) The modified gelatin / bioactive glass composite microgel suspension was injected into a cylindrical mold using a syringe and irradiated under 365nm ultraviolet light for 2 minutes to construct the modified gelatin / bioactive glass composite microgel assembly scaffold in situ.

[0055] Example 3

[0056] (1) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0.12g of chondroitin sulfate and dissolve them in 2mL of phosphate buffer solution to obtain modified gelatin / chondroitin sulfate composite aqueous solution.

[0057] (2) Add the modified gelatin / chondroitin sulfate composite aqueous solution to a continuous oil phase containing 1 mL Span 80 and 30 mL liquid paraffin, and mechanically stir at 300 rpm for 30 min to form modified gelatin / chondroitin sulfate droplets; then place the droplets under 365 nm ultraviolet light for 10 min to initiate free radical polymerization and achieve internal cross-linking of the droplets; then wash the liquid paraffin and Span 80 on the surface of the microgel with acetone, and filter through a stainless steel mesh screen to obtain a modified gelatin / chondroitin sulfate composite microgel with a certain diameter.

[0058] (3) Weigh 0.03g of carbon-carbon double bond and phenylboronic acid double-modified hyaluronic acid and 0.005g of I 2959 photoinitiator and dissolve them in 1mL of phosphate buffer solution to obtain modified hyaluronic acid aqueous solution. Then weigh 200mg of lyophilized modified gelatin / chondroitin sulfate composite microgel, rehydrate it and suspend it in modified hyaluronic acid solution, and put it into a 5mL syringe.

[0059] (4) The modified gelatin / chondroitin sulfate composite microgel suspension was injected into a cylindrical mold using a syringe and irradiated under 365nm ultraviolet light for 5 min to construct the modified gelatin / chondroitin sulfate composite microgel assembly scaffold in situ.

[0060] Example 4

[0061] (1) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0.02g of 45S5 bioactive glass and add them to 2mL of phosphate buffer solution to obtain modified gelatin / bioactive glass suspension aqueous solution;

[0062] (2) Add the modified gelatin / bioactive glass suspension aqueous solution to a continuous oil phase containing 1 mL Span 80 and 30 mL liquid paraffin, and mechanically stir at 300 rpm for 30 min to form modified gelatin / bioactive glass droplets; then place the droplets under 365 nm ultraviolet light for 10 min to initiate free radical polymerization and achieve internal cross-linking of the droplets; then wash the liquid paraffin and Span 80 on the surface of the microgel with petroleum ether, and filter through a stainless steel mesh screen to obtain microgels of a certain diameter.

[0063] (3) Weigh 0.02g of carbon-carbon double bond and phenylboronic acid double-modified hyaluronic acid and 0.005g of I 2959 photoinitiator and dissolve them in 1mL of phosphate buffer solution to obtain modified hyaluronic acid aqueous solution. Then weigh 200mg of lyophilized modified gelatin / bioactive glass composite microgel, rehydrate it and suspend it in modified hyaluronic acid solution, and put it into a 5mL syringe.

[0064] (5) The modified gelatin / bioactive glass composite microgel suspension was injected into a cylindrical mold using a syringe and irradiated under 365nm ultraviolet light for 1 min to construct the modified gelatin / bioactive glass composite microgel assembly scaffold in situ.

[0065] Example 5

[0066] (1) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0.06g of 45S5 bioactive glass and add them to 2mL of phosphate buffer solution to obtain modified gelatin / bioactive glass suspension aqueous solution;

[0067] (2) Weigh 0.4g of carbon-carbon double bond and dopamine-modified gelatin, 0.01g of I 2959 photoinitiator and 0.02g of chondroitin sulfate and dissolve them in 2mL of phosphate buffer solution to obtain modified gelatin / chondroitin sulfate composite aqueous solution.

[0068] (3) The modified gelatin / bioactive glass suspension and the modified gelatin / chondroitin sulfate composite aqueous solution were added to a continuous oil phase containing 1 mL Span 80 and 30 mL liquid paraffin, respectively. The mixture was mechanically stirred at 300 rpm for 30 min to form modified gelatin / bioactive glass droplets and modified gelatin / chondroitin sulfate droplets. The droplets were then placed under 365 nm ultraviolet light for 10 min to initiate free radical polymerization and achieve internal cross-linking of the droplets. The liquid paraffin and Span 80 on the surface of the microgel were then washed with petroleum ether. After filtration through a stainless steel mesh screen, modified gelatin / bioactive glass composite microgels and modified gelatin / chondroitin sulfate composite microgels with a certain diameter were obtained.

[0069] (4) Weigh 0.025g of carbon-carbon double bond and phenylboronic acid double-modified hyaluronic acid and 0.005g of I 2959 photoinitiator and dissolve them in 1mL of phosphate buffer solution to obtain modified hyaluronic acid aqueous solution. Weigh 120mg of modified gelatin / bioactive glass composite microgel, rehydrate it and suspend it in the modified hyaluronic acid solution, and put it into 5mL syringe A. Weigh 50mg of modified gelatin / chondroitin sulfate composite microgel, rehydrate it and suspend it in the modified hyaluronic acid aqueous solution, and put it into 5mL syringe B.

[0070] (5) First, the modified gelatin / bioactive glass composite microgel suspension in syringe A was injected into a cylindrical mold. Then, the modified gelatin / chondroitin sulfate composite microgel suspension in syringe B was injected onto the modified gelatin / bioactive glass composite microgel suspension. After irradiation with 365nm ultraviolet light for 2 minutes, a microgel assembly double-layer scaffold was constructed in situ.

[0071] Performance test (1):

[0072] The size distribution and injectability of the modified gelatin / chondroitin sulfate composite microgel prepared in Example 3 were characterized. Figure 1 As shown, the structure of the microgel prepared in Example 3 in the aqueous phase was observed by an inverted microscope, and the average diameter was 252.5 ± 80.35 μm, which was statistically analyzed by ImageJ software. The rheological properties of the modified gelatin / chondroitin sulfate composite microgel suspension were tested by a rheometer. The results showed that the viscosity of the suspension gradually decreased with the increase of shear rate, and the size of the prepared microgel could meet the injection requirements.

[0073] Performance testing (2):

[0074] The modified gelatin microgels, modified gelatin / bioactive glass microgels, and modified gelatin / chondroitin sulfate microgels prepared in Example 1 were characterized for inducing chondrogenic and osteogenic differentiation of bone MSCs. After co-culturing BMSCs and microgels in chondrogenic and osteogenic differentiation media for 7 days, the expression levels of Sox9 (a gene related to chondrogenesis) and Runx2 (a gene related to osteogenic differentiation) were analyzed by qRT-PCR. The results are as follows: Figure 2 As shown in a, after 7 days of culture, the expression level of Sox9 gene in the GC4 Ms group was significantly higher than that in the blank control group and other experimental groups. The expression levels of Runx2 gene in the GB1 Ms, GB2 Ms, and GB5 Ms groups were also significantly higher than those in the blank control group and the G Ms group. Figure 2 (b) However, there was no significant difference in the expression level of the Runx2 gene among the GB1 Ms, GB2 Ms, and GB5 Ms groups. These results indicate that GC4 Ms is more conducive to chondrogenic differentiation of BMSCs, while GB1 Ms, GB2 Ms, and GB5 Ms with added bioactive glass are more conducive to osteogenic differentiation of BMSCs.

[0075] Performance test (3):

[0076] The surface morphology and structure of the microgel-assembled bilayer scaffold prepared in Example 5 were characterized. The surface morphology of the microgel-assembled bilayer scaffold prepared in Example 5 was characterized using stereomicroscopy and scanning electron microscopy. Figure 3 As shown, the in-situ constructed microgel-assembled bilayer scaffold possesses a stable bilayer structure. The lower layer is a rhodamine-stained modified gelatin / bioactive glass composite microgel assembly layer, and the upper layer is a modified gelatin / chondroitin sulfate composite microgel assembly layer. SEM images reveal that the microstructure of the lyophilized scaffold exhibits an open porous structure.

[0077] Performance test (4):

[0078] Cell compatibility was evaluated using the microgel-assembled bilayer scaffold prepared in Example 5. First, 1 × 10⁻⁶ cells were used per well. 5Bone marrow mesenchymal stem cells (BMSCs) were seeded onto the surface of the microgel at a cell density of [number] cells. Then, a bilayer scaffold loaded with BMSCs was prepared using the method described in Example 5. After culturing for 1 day and 7 days, the BMSCs in the bilayer scaffold were double-stained for 30 minutes using a live-dead staining working solution, and then observed under a laser confocal microscope. Figure 4 As shown, from day 1 to day 7, the cells showed a proliferative trend with almost no dead cells, indicating that BMSCs can maintain good cell viability in the microgel-assembled bilayer scaffold.

[0079] Performance test (5):

[0080] The ability of the microgel-assembled bilayer scaffold prepared in Example 5 to induce bidirectional differentiation of BMSCs into chondrogenic and osteogenic processes was characterized. After culturing BMSCs within the microgel-assembled bilayer scaffold in chondrogenic and osteogenic differentiation media for 21 days, the scaffolds were washed with phosphate buffer and fixed with 4% formaldehyde. They were then incubated overnight at 4°C with primary antibody solutions of Col II (1:200) and Col I (1:200). The samples were washed three times with phosphate buffer for 5 min each time, and then incubated with goat anti-rabbit (Alexa Fluor 488) secondary antibody solution at room temperature for 1.5 h. The dye TRITCPhalloidin was diluted 1:200 with phosphate buffer and incubated with the samples for 1 h, followed by staining with DAPI staining solution for 15 min. Finally, the fluorescence images were observed using a laser scanning electron microscope (see [link to sample image]). Figure 5 Col II is a major component of the extracellular matrix of chondrocytes, and its protein expression level can characterize the chondrogenic differentiation capacity of bone mesenchymal stem cells (BMSCs). Col I is an important component of the calcium-mineralized bone matrix, and its protein expression level can characterize the osteogenic differentiation capacity of BMSCs. Figure 4 The immunofluorescence staining images show the expression of Col II and Col I within the scaffold, with Col II being expressed in greater quantities in the upper layer and Col I in greater quantities in the lower layer. These results indicate that microgel-assembled bilayer scaffolds can promote bidirectional differentiation of BMSCs, demonstrating great potential for integrated osteochondral applications. In the images, DAPI represents dye-stained cell nuclei; F-actin represents dye-stained cytoskeleton structures. "Merge" refers to the image displayed after overlaying DAPI and F-actin images.

Claims

1. A method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair, characterized in that, Includes the following steps: (1) In a buffer solution, a photoinitiator, carbon-carbon double bond and dopamine-modified gelatin and bioactive glass are added to obtain a modified gelatin / bioactive glass suspension aqueous solution; In a buffer solution, a photoinitiator, carbon-carbon double bond and dopamine-modified gelatin and chondroitin sulfate are added to obtain a modified gelatin / chondroitin sulfate composite aqueous solution. (2) The modified gelatin / bioactive glass suspension aqueous solution and the modified gelatin / chondroitin sulfate composite aqueous solution are respectively added to liquid paraffin containing Span 80, and stirred to form modified gelatin / bioactive glass droplets and modified gelatin / chondroitin sulfate droplets. The resulting droplets are then subjected to free radical polymerization under ultraviolet light to achieve internal cross-linking of the microgel. After washing and filtration, the modified gelatin / bioactive glass composite microgel and the modified gelatin / chondroitin sulfate composite microgel are obtained. (3) The modified gelatin / bioactive glass composite microgel is suspended in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing photoinitiator, and loaded into syringe A; the modified gelatin / chondroitin sulfate composite microgel is suspended in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing photoinitiator, and loaded into syringe B; (4) The two microgel suspensions were injected into the mold in sequence using a syringe, and the microgel assembly double scaffold was constructed in situ under ultraviolet light. The microgels obtained in step (2) have a diameter of 100 μm to 400 μm.

2. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, The chondroitin sulfate mentioned in step (1) is derived from bovine cartilage, has a molecular weight of 10-20 kDa, and a mass concentration of 10 mg / mL to 60 mg / mL in the buffer solution; the bioactive glass has a particle size of 10-200 nm and a mass concentration of 10 mg / mL to 50 mg / mL in the buffer solution.

3. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, The molecular weight of the hyaluronic acid used in step (3) is 1000 kDa to 1500 kDa; the mass concentration of the carbon-carbon double bond and phenylboronic acid double-modified hyaluronic acid in the buffer solution is 5 mg / mL to 3 mg / mL.

4. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, The mass concentration of the modified gelatin / bioactive glass composite microgel suspended in step (3) in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing photoinitiator is 50 mg / mL to 200 mg / mL; the mass concentration of the modified gelatin / chondroitin sulfate composite microgel suspended in a solution of carbon-carbon double bonds and phenylboronic acid double-modified hyaluronic acid containing photoinitiator is 50 mg / mL to 200 mg / mL.

5. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, In step (4), the syringes inject the two microgel suspensions into the mold in sequence. First, the modified gelatin / bioactive glass composite microgel suspension is injected with syringe A, and then the modified gelatin / chondroitin sulfate composite microgel suspension is injected with syringe B.

6. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, The photoinitiator mentioned in steps (1) and (3) is photoinitiator I 2959, with a mass concentration of 5 mg / mL in the buffer solution; the mass concentration of carbon-carbon double bond and dopamine-modified gelatin mentioned in step (1) is 200 mg / mL in the buffer solution; the volume ratio of the composite aqueous solution or suspension aqueous solution to liquid paraffin mentioned in step (2) is 1:15; and the volume ratio of Span 80 to liquid paraffin is 1:

30.

7. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, In step (2), the ultraviolet light irradiation time is 10 min, and in step (4), the ultraviolet light irradiation time is 1 min to 5 min.

8. The method for preparing a microgel-assembled bilayer scaffold for integrated osteochondral repair according to claim 1, characterized in that, The preparation of the carbon-carbon double bond and dopamine-modified gelatin in step (1) includes the following steps: Gelatin was dissolved in phosphate buffer solution, and methacrylic anhydride was added dropwise. After the reaction, the reaction was terminated with phosphate buffer solution and stirred until homogeneous. The reactants were dialyzed and then freeze-dried to obtain carbon-carbon double-bond modified gelatin. The carbon-carbon double-bond modified gelatin was then dissolved in morpholine ethanesulfonic acid buffer solution, and a carboxyl activator was added to activate the carboxyl groups. Under a nitrogen atmosphere, dopamine hydrochloride was added, and the reaction was carried out in the dark. The reactants were dialyzed and then freeze-dried to obtain carbon-carbon double-bond and dopamine-modified gelatin. The gelatin was derived from pigskin. The preparation of the carbon-carbon double bond and phenylboronic acid-modified hyaluronic acid in step (3) includes the following steps: Hyaluronic acid was dissolved in morpholine ethanesulfonic acid buffer, and 3-aminophenylboronic acid was added. After reacting in the dark, the reactants were dialyzed and then freeze-dried to obtain phenylboronic acid-modified hyaluronic acid. Then, phenylboronic acid-modified hyaluronic acid was dissolved in deionized water, and methacrylic anhydride was added dropwise to adjust the pH to 8-9. After reacting, the reactants were dialyzed and then freeze-dried to obtain carbon-carbon double bond and phenylboronic acid-modified hyaluronic acid.

9. A microgel-assembled bilayer scaffold for integrated osteochondral repair, prepared by the preparation method according to any one of claims 1-8.

Images