Probiotic film bubble functionalized gradient electrospun fiber scaffolds for osteochondral repair

Abstract

This invention discloses a probiotic vesicle-functionalized gradient electrospun fiber scaffold for osteochondral repair. The scaffold has a three-layer gradient structure, consisting of a cartilage layer, an intermediate calcified layer, and a subchondral bone layer from top to bottom. The cartilage layer is a PCL / Gel fiber-reinforced PVA / CS hydrogel. The intermediate calcified layer and the subchondral bone layer are PCL / Gel three-dimensional fiber scaffolds with strontium-doped hydroxyapatite deposited on their surfaces. The PCL / Gel three-dimensional fiber scaffold has a through-pore structure, and the SrHA deposition in the subchondral bone layer is greater than that in the intermediate calcified layer. The scaffold is modified with a cross-linking agent to contain PLGA nanospheres carrying Lactobacillus reuteri membrane vesicles. This invention uses a three-layer structure with a cartilage layer as a fiber-reinforced hydrogel, an intermediate calcified layer, and a subchondral bone layer as a gradient mineralized three-dimensional fiber scaffold, which promotes the directed differentiation of bone marrow mesenchymal stem cells, exhibits good biocompatibility, has a clear preparation process, and is easy to scale up for production.

Description

Technical Field

[0001] This invention relates to a fiber scaffold processing technology, and more particularly to a probiotic membrane vesicle functionalized gradient electrospun fiber scaffold for osteochondral repair. Background Technology

[0002] Articular cartilage is a hyaline cartilage tissue without blood vessels, lymphatic vessels, or nerves. It relies on synovial fluid for nutrition and has extremely limited self-repair capabilities. Repairing osteocartilage defects has become a major clinical challenge in orthopedics. Commonly used treatments include intra-articular drug injection, microfracture, drilling, autologous chondrocyte transplantation, osteocartilage transplantation, and joint replacement. However, these methods all have varying degrees of limitations: palliative treatments such as bone marrow stimulation can only relieve symptoms and cannot achieve tissue regeneration; allogeneic transplantation carries risks of immune rejection and disease transmission; and autologous chondrocyte transplantation faces problems such as limited donor tissue and secondary damage at the harvesting site. Therefore, there is an urgent need to develop alternative therapies for repairing osteocartilage defects.

[0003] In recent years, tissue engineering technology has provided a new direction for the repair of osteochondral defects by constructing functional scaffolds. Tissue-engineered scaffolds need to mimic the structure of the natural extracellular matrix, balancing mechanical support and cellular activity space. Electrospinning technology can prepare nanofiber scaffolds with high specific surface area and high porosity, forming biomimetic microporous structures. However, in traditional electrospun fiber membranes, the fibers are densely packed, resulting in limited membrane thickness and small pore sizes, hindering cell migration and tissue penetration. Furthermore, existing monophase or biphase scaffolds, due to their simple structure, cannot accurately match the complex heterogeneous structure of osteochondral, and therefore cannot induce synchronous regeneration and functional maturation of the entire tissue layer. Current research has moved from early monophase, single-factor scaffolds to multiphase gradient scaffolds, but how to manufacture gradient scaffolds with a continuous osteochondral interface and possessing physical and biological induction signals remains a challenge in tissue engineering.

[0004] An ideal osteocartilage scaffold, besides possessing structural biomimicry and mechanical adaptability, should also have immunomodulatory capabilities. Macrophages, as key immune cells in the osteocartilage microenvironment, are a significant cause of the continuous progression of cartilage damage. Existing scaffolds mostly focus on directly promoting stem cell differentiation, lacking sufficient ability to regulate the inflammatory microenvironment. Studies have shown that membrane vesicles secreted by *Lactobacillus reuteri* can accelerate tissue regeneration by regulating macrophage mitochondrial function, promoting their transformation into the anti-inflammatory and repair-promoting M2 phenotype. *Lactobacillus reuteri* membrane vesicles can also induce macrophage M2 polarization through the SPP1-JAK1-STAT3 signaling pathway, promoting bone regeneration. While traditional bioactive factors such as mesenchymal stem cell exosomes have shown potential in tissue repair, they face challenges such as difficult extraction and purification, high cost, poor stability, and difficulties in standardized production, limiting their clinical translation and failing to meet the complex requirements of full-thickness osteocartilage regeneration. Therefore, a probiotic membrane vesicle-functionalized gradient electrospun fiber scaffold for osteocartilage repair is needed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a probiotic vesicle functionalized gradient electrospun fiber scaffold for osteochondral repair.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: The scaffold of this invention has a three-layer gradient structure, consisting of a cartilage layer, an intermediate calcified layer, and a subchondral bone layer from top to bottom. The cartilage layer is a PCL / Gel fiber-reinforced PVA / CS hydrogel. The intermediate calcified layer and the subchondral bone layer are PCL / Gel three-dimensional fiber scaffolds with strontium-doped hydroxyapatite deposited on their surfaces. The PCL / Gel three-dimensional fiber scaffold has a through-pore structure, and the SrHA deposition amount in the subchondral bone layer is greater than that in the intermediate calcified layer. The scaffold is modified with a crosslinking agent to contain PLGA nanospheres carrying Lactobacillus reuteri membrane vesicles.

[0007] Furthermore, the crosslinking agent is Sulfo-SMCC.

[0008] On the other hand, a method for preparing a probiotic vesicle-functionalized gradient electrospun fiber scaffold for osteochondral repair includes the following steps: (1) Extraction of Lactobacillus reuteri membrane vesicles; (2) The Lactobacillus reuteri membrane vesicles were encapsulated in PLGA nanospheres to obtain PLGA nanospheres loaded with Lactobacillus reuteri membrane vesicles; (3) Preparation of PCL / Gel electrospun fiber membrane; (4) Disperse the PCL / Gel electrospun fiber membrane and form a three-dimensional fiber scaffold; (5) The three-dimensional fiber scaffold was subjected to mineralization treatment in strontium-doped simulated body fluid for different times to obtain an intermediate calcified layer scaffold and a subchondral bone layer scaffold with gradient SrHA deposition. (6) PCL / Gel short fibers are mixed with PVA / CS hydrogel precursor to prepare a cartilage layer, and the cartilage layer, intermediate calcified layer scaffold and subchondral bone layer scaffold are integrated into a three-layer gradient scaffold. (7) The PLGA nanospheres carrying Lactobacillus reuteri membrane vesicles are modified onto the three-layer gradient scaffold by a crosslinking agent.

[0009] Further, the preparation of PLGA nanospheres loaded with Lactobacillus reuteri membrane vesicles in step (2) includes: dissolving PLGA in dichloromethane at a concentration of 50 mg / mL as the oil phase; dissolving Lactobacillus reuteri membrane vesicles in a PBS solution containing 1% BSA and 5% sucrose at a protein concentration of 0.5 mg / mL as the inner aqueous phase; mixing the inner aqueous phase and the oil phase at a volume ratio of 1:5, and ultrasonically emulsifying under ice bath conditions at a power of 200 W for 3 min to form a first emulsion; adding the first emulsion dropwise into a 1% PVA aqueous solution and magnetically stirring for 30 min to form a second emulsion; collecting the nanospheres, washing them, and then freeze-drying them.

[0010] Further, the strontium-doped simulated body fluid mentioned in step (5) is a strontium-doped simulated body fluid with a concentration of 5 times, and its formula is: NaCl 40.0g, NaHCO3 1.75g, KCl 1.125g, K2HPO4・3H2O 1.14g, SrCl2・6H2O 0.75g, Na2SO4 0.45g, Tris 15.125g, CaCl2 1.8375g, adjusted to 1L, pH 7.4.

[0011] Further, the mineralization treatment in step (5) is as follows: the three-dimensional fiber scaffold is immersed in strontium-doped simulated body fluid, the intermediate calcified layer scaffold is mineralized for 24 hours, and the subchondral bone layer scaffold is mineralized for 48 hours. The beneficial effects of this invention are: (1) The present invention adopts a three-layer structure with the cartilage layer as fiber-reinforced hydrogel, the intermediate calcified layer and the subchondral bone layer as gradient mineralized three-dimensional fiber scaffold, which simulates natural bone and cartilage tissue in terms of composition and mechanical properties, and provides a differentiated growth microenvironment for chondrocytes and osteoblasts.

[0012] (2) In this invention, PLGA nanospheres carrying Lactobacillus reuteri membrane vesicles are covalently grafted onto the surface of a scaffold using a crosslinking agent. Lactobacillus reuteri membrane vesicles can promote macrophage polarization toward the M2 phenotype and reduce the secretion of pro-inflammatory factors. Strontium-doped hydroxyapatite is distributed in a gradient along the thickness of the scaffold, providing gradient bone induction signals. It works synergistically with the immune regulation of Lactobacillus reuteri membrane vesicles to promote the directed differentiation of bone marrow mesenchymal stem cells. It has good biocompatibility, a clear preparation process, and is easy to scale up. Attached Figure Description

[0013] Figure 1 This is a diagram showing the L-MV standardized extraction process and product performance characterization of the present invention; Figure 2 This is a diagram showing the entire process of LMP nanosphere emulsification-curing preparation and the performance characterization of the products in this invention; Figure 3 This is a diagram showing the preparation process and product performance characterization of the PCL / Gel composite electrospun fiber membrane of this invention. Figure 4 This is a diagram showing the cryo-thermal crosslinking preparation process and product performance characterization of the PCL / Gel three-dimensional fiber scaffold of this invention; Figure 5 Characterization diagram of the three-dimensional scaffold gradient mineralization process and SrHA gradient modified scaffold of the present invention; Figure 6 This is a diagram illustrating the precise integration process and structure of the three-layer osteochondral biomimetic gradient scaffold of the present invention. Figure 7 The image shows the characterization of the Sulfo-SMCC-mediated LMP covalent modification process and functionalized gradient scaffold of this invention. Detailed Implementation

[0014] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions herein are used to explain the present invention, but are not intended to limit the present invention.

[0015] In this invention, PCL represents polycaprolactone, Gel represents gelatin, PVA represents polyvinyl alcohol, CS represents chitosan, PLGA represents polylactic acid-glycolic acid copolymer, SrHA represents strontium-doped hydroxyapatite, Sulfo-SMCC represents sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, BSA represents bovine serum albumin, PBS represents phosphate buffer, GelMA represents methacrylated gelatin, TEM represents transmission electron microscopy, SEM represents scanning electron microscopy, DLS represents dynamic light scattering, and EDS represents energy dispersive spectroscopy.

[0016] In Example 1, this invention provides a probiotic vesicle-functionalized gradient electrospun fiber scaffold for osteochondral repair. This scaffold has a three-layer gradient structure, consisting of a cartilage layer, an intermediate calcified layer, and a subchondral bone layer from top to bottom. The cartilage layer is a PCL / Gel fiber-reinforced PVA / CS hydrogel, while the intermediate and subchondral bone layers are PCL / Gel three-dimensional fiber scaffolds with varying amounts of strontium-doped hydroxyapatite (SrHA) deposited on their surfaces. The entire scaffold is modified with a Sulfo-SMCC crosslinking agent to form PLGA nanospheres (LMPs) carrying Lactobacillus reuteri vesicles.

[0017] The specific preparation method is as follows: Step 1: Extraction and characterization of Lactobacillus reuteri membrane vesicles (L-MV) Lactobacillus reuteri culture: The Lactobacillus reuteri (NBRC15892) used in this invention was purchased from Wuhan Huanna Biotechnology Co., Ltd., and was cultured in MRS medium under anaerobic conditions at 37°C until the bacterial density reached 10⁹ CFU / mL.

[0018] Membrane vesicle extraction: Centrifuge at 3000 rpm for 10 min to collect the supernatant, centrifuge at 10000 g for 30 min to remove bacterial debris, and ultracentrifuge at 160000 g for 80 min to collect the precipitate, which is the crude L-MV extract.

[0019] Purification and characterization: The precipitate was resuspended in PBS, and its morphology was observed by transmission electron microscopy (TEM). The particle size distribution was determined by dynamic light scattering (DLS), the protein concentration was determined by BCA method, and its bioactive components were analyzed by proteomics.

[0020] like Figure 1 As shown, the standardized extraction process of L-MV is demonstrated. TEM characterization shows that L-MV has a circular / elliptical nanovesicle structure with clear boundaries. DLS detection shows that the particle size is concentrated in 50–200 nm and is uniformly distributed. BCA method determination shows that the protein concentration is stable in 0.5–1.0 mg / mL.

[0021] Step 2: Preparation of L-MV-loaded PLGA nanospheres (LMP) Solution preparation: Dissolve PLGA in dichloromethane to a concentration of 50 mg / mL; dissolve L-MV in PBS containing 1% BSA and 5% sucrose to a protein concentration of 0.5 mg / mL.

[0022] Emulsion preparation: The L-MV solution and PLGA solution were mixed at a volume ratio of 1:5 and ultrasonically emulsified under ice bath conditions (power 200W, time 3min) to form the first emulsion; the first emulsion was slowly dripped into a 1% PVA aqueous solution and magnetically stirred for 30min to form the second emulsion.

[0023] Collection and Characterization: Nanospheres were collected by centrifugation, washed three times with deionized water, and freeze-dried. Morphology was observed by TEM, particle size and zeta potential were determined by DLS, encapsulation efficiency and drug loading were determined by BCA method, and sustained-release performance was verified by in vitro release experiments.

[0024] like Figure 2 As shown, the entire process of emulsification and solidification preparation of LMP nanospheres is presented. A water-in-oil (W / O / W) double emulsification method was successfully used to prepare uniformly dispersed LMP nanospheres. TEM showed that the nanospheres were regularly spherical with smooth, non-adhesive surfaces; DLS measurements showed an average particle size of 180±30 nm and a zeta potential of -15±5 mV, indicating good stability; BCA measurements showed an L-MV encapsulation efficiency of ≥75% and a drug loading of 5–8 μg / mg; in vitro release experiments verified sustained release over 4 weeks without burst release, meeting the requirements for long-term scaffold modification.

[0025] Step 3: Preparation of PCL / Gel electrospun fiber membrane Preparation of spinning solution: Mix PCL and gelatin at a mass ratio of 3:7, dissolve in hexafluoroisopropanol to a total concentration of 15% (w / v), add 1% (v / v) glacial acetic acid, and stir at room temperature until completely dissolved.

[0026] Electrospinning: The spinning parameters were set as follows: voltage 20kV, receiving distance 15cm, flow rate 2mL / h, and receiving roller speed 500rpm. The spun film was collected and vacuum dried for 24h for later use.

[0027] like Figure 3 As shown, the process for preparing PCL / Gel composite electrospun fiber membranes was demonstrated. After optimizing the spinning parameters, high-porosity nanofiber membranes were successfully obtained. SEM observation showed that the fiber diameter was 200–800 nm, the fibers were continuous and unbroken, and they formed a three-dimensional interwoven network structure. The porosity was ≥85%, and the connectivity was excellent, which is conducive to subsequent shearing and dispersion into short fibers. The fiber membrane had good flexibility and mechanical strength suitable for the construction of three-dimensional scaffolds.

[0028] Step 4: Fabrication of the three-dimensional fiber optic scaffold Fiber dispersion: The PCL / Gel spinning membrane is cut into small pieces, suspended in tert-butanol, and sodium chloride pore-forming agent (particle size 200-300μm) is added. The mixture is then sheared at high speed (10000rpm, 5min) to form a uniform fiber suspension.

[0029] Freeze-drying: The fiber suspension is transferred to a cylindrical mold, pre-frozen at -80℃ for 2 hours, and then freeze-dried under vacuum for 48 hours.

[0030] Thermal crosslinking: The dried scaffold was placed in a vacuum oven at 120°C for 2 hours for thermal crosslinking. It was then soaked in deionized water to remove the sodium chloride porogen and freeze-dried again to obtain a three-dimensional fiber scaffold with a through-pore structure.

[0031] like Figure 4 As shown, the cryo-thermal crosslinking process for preparing a three-dimensional fiber scaffold is presented, successfully constructing a PCL / Gel three-dimensional scaffold with a through-pore structure. SEM shows that the scaffold has a pore size of 200–500 μm, the pore walls are composed of short fibers, and the pore connectivity is ≥90%. The compression test shows a compressive strength of 0.8–1.2 MPa, matching the osteochondral mechanical gradient. The scaffold is lightweight, has good elasticity, and allows for deep cell infiltration.

[0032] Step 5: Gradient mineralization treatment Preparation of 5-fold Sr-doped simulated body fluid (5Sr-SBF): Add NaCl 40.0g, NaHCO3 1.75g, KCl 1.125g, K2HPO4·3H2O 1.14g, SrCl2·6H2O 0.75g, Na2SO4 0.45g, and Tris 15.125g sequentially. Adjust the pH to 7.4 with 1M HCl. Finally, add CaCl2 1.8375g and bring the volume to 1L.

[0033] Gradient mineralization: The three-dimensional fiber scaffold was immersed in 5Sr-SBF, the middle layer was mineralized for 24 hours, the subchondral bone layer was mineralized for 48 hours, rinsed with deionized water 3 times, and then freeze-dried for later use.

[0034] like Figure 5 As shown, a gradient mineralization process was successfully used to prepare a three-dimensional scaffold modified with SrHA gradients through differentiated mineralization times. SEM showed that sparse nanoscale SrHA particles were deposited on the surface of the intermediate layer (24h), while the subchondral bone layer (48h) was covered with a dense and continuous SrHA coating. EDS confirmed that Sr and Ca elements were distributed in a gradient along the thickness of the scaffold, with higher Sr content in the subchondral bone layer. After mineralization, the osteogenic induction activity of the scaffold was enhanced, and the mechanical strength increased with mineralization time, matching the calcification gradient characteristics of natural osteochondrocytes.

[0035] Step 6: Preparation of cartilage layer hydrogel and integration of the three-layer scaffold Preparation of PVA / CS hydrogel precursor: PVA and chitosan were mixed at a mass ratio of 9:1, dissolved in deionized water, and stirred at 90℃ for 3h to obtain a 10% (w / v) mixed solution; PCL / Gel short fibers (mass fraction 5%) were added and stirred evenly.

[0036] Three-layer integration: The hydrogel precursor solution was injected into the mold and pre-crosslinked at 4°C for 30 min; the 1 mm thick intermediate scaffold and the 2 mm thick subchondral bone scaffold were wetted with methacrylated gelatin (GelMA), stacked and adhered to the hydrogel surface, and cured under ultraviolet light for 10 min; the three-layer gradient scaffold with a diameter of 5 mm and a height of 4 mm was obtained by cutting and trimming.

[0037] like Figure 6As shown, a precise integration process of the three-layer scaffold was successfully used to construct a biomimetic gradient osteochondral scaffold with continuous structure and stable interface. The cartilage layer is made of PCL / Gel fiber-reinforced PVA / CS hydrogel, which has lubricity and elasticity; the middle layer (1mm) and the subchondral bone layer (2mm) are gradient mineralized fiber scaffolds with increasing mechanical and osteogenic activity; GelMA UV curing ensures that the three layers are not delaminated and are firmly bonded; the overall size of the scaffold is regular and meets the implantation requirements.

[0038] Step 7: LMP Modification of Gradient Scaffold Scaffold activation: The three-layer gradient scaffold was immersed in 2 mg / mL Sulfo-SMCC solution and incubated at room temperature for 1 h, followed by washing with PBS 3 times.

[0039] LMP grafting: The activated scaffold was immersed in 0.1 mg / mL LMP solution, incubated at 37°C for 2 h, washed 3 times with PBS, and freeze-dried to obtain the final probiotic vesicle functionalized gradient electrospun fiber scaffold.

[0040] like Figure 7 As shown, the Sulfo-SMCC-mediated LMP covalent modification process successfully yielded a probiotic vesicle-functionalized gradient scaffold. Sulfo-SMCC crosslinking ensures stable covalent bonding of LMP to the scaffold surface, preventing detachment. SEM images show that LMP is uniformly distributed on the scaffold surface and within the pores, without aggregation. The modified scaffold maintains its original gradient structure and mechanical properties while also possessing immunomodulatory and long-lasting anti-inflammatory activities, achieving an integrated design of structural biomimicry, immunomodulation, and differentiation promotion, meeting the requirements of full-thickness osteochondral repair technology.

[0041] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A probiotic-functionalized gradient electrospun fiber scaffold for osteochondral repair, characterized in that, The scaffold has a three-layer gradient structure, consisting of a cartilage layer, an intermediate calcified layer, and a subchondral bone layer from top to bottom. The cartilage layer is a PCL / Gel fiber-reinforced PVA / CS hydrogel. The intermediate calcified layer and the subchondral bone layer are PCL / Gel three-dimensional fiber scaffolds with strontium-doped hydroxyapatite deposited on their surfaces. The PCL / Gel three-dimensional fiber scaffold has a through-pore structure, and the SrHA deposition amount in the subchondral bone layer is greater than that in the intermediate calcified layer. The scaffold is modified with a crosslinking agent to contain PLGA nanospheres carrying Lactobacillus reuteri membrane vesicles.

2. The probiotic-functionalized gradient electrospun fiber scaffold for osteochondral repair according to claim 1, characterized in that, The crosslinking agent is Sulfo-SMCC.

3. A method for preparing a probiotic-functionalized gradient electrospun fiber scaffold for osteochondral repair as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Extraction of Lactobacillus reuteri membrane vesicles; (2) The Lactobacillus reuteri membrane vesicles were encapsulated in PLGA nanospheres to obtain PLGA nanospheres loaded with Lactobacillus reuteri membrane vesicles; (3) Preparation of PCL / Gel electrospun fiber membrane; (4) Disperse the PCL / Gel electrospun fiber membrane and form a three-dimensional fiber scaffold; (5) The three-dimensional fiber scaffold was subjected to mineralization treatment in strontium-doped simulated body fluid for different times to obtain an intermediate calcified layer scaffold and a subchondral bone layer scaffold with gradient SrHA deposition. (6) PCL / Gel short fibers are mixed with PVA / CS hydrogel precursor to prepare a cartilage layer, and the cartilage layer, intermediate calcified layer scaffold and subchondral bone layer scaffold are integrated into a three-layer gradient scaffold. (7) The PLGA nanospheres carrying Lactobacillus reuteri membrane vesicles are modified onto the three-layer gradient scaffold by a crosslinking agent.

4. The method for preparing the probiotic membrane vesicle functionalized gradient electrospun fiber scaffold for osteochondral repair according to claim 3, characterized in that, The preparation of PLGA nanospheres loaded with Lactobacillus reuteri membrane vesicles in step (2) includes: dissolving PLGA in dichloromethane at a concentration of 50 mg / mL as the oil phase; dissolving Lactobacillus reuteri membrane vesicles in a PBS solution containing 1% BSA and 5% sucrose at a protein concentration of 0.5 mg / mL as the inner aqueous phase; mixing the inner aqueous phase and the oil phase at a volume ratio of 1:5, and ultrasonically emulsifying under ice bath conditions at a power of 200 W for 3 min to form a first emulsion; adding the first emulsion dropwise into a 1% PVA aqueous solution and magnetically stirring for 30 min to form a second emulsion; collecting the nanospheres, washing them, and then freeze-drying them.

5. The method for preparing the probiotic membrane vesicle functionalized gradient electrospun fiber scaffold for osteochondral repair according to claim 3, characterized in that, The strontium-doped simulated body fluid mentioned in step (5) is a strontium-doped simulated body fluid with a concentration of 5 times. Its formula is: NaCl 40.0g, NaHCO3 1.75g, KCl 1.125g, K2HPO4・3H2O 1.14g, SrCl2・6H2O 0.75g, Na2SO4 0.45g, Tris 15.125g, CaCl2 1.8375g, adjusted to 1L, pH 7.

4.

6. The method for preparing a probiotic-functionalized gradient electrospun fiber scaffold for osteochondral repair according to claim 3, characterized in that, The mineralization process described in step (5) is as follows: the three-dimensional fiber scaffold is immersed in strontium-doped simulated body fluid, the intermediate calcified layer scaffold is mineralized for 24 hours, and the subchondral bone layer scaffold is mineralized for 48 hours.

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