Enzyme-responsive self-assembly of polypeptide-modified methacrylated polymers, methods of making and uses thereof

Abstract

The application discloses an enzyme response self-assembly polypeptide modified methacrylating polymer and a preparation method and application thereof, and the enzyme response self-assembly polypeptide modified methacrylating polymer has a structure of formula 1. The application converts an endogenous tissue-specific marker alkaline phosphatase concentration gradient into a dynamic gradient evolution of the mechanical properties of a scaffold. By introducing an enzyme response self-assembly polypeptide into a methacrylating polymer structure, the scaffold is subjected to molecular level dynamic self-assembly under the catalysis of phosphatase, and a continuous mechanical gradient is formed from a cartilage region to a subchondral bone region. The process does not require external intervention and is completely regulated by a local microenvironment, thereby real-time adapting to the dynamic requirements of 'early proliferation-late differentiation' of bone cartilage repair. Compared with the prior art, the application not only breaks through the mismatch between a static gradient and a dynamic microenvironment, but also precisely regulates mechanical signals at a molecular scale, and significantly improves the spatial specificity and time synchronism of stem cell differentiation.

Description

Technical Field

[0001] This application relates to the field of bone repair technology, and in particular to an enzyme-responsive self-assembling polypeptide-modified methacrylamide polymer, its preparation method, and its application. Background Technology

[0002] Osteochondrial tissue consists of two highly heterogeneous regions, the cartilage layer and the bone layer, which are structurally and functionally distinct. This significant difference makes the repair of osteochondral defects spanning these two tissues extremely challenging. Traditional treatments, such as microfractures and autologous chondrocyte transplantation, often fail to regenerate tissue with its native biphasic structure, leading to fibrocartilage disorder and poor tissue integration.

[0003] In recent years, tissue engineering techniques, by combining mesenchymal stem cells (MSCs) with biomaterial scaffolds, have emerged as a promising repair strategy. For example, collagen- or hyaluronic acid-based hydrogel scaffolds can provide a three-dimensional support microenvironment for MSCs, promoting osteochondral tissue regeneration. However, the unique structure of natural osteochondral units—exhibiting a continuous mechanical gradient—gradually transitioning from a soft cartilage layer to a rigid bone layer, not only provides essential mechanical support for joints but also mediates stem cell fate determination through dynamic mechanical signals, guiding their differentiation towards cartilage or bone, thus achieving functional tissue reconstruction.

[0004] Most traditional tissue engineering scaffolds currently available are limited by their static and singular mechanical properties, making it difficult to reconstruct this natural mechanical gradient structure and meet the dynamic mechanical regulation requirements of the "early cell proliferation to late tissue differentiation" process in osteochondral repair. Although some dynamic scaffolds have been able to achieve changes in mechanical properties in recent years, most of them rely on external physical stimuli (such as magnetic or electric fields) for regulation, making it difficult to accurately respond to the spatiotemporal distribution of endogenous biological signals in the body.

[0005] Therefore, developing a gradient self-assembly scaffold capable of autonomously sensing and precisely regulating mechanical signals to achieve spatial specificity and temporal synchronization of stem cell differentiation has become a key technical challenge that urgently needs to be overcome in the field of osteochondral tissue engineering. Summary of the Invention

[0006] In view of this, this application provides an enzyme-responsive self-assembling peptide-modified methacrylamide polymer, which, through a binary double crosslinked network structure design, enables the dynamic evolution of the mechanical properties of the hydrogel scaffold and the precise regulation of stem cell behavior.

[0007] This application provides an enzyme-responsive self-assembling peptide-modified methacrylamide polymer having the structure of Formula 1:

[0008] Formula 1;

[0009] Where X is a lysine ε-amino side chain residue linked to an amide bond in methacrylamide gelatin, a hydroxypropyl amino side chain residue linked to an amide bond in methacrylamide silk fibroin, or a hydroxyl residue linked to an amide bond on the polysaccharide backbone in methacrylamide hyaluronic acid, and R is a cysteine ​​thiol side chain residue in an enzyme-responsive self-assembled polypeptide, which forms a CS bond through an addition reaction with a carbon-carbon double bond.

[0010] In some specific implementations, R is Ar–(AA). n –Yp–C;

[0011] Where Ar is a hydrophobic aromatic group, (AA) n It is a polypeptide fragment containing 0–10 amino acid residues;

[0012] Yp represents phosphorylated amino acid residues; C represents cysteine ​​residues.

[0013] The enzyme-responsive self-assembling peptide-modified methacrylamide polymer exhibits phosphatase responsiveness. Its β-sheet nanofibers can dynamically reconstruct their mechanical properties according to the phosphatase concentration gradient, realizing the intelligent transformation of endogenous biochemical signals into biomimetic mechanical cues. This dynamic mechanical enhancement can meet the needs of promoting cell proliferation in the initial cell proliferation stage with lower stiffness, and later forming a mechanical gradient scaffold to induce multi-directional differentiation of stem cells, adapting to the dynamic evolution of cells during the repair process, and promoting efficient full-thickness repair of osteochondrium.

[0014] This application also provides a method for preparing an enzyme-responsive self-assembled peptide-modified methacrylated polymer, comprising:

[0015] Methacrylamide polymers were subjected to a Michael addition reaction with an enzyme-responsive self-assembly peptide of Formula 2 to obtain methacrylamide polymers modified with enzyme-responsive self-assembly peptides.

[0016] The methacrylamide polymer includes any one of methacrylamide gelatin, methacrylamide hyaluronic acid, or methacrylamide silk fibroin.

[0017] The enzyme-responsive self-assembled polypeptide structure of formula 2 is as follows:

[0018] Ar–(AA) n –Yp–C Formula 2;

[0019] Ar is a hydrophobic aromatic group;

[0020] (AA) n It is a polypeptide fragment containing 0–10 amino acid residues;

[0021] Yp represents phosphorylated amino acid residues;

[0022] C represents a cysteine ​​residue.

[0023] Ar is a hydrophobic aromatic group: Nap = naphthoyl, Fmoc = 9-fluorenylmethoxycarbonyl;

[0024] Ar = or ;

[0025] (AA) n This represents a polypeptide fragment containing 0–10 amino acid residues (n is an integer from 0 to 10):

[0026] Short peptide backbones are generally chosen to regulate secondary structure, typically using aromatic residues F = Phe (phenylalanine); Y = Tyr (tyrosine); and charged residues D = Asp (aspartic acid); G = Gly (glycine) to regulate water solubility and β-sheet tendency.

[0027] Yp represents phosphorylated amino acid residues that can be dephosphorylated by alkaline phosphatase.

[0028] C represents cysteine ​​undergoing a Michael addition reaction with the double bond.

[0029] Therefore, the enzyme-responsive self-assembling polypeptide of Formula 2 can be NapFFYpC (Formula 2-1), NapGDFDFYpC (Formula 2-2), FmocYpC (Formula 2-3), or FmocFFYpC (Formula 2-4).

[0030] .

[0031] In some specific implementations, the molar ratio of the methacrylamide polymer to the compound of formula 2 is 1:(2~70); more preferably 1:(10~45); and most preferably 1:30; the solvent includes N,N-dimethylformamide; the temperature of the Michael addition reaction is 45 °C to 55 °C; and the time of the Michael addition reaction is 10 h to 48 h, preferably 24 h. The Michael addition reaction is carried out under conditions of pH 7-9, preferably pH 8. After the reaction, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis for 3 days. After dialysis, the product solution in the dialysis bag is taken out and freeze-dried to obtain the enzyme-responsive self-assembled peptide-modified methacrylamide polymer. The Michael addition reaction is a conjugated addition reaction between the thiol group of the compound of formula 2 under alkaline conditions, forming a nucleophilic thioanion, and the electrophilic carbon-carbon double bond of the methacrylamide polymer to form a CS bond.

[0032] In some specific implementations, the method for preparing the methacrylamide gelatin includes:

[0033] Gelatin was mixed with methacrylic anhydride and reacted to obtain methacrylated gelatin;

[0034] The method for preparing the methacrylamide hyaluronic acid includes:

[0035] Hyaluronic acid is mixed with methacrylic anhydride and reacted to obtain methacrylated hyaluronic acid;

[0036] The method for preparing the methacrylamide silk fibroin includes:

[0037] Silk fibroin was mixed with glycidyl methacrylate and reacted to obtain methacrylated silk fibroin;

[0038] The reaction was carried out at a pH of 8 to 9.

[0039] In some specific implementations, the enzyme-responsive self-assembling polypeptide of Formula 2 is NapFFYpC, and its preparation method is as follows:

[0040] First, weigh 2-Chlorotrityl Chloride resin (degree of substitution 1.1 mmol / g) and add it to the reaction tube, adding 15 mL of [unspecified ingredient] per gram of resin. N,N The resin was swollen with dimethylformamide (DMF) and shaken at room temperature for 60 minutes to fully activate it. After swelling, the solvent was removed, and 3 equivalents of Fmoc-protected C-terminal amino acids, 10 equivalents of diisopropylethylamine (DIEA), and 10 mL / g DMF were added to the reaction system. The mixture was shaken at room temperature for 30 minutes to achieve grafting of the initial amino acid. After grafting, the residual active groups were capped with 10 mL / g methanol, and the reaction was allowed to proceed for 30 minutes.

[0041] Next, Fmoc deprotection was performed by treating the resin twice with 20% piperidine / DMF solution. The first treatment was with a concentration of 15 mL / g for 5 minutes, and the second treatment was with a concentration of 15 mL / g for 15 minutes to completely remove the Fmoc groups. After deprotection, the exposure of the resin's amino groups was detected by colorimetric analysis using Kaiser's reagent. The reaction conditions were heating at 105–110 °C for 5 minutes; a deep blue color indicated a positive result. Subsequently, the resin was washed twice with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g) to remove residual reagents.

[0042] The stepwise condensation of amino acids was performed under the following conditions: For each condensation, 3 equivalents of Fmoc-protected amino acid, 3 equivalents of HBTU, and 10 equivalents of DIEA were added, dissolved in 10 mL / g DMF, and the reaction was carried out with shaking at room temperature for 45 minutes. After condensation, the amino group was confirmed by Kaiser's reagent; a colorless result indicated a negative result, signifying complete condensation. The above deprotection and condensation steps were performed alternately, stepwise extending the peptide chain along the target sequence until all amino acids were linked. After removing Fmoc protection from the peptide chain, 3 equivalents of 2-naphthaleneacetic acid were introduced as a C-terminal blocking group. Condensation was again promoted with 3 equivalents of HBTU and 10 equivalents of DIEA under the same reaction conditions.

[0043] After synthesis, the resin was thoroughly washed twice with DMF (10 mL / g), three times with DCM (10 mL / g), and four times with methanol (10 mL / g). Finally, the resin was dried for 10 minutes to remove residual solvent. The treated resin was then treated with a cutting solution prepared by mixing TFA, triisobutylsilane (TIS), 1,2-ethylidene dithiol (EDT), and water in a volume ratio of 95:1:2:2, using 10 mL / g of resin. The reaction was carried out at room temperature for 180 minutes to release the target peptide.

[0044] After the pyrolysis reaction, the organic phase was dried to near dryness using nitrogen. Anhydrous diethyl ether was added to induce peptide precipitation. The precipitate was centrifuged at 8000 rpm for 3 minutes, and the supernatant was discarded. The precipitate was then washed six times with anhydrous diethyl ether to remove organic residues and dried at room temperature to obtain the crude peptide product. The crude product was dissolved in a 0.1% trifluoroacetic acid (TFA) water / acetonitrile system and purified using C18 reversed-phase preparative high-performance liquid chromatography (HPLC). The mobile phase consisted of water containing 0.1% TFA (phase A) and acetonitrile containing 0.1% TFA (phase B). The detection wavelength was 220 nm, the column temperature was 25℃, the flow rate was 15 mL / min, and the injection volume was 20 mL. The target peak was collected, and samples were taken for mass spectrometry analysis to confirm its molecular weight. The purity was determined by HPLC. The purified target peptide solution was freeze-dried to obtain the compound with the structure of formula 2-1.

[0045] In some specific implementations, the methacrylated polymer is methacrylated gelatin, and its preparation method includes:

[0046] Gelatin is mixed with methacrylic anhydride and reacted to obtain methacrylamide gelatin (GelMA).

[0047] The reaction is carried out at a pH of 8 to 9, at a temperature of 45°C to 55°C, preferably 50°C, and for a time of 2 to 4 hours, preferably 3 hours. In some specific implementations, the molar ratio of gelatin (100-200 kDa, based on a lysine content of 0.3–0.6 mmol / g in the gelatin) to methacrylic anhydride is preferably 1:(3~6), more preferably 1:5. In some specific implementations, the pH is adjusted by adding sodium hydroxide solution. After the reaction, hydrochloric acid is added to adjust the pH to 7 to terminate the reaction; the reaction product is transferred to a centrifuge tube and centrifuged at 722.75 × g for 5-10 minutes to separate unreacted methacrylic anhydride from the product and remove residual methacrylic anhydride. After centrifugation, the supernatant is collected and placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000, and dialyzed in sufficient deionized water. The deionized water is changed regularly during dialysis to ensure the dialysis effect. After dialysis, the solution in the dialysis bag is removed and freeze-dried to obtain GelMA. In some specific implementations, the dialysis time is 1 to 5 days, preferably 3 days.

[0048] This application also provides a phosphatase-responsive hydrogel, which is obtained by cross-linking a methacrylamide polymer modified with the enzyme-responsive self-assembled peptide described above or a methacrylamide polymer modified with an enzyme-responsive self-assembled peptide prepared by the above preparation method.

[0049] This application also provides a method for preparing a phosphatase-responsive hydrogel, comprising:

[0050] A phosphatase-responsive hydrogel was obtained by cross-linking a methacrylated polymer modified with an enzyme-responsive self-assembled peptide with phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0051] The enzyme-responsive self-assembly peptide-modified methacrylamide polymer is the enzyme-responsive self-assembly peptide-modified methacrylamide polymer described above or the enzyme-responsive self-assembly peptide-modified methacrylamide polymer prepared by the above preparation method.

[0052] In some specific implementations, the cross-linking reaction is carried out under blue light irradiation; the cross-linking reaction is carried out in a phosphate buffer solution. In some specific implementations, the wavelength of the blue light irradiation is 400 nm to 500 nm, preferably 405 nm. The mass ratio of the enzyme-responsive self-assembled polypeptide-modified methacryloyl polymer to phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is (5-10):1.

[0053] This application also provides a self-assembled gradient hydrogel scaffold, which is obtained by mixing and co-incubating the phosphatase-responsive hydrogel prepared by the above-described method with phosphatase.

[0054] In some specific implementations, the incubation time is 4 hours to 48 hours.

[0055] This application transforms the concentration gradient of alkaline phosphatase, an endogenous tissue-specific marker, into a dynamic gradient evolution of scaffold mechanical properties. By introducing enzyme-responsive peptides (phosphorylated tyrosine sequences), the scaffold undergoes molecular-level dynamic self-assembly under the catalysis of phosphatase, forming a continuous mechanical gradient from the cartilage region to the subchondral bone region. This process requires no external intervention and is entirely regulated by the local microenvironment, thereby adapting in real time to the dynamic needs of "early proliferation-late differentiation" in osteochondral repair. Compared to existing technologies, this application not only overcomes the mismatch between static gradients and dynamic microenvironments but also significantly improves the spatial specificity and temporal synchronization of stem cell differentiation through precise molecular-scale regulation of mechanical signals. Attached Figure Description

[0056] Figure 1 The GelMA, Nap-FFYpC, and G- provided in Embodiment 1 of this application g -NapFFYpC's proton NMR spectrum;

[0057] Figure 2 The GelMA, Nap-FFYpC, and G- provided in Embodiment 1 of this application g -Infrared spectrum of NapFFYpC;

[0058] Figure 3 This is a conformational percentage diagram of the secondary structure calculated from the circular dichroism chromatogram;

[0059] Figure 4 Compression modulus diagrams of the self-assembled gradient hydrogel scaffolds provided in Examples 2-7;

[0060] Figure 5 Cryo-electron microscopy scan of GelMA provided in Example 1;

[0061] Figure 6 This is a cryo-electron microscopy image of the phosphatase-responsive hydrogel provided in Example 1;

[0062] Figure 7 This is a cryo-electron microscopy image of the phosphatase-catalyzed self-assembled gradient hydrogel scaffold provided in Example 7;

[0063] Figure 8 Image showing the live / dead staining results of BMSCs after 3 days of three-dimensional culture in GelMA;

[0064] Figure 9 For BMSCs in G- g - Image showing the live / dead staining results after three days of three-dimensional culture in NapFFYpC hydrogel;

[0065] Figure 10 For BMSCs after phosphatase catalysis G- g - Image showing the live / dead staining results after three days of three-dimensional culture in NapFFYpC hydrogel;

[0066] Figure 11 A graph showing the migration of BMSCs after 3 days of three-dimensional culture in GelMA;

[0067] Figure 12 For BMSCs in G- g -Migration pattern in NapFFYpC hydrogel after 3 days of three-dimensional culture;

[0068] Figure 13 For BMSCs after phosphatase catalysis G- g -Migration pattern in NapFFYpC hydrogel after 3 days of three-dimensional culture;

[0069] Figure 14 This image shows the effect of repair and regeneration of the full-thickness osteochondral defect area 8 weeks after implantation of the hydrogel scaffold. Detailed Implementation

[0070] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0071] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0072] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions may be performed simultaneously.

[0073] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0074] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0075] This application provides an enzyme-responsive self-assembling peptide-modified methacrylamide polymer having the structure of Formula 1:

[0076] Formula 1;

[0077] Where X is a lysine ε-amino side chain residue linked to an amide bond in methacrylated gelatin, a hydroxypropyl amino side chain residue linked to an amide bond in methacrylated silk fibroin, or a hydroxyl residue linked to an amide bond on the polysaccharide backbone in methacrylated hyaluronic acid, and R is a cysteine ​​thiol side chain residue in an enzyme-responsive self-assembled polypeptide.

[0078] This application transforms the concentration gradient of alkaline phosphatase, an endogenous tissue-specific marker, into a dynamic gradient evolution of scaffold mechanical properties. By introducing methacrylamide polymers modified with enzyme-responsive self-assembling peptides (phosphotyrosine sequences), the scaffold undergoes molecular-level dynamic self-assembly under the catalysis of phosphatase, forming a continuous mechanical gradient from the cartilage region to the subchondral bone region. This process requires no external intervention and is entirely regulated by the local microenvironment, thereby adapting in real time to the dynamic needs of "early proliferation-late differentiation" in osteochondral repair. Compared to existing technologies, this application not only overcomes the mismatch between static gradients and dynamic microenvironments but also significantly improves the spatial specificity and temporal synchronization of stem cell differentiation through precise molecular-scale regulation of mechanical signals.

[0079] The present application is further illustrated below with reference to embodiments. The scope of protection of the present application is not limited to the following embodiments.

[0080] Example 1

[0081] This embodiment provides a methacrylamide gelatin modified with the enzyme-responsive self-assembling polypeptide NapFFYpC:

[0082] The method for preparing the enzyme-responsive self-assembled polypeptide NapFFYpC-modified methacrylamide gelatin includes:

[0083] Synthesis of methacrylated gelatin: At 50 °C, 1 g of gelatin was slowly added to 100 mL of phosphate-buffered saline (PBS), with continuous stirring until the gelatin was completely dissolved, forming a homogeneous solution. Then, 1 mL of methacrylic anhydride solution was added dropwise to the above solution, ensuring it was fully dispersed in the system. Immediately afterwards, 1 mol L... -1 The pH of the reaction solution was adjusted using a sodium hydroxide solution, carefully controlled to maintain a stable pH between 8.5 and 9. Under these conditions, the reaction was allowed to proceed for 3 hours. After 3 hours, the pH of the reaction solution was immediately adjusted to 7.4 using a suitable acid solution (such as hydrochloric acid) to terminate the reaction. The reaction product was then transferred to centrifuge tubes and centrifuged at 722.75 × g for 5 minutes to separate unreacted methacrylic anhydride from the product, removing residual methacrylic anhydride. After centrifugation, the supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000. Dialysis was then performed in sufficient deionized water for 3 days, with the deionized water changed regularly to ensure dialysis effectiveness. After dialysis, the solution in the dialysis bag was removed and freeze-dried to obtain methacrylamide gelatin (GelMA) product.

[0084] Synthase-responsive self-assembled peptide NapFFYpC-modified methacrylamide gelatin: Weigh 0.605 g of GelMA and place it in 50 mL of ultrapure water. Stir at 50 °C until the GelMA is completely dissolved, forming a homogeneous and transparent solution. Separately, take 0.1 g of peptide NapFFYpC and add it to 3 mL of N,N-dimethylformamide, shaking thoroughly to ensure complete dissolution. Slowly pour the dissolved NapFFYpC solution into the dissolved GelMA solution and mix well. Next, add triethylamine dropwise to the mixture while simultaneously monitoring the pH with a pH meter and precisely adjusting the pH to 8. After pH adjustment, seal the reaction vessel and stir overnight at room temperature to allow the reaction to proceed fully. The next day, the reaction is complete. Transfer the reaction solution to a pre-activated dialysis bag with a molecular weight cutoff of 3500 Da and dialyze in sufficient ultrapure water. During dialysis, the pH of the dialysate was measured periodically and adjusted to 7.4. Dialysis continued for three days. After dialysis, the product solution was removed from the dialysis bag and freeze-dried to obtain methacrylated gelatin (G-) modified with the enzyme-responsive self-assembled peptide NapFFYpC. g -NapFFYpC).

[0085] This embodiment also provides a phosphatase-responsive hydrogel, the preparation method of which includes:

[0086] In a light-protected environment, weigh 2.5 mg of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and dissolve it in phosphate buffered saline (PBS). Then, weigh 80 mg of G- g -NapFFYpC, added to a solution system containing LAP. Through vortex oscillation and ultrasonic treatment, G- g -NapFFYpC was fully dissolved to obtain a polymer pre-crosslinked solution. An appropriate amount of this pre-crosslinked solution was taken and transferred to a mold, and irradiated with 405 nm blue light to initiate a crosslinking reaction, thus preparing a phosphatase-responsive hydrogel.

[0087] The GelMA, Nap-FFYpC, and G- provided in this embodiment g -NapFFYpC's proton NMR spectrum is shown below. Figure 1 As shown.

[0088] The GelMA, Nap-FFYpC, and G- provided in this embodiment g The infrared spectrum of -NapFFYpC is as follows Figure 2 As shown.

[0089] Example 2

[0090] This embodiment provides a self-assembled gradient hydrogel scaffold, the preparation method of which includes:

[0091] The phosphatase-responsive hydrogel provided in Example 1 was co-incubated with phosphatase for 0 h.

[0092] Example 3

[0093] This embodiment provides a self-assembled gradient hydrogel scaffold, the preparation method of which includes:

[0094] The phosphatase-responsive hydrogel provided in Example 1 was co-incubated with phosphatase for 4 hours.

[0095] Example 4

[0096] This embodiment provides a self-assembled gradient hydrogel scaffold, the preparation method of which includes:

[0097] The phosphatase-responsive hydrogel provided in Example 1 was co-incubated with phosphatase for 8 hours.

[0098] Example 5

[0099] This embodiment provides a self-assembled gradient hydrogel scaffold, the preparation method of which includes:

[0100] The phosphatase-responsive hydrogel provided in Example 1 was co-incubated with phosphatase for 12 h.

[0101] Example 6

[0102] This embodiment provides a self-assembled gradient hydrogel scaffold, the preparation method of which includes:

[0103] The phosphatase-responsive hydrogel provided in Example 1 was co-incubated with phosphatase for 24 hours.

[0104] Example 7

[0105] This embodiment provides a self-assembled gradient hydrogel scaffold, the preparation method of which includes:

[0106] The phosphatase-responsive hydrogel provided in Example 1 was co-incubated with phosphatase for 48 h.

[0107] After co-incubation of the phosphatase-responsive hydrogel with phosphatase, the self-assembled gradient hydrogel scaffold was subjected to circular dichroism chromatographic analysis with increasing phosphatase catalysis time (0 h, 4 h, 8 h, 12 h, 24 h, 48 h). The calculated conformational percentages of the secondary structures are shown in the figure below. Figure 3 As shown.

[0108] The microstructure and mechanical properties of the self-assembled gradient hydrogel scaffolds obtained in Examples 2-7 were characterized.

[0109] Phosphatase-responsive hydrogels were prepared using the above-described photocrosslinking method. Phosphatase was added, and the reaction was carried out at 37 °C. Samples were taken at 0, 4, 8, 12, 24, and 48 h for microscopic morphology characterization using cryo-scanning electron microscopy and compression testing using a universal testing machine. The compression modulus diagrams of the self-assembled gradient hydrogel scaffolds obtained in Examples 2-7 are shown below. Figure 4 As shown, this phenomenon is due to the transformation of the secondary structure within the hydrogel system under the catalysis of phosphatase, with a gradual increase in the content of α-helices and β-sheets, thereby significantly improving the mechanical properties of the hydrogel. To further investigate the microstructural changes before and after phosphatase catalysis, GelMA and G- g -NapFFYpC and the self-assembled gradient hydrogel scaffold catalyzed by phosphatase were subjected to cryo-electron microscopy (cryo-EM) scanning. The cryo-EM images of GelMA are shown below. Figure 5 As shown, the cryo-electron microscopy image of the phosphatase-responsive hydrogel is as follows: Figure 6As shown, the cryo-electron microscopy image of the self-assembled gradient hydrogel scaffold after the addition of phosphatase catalysis is as follows. Figure 7 As shown, the results indicate that G- g The NapFFYpC hydrogel exhibits a more compact network structure. Furthermore, under the catalysis of phosphatase, short peptides self-assemble, forming even denser network regions, further enhancing the hydrogel's mechanical properties. Microstructural changes observed under electron microscopy further validate the successful construction of the phosphatase-responsive allosteric mechanical enhancement system.

[0110] Bone marrow mesenchymal stem cells (BMSCs) were thoroughly mixed with a polymer pre-crosslinking solution and photocrosslinked. Afterward, they were cultured in complete culture media (α-MEM solution containing 10% fetal bovine serum and 1% penicillin / streptomycin) containing and without phosphatase. Samples were taken on day 3 for live / dead staining. The results of live / dead staining of BMSCs after 3 days of three-dimensional culture in GelMA are shown in the figure below. Figure 8 As shown in the figure, the live / dead staining results of BMSCs after three days of three-dimensional culture in a phosphatase-responsive hydrogel are as follows. Figure 9 As shown, BMSCs after G- phosphatase catalysis g -The results of live / dead staining after 3 days of three-dimensional culture in NapFFYpC hydrogel are shown in the figure below. Figure 10 As shown, no dead cells (red) were observed at any time point, and the cells maintained good proliferative capacity, demonstrating their good biocompatibility. The phosphatase-responsive hydrogel invasion assay (hBM-MSCs were seeded on the hydrogel surface and co-cultured for 3 days. Live cells were labeled using a live / dead detection kit, and 3D confocal imaging was used to observe cell migration and penetration into the hydrogel) also verified that BMSCs could migrate and penetrate well into the entire hydrogel matrix. The migration of BMSCs after 3 days of three-dimensional culture in GelMA is shown in the figure below. Figure 11 As shown in the figure, the migration of BMSCs after 3 days of three-dimensional culture in a phosphatase-responsive hydrogel is as follows. Figure 12 As shown, BMSCs after G- phosphatase catalysis g The migration pattern in the NapFFYpC hydrogel after 3 days of three-dimensional culture is shown in the figure below. Figure 13 As shown.

[0111] Using 3-4 month old skeletally mature New Zealand white rabbits, after anesthesia, the joint capsule was incised along the medial side of the knee joint to expose the femoral condyle. A trephine was used to create a model of the osteochondral defect in the femoral condyle, with a diameter of 5 mm and a depth of 5 mm. No further treatment was performed on the defect model after creation; sutures were then made. In the control group (homogeneous GelMA hydrogel implantation group) and the phosphatase-responsive hydrogel implantation group, a prepolymerized solution of liquid hydrogel and BMSCs was injected to fill the defect, and the area was irradiated with blue light until gelation occurred. Eight weeks post-surgery, some New Zealand white rabbits were euthanized, and femoral condyle specimens were collected. Micro-CT scans and three-dimensional reconstructions were performed on the collected specimens to evaluate the repair effect of the osteochondral defect. Micro-CT analysis of images of the full-thickness osteochondral defect area 8 weeks after hydrogel scaffold implantation showed that, compared with the simple defect group and the control homogeneous gradient GelMA group, G- g The NapFFYpC self-assembled gradient hydrogel group showed the best results, with full-thickness repair and regeneration of the osteochondral defect area 8 weeks after implantation of the hydrogel scaffold shown in the following full-thickness repair and regeneration diagram. Figure 14 As shown. From Figure 14 As shown in the results of osteochondral full-thickness repair and regeneration, compared with the simple defect group and the control uniform gradient GelMA group, the phosphatase-responsive hydrogel group exhibited the best full-thickness repair and regeneration effect.

[0112] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and application concept of this application, should be included within the scope of protection of this application.

Claims

1. A methacrylamide polymer modified with an enzyme-responsive self-assembling polypeptide, characterized in that, It has the structure of Formula 1: Formula 1; Where X is the lysine ε-amino side chain residue linked to the amide bond in methacrylamide gelatin, and R is the cysteine ​​thiol side chain residue in the enzyme-responsive self-assembled polypeptide. R is Ar–(AA) n –Yp–C; Where Ar is a hydrophobic aromatic group, (AA) n It is a polypeptide fragment containing 0–10 amino acid residues; Yp represents phosphorylated amino acid residues; C represents cysteine ​​residues.

2. A method for preparing a methacrylated polymer modified with an enzyme-responsive self-assembled polypeptide as described in claim 1, characterized in that, include: Methacrylamide polymers and enzyme-responsive self-assembly peptides of Formula 2 were subjected to Michael addition reactions to obtain enzyme-responsive self-assembly peptide-modified methacrylamide polymers. The methacrylamide polymer is methacrylamide gelatin; Ar–(AA) n –Yp–C formula 2; Ar is a hydrophobic aromatic group; (AA) n It is a polypeptide fragment containing 0–10 amino acid residues; Yp represents phosphorylated amino acid residues; C represents a cysteine ​​residue.

3. The preparation method according to claim 2, characterized in that, The enzyme-responsive self-assembling polypeptide has any one of the structures of Formula 2-1 to Formula 2-4; 。 4. The preparation method according to claim 2, characterized in that, The mass ratio of the methacrylamide polymer to the compound of formula 2 is 1:(2~70); the Michael addition reaction is carried out in a solvent; the solvent includes N,N-dimethylformamide; the temperature of the Michael addition reaction is 45 °C to 55 °C; and the time of the Michael addition reaction is 10 h to 48 h.

5. The preparation method according to claim 2, characterized in that, The method for preparing the methacrylamide gelatin includes: Gelatin was mixed with methacrylic anhydride and reacted to obtain methacrylated gelatin; The reaction was carried out at a pH of 8 to 9.

6. A phosphatase-responsive hydrogel, characterized in that, The methacrylamide polymer modified with the enzyme-responsive self-assembly peptide according to claim 1 or the methacrylamide polymer modified with the enzyme-responsive self-assembly peptide according to any one of claims 2 to 5 is obtained by cross-linking reaction.

7. A method for preparing a phosphatase-responsive hydrogel, characterized in that, include: A phosphatase-responsive hydrogel was obtained by cross-linking a methacrylated polymer modified with an enzyme-responsive self-assembled peptide with phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. The enzyme-responsive self-assembly peptide-modified methacrylamide polymer is the enzyme-responsive self-assembly peptide-modified methacrylamide polymer of claim 1 or the enzyme-responsive self-assembly peptide-modified methacrylamide polymer prepared by any one of claims 2 to 5.

8. The preparation method according to claim 7, characterized in that, The cross-linking reaction is carried out under blue light irradiation; the cross-linking reaction is carried out in phosphate buffer solution; the mass ratio of the enzyme-responsive self-assembled polypeptide modified methacrylamide polymer to phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is (5-10):

1.

9. A self-assembled gradient hydrogel scaffold, characterized in that, The phosphatase-responsive hydrogel prepared by the method described in claim 6 or the method described in claim 7 or 8 is obtained by co-incubating with phosphatase.

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