Acellular Scaffold for Cartilage Repair, Its Preparation Method and Application

Abstract

This application relates to a decellularized scaffold for cartilage repair in the biomedical field, its preparation method, and application. The preparation method comprises: preparing a decellularized matrix from animal cartilage; chemically bonding the decellularized matrix with a lipid compound to obtain a double-bond-modified decellularized matrix; copolymerizing and cross-linking the double-bond-modified decellularized matrix to obtain a decellularized matrix hydrogel; and immersing the decellularized matrix hydrogel in a dipeptide solution for modification to obtain the decellularized scaffold. The decellularized scaffold of this application introduces both mechanical and electrical stimulation effects into the scaffold, addressing deficiencies in previous research and demonstrating good biocompatibility and repair effectiveness in both in vivo and in vitro experiments.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a decellularized scaffold for cartilage repair, and a preparation method and application thereof. Background Art

[0002] Cartilage is avascular and anervous, with limited intrinsic regenerative capacity, so external intervention is necessary after injury. Patients often experience pain, and untreated cases can develop osteoarthritis, severely affecting mobility and placing a heavy burden on families. Current clinical strategies mainly focus on relieving symptoms through analgesics and anti-inflammatory drugs.

[0003] Bioelectricity plays an important role not only in neural function but also in various physiological processes. Collagen, the main protein in the extracellular matrix (ECM) of bone and cartilage, has inherent piezoelectric properties with a piezoelectric coefficient between 0.2 and 2.0 pC / N. Although tissue engineering and microfracture therapy for cartilage repair complement each other, there are still huge challenges in optimizing the delivery of biological factors and chemical modification. Biomechanical factors have not received enough attention, and their inherent bioelectrical properties have been even more neglected. In addition, existing materials have poor biocompatibility and high toxicity, making them unable to be used clinically. Summary of the Invention

[0004] In view of the problems existing in the background technology, the present application provides a decellularized scaffold for cartilage repair, its preparation method and application. The decellularized scaffold is based on dipeptides such as phenylalanine dipeptide or phenylalanine-tryptophan peptide, and introduces mechanical-electrical stimulation effects into the scaffold at the same time. It not only has excellent piezoelectric properties, but also has good biocompatibility and repair effects.

[0005] According to a first aspect of the present invention, a method for preparing a decellularized scaffold for cartilage repair is provided, comprising: preparing a decellularized matrix from animal cartilage; chemically combining the decellularized matrix with a lipid compound to obtain a double-bond modified decellularized matrix; copolymerizing and cross-linking the double-bond modified decellularized matrix to obtain a decellularized matrix hydrogel; and immersing the decellularized matrix hydrogel in a dipeptide solution for modification to obtain a decellularized scaffold.

[0006] The decellularized scaffold of the present invention is based on dipeptide. The animal cartilage decellularized matrix is combined with lipid compounds to obtain a double-bond modified decellularized matrix, and the double-bond modified decellularized matrix is copolymerized and cross-linked to obtain a decellularized matrix hydrogel, which is further modified with dipeptide to finally obtain a piezoelectric decellularized scaffold, realizing the simultaneous introduction of mechanical and electrical stimulation effects into the scaffold. It not only has excellent piezoelectric properties, but also has good biocompatibility and repair effects.

[0007] In some embodiments of the present invention, the preparation of the decellularized matrix includes: first digesting the animal cartilage in a trypsin solution, then adding DNase and RNase for treatment, then further treating the animal cartilage with a sodium dodecyl sulfate solution and a cell lysis solution, and rinsing it to obtain the decellularized matrix.

[0008] In some embodiments of the present invention, the animal cartilage is porcine femoral knee cartilage.

[0009] In some embodiments of the present invention, the lipid compound includes methacrylic anhydride or glyceryl methacrylate.

[0010] In some embodiments of the present invention, chemically combining a decellularized matrix with a lipid compound to obtain a double-bond modified decellularized matrix comprises: first, dissolving the decellularized matrix in deionized water and cooling it in an ice-water bath to obtain a mixed solution; then adjusting the pH of the mixed solution to alkaline; then, dripping the lipid compound into the solution under stirring to react; and then performing dialysis separation to obtain the double-bond modified decellularized matrix.

[0011] In some embodiments of the present invention, the cut-off molecular weight of the dialysis separation is 7000 Da.

[0012] In some embodiments of the present invention, copolymerizing and cross-linking the double-bond modified decellularized matrix to obtain a decellularized matrix hydrogel includes: first dissolving the double-bond modified decellularized matrix in a PBS solution, then adding an initiator, and then irradiating the system under blue light to complete gelation to obtain the decellularized matrix hydrogel.

[0013] In some embodiments of the present invention, the dipeptide comprises a phenylalanine dipeptide or a phenylalanine-tryptophan peptide.

[0014] In some embodiments of the present invention, immersing the decellularized matrix hydrogel in a dipeptide solution for modification to obtain a decellularized scaffold includes: preparing a mixed solvent of methanol and water; dissolving a dipeptide in the mixed solvent to obtain a dipeptide solution; immersing the decellularized matrix hydrogel in the dipeptide solution for reaction, then removing the hydrogel and freeze-drying the hydrogel; repeating the process of immersing the decellularized matrix hydrogel in the dipeptide solution, removing the hydrogel and freeze-drying the hydrogel multiple times; and obtaining the decellularized scaffold.

[0015] In some embodiments of the present invention, the concentration of the phenylalanine dipeptide solution is 8 mg / mL.

[0016] According to a second aspect of the present invention, a decellularized scaffold for cartilage repair prepared by the above-mentioned preparation method is provided.

[0017] According to a third aspect of the present invention, there is provided a decellularized scaffold prepared by the above-mentioned preparation method or use of the above-mentioned decellularized scaffold in preparing a cartilage repair material. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present application. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0019] Figure 1 This is the pressure-current test diagram of different decellularized scaffold materials of the present invention. Figure 1 (A) The decellularized scaffold material was not piezoelectrically modified. Figure 1 (B) Decellularized scaffold material was modified with piezoelectricity;

[0020] Figure 2 This is a pressure-voltage test diagram of the decellularized scaffold modified with phenylalanine dipeptide according to the present invention;

[0021] Figure 3 This is a biocompatibility test diagram of different decellularized scaffold materials of the present invention. Figure 3 (A) The decellularized scaffold material was not piezoelectrically modified. Figure 3 (B) Decellularized scaffold material was modified with piezoelectricity;

[0022] Figure 4 This is a test diagram of the implantation effect of different decellularized scaffold materials of the present invention. Figure 4 (A) The decellularized scaffold material was not piezoelectrically modified. Figure 4 (B) Decellularized scaffold material was modified with piezoelectric technology. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely intended to explain the present invention and are not intended to limit the scope of the invention. Those skilled in the art will readily recognize multiple non-critical parameters that can be varied or modified with substantially the same results.

[0024] Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or in the product specifications were used. Reagents or instruments used without manufacturer specified were all conventional products available through regular channels. The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all commercially available products.

[0025] Commonly used inorganic materials in contemporary research include salts containing heavy metal ions, such as barium titanate (BaTiO3), which has excellent piezoelectric properties. However, its poor biodegradability and low doping level limit its applicability for in vivo implantation. Similarly, the piezoelectric properties of poly (L-lactic acid) (PLLA) are not ideal, and the acidic metabolites produced by its decomposition can induce an inflammatory response at the implant site, thereby affecting the efficacy. In addition, traditional piezoelectric polymers, such as poly (vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), require high-voltage polarization and are non-degradable.

[0026] Phenylalanine, an essential amino acid, can self-assemble into diphenylalanine (FF) to form a hexagonal structure with strong piezoelectric properties, with a piezoelectric coefficient d33 of 18 pm / V. Phenylalanine dipeptide is used in sensors and, due to its biocompatibility and biodegradability, has yet to be explored in the medical field, but has great potential for clinical application.

[0027] A first aspect of the present invention provides a method for preparing a decellularized scaffold for cartilage repair, the method comprising the following steps:

[0028] 1) Decellularization of materials.

[0029] The animal cartilage is taken to prepare the decellularized matrix: the animal cartilage is first digested in a trypsin solution, and then treated with DNase and RNase. The animal cartilage is then further treated with a sodium dodecyl sulfate solution and a cell lysis solution, and rinsed to obtain the decellularized matrix.

[0030] In some embodiments of the present invention, the animal cartilage includes but is not limited to porcine femoral knee cartilage.

[0031] Furthermore, the pig femur is preferably fresh pig femur, specifically taken from pig femurs after slaughter in a slaughterhouse.

[0032] In some embodiments of the present invention, after the animal cartilage is treated with the sodium lauryl sulfate solution and the cell lysis solution, it is rinsed with pure water until there is no foam.

[0033] Furthermore, pure water includes but is not limited to distilled water, deionized water, etc.

[0034] Furthermore, the animal cartilage was stirred using a magnetic stirrer at a rotation speed of 210 rpm.

[0035] In some embodiments of the present invention, the animal cartilage is rinsed, freeze-dried, and ground into powder using a mechanical grinder.

[0036] 2) Modified with methacrylic anhydride or glycerol methacrylate.

[0037] The decellularized matrix is chemically bonded with lipid compounds such as methacrylic anhydride or methacrylate glycerol to obtain a double-bond modified decellularized matrix: first, the decellularized matrix is dissolved in deionized water and cooled in an ice-water bath to obtain a mixed solution; then, the pH of the mixed solution is adjusted to alkaline; then, under stirring, methacrylic anhydride or methacrylate glycerol is dropped into the solution for reaction; and then, dialysis separation is performed to obtain the double-bond modified decellularized matrix.

[0038] In some embodiments of the present invention, the pH value of the mixed solution is adjusted by sodium hydroxide.

[0039] Furthermore, the pH value of the mixed solution was adjusted to 8.

[0040] In some embodiments of the present invention, the molecular weight cut-off for dialysis separation is 7000 Da.

[0041] 3) Preparation of decellularized extracellular matrix hydrogel.

[0042] Decellularized matrix hydrogels were prepared using gradient freezing and photocrosslinking: first, the double-bond modified decellularized matrix was dissolved in PBS solution, then an initiator was added, and then the system was irradiated under blue light to complete gelation, thereby obtaining a decellularized matrix hydrogel.

[0043] In some embodiments of the present invention, a bridge agent is added to the PBS solution to which the double-bond-modified decellularized matrix is added as an initiator.

[0044] Furthermore, the bridge material is phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0045] In some embodiments of the present invention, the PBS solution to which the double-bond modified acellular matrix is added is placed in a freezing environment to form a freezing system, and the freezing system is irradiated under blue light.

[0046] Furthermore, the gradient freezing system is first at 4°C for 30 min, -20°C for 2 h, and then at -80°C for 12 h after photocrosslinking and sufficient swelling.

[0047] 4) Dipeptide modification.

[0048] The decellularized matrix hydrogel is immersed in a dipeptide solution such as phenylalanine dipeptide or phenylalanine-tryptophan peptide for modification to obtain a piezoelectric decellularized scaffold: a mixed solvent of methanol and water is prepared; phenylalanine dipeptide or phenylalanine-tryptophan peptide is dissolved in the mixed solvent to obtain a phenylalanine dipeptide solution or a phenylalanine-tryptophan peptide solution; the decellularized matrix hydrogel is immersed in the phenylalanine dipeptide solution or the phenylalanine-tryptophan peptide solution for reaction, and then removed and freeze-dried; the process of immersing the decellularized matrix hydrogel in the phenylalanine dipeptide solution or the phenylalanine-tryptophan peptide solution, removing, and freeze-drying is repeated three times; and a piezoelectric decellularized scaffold is obtained.

[0049] In some embodiments of the present invention, the concentration of the phenylalanine dipeptide solution is 8 mg / mL.

[0050] The piezoelectric decellularized scaffold of the present invention is based on phenylalanine dipeptide or phenylalanine-tryptophan peptide. The animal cartilage decellularized matrix is combined with methacrylic anhydride or methacrylate glycerol to obtain a double-bond modified decellularized matrix, and the double-bond modified decellularized matrix is copolymerized and cross-linked to obtain a decellularized matrix hydrogel, which is further modified with phenylalanine dipeptide or phenylalanine-tryptophan peptide to finally obtain a piezoelectric decellularized scaffold, which realizes the simultaneous introduction of mechanical and electrical stimulation effects into the scaffold. It not only has excellent piezoelectric properties, but also has good biocompatibility and repair effects.

[0051] The second aspect of the present invention provides a decellularized scaffold for cartilage repair, which is prepared by the preparation method provided by the first aspect of the present invention.

[0052] The third aspect of the present invention provides an application of a decellularized scaffold for cartilage repair in the preparation of cartilage repair materials, which is the decellularized scaffold prepared by the preparation method provided by the first aspect of the present invention or the decellularized scaffold provided by the second aspect of the present invention.

[0053] The preparation method of the acellular scaffold for cartilage repair in the present invention will be further described below with reference to specific examples.

[0054] Example 1

[0055] Preparation of Piezoelectric Decellularized Porcine Femoral Knee Cartilage Scaffolds

[0056] (1) Material decellularization process: Fresh pig femoral knee cartilage was obtained from slaughterhouses and digested with 0.2% trypsin at 4°C for 24 hours. 0.05‰ DNase and 0.05‰ RNase were added and treated at 37°C for 24 hours. The cartilage was then treated with 1% (w / v) sodium dodecyl sulfate (SDS) and 1% Trion X-100 for 72 hours. The cartilage was rinsed with distilled water until there was no foam. The decellularized cartilage slices were then stirred with a magnetic stirrer at 210 rpm.

[0057] (2) Double bond cross-linking modification process: 3 g of dECM (decellularized matrix) powder was dissolved in 100 ml of deionized water and cooled in an ice water bath. The pH value of the mixed solution was adjusted to 8 with NaOH. Under stirring, 3 mL of methacrylic anhydride was added dropwise to the solution. The reaction was stopped after 24 hours. The solution was then dialyzed for 3 days with a cutoff molecular weight of 7000 Da. After freeze-drying, double bond-modified dECM was obtained. Then, 3 g of modified dECM powder was dissolved in 300 ml of PBS solution, and 0.3 g of the bridge was added as an initiator. The frozen system was then placed under 405 nm blue light with a power of 5 W for 5 minutes to complete gelation and obtain a hydrogel.

[0058] (3) Modification of phenylalanine dipeptide: A mixed solvent of methanol and water was prepared with a ratio of methanol to water equal to 3:1 to dissolve phenylalanine dipeptide. The concentration of phenylalanine dipeptide was 8 mg / mL. After the hydrogel was formed and freeze-dried, the double-bond modified cross-linked acellular matrix (hydrogel) was immersed in the phenylalanine dipeptide solution for 6 hours. Subsequently, they were taken out and freeze-dried again. This process was repeated three times.

[0059] Example 2

[0060] Material piezoelectric performance test

[0061] The piezoelectric decellularized scaffold of porcine femoral knee cartilage prepared in Example 1 and the decellularized scaffold of porcine femoral knee cartilage prepared only by steps (1) and (2) of Example 1 were taken to test the piezoelectric properties of the materials.

[0062] To measure electrical conductivity, fully swollen hydrogels (d = 15 mm, h = 4 mm) were placed in a compression apparatus and subjected to compression tests at a frequency of 1 Hz and a strain rate of 20%. Voltage and current outputs were collected using an electrometer (Tektronix 6514, China). To test the stability of the hydrogels' piezoelectric output, they were immersed in a 37°C PBS solution. The materials were then removed at 0, 7, and 14 days, and the piezoelectric tests were repeated under cyclic compression conditions.

[0063] like Figure 1 As shown, Figure 1(A) In the absence of phenylalanine dipeptide modification, the material under pressure exhibited a small fluctuation range in current output, indicating no significant effect. Figure 1 (B) After modification with the phenylalanine dipeptide group, the current output increases rapidly and remains essentially stable, demonstrating a significant improvement in electrical output.

[0064] like Figure 2 As shown, Figure 2 The hydrogel modified with phenylalanine dipeptide showed a stable voltage range on the pressure-electric output meter, and was able to maintain piezoelectric properties for a long time when treated at a frequency of 1 Hz, indicating the stability of the modification.

[0065] Example 3

[0066] Biocompatibility testing of piezoelectrically modified hydrogels

[0067] The piezoelectric decellularized scaffold of porcine femoral knee cartilage prepared in Example 1 and the decellularized scaffold of porcine femoral knee cartilage prepared only by steps (1) and (2) of Example 1 were subjected to biocompatibility testing.

[0068] like Figure 3 As shown, Figure 3 (A) Unmodified hydrogel (B) Phenylalanine dipeptide-modified hydrogel. There is no significant difference in the cell migration effect between the two, indicating that Phenylalanine dipeptide has good biocompatibility.

[0069] Example 4

[0070] Implantation effect test of piezoelectric modified hydrogel in miniature pig knee joint

[0071] The piezoelectric decellularized scaffold of porcine femoral knee cartilage prepared in Example 1 and the decellularized scaffold of porcine femoral knee cartilage prepared only by steps (1) and (2) of Example 1 were used to test the implantation effect.

[0072] like Figure 4 As shown, Figure 4 (A) The repair effect of the hydrogel without piezoelectric modification on the large defect of the miniature pig knee joint after 24 weeks, where the presence of cavities can be clearly observed; Figure 4 (B) The piezoelectric hydrogel not only filled the defect but also achieved the healing of new cartilage 24 weeks after implantation.

[0073] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing an acellular scaffold for cartilage repair, characterized in that: include: Animal cartilage was taken to prepare acellular matrix; The decellularized matrix is chemically bonded with a lipid compound to obtain a double-bond modified decellularized matrix: first, the decellularized matrix is dissolved in deionized water and cooled in an ice water bath to obtain a mixed solution; then, the pH of the mixed solution is adjusted to alkaline; Then, the lipid compound is dropped into the solution for reaction under stirring; Then, dialysis separation is performed, and the cut-off molecular weight of the dialysis separation is 7000 Da; obtaining the double bond-modified acellular matrix; The double-bond-modified acellular matrix was prepared into an acellular matrix hydrogel by gradient freezing and photocrosslinking: the double-bond-modified acellular matrix was first dissolved in PBS solution, and then an initiator was added. The PBS solution with the double-bond-modified acellular matrix was placed in a freezing environment to form a freezing system. The freezing system was then irradiated with blue light for 5 minutes to complete gelation, thereby obtaining the acellular matrix hydrogel. The gradient freezing process was first at 4°C for 30 minutes, then at -20°C for 2 hours, and then at -80°C for 12 hours after photocrosslinking and sufficient swelling. The decellularized matrix hydrogel is immersed in a dipeptide solution for modification to obtain a decellularized scaffold: a mixed solvent of methanol and water is prepared with a methanol:water ratio of 3:1; a dipeptide is dissolved in the mixed solvent to obtain a dipeptide solution, wherein the dipeptide includes a phenylalanine dipeptide or a phenylalanine-tryptophan peptide; the concentration of the phenylalanine dipeptide solution is 8 mg / mL; the decellularized matrix hydrogel is immersed in the dipeptide solution for 6 hours, then removed and freeze-dried; the process of immersing the decellularized matrix hydrogel in the dipeptide solution, removing, and freeze-drying is repeated multiple times to obtain the decellularized scaffold.

2. The preparation method according to claim 1, characterized in that The preparation of the decellularized matrix comprises: First, the animal cartilage is digested in a trypsin solution, and then treated with DNA enzyme and RNA enzyme. Thereafter, the animal cartilage is further treated with a sodium dodecyl sulfate solution and a cell lysis solution, and then rinsed to obtain the acellular matrix.

3. The preparation method according to claim 1, characterized in that The lipid compound includes methacrylic anhydride or glyceryl methacrylate.

4. A decellularized scaffold for cartilage repair prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the decellularized scaffold prepared by the preparation method according to any one of claims 1 to 3 or the decellularized scaffold according to claim 4 in the preparation of cartilage repair materials.

Images