Recombinant protein, printing ink, 4D scaffold and preparation method and application of 4D scaffold

Abstract

The invention relates to a recombinant protein, printing ink, a 4D scaffold and a preparation method and application thereof, and belongs to the technical field of biology. The recombinant protein provided by the invention can provide extracellular matrix-like structure support, catalyzes generation of dopamine groups, endows the recombinant protein with bionic adhesion performance, and meanwhile can regulate and control the activity of matrix metalloproteinase, maintain balance of scaffold degradation and tissue regeneration, target inflammatory factor genes and relieve local inflammatory response. The recombinant protein, the P (NIPAM-co-AA) copolymer and the KLT peptide-loaded mesoporous SiO2 jointly form the printing ink, and the printing ink can be used for printing a 4D biological scaffold, not only has the effect of the recombinant protein, but also has the effects of temperature responsiveness and promoting cell proliferation and vascularization. Meanwhile, due to the fact that a TPMS structure model is adopted in the 4D stent, directional curling can be achieved, and the better mechanical property is achieved; the hydrogel can be used for tissue repair and filling, tissue regeneration is promoted, and wounds are rapidly healed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a recombinant protein, printing ink and 4D scaffold, as well as their preparation methods and applications. Background Technology

[0002] 4D printing technology is based on the principles of 3D printing. The most important core component of 4D printing is smart materials. Smart materials can sense external stimuli such as force, heat, electricity, light, and sound, giving 4D-printed objects intelligence. They are commonly used in the printing of tissue-engineered biomaterials. Tissue-engineered bioscaffold materials are artificial extracellular matrices that can bind to living cells and be implanted into organisms to replace or repair the function of diseased tissues and organs.

[0003] In the design and fabrication of bioscaffolds, proteins are a key component of biomaterials. Their core value lies in mimicking the structure and function of the natural extracellular matrix, providing a suitable microenvironment for cell growth, proliferation, and differentiation. Currently commonly used proteins include collagen, fibronectin, and laminin, but they often suffer from low stability, poor mechanical properties, and insufficient biomimetic performance, resulting in poor tissue repair capabilities. Therefore, there is an urgent need to improve the performance of proteins in bioscaffolds to achieve better repair effects.

[0004] Musse Adhesive Protein (MAP) is a natural adhesive protein derived from marine byssal glands. Rich in lysine and 3,4-dihydroxyphenylalanine (DOPA), this protein can bind tightly to negatively charged cells and tissues in the human body through electrostatic interactions. Furthermore, the oxidative cross-linking of the DOPA groups can form membrane or network structures, which enhances the adhesion stability of the material. However, current applications of musse adhesive protein are mostly limited to solutions, lyophilized powders, or simple coatings, primarily for microscopic cell adhesion and tissue bonding. Due to its short degradation cycle, it is difficult to support long-term tissue regeneration. Therefore, there are no reports of using musse adhesive protein in tissue regeneration and repair materials.

[0005] Current biological scaffolds suffer from several performance defects besides the poor performance of proteins. These include: static structures, failing to dynamically adapt to structural evolution during tissue regeneration and adjust pore size and mechanical properties according to microenvironments such as temperature changes; lack of bioactivity, for example, hyaluronic acid fillers only physically occupy space and lack collagen regeneration capabilities; poor degradation performance, such as non-degradable metal scaffolds causing secondary stenosis, and polylactic acid scaffold degradation products leading to local acidosis and delaying healing; and insufficient biomimetic properties, with most scaffolds only mimicking the macroscopic structure of tissue and failing to achieve the natural chemical microenvironment and bioadhesion characteristics, resulting in low cell colonization efficiency, difficulty in controlling differentiation direction, and impact on the quality of functional recovery of regenerated tissue. Therefore, there is an urgent need in this field to provide a biological scaffold with microenvironment responsiveness, better mechanical and biomimetic properties, and thus better promotion of wound healing. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a recombinant protein, printing ink, and a 4D scaffold, as well as their preparation method and application. The recombinant protein possesses biomimetic properties similar to mussel adhesive protein, and the 4D scaffold prepared with the printing ink containing the recombinant protein exhibits excellent mechanical and biomimetic adhesive properties, as well as temperature responsiveness, enabling directional curling and resulting in better tissue repair and dermal filling effects.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a recombinant protein, the gene sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a printing ink comprising the recombinant protein, P(NIPAM-co-AA) copolymer, and KLT peptide@mesoporous SiO2, wherein the weight ratio of the recombinant protein, P(NIPAM-co-AA) copolymer, and KLT peptide@mesoporous SiO2 is (0.3~0.5):(1.3~1.5):(0.1~0.2). The KLT peptide@mesoporous SiO2 is obtained by loading the KLT peptide into mesoporous SiO2.

[0009] Preferably, the P(NIPAM-co-AA) copolymer is obtained by free radical copolymerization of N-isopropylacrylamide and acrylic acid in the presence of azobisisobutyronitrile, wherein the molar ratio of N-isopropylacrylamide, acrylic acid and azobisisobutyronitrile is (6.8~7.2):(2.8~3.2):(0.048~0.052).

[0010] Preferably, the reaction temperature of the free radical copolymerization reaction is 69~71℃, and the reaction time is 7.8~8.2h.

[0011] Preferably, the KLT peptide loading rate of the KLT peptide@mesoporous SiO2 is 14~16 wt%.

[0012] The present invention provides a method for preparing the printing ink, comprising the following steps: mixing P(NIPAM-co-AA) copolymer with PBS buffer; then adding recombinant protein and KLT peptide@mesoporous SiO2 and stirring until the solute is dispersed.

[0013] The present invention also provides the application of the recombinant protein, the printing ink, or the method for preparing the printing ink in the preparation of 4D scaffolds.

[0014] The present invention also provides a 4D scaffold, which is printed using the printing ink described above or the printing ink prepared by the preparation method described above.

[0015] The present invention also provides a method for preparing the 4D scaffold, which is based on a Gyroid-type TPMS structure model to print printing ink. The construction of the Gyroid-type TPMS structure model includes: setting the relative density of the center point to 40%, the relative density of the edge region to 15%, and the relative density from the edge region to the center point to be linearly gradient, wherein the rate of change of the linear gradient is 0.05-0.10 relative density / mm; and the cell size is 2.0 mm.

[0016] The present invention also provides the application of the recombinant protein, the printing ink, the method for preparing the printing ink, the 4D scaffold, or the method for preparing the 4D scaffold in the preparation of tissue repair and / or dermal filler products.

[0017] The beneficial effects of this invention are: The recombinant protein provided by this invention has biomimetic properties of mussel adhesive protein, which can provide extracellular matrix-like structural support and catalyze the production of dopamine groups, giving it biomimetic adhesion properties. At the same time, it can regulate the activity of matrix metalloproteinases, maintain the balance between scaffold degradation and tissue regeneration, and can also target inflammatory factor genes to reduce local inflammatory response.

[0018] This invention uses recombinant protein, P(NIPAM-co-AA) copolymer, and KLT peptide@mesoporous SiO2 to form a printing ink, which can be used to print 4D bioscaffolds. The resulting 4D scaffolds not only possess the functions of the recombinant protein but also exhibit temperature responsiveness, undergoing a phase transition near body temperature, causing the scaffold to shrink or expand in volume. Simultaneously, the addition of KLT peptide@mesoporous SiO2 enables the slow release of growth factors, delaying their action time and promoting cell proliferation and angiogenesis.

[0019] This invention, during the fabrication of a 4D scaffold, utilizes a TPMS structural model based on the differentiated shrinkage characteristics of leaf veins. By designing the material density and composition ratio of different regions of the scaffold, the scaffold undergoes pre-defined anisotropic deformation under temperature stimulation, achieving morphological changes such as directional curling and pore adjustment. Simultaneously, the 4D scaffold possesses a continuous and interconnected pore network and excellent structural stability. By optimizing the TPMS unit parameters and arrangement, the scaffold maintains structural integrity during response, while achieving dynamic matching of mechanical properties. The 4D scaffold provided by this invention can be used for tissue repair and dermal filling, promoting tissue regeneration and accelerating wound healing. Attached Figure Description

[0020] Figure 1 This is a plasmid map of pPIC9K-COL1α1-DOPAsynthase-MMP2-siRNA.

[0021] Figure 2 This is an SDS-PAGE gel image of the recombinant protein from Example 1.

[0022] Figure 3 The image shows a SEM image of the P(NIPAM-co-AA) copolymer hydrogel prepared in Example 1.

[0023] Figure 4 The results are from a temperature scan test of the printing ink.

[0024] Figure 5 The results of the time-scan test for printing ink. Detailed Implementation

[0025] The present invention provides a recombinant protein, the gene sequence of which is shown in SEQ ID NO.1.

[0026] The recombinant protein of this invention is obtained by constructing the gene sequence shown in SEQ ID NO.1 into an expression vector, expressing it in Pichia pastoris, and then isolating and purifying it. This invention does not have specific limitations on the methods for constructing the expression vector, expressing it in Pichia pastoris cells, and isolating and purifying it; conventional methods in the art can be used. The recombinant protein of this invention possesses biomimetic properties of mussel adhesive proteins, providing extracellular matrix-like structural support and catalyzing the production of dopamine groups, thus endowing it with biomimetic adhesion properties. Simultaneously, it can regulate matrix metalloproteinase activity, maintain the balance between scaffold degradation and tissue regeneration, and target inflammatory factor genes, alleviating local inflammatory responses.

[0027] The present invention also provides a printing ink comprising the recombinant protein, P(NIPAM-co-AA) copolymer and KLT peptide@mesoporous SiO2, wherein the weight ratio of the recombinant protein, P(NIPAM-co-AA) copolymer and KLT peptide@mesoporous SiO2 is (0.3~0.5):(1.3~1.5):(0.1~0.2); preferably (0.35~0.45):(1.35~1.45):(0.12~0.18), and more preferably 0.4:1.4:0.15. In this invention, the printing ink is preferably prepared using PBS buffer as a solvent, and the ratio of the recombinant protein, P(NIPAM-co-AA) copolymer, KLT peptide@mesoporous SiO2 and printing ink is preferably (0.3~0.5) g : (1.3~1.5) g : (0.1~0.2) g : (9~11) mL; more preferably (0.35~0.45) g : (1.35~1.45) g : (0.12~0.18) g : (9.5~10.5) mL, and even more preferably 0.4 g : 1.4 g : 0.15 g : 10 mL.

[0028] The KLT peptide@mesoporous SiO2 is obtained by loading KLT peptide into mesoporous SiO2. The KLT peptide loading rate of the KLT peptide@mesoporous SiO2 is preferably 14-16 wt%, and in some embodiments, it can be 14 wt%, 15 wt%, or 16 wt%. This invention does not impose any particular limitations on the method of loading KLT peptide into mesoporous SiO2, or on the concentration and amount of KLT peptide and mesoporous SiO2. Conventional methods in the art that can meet the loading rate requirements of this invention can be used. In one embodiment, the loading method can be: "Dispersing 0.6 g of mesoporous SiO2 in 15 mL of 0.8 mg / mL KLT peptide solution, stirring at room temperature for 20 h; collecting by centrifugation, freeze-drying, and obtaining KLT peptide@mesoporous SiO2."

[0029] This invention does not have any particular limitation on the source of KLT peptide and PBS buffer; conventional commercially available products in the art can be used. In this invention, the KLT peptide was purchased from NovoPro. This invention does not have any particular limitation on the source of mesoporous SiO2; conventional commercially available products in the art or self-prepared products can be used. When self-prepared, in some embodiments, the sol-gel method can be selected. This invention does not have any particular limitation on the preparation steps of the sol-gel method; conventional sol-gel preparation steps in the art can be used. In some embodiments, it can be: "Dissolve 1.2g CTAB (hexadecyltrimethylammonium bromide) in 60mL of deionized water, add 6mL of ammonia water, stir for 10min; slowly add 6mL of TEOS (tetraethoxysilane), stir at room temperature for 24h, centrifuge to collect the precipitate, wash 3 times with ethanol, calcine at 550℃ for 6h to remove the template agent, and obtain mesoporous SiO2."

[0030] The P(NIPAM-co-AA) copolymer is preferably obtained by free radical copolymerization of N-isopropylacrylamide and acrylic acid in the presence of azobisisobutyronitrile (AIBN). In this invention, AIBN is preferably used as a free radical initiator to initiate the copolymerization reaction. The molar ratio of N-isopropylacrylamide, acrylic acid, and AIBN is preferably (6.8~7.2):(2.8~3.2):(0.048~0.052), more preferably (6.9~7.1):(2.9~3.1):(0.049~0.051), and even more preferably 7:3:0.05. The reaction temperature of the free radical copolymerization reaction is preferably 69~71℃, more preferably 69.5~70.5℃, and even more preferably 70℃; the reaction time is preferably 7.8~8.2h, more preferably 7.9~8.1h, and even more preferably 8h. This invention does not impose any special limitations on the specific steps of the free radical copolymerization reaction or the solvents used; conventional steps for the free radical copolymerization reaction of N-isopropylacrylamide and acrylic acid in this field can be used.

[0031] The present invention provides a method for preparing the printing ink, comprising the following steps: mixing P(NIPAM-co-AA) copolymer with PBS buffer; then adding recombinant protein and KLT peptide@mesoporous SiO2 and stirring until the solute is dispersed.

[0032] In this invention, the preparation of the printing ink is preferably carried out in a sterile environment. The weighed P(NIPAM-co-AA) copolymer is mixed with PBS buffer, and the mixture is preferably stirred at a temperature of 36.8℃~37.2℃ until dissolved. The temperature is more preferably 36.8℃, 36.9℃, 37℃, 37.1℃ or 37.2℃. This invention does not have a special limitation on the stirring method. Conventional stirring methods in the art can be used. In some embodiments, stirring at 500 rpm for 2~3 hours, such as 2 hours, 2.5 hours or 3 hours, can be selected to completely dissolve the P(NIPAM-co-AA) copolymer and obtain a transparent and clear solution. After dissolution, the solution is preferably stirred at 24.8℃~25.2℃ for 28~32 minutes to ensure the solution temperature uniformly decreases to the printing temperature. More preferably, the temperature is 24.8℃, 24.9℃, 25℃, 25.1℃, or 25.2℃, and the stirring time is more preferably 28 minutes, 29 minutes, 30 minutes, 31 minutes, or 32 minutes. This invention does not specifically limit the stirring rate; any stirring rate that ensures the solution temperature uniformly decreases to the printing temperature is acceptable. Then, recombinant protein and KLT peptide@mesoporous SiO2 are added and stirred until the solute is dispersed. The preferred order of addition is sequential: recombinant protein and KLT peptide@mesoporous SiO2. The stirring speed is preferably 200 rpm to avoid generating excessive bubbles. This invention does not specifically limit the stirring time; any stirring time that allows for sufficient dispersion of the recombinant protein and KLT peptide@mesoporous SiO2 is acceptable. In this invention, if the volume of printing ink after stirring is insufficient, it is preferably supplemented with PBS buffer. The printing ink is preferably degassed before printing. The present invention does not have any special limitation on the degasing step, and conventional degasing steps in the art can be used.

[0033] This invention also provides the application of the recombinant protein, the printing ink, or the printing ink prepared by the method described above in the fabrication of 4D scaffolds. The recombinant protein or printing ink described in this invention, when used to fabricate 4D scaffolds, not only possesses the efficacy of the recombinant protein but also exhibits temperature responsiveness, undergoing a phase transition near body temperature, causing the scaffold to shrink or expand in volume. Simultaneously, the addition of KLT peptide@mesoporous SiO2 enables the slow release of growth factors, delaying the duration of growth factor action, promoting cell proliferation and angiogenesis, thereby accelerating tissue repair.

[0034] The present invention also provides a 4D scaffold, which is printed using the printing ink or the printing ink prepared by the preparation method.

[0035] This invention also provides a method for fabricating the 4D scaffold, which involves printing ink based on a Gyroid-type TPMS structural model. The construction of the Gyroid-type TPMS structural model includes: setting the relative density at the center point to 40% (high material content, high rigidity, strong deformation constraint), and the relative density at the edge region to 15% (low material content, high flexibility, easy deformation). The relative density from the edge region to the center point exhibits a linear gradient; the rate of change of this linear gradient is 0.05-0.10 relative density / mm, preferably 0.05, 0.08, or 0.10 relative density / mm; the cell size is 2.0 mm. This invention does not impose any special limitations on other printing settings and specific steps; conventional steps and parameters in the art that match the Gyroid-type TPMS structural model of this invention can be used. In some embodiments, printing parameters can be set as follows: nozzle temperature 22℃; platform temperature gradient 25-28℃ (center point 25℃, transition area 26℃, edge area 28℃); printing speed: edge area 10mm / s, center point 6mm / s, transition area 7mm / s; layer thickness 0.2mm. The transition area is the area between the center point and the edge area. In this invention, based on the differentiated shrinkage characteristics of the vein structure of dead leaves, a TPMS structural model is designed. By designing the material density of different areas of the scaffold, the scaffold produces a preset anisotropic deformation under temperature stimulation, realizing morphological changes such as directional curling and pore adjustment; at the same time, the 4D scaffold has a continuous and interconnected pore network and excellent structural stability. By optimizing the TPMS unit parameters and arrangement, the scaffold maintains structural integrity during the response process, while achieving dynamic matching of mechanical properties. It can be used for tissue repair and dermal filling, promoting tissue regeneration and enabling rapid wound healing.

[0036] This invention also provides the application of the recombinant protein, the printing ink, the method for preparing the printing ink, the 4D scaffold, or the method for preparing the 4D scaffold in the preparation of tissue repair and / or dermal filler products. The prepared 4D scaffold, when implanted into tissue, can provide support and filling, while simultaneously promoting rapid tissue repair, tissue regeneration, and rapid wound healing.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1 1. Preparation of recombinant proteins

[0041] 1.2 The above gene sequence was digested using EcoRI+NotI double restriction sites and BamHI single restriction site, and then sent to Shanghai Bioengineering Co., Ltd. After full gene synthesis, it was cloned into plasmid pPIC9K to obtain the recombinant plasmid pPIC9K-COL1α1-DOPAsynthase-MMP2-siRNA. The plasmid map is shown below. Figure 1 As shown.

[0042] 1.3 Preparation of Pichia pastoris competent cells: Single colonies were picked and inoculated into YPD liquid medium and cultured overnight (18 h) at 30℃ and 220 rpm to fully activate the strain. Then, 100 μL of the bacterial culture was inoculated into 100 mL of YPD liquid medium and cultured at 30℃ and 220 rpm until OD500 was reached. 600 =1.0-1.5. Divide 100 mL of bacterial culture into two equal portions, centrifuge at 5000 rpm for 5 min, and remove the supernatant. Add 20 mL of pre-chilled sterile water, centrifuge at 5000 rpm for 5 min, and remove the supernatant. Repeat twice. Resuspend the bacterial cells in 20 mL of ice-chilled 1M sorbitol solution, centrifuge at 5000 rpm for 5 min, and remove the supernatant. Resuspend the yeast in 400 μL of pre-chilled 1M sorbitol solution, aliquot the bacterial culture into 80 μL tubes, and store at -80°C.

[0043] 1.4 Plasmid linearization: The plasmid obtained in step 1.2 was linearized using restriction endonuclease SaII, and the plasmid was recovered using a gel extraction kit.

[0044] 1.5 Electroporation of Pichia pastoris: 2 μg plasmid (obtained in step 1.4) + 80 μL competent cells (obtained in step 1.3) were mixed and pre-cooled on ice for 5 min. Electroporation was performed using an electroporator (parameters: constant voltage 1.5 kV, resistance 200 Ω, capacitance 25 μf, electric shock 5 msec). The cells were suspended in 1 M sorbitol solution and spread on resistant or auxotrophic MD plates. The plates were incubated statically at 30 °C for 60 h until obvious colonies appeared.

[0045] 1.6 PCR Identification Single colonies were picked and incubated in sterile water at 30°C for 30 min, followed by heating at 95°C for 10 min in a PCR instrument to fully release the yeast DNA, which was then used as a DNA template for PCR detection. 3 μL of the prepared DNA template was added to a 17 μL PCR reaction system, as shown in Table 1 below.

[0046] Table 1: PCR reaction system

[0047] Set the PCR reaction parameters as follows: STEP1 (initial denaturation): 94℃ for 3 min; STEP2 (denaturation): 94℃ for 30 sec; STEP3 (annealing): 55℃ for 30 sec; STEP4 (extension): 72℃ for 1 min / kb; STEP5 (cycles): 35 cycles; STEP6 (final extension): 72℃ for 10 min; STEP7 (temporary storage): 4℃ forever, to obtain the PCR product.

[0048] 1.7 Identification of PCR bands by agarose gel electrophoresis: 1% agarose gel was used for identification. 10 μL of PCR product (obtained in step 1.6) was loaded onto the gel, and electrophoresis was performed at a constant voltage of 120V for 30 min. The images were then taken under blue light to observe whether there were any target bands.

[0049] 1.8 Recombinant protein induction expression: Positive clone transformants were activated, and bacterial culture was used to control OD600 to 1. 100 mL of BMGY was inoculated, and methanol was added to the final concentration of 0.5%. Expression was induced for 120 h. The sample was collected and centrifuged (10,000 rpm for 10 min), and the supernatant was retained and stored at -80℃.

[0050] 1.9 Supernatant Purification: The supernatant obtained in step 1.8 was first subjected to fractional salting out with ammonium sulfate (according to the initial ammonium sulfate concentration table), with ammonium sulfate concentrations of 20%, 40%, 60%, 80%, and 100%. After each concentration was added, the mixture was allowed to stand and then centrifuged at 10,000 rpm for 30 min. The precipitates obtained at each ammonium sulfate concentration were collected and resuspended in 1 mL of purified water to obtain crude protein. The crude protein was desalted by dialysis and then further purified using an anion exchange column. A Q-Sepharose FastFlow anion exchange resin pre-packed column was selected and purified using a purifier. The flow rate was set to 2 mL / min, and the column was equilibrated with 5 column volumes of anion exchange column binding buffer (20 mM Tris-HCl + 1 mM DTT + 50 mM NaCl). The dialysis solution was taken and loaded at 100%. The overflow was collected, and the baseline was leveled with binding buffer. Elution was performed using a combination of binding buffer and anion elution buffer as the mobile phase. Anion elution buffer (20 mM Tris-HCl + 1 mM DTT + 50 mM NaCl) was used. The recombinant protein was eluted with a gradient of 20%, 30%, 40%, 60%, 80%, and 100% using Tris-HCl + 1mM DTT + 2M NaCl. The eluent was collected at the absorption peak at 280 nm. After further removal of endotoxin by gel filtration chromatography, the eluent was concentrated and freeze-dried to obtain pure recombinant protein with a purity >90%.

[0051] 2. Synthesis of P(NIPAM-co-AA) copolymer 7 mmol N-isopropylacrylamide, 3 mmol acrylic acid, and 0.05 mmol azobisisobutyronitrile were added to a 50 mL three-necked flask; 25 mL DMSO was added as solvent, and nitrogen gas was purged for 30 min to remove oxygen; the mixture was reacted in an oil bath at 70 °C for 8 h, and the product was poured into excess diethyl ether to precipitate; after vacuum drying, a white powdery copolymer was obtained. 1 The composition was determined by 1H NMR, and its LCST (lower critical solution temperature) was determined by DSC to be 32℃.

[0052] 3. Preparation of KLT peptide@mesoporous SiO2 3.1 Preparation of mesoporous SiO2 by sol-gel method: 1.2 g CTAB was dissolved in 60 mL deionized water, 6 mL ammonia water was added, and the mixture was stirred for 10 min; 6 mL TEOS was slowly added dropwise, and the mixture was stirred at room temperature for 24 h; 3.2 The precipitate was collected by centrifugation, washed three times with ethanol, and calcined at 550℃ for 6 hours to remove the template agent, yielding mesoporous SiO2. 3.3 Disperse 0.6 g of mesoporous SiO2 in 15 mL of 0.8 mg / mL KLT peptide solution and stir at room temperature for 20 h; 3.4 Centrifuge and freeze-dry to obtain KLT peptide@mesoporous SiO2 with a loading of 15 wt%.

[0053] 4. Fabrication of 4D scaffolds 4.1 Printing Ink Preparation (1) In a sterile environment, accurately weigh 1.4g of P(NIPAM-co-AA) copolymer, add it to 5mL of PBS buffer, place the mixture on a magnetic stirrer at 37℃, and stir at 500rpm for 2.5h until the polymer is completely dissolved and a clear solution is obtained.

[0054] (2) Take the above solution out of the 37°C water bath and transfer it to a 25°C constant temperature water bath. Continue stirring for 30 minutes to ensure that the solution temperature drops uniformly to the printing temperature.

[0055] (3) Add 0.4g of recombinant protein and 0.15g of KLT peptide@mesoporous SiO2 to the cooled solution in sequence, stir at a speed of 200rpm to avoid generating too many bubbles and to ensure that the solution is fully dispersed.

[0056] (4) Use PBS buffer to make up the total volume of the mixed solution to 10 mL to obtain printing ink.

[0057] (5) Transfer the printing ink into a syringe and place it in a centrifuge degasser. Centrifuge at 2000 rpm for 3 min at 25°C to remove air bubbles introduced during stirring. Store at 4°C for a short period.

[0058] 4.2 Structural design and 4D printing of 4D support frame (1) Based on the principle of leaf curling, a Gyroid-type TPMS structure model was constructed in nTopoLogy software. The relative density was set to 15%~40% (where the relative density at the center point was controlled at 40%; the relative density at the edge region was controlled at 15%; the relative density from the edge region to the center point was linearly gradient (from 40% to 15%), with a linear gradient rate of 0.10 relative density / mm and a cell size of 2.0 mm, generating 10 10 A 10mm 3D model is created and an STL file is generated. The model information is then converted into G-code for printing control using the 3D model slicing software SLic3r. Subsequently, the G-code is imported into the 3D printing control software for printing.

[0059] (2) Before the actual printing, attach the syringe containing printing ink to the 3D printer and print using a needle with an inner diameter of 300μm at an ambient temperature (25℃) with a fill rate of 100%.

[0060] (3) The specific printing parameters are set as follows: nozzle temperature 22℃; platform temperature gradient 25-28℃ (center point 25℃, transition area 26℃, edge area 28℃); printing speed: edge area 10mm / s, center point 6mm / s, transition area 7mm / s; layer thickness 0.2mm; (4) Post-processing: The printed scaffold was placed in a 4 mmol / L NaIO4 solution for 2 h for cross-linking, washed with PBS and then freeze-dried to obtain a 4D scaffold.

[0061] Example 2 1. Preparation of recombinant proteins Same as step 1 in Example 1.

[0062] 2. Synthesis of P(NIPAM-co-AA) copolymer 6.8 mmol N-isopropylacrylamide, 2.8 mmol acrylic acid, and 0.048 mmol azobisisobutyronitrile were added to a 50 mL three-necked flask; 25 mL DMSO was added as solvent, and nitrogen gas was purged for 30 min to remove oxygen; the mixture was reacted in an oil bath at 69 °C for 7.8 h, and the product was poured into excess diethyl ether to precipitate; after vacuum drying, a white powdery copolymer was obtained. 1 The composition was determined by 1H NMR, and its LCST (lower critical solution temperature) was determined by DSC to be 32℃.

[0063] 3. Preparation of KLT peptide@mesoporous SiO2 Same as step 3 in Example 1.

[0064] 4. Fabrication of 4D scaffolds 4.1 Printing Ink Preparation (1) In a sterile environment, accurately weigh 1.3g of P(NIPAM-co-AA) copolymer, add it to 5mL of PBS buffer, place the mixture on a magnetic stirrer at 36.8℃, and stir at 500rpm for 2h until the polymer is completely dissolved and a clear solution is obtained.

[0065] (2) Take the above solution out of the 36.8℃ water bath and transfer it to a 24.8℃ constant temperature water bath. Continue stirring for 28 minutes to ensure that the solution temperature drops uniformly to the printing temperature.

[0066] (3) Add 0.3g of recombinant protein and 0.1g of KLT peptide@mesoporous SiO2 to the cooled solution in sequence, stir at a speed of 200rpm to avoid generating too many bubbles and to ensure that the solution is fully dispersed.

[0067] (4) Use PBS buffer to make up the total volume of the mixed solution to 9 mL to obtain printing ink.

[0068] (5) Transfer the printing ink into a syringe and place it in a centrifuge degasser. Centrifuge at 2000 rpm for 2 min at 24.8℃ to remove air bubbles introduced during stirring. Store at 4℃ for a short period.

[0069] 4.2 Structural design and 4D printing of 4D support frame (1) Based on the principle of leaf curling, a Gyroid-type TPMS structure model was constructed in nTopoLogy software. The relative density was set to 15%~40% (where the relative density at the center point was controlled at 40%; the relative density at the edge region was controlled at 15%; in the transition region, the relative density from the edge region to the center point gradually changed linearly (from 40% to 15%), with a linear change rate of 0.05 relative density / mm and a cell size of 2.0mm, generating 10 10 A 10mm 3D model is created and an STL file is generated. The model information is then converted into G-code for printing control using the 3D model slicing software SLic3r. Subsequently, the G-code is imported into the 3D printing control software for printing.

[0070] (2) Before the actual printing, attach the syringe containing printing ink to the 3D printer and print using a needle with an inner diameter of 300μm at an ambient temperature (24.8℃) with a fill rate of 100%.

[0071] (3) The specific printing parameters are set as follows: nozzle temperature 22℃; platform temperature gradient 25-28℃ (center point 25℃, transition area 26℃, edge area 28℃); printing speed: edge area 10mm / s, center point 6mm / s, transition area 7mm / s; layer thickness 0.2mm; (4) Post-processing: The printed scaffold was placed in a 4 mmol / L NaIO4 solution for cross-linking for 2 h, washed with PBS and then freeze-dried to obtain a 4D scaffold.

[0072] Example 3 1. Preparation of recombinant proteins Same as step 1 in Example 1.

[0073] 2. Synthesis of P(NIPAM-co-AA) copolymer 7.2 mmol N-isopropylacrylamide, 3.2 mmol acrylic acid, and 0.052 mmol azobisisobutyronitrile were added to a 50 mL three-necked flask; 25 mL DMSO was added as solvent, and nitrogen gas was purged for 30 min to remove oxygen; the mixture was reacted in an oil bath at 71 °C for 8.2 h, and the product was poured into excess diethyl ether to precipitate; after vacuum drying, a white powdery copolymer was obtained. 1 The composition was determined by 1H NMR, and its LCST (lower critical solution temperature) was determined by DSC to be 32℃.

[0074] 3. Preparation of KLT peptide@mesoporous SiO2 Same as step 3 in Example 1.

[0075] 4. Fabrication of 4D scaffolds 4.1 Printing Ink Preparation (1) In a sterile environment, accurately weigh 1.5g of P(NIPAM-co-AA) copolymer, add it to 5mL of PBS buffer, place the mixture on a magnetic stirrer at 37.2℃, and stir at 500rpm for 3h until the polymer is completely dissolved and a clear solution is obtained.

[0076] (2) Take the above solution out of the 37.2℃ water bath and transfer it to a 25.2℃ constant temperature water bath. Continue stirring for 32 minutes to ensure that the solution temperature drops uniformly to the printing temperature.

[0077] (3) Add 0.5g of recombinant protein and 0.2g of KLT peptide@mesoporous SiO2 to the cooled solution in sequence, stir at a speed of 200rpm to avoid generating too many bubbles and to ensure that the solution is fully dispersed.

[0078] (4) Use PBS buffer to make up the total volume of the mixed solution to 11 mL to obtain printing ink.

[0079] (5) Transfer the printing ink into a syringe and place it in a centrifuge degasser. Centrifuge at 2000 rpm for 2 min at 25.2℃ to remove air bubbles introduced during stirring. Store at 4℃ for a short period.

[0080] 4.2 Structural design and 4D printing of 4D support frame (1) Based on the principle of leaf curling, a Gyroid-type TPMS structure model was constructed in nTopoLogy software, with a relative density of 15%~40%; the relative density of the edge region was controlled at 15%; the relative density from the edge region to the center point was linearly gradual (from 40% to 15%), with a linear change rate of 0.08 relative density / mm and a cell size of 2.0mm. A 10×10×10mm three-dimensional model was generated and an STL file was generated. The model information was converted into G code for printing control using the three-dimensional model slicing software SLic3r. Subsequently, the G code was imported into the 3D printing control software for printing.

[0081] (2) Before the actual printing, attach the syringe containing printing ink to the 3D printer and print using a needle with an inner diameter of 300μm at an ambient temperature (25.2℃) with a fill rate of 100%.

[0082] (3) The specific printing parameters are set as follows: nozzle temperature 22℃; platform temperature gradient 25-28℃ (center point 25℃, transition area 26℃, edge area 28℃); printing speed: edge area 10mm / s, center point 6mm / s, transition area 7mm / s; layer thickness 0.2mm; (4) Post-processing: The printed scaffold was placed in a 4 mmol / L NaIO4 solution for cross-linking for 2 h, washed with PBS and then freeze-dried to obtain a 4D scaffold.

[0083] Comparative Example 1 The only difference from Example 1 is that step 1 is omitted, and the 0.4g recombinant protein in step 4.1 is replaced with an equal amount of type I collagen (purchased from Guangzhou Huayun Biotechnology Co., Ltd.). All other aspects are the same as in Example 1.

[0084] Comparative Example 2 The only difference from Example 1 is that step 1 is omitted, and the 0.4g recombinant protein in step 4.1 is replaced with an equal amount of fibronectin (purchased from Guangzhou Huayun Biotechnology Co., Ltd.). All other aspects are the same as in Example 1.

[0085] Comparative Example 3 The only difference from Example 1 is that step 1 is omitted, and the 0.4g recombinant protein in step 4.1 is replaced with an equal amount of laminin (purchased from Guangzhou Huayun Biotechnology Co., Ltd.). All other aspects are the same as in Example 1.

[0086] Experimental Example 1 1. Detection of recombinant proteins Example 1: The recombinant protein pPIC9K-COL1α1-DOPA synthase-MMP2-siRNA (pPIC9K-CLO-DOPA-MMP2) prepared in step 1 has a theoretical molecular weight of 33 kDa. SDS-PAGE electrophoresis showed that the molecular weight of the recombinant protein was around 50 kDa, close to the theoretical molecular weight. The SDS-PAGE gel image is shown below. Figure 2 As shown.

[0087] 2. Morphological characterization results of the P(NIPAM-co-AA) copolymer hydrogel prepared in Example 1 The P(NIPAM-co-AA) copolymer was sent to a testing institution for morphological analysis. Scanning electron microscopy was used for testing, and the results are as follows: Figure 3 As shown, the hydrogels all exhibit a honeycomb structure inside, and the structure is evenly distributed.

[0088] 3. Rheological testing of printing ink 3.1 Rheological Testing Methods (1) Before testing, place the sample (printing ink, prepared in step 4.1 of Example 1) in a 6°C refrigerator for 30 min. Take the sample precursor solution (printing ink is in a fluid state at 4°C and in a hydrogel state (similar to a solid) at human body temperature (37°C), so it usually needs to be stored at low temperature to restore the fluid state before use for the convenience of the experiment) and add it to the parallel plate of the rheometer.

[0089] (2) Temperature scanning test: The test mode was set to modulus test, the modulus test was fixed strain, the constant frequency was 1Hz, the constant strain (stress) was 0.5Pa, the test temperature range was room temperature to 200℃, the pH value was 4.83, and the changes in the storage modulus and loss modulus of the hydrogel within 1 hour were explored. (3) Time scan test: The test mode is set to modulus test, the modulus test is fixed strain, the constant frequency is 1Hz, the constant strain (stress) is 0.5Pa, the test time is 1 hour, and the changes in the storage modulus and loss modulus of the hydrogel within 1 hour are explored.

[0090] 3.2 Rheological test results (1) Temperature scan test results The temperature-sensitive solution-gel transition behavior of P(NIPAM-co-AA) printing ink was observed through temperature scanning tests. The results are as follows: Figure 4 As shown, at low temperatures (<25℃), the loss modulus (G) Slightly higher than the energy storage modulus (G The system exhibits a viscous liquid state with good fluidity. As the temperature increases, G... With G All began to rise rapidly and crossed at 26°C (G >G This temperature point is defined as the gel point. Afterwards, G... The sustained and significant increase indicates that a robust three-dimensional network structure is forming. At 37°C (human body temperature), G... The value reached approximately 15 Pa, indicating that the gel has sufficient mechanical strength.

[0091] (2) Time scan test results The kinetics and stability of the printing ink were tested using time-scan testing. The results are as follows: Figure 5 As shown, when the temperature jumps from 25℃ to 37℃, the storage modulus (G) increases. The modulus increased rapidly within 10 minutes and reached equilibrium within 15 minutes, indicating that the sample has rapid gel kinetics; the modulus remained stable and showed no decay phenomenon after continuous measurement at 37°C for 30 minutes, indicating that the sample has good thermal stability.

[0092] 4. 4D Bionic Scaffold Dynamic Deformation Performance Test Results The dynamic deformation performance of the 4D biomimetic scaffold was tested, examining the curling angle, equilibrium time, and deformation reversibility of the 4D scaffold prepared in Example 1 at different temperatures. A morphological tracking method under temperature control was employed, placing the scaffold in constant temperature incubators at different temperatures (25℃, 32℃, 37℃, and 40℃), and recording the scaffold morphological changes at regular intervals. The curling angle was measured using ImageJ image analysis software. The experimental results are shown in Table 2.

[0093] Table 2 Dynamic Deformation Performance Data of 4D Bionic Scaffold

[0094] According to Table 2, the stent is initially planar at 25℃; after 30 minutes at 37℃, the stent curls up to a 65° shape and returns to a planar shape within 15 minutes after cooling down to 25℃; after 20 minutes at 40℃, the stent curls up to a 90° shape and returns to a planar shape within 20 minutes after cooling down to 25℃.

[0095] 5. Test results of mechanical properties of the 4D bionic scaffold Mechanical properties of the 4D biomimetic scaffold were tested, including the compressive modulus, mechanical strength loss rate during the curling process, and pore connectivity of the 4D scaffolds prepared in Example 1 and Comparative Examples 1-3. A uniaxial compression test was conducted, with the scaffold sample (10mm × 10mm × 2mm) placed between the upper and lower clamps of a mechanical testing machine, and a compressive load applied at a rate of 1mm / min. The test data were recorded. A fluid permeation method was used, measuring the amount of liquid permeating through the scaffold under constant pressure, and calculating the proportion of connected pores based on the scaffold volume. The results are shown in Table 3.

[0096] Table 3. Test results of mechanical properties of the 4D bionic scaffold

[0097] As shown in Table 3, the porosity, pore size, and pore connectivity of all experimental groups remained highly consistent and at an excellent level. However, the compressive modulus of the 4D scaffold prepared by this invention (38 MPa) was significantly higher than that of all other 4D scaffolds prepared by proteins. This indicates that the unique structural domains (especially hydrophobic domains) in the recombinant protein and the temperature-sensitive matrix in this invention have a synergistic effect at the molecular level, which strengthens the polymer network structure. The mechanical strength loss rate during the curling process (10.12%) was the only group below 15%, especially the 4D scaffold prepared by laminin, which had a mechanical strength loss rate as high as 50.46% during the curling process. This indicates that this invention can adapt to and stabilize the dynamic shape change process caused by temperature changes.

[0098] 6. KLT peptide release performance test results The release performance of KLT peptide was assessed, and the cumulative release rate of KLT peptide from the 4D scaffold prepared in Example 1 was determined at different time points. High-performance liquid chromatography (HPLC) was used (equipment: Shimadzu LC-2050). The scaffold sample was placed in 5 mL of PBS buffer (pH 7.4, 37℃) for the release experiment. Samples were taken periodically (with an equal volume of fresh buffer added simultaneously). The concentration of KLT peptide in the sample was detected by HPLC, and the cumulative release rate (total release at each time point / initial loading) was calculated. The results are shown in Table 4.

[0099] Table 4. Cumulative release rate of KLT peptide

[0100] As can be seen from Table 4, the KLT peptide release rate of the 4D scaffold in Example 1 showed a slow upward trend, with a cumulative release rate of 82.5% after 28 days, and no obvious burst release phenomenon, indicating that the 4D scaffold of the present invention has good sustained-release performance of KLT peptide.

[0101] 7. Cell compatibility test results Cell compatibility testing was performed to detect the proliferation rate of human dermal fibroblasts. The CCK-8 assay was used to analyze human dermal fibroblasts at a concentration of 1×10⁻⁶ cells / cells. 4 Cells were seeded at densities of 200 μL / well on 4D scaffolds prepared in Examples 1, 1, 2, and 3, respectively. After culturing for 1, 3, 7, and 14 days, CCK-8 reagent (10 μL / well) was added and the cells were incubated at 37°C for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. The proliferation rate was calculated using the blank group (simple cell suspension) as a control.

[0102] Table 5. Cell compatibility test data (proliferation rate of scaffolds in BMSCs)

[0103] The results are shown in Table 5. The proliferation rate of the 4D scaffold in Example 1 was significantly higher than that of all comparative examples and the blank group on day 14, indicating that the 4D scaffold of the present invention has a strong ability for long-term cell proliferation and can provide a microenvironment for rapid cell growth.

[0104] 8. Results of rat skin wound repair experiment Fifteen rats were selected and divided into three groups of five each: a control group, Example 1 group, and Comparative Example 3 group. In the control group, no treatment was performed on the 1cm × 1cm skin defect. In Example 1 group and Comparative Example 3 group, the scaffolds from Example 1 and Comparative Example 3 were implanted into the 1cm × 1cm skin defect, respectively. The appearance of the wound was observed at different time points after implantation. The results showed that the wound in Example 1 group shrank to 0.15cm after 7 days. 2 The wound healed completely in 14 days; in the control group (group 3), 0.1 cm of wound remained after 14 days. 2 Masson staining of the skin tissue after healing showed that the collagen fibers in the experimental group were neatly arranged, close to normal skin, while the control group showed disordered collagen deposition (scarring). The wound healing rate (healed area / initial wound area × 100%) was calculated, and the results are shown in Table 6. The healing rate of Example 1 group was 100% after 14 days, while that of Comparative Example 3 group was 90%, indicating that the wound healing rate of the scaffold of the present invention was significantly higher than that of the 4D scaffold prepared in Comparative Example 3.

[0105] Table 6 Healing rate of skin wound repair scaffolds in rats

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant protein, characterized in that, The gene sequence of the recombinant protein is shown as SEQ ID NO.

1.

2. A printing ink, characterized by The printing ink comprises the recombinant protein, the P(NIPAM-co-AA) copolymer and the KLT peptide@mesoporous SiO2 according to claim 1, and the weight ratio of the recombinant protein, the P(NIPAM-co-AA) copolymer and the KLT peptide@mesoporous SiO2 is (0.3-0.5):(1.3-1.5):(0.1-0.2). The KLT peptide@mesoporous SiO2 is obtained by loading the KLT peptide into the mesoporous SiO2.

3. The printing ink according to claim 2, characterized in that, The P(NIPAM-co-AA) copolymer is obtained by radical copolymerization of N-isopropyl acrylamide and acrylic acid under the action of azobisisobutyronitrile, and the molar concentration ratio of the N-isopropyl acrylamide, the acrylic acid and the azobisisobutyronitrile is (6.8-7.2):(2.8-3.2):(0.048-0.052).

4. The printing ink according to claim 3, characterized in that, The reaction temperature of the radical copolymerization is 69-71℃, and the reaction time is 7.8-8.2h.

5. The printing ink according to claim 2, characterized in that, The KLT peptide loading rate of the KLT peptide@mesoporous SiO2 is 14-16wt%.

6. A method of preparing a printing ink according to any one of claims 2 to 5, characterised in that, The method comprises the following steps: The P(NIPAM-co-AA) copolymer is mixed with the PBS buffer solution, and then the recombinant protein and the KLT peptide@mesoporous SiO2 are added and stirred until the solute is dispersed.

7. The application of the recombinant protein according to claim 1, the printing ink according to any one of claims 2-5 or the printing ink prepared by the method according to claim 6 in the preparation of a 4D scaffold.

8. A 4D stent, characterized in that, The printing ink is printed by using the printing ink according to any one of claims 2-5 or prepared by the method according to claim 6.

9. The method of claim 8, wherein the 4D scaffold is prepared by, The printing ink is printed based on a Gyroid-type TPMS structure model, and the construction of the Gyroid-type TPMS structure model comprises: setting the relative density of the center point as 40%, the relative density of the edge region as 15%, the relative density of the edge region to the center point as linearly changing, and the change rate of the linear change as 0.05-0.10 relative density / mm; and the unit cell size as 2.0mm.

10. The application of the recombinant protein according to claim 1, the printing ink according to any one of claims 2-5, the printing ink according to claim 6, the method for preparing the 4D scaffold according to claim 8 or the method for preparing the 4D scaffold according to claim 9 in the preparation of a tissue repair and / or dermal filling product.

Images