A polyamino acid-based in-situ cartilage regeneration porous gel scaffold and a preparation method thereof

Abstract

This invention relates to a polyamino acid-based in-situ cartilage regeneration porous gel scaffold and its preparation method. Phenylboronic acid ester is esterified and linked with polyethylene glycol-modified Kartogenin to obtain phenylboronic acid ester-modified PEGKGN. The PEGKGN modified with phenylboronic acid ester is then grafted onto the side chains of polyamino acids with allyl polyethylene glycol to obtain a graft copolymer PLBPKA. The graft copolymer PLBPKA is blended with small molecules of Kartogenin and a silk fibroin solution to form a drug-loaded self-assembly network under shear force. Finally, the polyamino acid-based in-situ cartilage regeneration porous gel scaffold is constructed through ultraviolet light crosslinking and freeze-phase separation, which is a differentially released drug hydrogel scaffold. Compared with existing technologies, the scaffold of this invention has good biocompatibility and biocontrollable degradation, and can rapidly release drugs in inflammatory environments to promote stem cell migration; while in normal physiological environments, it slowly releases drugs to promote the differentiation of stem cells into cartilage. Therefore, it can promote in-situ regeneration of articular cartilage in the absence of exogenous stem cells.

Description

Technical Field

[0001] This invention belongs to the field of articular cartilage tissue engineering scaffold preparation technology, and in particular relates to a polyamino acid-based in situ cartilage regeneration porous gel scaffold and its preparation method. Background Technology

[0002] The use of endogenous stem cells (ESCs) to regenerate cartilage has become a new trend in the clinical application of cartilage tissue engineering technology. Direct recruitment of ESCs not only avoids the loss of cell viability during the lengthy and complicated in vitro culture process, but also reduces the risk of disease transmission during subsequent transplantation. However, this technology has higher requirements for materials, especially those with the ability to recruit ESCs and the induction activity to initiate cartilage differentiation.

[0003] Because ESCs have a short recruitment window, rapid drug release is necessary; however, the induction phase lasts throughout the entire repair process, requiring sustained drug release. Studies have shown that sequential injection of chemokines and growth factors promotes stem cell migration and differentiation better than simultaneous injection, resulting in a higher degree of tissue regeneration. Therefore, designing and optimizing the on-demand release of KGN is crucial. Constructing an intelligent, responsive, in-situ cartilage tissue engineering scaffold that can function appropriately at different stages to achieve on-demand drug release at the defect site will have significant research and clinical value.

[0004] Studies have shown that cartilage injury sites present a localized inflammatory, slightly acidic environment accompanied by excessive enzyme and reactive oxygen species (ROS) expression, significantly different from normal tissue (Frapin et al., 2020; Hwang et al., 2015; Wenet et al., 2019; Wu et al., 2020; Zhang et al., 2020). Intelligent response systems can trigger the breakage of response units by sensing changes in the microenvironment of the inflammatory lesion site, achieving rapid drug release. However, pH- and enzyme-based ester bond hydrolysis exhibits low selectivity and sensitivity, failing to meet this requirement. ROS in the inflammatory environment show high sensitivity and specificity to the dissociation of phenylboronic acid esters, thus serving as a trigger switch for rapid drug release. However, its application in in-situ cartilage tissue engineering is rarely reported (Frapin et al., 2020; Hwang et al., 2015; Wen et al., 2019; Wu et al., 2020; Zhang et al., 2020). Meanwhile, phenylboronic acid esters have excellent biocompatibility and can carry a wealth of functional groups, which can meet the modification needs in practical applications.

[0005] Non-covalent bonding of drugs to materials is a simple and efficient method of drug delivery. Silk fibroin (SF) is a natural fibrous protein derived from silkworm cocoons and is also an FDA-approved biomedical polymer commonly used in wound closure, tissue regeneration, and drug delivery systems. SF has multiple conformations (random, helical, and folded). When its conformation changes to a β-sheet, the peptide chain Gly-Ala-GlyAla-Gly-Ser combines with other amino acids through hydrogen bonds to form hydrophobic self-assembled microdomains. This is often used to enhance the mechanical properties of SF-based materials and regulate their degradation rate (Hasturk et al., 2020; McNamara et al., 2021; Su et al., 2017; Wang et al., 2021). Simultaneously, small hydrophobic molecules can be incorporated into the hydrophobic network during SF self-assembly through hydrophobic aggregation, achieving long-term sustained release (Zhou et al., 2014).

[0006] Developing in-situ tissue engineering scaffolds that can promote endogenous stem cell migration and cartilage differentiation, thereby enhancing the in-situ regeneration capacity of articular cartilage, is a key technology and a major challenge in the field of tissue engineering. Summary of the Invention

[0007] Given the current lack of high-performance in-situ tissue engineering scaffolds that can promote endogenous stem cell migration and cartilage differentiation in existing technologies, this invention provides a polyamino acid-based in-situ cartilage regeneration porous gel scaffold and its preparation method.

[0008] Specifically, the polyamino acid-based in situ cartilage regeneration porous gel scaffold provided by this invention can promote the migration of endogenous stem cells and cartilage differentiation, ultimately achieving in situ regeneration of articular cartilage.

[0009] The polyamino acid-based in-situ cartilage regeneration porous gel scaffold provided by this invention is a smart polyamino acid hydrogel scaffold that can promote articular cartilage regeneration. This scaffold possesses a differentiated KGN release function to achieve a differentiated window of recruitment of endogenous stem cells (ESCs) and cartilage induction, ultimately promoting articular cartilage regeneration. Currently, there are no reports in the prior art regarding smart polyamino acid hydrogel scaffolds and methods for promoting articular cartilage regeneration.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] This invention first provides a method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold with on-demand drug release function, comprising the following steps:

[0012] Phenylboronic acid ester (PBE) was esterified and linked with polyethylene glycol-modified Kartogenin (PEGKGN) to obtain phenylboronic acid ester-modified PEGKGN (i.e., PB-PEGKGN);

[0013] Boronate-modified PEGKGN and allyl polyethylene glycol (APEG) were co-grafted onto the side chains of polyamino acids to obtain the graft copolymer PLBPKA.

[0014] The graft copolymer PLBPKA was blended with small molecules of Kartogenin (KGN) and silk fibroin (SF) solution to form a drug-loaded self-assembly network under shear force. Finally, a polyamino acid-based in-situ cartilage regeneration porous gel scaffold was constructed by ultraviolet (UV) crosslinking and freezing phase separation. It is a differential drug-release hydrogel scaffold.

[0015] In one embodiment of the present invention, the polyamino acid is selected from one or a combination of several of poly-L-glutamic acid, poly-γ-glutamic acid, polyleucine, polyaspartic acid, or polylysine. The polyamino acid-based in-situ cartilage regeneration porous gel scaffold of the present invention is a biocompatible and biodegradable intelligent porous gel scaffold, with polyamino acid as the main material.

[0016] In one embodiment of the present invention, the number-average molecular weight of the polyamino acid is 30,000 to 160,000.

[0017] In one embodiment of the present invention, the polyethylene glycol-modified Kartogenin (PEGKGN) is an active drug PEGKGN obtained through hydrophilic modification, and the preparation method of the polyethylene glycol-modified Kartogenin is as follows:

[0018] KGN was dissolved in N,N-dimethylformamide, and after clarification, EDC·HCl was added. The mixture was continuously stirred in an ice bath to activate the carboxyl groups in KGN.

[0019] Aminated polyethylene glycol (H2N-PEG) was then added to the reaction solution and stirred at room temperature for 2–3 days. After the reaction was completed, PEGKGN was obtained by separation.

[0020] In one embodiment of the present invention, the method for separating and obtaining PEGKGN after the reaction is completed is as follows: after the reaction is completed, excess chloroform is added to the solution and mixed evenly, and the solution is washed several times with saturated saline solution. The lower organic phase is collected using a separatory funnel, and sufficient anhydrous MgSO4 is added and dried overnight. Then, the filtered clear organic solution is concentrated by rotary evaporation and poured into excess ice-cold ether for precipitation. The light yellow powder PEGKGN is obtained by suction filtration and vacuum drying.

[0021] In one embodiment of the present invention, the polyamino acid exists in solution form, and the concentration of the polyamino acid solution is 5% to 30%.

[0022] In one embodiment of the present invention, the method for obtaining phenylboronic acid ester modified PEGKGN by esterification linking of phenylboronic acid ester (PBE) with polyethylene glycol modified Kartogenin (PEGKGN) is as follows:

[0023] PEGKGN was dissolved together with excess succinic anhydride in dichloromethane, and then a small amount of pyridine was added to catalyze the reaction. The mixture was stirred continuously at room temperature for 2-3 days. After the reaction was completed, carboxylated modified PEGKGN (HOOC-PEGKGN) was obtained by separation.

[0024] Phenylboronic acid ester (PBE) was dissolved in tetrahydrofuran and the solution was cooled in an ice bath. Then, a DMSO solution of HOOC-PEGKGN, EDC·HCl and DMAP was added dropwise to the stirred PBE tetrahydrofuran solution. After the addition was completed, the system was allowed to react in an ice bath for a period of time, and then the reaction was continued overnight at room temperature. After the reaction was completed, PEGKGN modified with phenylboronic acid ester was obtained by separation.

[0025] In one embodiment of the present invention, the number-average molecular weight of PEG in the phenylboronic acid ester modified PEGKGN is 500 to 2500.

[0026] In one embodiment of the present invention, the method for separating and obtaining carboxylated modified PEGKGN after the reaction is as follows: After the reaction, the mixture is washed multiple times with saturated NaCl solution in a separatory funnel to separate the organic phase, and sufficient anhydrous MgSO4 is added to it for overnight drying. The dried organic phase is filtered, concentrated, and then precipitated with ice-cold diethyl ether. Finally, residual solvent is removed by vacuum drying to obtain carboxylated modified PEGKGN (HOOC-PEGKGN).

[0027] In one embodiment of the present invention, the method for separating and obtaining phenylboronic acid ester modified PEGKGN after the reaction is as follows: after the reaction is completed, the product in the reaction system is extracted into chloroform, concentrated and then poured into excess ice-cold ether for precipitation, and finally dried under vacuum to obtain the product PB-PEGKGN.

[0028] In one embodiment of the present invention, the method for grafting borate-modified PEGKGN and allyl polyethylene glycol (APEG) onto the side chains of polyamino acids to obtain the graft copolymer PLBPKA is as follows:

[0029] Polyamino acid and borate-modified PEGKGN were dissolved together in dimethyl sulfoxide. After the solution became clear, EDC·HCl and DMAP were added. The mixture was stirred continuously at room temperature for a period of time. Then, excess allyl polyethylene glycol (APEG) was added and the reaction was continued for 2 to 3 days. After the reaction was completed, the final product, graft copolymer PLBPKA, was obtained.

[0030] In one embodiment of the present invention, the number average molecular weight of the allyl polyethylene glycol (APEG) is 500 to 3000.

[0031] In one embodiment of the present invention, the method for obtaining the final product graft copolymer PLBPKA after the reaction is as follows: the solution is poured into a dialysis bag, dialyzed with deionized water, and finally, the product solution in the dialysis bag is freeze-dried to obtain the final product graft copolymer PLBPKA.

[0032] In one embodiment of the present invention, the method for forming a drug-loaded self-assembled network by blending the graft copolymer PLBPKA with small molecules of Kartogenin (KGN) and silk fibroin (SF) solution under shear force is as follows:

[0033] PLBPKA and photoinitiator I2959 were dissolved together in a silk fibroin solution to prepare a mixed solution. Then, the active small molecule drug KGN was added to the mixed solution. After thorough shaking, the reaction solution was poured into a cylindrical mold. Then, the reaction solution was subjected to high-speed shearing using an emulsifier to promote the conformational transformation of silk fibroin to β-sheet and self-assembly, forming a system containing a drug-loaded self-assembly network.

[0034] In one embodiment of the present invention, the mass concentration of silk fibroin in the silk fibroin solution is above 3%.

[0035] In one embodiment of the invention, the PLBPKA solid content in the mixed solution is 5-30 wt%. For example, it is selected as 15 wt%, 20 wt%, 25 wt%, and 30 wt%.

[0036] In one embodiment of the present invention, the method for constructing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold by ultraviolet (UV) crosslinking and freezing phase separation is as follows:

[0037] Subsequently, the system containing the drug-loaded self-assembly network was subjected to UV irradiation to crosslink and solidify it. The resulting hydrogel was dialyzed with deionized water to remove the catalyst introduced in the reaction, and then freeze-dried to obtain a polyamino acid-based in-situ cartilage regeneration porous gel scaffold. The polyamino acid-based in-situ cartilage regeneration porous gel scaffold is a tissue engineering scaffold that can promote in-situ regeneration of articular cartilage.

[0038] The present invention further provides a polyamino acid-based in situ cartilage regeneration porous gel scaffold prepared based on the above preparation method.

[0039] In one embodiment of the present invention, the pore size of the polyamino acid-based in situ cartilage regeneration porous gel scaffold is 100-500 μm.

[0040] In one embodiment of the present invention, the compressive modulus of the polyamino acid-based in situ cartilage regeneration porous gel scaffold is 70-200 kPa.

[0041] In one embodiment of the present invention, the polyamino acid-based in situ cartilage regeneration porous gel scaffold can accelerate the release of drugs under the action of matrix metalloproteinases, with a concentration of 4 μM or higher.

[0042] In one embodiment of the present invention, the polyamino acid-based in situ cartilage regeneration porous gel scaffold can slowly release drugs in PBS at a concentration of 100 nM or higher.

[0043] The present invention further provides the application of polyamino acid-based in situ cartilage regeneration porous gel scaffold. The polyamino acid-based in situ cartilage regeneration porous gel scaffold has endogenous stem cell recruitment and cartilage induction functions. After being implanted into the articular cartilage defect site in animals, the matrix structure and composition of the articular cartilage are reconstructed through in situ regeneration in vivo.

[0044] In this invention, polyamino acids mimic the main components of proteins in terms of composition, exhibiting no immunogenicity or toxic side effects in vivo. Silk fibroin (SF) is a natural fibrous protein derived from silkworm cocoons and is also an FDA-approved biomedical polymer commonly used in wound suturing, tissue regeneration, and drug delivery systems. Kartogenin (KGN) is a non-protein, hydrophobic small molecule drug that releases CBF-β from filament A, enters the cell nucleus, binds to RUNX1, and activates the transcription of cartilage matrix proteins. Therefore, KGN can promote chondrogenic differentiation of stem cells and also protect cartilage, thereby stimulating the repair of cartilage defects. Simultaneously, KGN has the potential to recruit stem cells; endogenous bone marrow stem cells, due to their chemotaxis, sense the concentration gradient of KGN and migrate to the defect site. Furthermore, compared to protein-based bioactive factors, KGN is more stable, easier to store, and easier to modify. These advantages give KGN an important place in the field of cartilage regeneration. Therefore, the application of KGN can simultaneously endow scaffold cell recruitment and chondrogenic induction functions.

[0045] This invention utilizes phenylboronic acid esters as an inflammatory-responsive drug release unit, branching the active drug KGN onto the polyamino acid side links; simultaneously, it encapsulates the hydrophobic drug KGN through the hydrophobic aggregation of the β-sheet conformation of silk fibroin (SF), constructing a smart polyamino acid hydrogel scaffold with ROS-responsive / SF sustained-release differential drug release function. The β-sheet SF significantly improves the mechanical properties of the hydrogel scaffold, increasing the compressive modulus from 90 kPa (α-helix) to 150 kPa (β-sheet). The scaffold also exhibits good biodegradability, degrading by 45% in 5 weeks. In a reactive oxygen species (ROS) environment, the scaffold demonstrates sensitive responsive drug release behavior, with a cumulative release of 52% after 3 days, effectively promoting stem cell migration, with the number of migrating cells being 10.3 times that of the blank scaffold; the β-sheet SF has a long-term sustained-release effect on KGN, with a cumulative release of 73% after 30 days, and the sustained-release KGN significantly increases the expression of SOX9, AGG, and COLII genes in stem cells.

[0046] Based on the research and development progress of articular cartilage tissue engineering scaffolds at home and abroad, this invention successfully developed a tissue-engineered scaffold with biocompatibility, non-degradability, and the ability to promote the recruitment of endogenous stem cells and cartilage differentiation. The prepared scaffold has good mechanical properties, with a maximum compressive modulus of 160 kPa. In the early stage of cartilage injury with active inflammatory response, KGN can be released more rapidly through ROS response, promoting the recruitment of ESPCs; after the inflammation subsides, KGN can be released slowly through SF, enhancing the cartilage differentiation of ESPCs.

[0047] Compared with existing technologies, the polyamino acid-based in-situ cartilage regeneration porous gel scaffold prepared in this invention is a polyamino acid / silk fibroin porous hydrogel scaffold that places greater emphasis on the network to achieve differentiation in drug loading and release mechanisms. This scaffold exhibits good biocompatibility and biocontrollable degradation, and in inflammatory environments, it can accelerate drug release to promote stem cell migration; while in normal physiological environments, it slowly releases drugs to promote stem cell cartilage differentiation. Therefore, it can promote in-situ regeneration of articular cartilage in the absence of exogenous stem cells.

[0048] The ROS-responsive / SF-controlled differentially released KGN smart hydrogel scaffold prepared in this invention can achieve cartilage regeneration in rat knee joints via microfracture technology. The newly formed cartilage is clearly layered with the underlying bone, exhibits a distinct lacunar structure, and is rich in COL II and GAG in the matrix. Attached Figure Description

[0049] Figure 1 (A) Photographs of the SF self-assembly process before and after shearing of the raw material system and the gel after cross-linking; (B) Photographs of the appearance of the scaffold constructed in this invention; (C) The scaffold has good swelling properties and shape recovery ability.

[0050] Figure 2 (A) Macroscopic appearance of the scaffold; (B) Scanning electron micrographs of scaffolds with different proportions; (C) Aperture dimensions of scaffolds with different proportions; (D) Aperture wall thickness of scaffolds with different proportions. Group α consists of scaffolds prepared using unsheared α-helical SF conformation; Group β consists of scaffolds prepared using sheared β-folded SF conformation.

[0051] Figure 3 (AD) Compressive stress-strain curves of stents with different proportions; (E) Compressive strength of stents with different proportions. Group α consists of stents prepared using unsheared α-helical SF; Group β consists of stents prepared using sheared β-folded SF.

[0052] Figure 4 (A) Responsive release behavior of drug-loaded scaffolds in simulated inflammatory environments; (B) Sustained-release behavior of drug-loaded scaffolds in simulated normal physiological environments; (CG) Transwell assays of cell migration under different environments; (HK) In vitro RT-PCR assays for chondrogenesis. Group α consisted of scaffolds prepared using unshorn α-helix SF conformation; Group β consisted of scaffolds prepared using shorn β-sheet SF conformation.

[0053] Figure 5 (AF) shows gross photographs of in situ cartilage regeneration in rats after 1 month and 3 months of treatment in the repair group, control group, and blank group, respectively; (GL) shows histological staining of in situ cartilage regeneration in rats after 1 month and 3 months of treatment in the repair group, control group, and blank group, respectively; (MR) shows safranin-fast green staining of in situ cartilage regeneration in rats after 1 month and 3 months of treatment in the repair group, control group, and blank group, respectively. Detailed Implementation

[0054] This invention provides a polyamino acid-based porous gel scaffold for in-situ cartilage regeneration that promotes endogenous stem cell recruitment and chondrogenic differentiation, thereby enhancing in-situ regeneration of articular cartilage. The design primarily relies on a ROS-responsive / SF sustained-release differential drug release system to differentiate the window of endogenous stem cell recruitment and chondrogenic induction.

[0055] This invention provides a method for preparing a polyamino acid-based in situ cartilage regeneration porous gel scaffold that can promote endogenous stem cell recruitment and chondrogenic differentiation, comprising the following steps:

[0056] Synthesis of the hydrophilic drug PEGKGN: KGN was dissolved in N,N-dimethylformamide, and after clarification, EDC·HCl was added. The mixture was continuously stirred in an ice bath to activate the carboxyl groups in KGN. Aminated polyethylene glycol (H2N-PEG) was then added to the reaction solution, and the reaction was stirred at room temperature for 2–3 days. After the reaction was complete, excess chloroform was added to the solution and mixed thoroughly, followed by washing several times with saturated brine. The lower organic phase was collected using a separatory funnel and dried overnight with sufficient anhydrous MgSO4. The clarified organic solution was then concentrated by rotary evaporation and settled in ice-cold diethyl ether. PEGKGN was obtained as a pale yellow powder by filtration and vacuum drying.

[0057] Synthesis of carboxylated PEGKGN: PEGKGN was dissolved in dichloromethane with excess succinic anhydride, followed by the addition of a small amount of pyridine to catalyze the reaction. The mixture was stirred continuously at room temperature for 2–3 days. After the reaction was complete, the solution was washed multiple times with saturated NaCl solution in a separatory funnel to separate the organic phase, which was then dried overnight with sufficient anhydrous MgSO4. The dried organic phase was filtered, concentrated, and then precipitated with ice-cold diethyl ether. Finally, residual solvent was removed by vacuum drying to obtain carboxylated modified PEGKGN (HOOC-PEGKGN).

[0058] Synthesis of PEGKGN modified with phenylboronic acid ester: PBE was dissolved in tetrahydrofuran and the solution was cooled in an ice bath. Then, a solution of HOOC-PEGKGN, EDC·HCl, and DMAP in DMSO was added dropwise to the stirred PBE tetrahydrofuran solution. After the addition was complete, the system was allowed to react in an ice bath for a period of time, and then the reaction continued overnight at room temperature. After the reaction was complete, the product was extracted into chloroform, concentrated, and then settled in excess ice-cold diethyl ether. Finally, the product was obtained by vacuum drying.

[0059] Synthesis of the graft copolymer PLBPKA: Polyamino acid and PB-PEGKGN were dissolved together in dimethyl sulfoxide. After the solution became clear, EDC·HCl and DMAP were added. The mixture was stirred continuously at room temperature for 5 hours. Subsequently, excess allyl polyethylene glycol (APEG) was added, and the reaction continued for 2–3 days. After the reaction was completed, the solution was poured into a dialysis bag and dialyzed with deionized water. Finally, the product solution in the dialysis bag was lyophilized to obtain the final product PLBPKA.

[0060] Preparation of in-situ tissue engineering scaffolds: PLBPKA and a small amount of photoinitiator I2959 were dissolved in SF solution to prepare mixed solutions with PLBPKA solid contents of 15wt%, 20wt%, 25wt%, and 30wt%. Subsequently, the active small molecule drug KGN was added to the mixed solution, and after thorough shaking, the reaction solution was poured into a cylindrical mold. Then, a high-speed shearing process was performed on the reaction solution using an emulsifier to promote the conformational transformation of SF to β-sheet, leading to self-assembly. The system was then subjected to UV irradiation to crosslink and solidify. The resulting hydrogel was dialyzed with deionized water to remove the catalyst introduced during the reaction, and then freeze-dried to obtain a tissue engineering scaffold capable of promoting in-situ regeneration of articular cartilage.

[0061] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0062] Example 1:

[0063] This embodiment provides the synthesis of raw materials for constructing tissue-engineered scaffolds that promote in-situ regeneration of articular cartilage, including the following steps:

[0064] Synthesis of the hydrophilic drug PEGKGN: KGN was dissolved in N,N-dimethylformamide, and after clarification, EDC·HCl was added. The mixture was continuously stirred in an ice bath to activate the carboxyl groups in KGN. Aminated polyethylene glycol (H2N-PEG) was then added to the reaction solution, and the reaction was stirred at room temperature for 2 days. After the reaction was complete, excess chloroform was added to the solution and mixed thoroughly, followed by washing several times with saturated brine. The lower organic phase was collected using a separatory funnel and dried overnight with sufficient anhydrous MgSO4. The filtered clear organic solution was then concentrated by rotary evaporation and settled in ice-cold diethyl ether. PEGKGN was obtained as a pale yellow powder by filtration and vacuum drying.

[0065] Synthesis of carboxylated PEGKGN: PEGKGN was dissolved in dichloromethane with excess succinic anhydride, followed by the addition of a small amount of pyridine to catalyze the reaction. The mixture was stirred continuously at room temperature for 2 days. After the reaction was complete, the solution was washed multiple times with saturated NaCl solution in a separatory funnel to separate the organic phase, which was then dried overnight with sufficient anhydrous MgSO4. The dried organic phase was filtered, concentrated, and then precipitated with ice-cold diethyl ether. Finally, residual solvent was removed by vacuum drying to obtain carboxylated modified PEGKGN (HOOC-PEGKGN).

[0066] Synthesis of PEGKGN modified with phenylboronic acid ester: PBE was dissolved in tetrahydrofuran and the solution was cooled in an ice bath. Then, a solution of HOOC-PEGKGN, EDC·HCl, and DMAP in DMSO was added dropwise to the stirred PBE tetrahydrofuran solution. After the addition was complete, the system was allowed to react in an ice bath for a period of time, and then the reaction continued overnight at room temperature. After the reaction was complete, the product was extracted into chloroform, concentrated, and then settled in excess ice-cold diethyl ether. Finally, the product was obtained by vacuum drying.

[0067] Synthesis of the graft copolymer PLBPKA: Poly-L-glutamic acid (PLGA) and PB-PEGKGN were co-dissolved in dimethyl sulfoxide. After the solution became clear, EDC·HCl and DMAP were added. The mixture was stirred continuously at room temperature for 5 hours. Subsequently, excess allyl polyethylene glycol (APEG) was added, and the reaction continued for 2 days. After the reaction was completed, the solution was poured into a dialysis bag and dialyzed with deionized water. Finally, the product solution in the dialysis bag was lyophilized to obtain the final product PLBPKA.

[0068] Example 2:

[0069] Scaffold Formation: The PLBPKA obtained in Example 1 was dissolved in SF solution with 2 mg of photoinitiator I2959 to prepare a mixed solution with a PLBPKA solid content of 15 wt%. Then, 3 mg of the small molecule drug KGN was added to the mixed solution, and after thorough shaking, the reaction solution was poured into a cylindrical mold. The reaction solution was then subjected to high-speed shearing (25000 rpm) using an emulsifier to promote the conformational transformation of SF to β-sheet for self-assembly. The system was then subjected to UV irradiation (365 nm) to crosslink and cure (5 min). Figure 1 Image A (from left to right) shows photographs of the reaction solution before high-speed shearing, after high-speed shearing using an emulsifier, and the hydrogel obtained after UV irradiation and crosslinking in Example 2. It can be seen that high-speed shearing of the reaction solution using an emulsifier promotes the conformational transformation of SF to β-sheet, leading to self-assembly. UV irradiation of the system allows for crosslinking and curing.

[0070] The prepared hydrogel was dialyzed with deionized water to remove the catalyst introduced during the reaction, and then freeze-dried to obtain a tissue-engineered scaffold capable of promoting in-situ regeneration of articular cartilage. Its appearance is shown in the photograph. Figure 1 As shown in (B), the pore size is 300-350 μm, the pore wall thickness is 7-10 μm, and the compressive modulus is 70-85 kPa. Pressing the tissue-engineered scaffold obtained in this embodiment reveals that the scaffold exhibits good swelling properties and shape recovery ability. (See Figure B.) Figure 1 (C).

[0071] Depend on Figure 1 It can be seen that the scaffold constructed in this embodiment has a dual physical / chemical network structure, which has good structural stability and mechanical properties.

[0072] Example 3:

[0073] Scaffold Formation: The PLBPKA obtained in Example 1 was dissolved in an SF solution with a small amount of photoinitiator I2959 to prepare a mixed solution with a PLBPKA solid content of 20 wt%. Then, the small molecule drug KGN was added to the mixed solution, and after thorough shaking, the reaction solution was poured into a cylindrical mold. The reaction solution was then subjected to high-speed shearing using an emulsifier to promote the conformational transformation of SF to β-sheet, leading to self-assembly. The system was then subjected to UV irradiation to crosslink and solidify. The resulting hydrogel was dialyzed with deionized water to remove the catalyst introduced during the reaction, and then freeze-dried to obtain a tissue-engineered scaffold capable of promoting in-situ regeneration of articular cartilage. Its pore size was 200-250 μm, pore wall thickness was 11-14 μm, and compressive modulus was 90-110 kPa.

[0074] Similar to Example 2, the scaffold constructed in this example also has a dual physical / chemical network structure, exhibiting good structural stability and mechanical properties.

[0075] Example 4:

[0076] Scaffold Formation: The PLBPKA obtained in Example 1 was dissolved in an SF solution with a small amount of photoinitiator I2959 to prepare a mixed solution with a PLBPKA solid content of 25 wt%. Then, the small molecule drug KGN was added to the mixed solution, and after thorough shaking, the reaction solution was poured into a cylindrical mold. The reaction solution was then subjected to high-speed shearing using an emulsifier to promote the conformational transformation of SF to β-sheet, leading to self-assembly. The system was then subjected to UV irradiation to crosslink and solidify. The resulting hydrogel was dialyzed with deionized water to remove the catalyst introduced during the reaction, and then freeze-dried to obtain a tissue-engineered scaffold capable of promoting in-situ regeneration of articular cartilage. Its pore size was 150-200 μm, pore wall thickness was 20-25 μm, and compressive modulus was 120-150 kPa.

[0077] Similar to Example 2, the scaffold constructed in this example also has a dual physical / chemical network structure, exhibiting good structural stability and mechanical properties.

[0078] Example 5:

[0079] Scaffold Formation: The PLBPKA obtained in Example 1 was dissolved in an SF solution with a small amount of photoinitiator I2959 to prepare a mixed solution with a PLBPKA solid content of 30 wt%. Then, the small molecule drug KGN was added to the mixed solution, and after thorough shaking, the reaction solution was poured into a cylindrical mold. The reaction solution was then subjected to high-speed shearing using an emulsifier to promote the conformational transformation of SF to β-sheet, leading to self-assembly. The system was then subjected to UV irradiation to crosslink and solidify. The resulting hydrogel was dialyzed with deionized water to remove the catalyst introduced during the reaction, and then freeze-dried to obtain a tissue-engineered scaffold capable of promoting in-situ regeneration of articular cartilage. Its pore size was 200-220 μm, pore wall thickness was 9-12 μm, and compressive modulus was 100-120 kPa.

[0080] Similar to Example 2, the scaffold constructed in this example also has a dual physical / chemical network structure, exhibiting good structural stability and mechanical properties.

[0081] Examples 2, 3, 4, and 5 above correspond to stents prepared with PLBPKA solid content of 15, 20, 25, and 30 wt%, respectively, and are respectively prepared with P 15 SF, P 20 SF, P 25 SF, P 30 SF represents the scaffolds. The α group consists of scaffolds prepared using unsheared α-helical SF conformation; the β group consists of scaffolds prepared using β-folded SF after shearing. Figure 2 (B) shows scanning electron microscope images of scaffolds with different proportions of PLBPKA solid content corresponding to Examples 2, 3, 4 and 5; Figure 2 (C) shows the aperture sizes of the stents with different proportions of PLBPKA solid content corresponding to Examples 2, 3, 4, and 5; Figure 2 (D) illustrates the pore wall thickness of the stents with different PLBPKA solid content ratios corresponding to Examples 2, 3, 4, and 5. Figure 2 It can be seen that the scaffold constructed by the present invention has a controllable and interconnected microporous structure.

[0082] Figure 3 The images in Figures A, D, and E respectively show stents with different proportions of PLBPKA solid content (P) corresponding to Examples 2, 3, 4, and 5. 15 SF, P 20 SF, P 25 SF, P 30 The compressive stress-strain curves of SF. The α group consists of scaffolds prepared using unsheared α-helical SF; the β group consists of scaffolds prepared using sheared β-folded SF.

[0083] Figure 3(E) Shows stents with different proportions of PLBPKA solid content corresponding to Examples 2, 3, 4, and 5 (P 15 SF, P 20 SF, P 25 SF, P 30 The compressive strength of SF. Group α consists of scaffolds prepared using unsheared α-helical SF; group β consists of scaffolds prepared using sheared β-folded SF. Figure 3 It can be seen that the scaffold constructed by the present invention has good mechanical properties.

[0084] Figure 4 (A) is the stent obtained in Example 4 (P) 25 SF) responsive release behavior in simulated inflammatory environments; Figure 4 (B) The stent obtained in Example 4 (P) 25 SF's sustained-release behavior under simulated normal physiological conditions; Figure 4 (CG) is the stent obtained in Example 4 (P) 25 Transwell experiments on cell migration under different environments (SF); Figure 4 ((HK) is the stent obtained in Example 4 (P) 25 SF) In vitro chondrogenic RT-PCR experiment. Group α used scaffolds prepared with uncut α-helix SF; Group β used scaffolds prepared with cut β-sheet SF.

[0085] Depend on Figure 4 It is known that the scaffold constructed in this invention can release drugs responsively in a verification environment and effectively promote the migration ability of stem cells, while the sustained release of drugs in a normal physiological environment can enhance the differentiation degree of stem cell cartilage.

[0086] The drug-loaded stent (P) obtained in Example 4 25 SF β As the repair group, using unmedicated stents (Control-P) 25 SF β The control group consisted of mice with simple defects (without implanted material) and the blank group, which were used for in situ cartilage regeneration experiments in rats. The specific experimental procedure was as follows: A 2mm diameter, 5mm deep osteochondral defect was drilled in the femoral plateau of the right leg of the rat. Repair, control, and blank groups were established according to a pre-set plan. After suturing the wound, the mice were fed normally. One and three months post-surgery, the mice were euthanized, and tissue samples were collected for gross observation, histology, immunohistochemistry, and safranin-fast green staining. The experimental results were then evaluated.

[0087] Figure 5 (AF) refers to the support obtained in Example 4 (P) respectively. 25 SFβ Gross photographs of in situ cartilage regeneration in rats at 1 month and 3 months in the control group and blank group; Figure 5 (GL) represents the stents obtained in Example 4 (P) respectively. 25 SF β Histological staining of in situ cartilage regeneration in rats at 1 month and 3 months in the control group and blank group; Figure 5 (MR) represents the stents obtained in Example 4 (P) respectively. 25 SF β Safranin-Fix-Green staining of in situ cartilage regeneration in rats at 1 month and 3 months in the control group and blank group.

[0088] Depend on Figure 5 It is known that the scaffold constructed in this invention can successfully regenerate the unique layered structure and cartilage matrix of hyaline cartilage in rats through in situ tissue engineering.

[0089] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold, characterized in that, Includes the following steps: Phenylboronic acid ester was esterified and linked with polyethylene glycol-modified Kartogenin to obtain phenylboronic acid ester modified PEGKGN; PEGKGN modified with phenylboronic acid ester was co-grafted with allyl polyethylene glycol onto the side chains of polyamino acids to obtain the graft copolymer PLBPKA. The graft copolymer PLBPKA was blended with small molecules of Kartogenin and silk fibroin solution to form a drug-loaded self-assembly network under shear force. Finally, a polyamino acid-based in situ cartilage regeneration porous gel scaffold was constructed by UV crosslinking and freezing phase separation.

2. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The polyamino acid is selected from one or a combination of several of poly-L-glutamic acid, poly-γ-glutamic acid, polyleucine, polyaspartic acid, or polylysine. The number average molecular weight of the polyamino acid is 30,000 to 160,000.

3. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The polyethylene glycol-modified Kartogenin is an active drug PEGKGN obtained through hydrophilic modification. The preparation method of the polyethylene glycol-modified Kartogenin is as follows: KGN was dissolved in N,N-dimethylformamide, and after clarification, EDC·HCl was added. The mixture was continuously stirred in an ice bath to activate the carboxyl groups in KGN. Aminated polyethylene glycol was then added to the reaction solution, and the mixture was stirred at room temperature for 2-3 days. After the reaction was completed, PEGKGN was obtained by separation.

4. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The polyamino acid exists in solution form, and the concentration of the polyamino acid solution is 5% to 30%.

5. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The method for obtaining phenylboronic acid ester-modified PEGKGN by esterification linking of phenylboronic acid ester with polyethylene glycol-modified Kartogenin is as follows: PEGKGN and excess succinic anhydride were dissolved together in dichloromethane, followed by the addition of pyridine to catalyze the reaction. The mixture was stirred continuously at room temperature for 2-3 days. After the reaction was completed, the carboxylated modified PEGKGN was obtained by separation. Phenylboronic acid ester was dissolved in tetrahydrofuran and the solution was cooled in an ice bath. Then, a DMSO solution of HOOC-PEGKGN, EDC·HCl and DMAP was added dropwise to the stirred PBE tetrahydrofuran solution. After the addition was completed, the system was allowed to react for a period of time in an ice bath, and then the reaction was continued overnight at room temperature. After the reaction was completed, PEGKGN modified with phenylboronic acid ester was obtained by separation.

6. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The number-average molecular weight of PEG in the phenylboronic acid ester modified PEGKGN is 500~2500.

7. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The method for obtaining the graft copolymer PLBPKA by co-grafting phenylboronic acid modified PEGKGN and allyl polyethylene glycol onto the side chains of polyamino acids is as follows: Polyamino acid and phenylboronic acid ester modified PEGKGN were dissolved together in dimethyl sulfoxide. After the solution was clear, EDC·HCl and DMAP were added. The mixed solution was stirred and reacted at room temperature for a period of time. Then, excess allyl polyethylene glycol was added and the reaction was continued for 2-3 days. After the reaction was completed, the final product graft copolymer PLBPKA was obtained.

8. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 1, characterized in that, The method for forming a drug-loaded self-assembled network by blending the graft copolymer PLBPKA with small molecules of Kartogenin and silk fibroin solution under shear force is as follows: PLBPKA and photoinitiator I2959 were dissolved together in a silk fibroin solution to prepare a mixed solution. Then, the active small molecule drug KGN was added to the mixed solution. After thorough shaking, the reaction solution was poured into a mold. Then, the reaction solution was subjected to high-speed shearing using an emulsifier to promote the conformational transformation of silk fibroin to β-sheet and self-assembly, forming a system containing a drug-loaded self-assembly network.

9. The method for preparing a polyamino acid-based in-situ cartilage regeneration porous gel scaffold according to claim 8, characterized in that, The silk fibroin solution contains a silk fibroin concentration of 3% or higher. The PLBPKA solid content in the mixed solution is 5-30 wt%.

10. A polyamino acid-based in-situ cartilage regeneration porous gel scaffold prepared by the preparation method according to any one of claims 1-9, characterized in that, The pore size of the polyamino acid in situ cartilage regeneration porous gel scaffold is 100~500μm, and the compressive modulus of the polyamino acid in situ cartilage regeneration porous gel scaffold is 70~200 kPa.

Images