Functional combined peptide for bone tissue repair as well as preparation method and application of functional combined peptide

Abstract

The invention discloses a functional combined peptide for bone tissue repair as well as a preparation method and application of the functional combined peptide. The functional peptide mainly solves the technical problems that in the existing bone tissue repair field, the function of a single short peptide is limited, the multi-link cooperative regulation capacity is insufficient, the preparation process efficiency of part of short peptides is low, and the product stability is poor, and the like, according to the technical scheme, the functional peptide for bone tissue repair is composed of a KTTKS short peptide, an FVAPFP short peptide and a VGVAPG short peptide according to the mass ratio of 1: 1: 1; according to the preparation method, an Fmoc solid-phase synthesis method is adopted and is used for synthesizing three short peptides, namely KTTKS, FVAPFP and VGVAPG. The three oligopeptides are used for promoting cell proliferation and differentiation, and an environment beneficial to bone tissue repair is created.

Description

Technical Field

[0001] This invention relates to the intersection of peptide preparation methods and applications, specifically a functional combinatorial peptide for bone tissue repair, its preparation method, and its applications. This functional peptide can be applied to the functional optimization of bone tissue engineering scaffold materials, the development of bone defect repair implants, and the production of bioactive preparations related to bone repair. It aims to solve technical challenges in the current field of bone repair, such as insufficient bioactivity of materials, limited cell regulation capabilities, and poor compatibility with the human physiological environment, providing a highly efficient and safe bioactive molecular system for bone tissue repair. Background Technology

[0002] In bone tissue engineering research, the synergistic effect of scaffold materials and bioactive molecules is the core factor determining the bone repair effect. Self-assembled short peptide hydrogels have become an important research direction for bone tissue engineering scaffold materials due to their unique advantages. Their porous structure formed by interwoven nanofibers has a pore size similar to that of the natural extracellular matrix (ECM), which can provide suitable channels for cell migration, nutrient exchange and metabolic product excretion; moreover, its main component is amino acids, and its degradation products are also amino acids, which can be naturally absorbed and utilized by the human body, without immunogenicity, and can effectively avoid the occurrence of local or systemic adverse reactions in the host. More importantly, the degradation rate of self-assembled short peptide hydrogels can be achieved by regulating the peptide sequence and structure, so that it can be precisely matched with the bone tissue growth rate, providing continuous support for the gradual formation of new bone tissue (Zhang Feng, Ren Lingfei, Ying Yongfang, et al. Study on osteoblast culture on the surface of seripro- ... In addition, this type of hydrogel can further induce osteoblast differentiation and new bone formation by introducing bioactive molecules or applying mechanical stimulation, and has good functional plasticity.

[0003] Existing research has shown that various short peptides have the potential to regulate cell function or promote tissue repair, but the function of a single short peptide in bone repair still has limitations. For example, KTTKS (lysine-threonine-threonine-lysine-serine), a pentapeptide derived from human type I collagen, can significantly promote chondrocyte proliferation and stimulate collagen and proteoglycan synthesis after lipolysis modification. It has been attempted for the treatment of osteoarthritis (Zhuang Guosheng. Use of penta / hexapeptide KTTKS(F) in the treatment of osteoarthritis [P]. Chinese Patent, A, 201610623696. 2, 2018-02-09.). In vitro experiments have also shown that KTTKS and C16-KTTKS can effectively promote osteoblast secretion of type I collagen. As the main structural component of bone tissue, the amount of collagen synthesized directly affects the strength and repair efficiency of bone tissue (Katayama, K., Armendariz, Borunda, J., Raghow, R., et al. A Pentapeptide from Type I Procollagen Promotes Extracelullar Matrix Production [J]. Biol. Chem. 1993, 268: 994). To enhance the activity of KTTKS, researchers designed derivatives such as SKTTKS-V4A3-SKTTKS (P3) and SKTTK-V4A2-KTTKS (P5) through molecular modeling. These derivatives can maintain the surface electrostatic distribution required for structural stability and function, have low toxicity to human adipose-derived stem cells (hADSCs), and can increase the yield of collagen and fibronectin in a concentration-dependent manner. However, the preparation process of such derivatives is complex and costly, making it difficult to apply on a large scale (Behzad, Seyfi., Nasser, Fatouraee., Abbas, Samani. A novel micro-to-macro structural approach for mechanical characterization of adipose tissue extracellular matrix[J]. Journal of the Mechanical Behavior of Biomedical Materials. 2018, 77.).

[0004] FVAPFP (phenylalanine-valine-alanine-proline-phenylalanine-proline) is a hexapeptide isolated from yeast extract and purified via solid-phase synthesis. Studies have shown that this peptide has no significant toxicity in normal human lung and skin fibroblasts and can promote Nrf2 nucleus accumulation, increase proteasome protein subunit expression levels and peptidase activity, thereby activating fibroblast repair function by regulating the protein homeostasis network (Aimilia, Sklirou, D., Marianna, Ralli., Ioannis, P. Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts[J]. Redox Biology. 2015, 5: 205). Fibroblasts are key cells for soft tissue repair surrounding bone, and their functional activation can create a favorable local microenvironment for bone repair. However, the regulatory effect of a single FVAPFP peptide on osteoblast differentiation is weak and cannot meet the multi-cell synergistic needs of bone tissue repair.

[0005] VGVAPG (valine-glycine-valine-alanine-proline-glycine) is a core repetitive hexapeptide sequence in elastin molecules. It binds with high affinity to elastin-binding protein (EBP) on the cell surface, transducing signals into the cell to regulate cell migration, chemotaxis, and mitosis. As a typical matrix-derived signal peptide, VGVAPG participates in various physiological processes such as inflammatory responses, wound repair, and angiogenesis. Experiments have shown that it can increase the expression of matrix metalloproteinases (MMPs) in connective tissue. MMPs play a key role in extracellular matrix remodeling, providing space for new bone formation by degrading old matrix (Michael, Wells, J., Amit, Gaggar. MMP generatedmatrikines[J]. Matrix Biology. 2015, 3: 44.). In addition, elastin mimic peptides containing VGVAPG (such as EM-19 and EM-23) can mimic the in vivo elastic fiber assembly mechanism through the synergistic effect of EBP and αvβ3 integrin, promoting smooth muscle cell adhesion and elastin deposition. However, the ability of such peptides to bind specifically to bone tissue and induce osteogenic formation still needs to be improved (Dhaval, Patel., Susan, Vandromme, E., Michael, Reid, E., et al. Activity of αvβ3 Integrins and the Elastin Binding Protein Enhance Cell-Matrix Interactions on Bioactive Hydrogel Surfaces[J]. Biomacromolecules. 2012, 13(5): 1420; Xing, Zhang., Bin, Xu., Yan, Wu., et al. Integrating valve-inspired design features into poly(ethylene glycol) hydrogelscaffolds for heart valvetissue engineering[J]. Acta Biomaterialia. 2015, 14: 11.).

[0006] In summary, existing technologies using single short peptides cannot simultaneously meet the multi-stage requirements of bone repair, including collagen synthesis, fibroblast activation, and extracellular matrix remodeling. Furthermore, some short peptide preparation processes suffer from low coupling efficiency, unstable product purity, and high costs. Moreover, achieving synergistic effects among multiple functional short peptides and ensuring their stable activity within the bone repair microenvironment remains a critical technological bottleneck. Therefore, developing a novel functional peptide system that is synergistically functional, efficiently prepared, and cost-effective is of great significance for promoting the clinical translation of bone tissue repair technologies. Summary of the Invention

[0007] Addressing the limitations of single short peptides in existing bone tissue repair technologies, such as insufficient synergistic regulation of multiple processes, low efficiency in the preparation of some short peptides, and poor product stability, the core objective of this invention is to provide a functional combination peptide for bone tissue repair, its preparation method, and its applications. By screening and combining three short peptides with complementary functions—KTTKS, FVAPFP, and VGVAPG—synergistic regulation of cell adhesion, proliferation, differentiation, and extracellular matrix synthesis during bone repair is achieved. Simultaneously, the Fmoc solid-phase synthesis process is optimized to improve peptide preparation efficiency and product purity, reduce production costs, and ultimately provide a functional peptide system with high bioactivity, strong adaptability, and scalable application for bone defect repair.

[0008] The technical solution of this invention is as follows: the functional combination peptide for bone tissue repair is composed of three short peptides, KTTKS, FVAPFP, and VGVAPG, combined in a specific ratio. The three short peptides have complementary functions and form a synergistic mechanism, as detailed below: KTTKS short peptide: The amino acid sequence is lysine-threonine-threonine-lysine-serine, and the chemical formula is C1. 23 H 45 N7O9, with a precise molecular weight of 563.64, is a peptide that specifically promotes the synthesis of type I collagen in tissues, while stimulating the secretion of extracellular matrix proteins such as fibronectin. This provides structural support for bone tissue and enhances cell-matrix adhesion, laying the foundation for cell proliferation and differentiation.

[0009] FVAPFP short peptide: The amino acid sequence is phenylalanine-valine-alanine-proline-phenylalanine-proline, and the chemical formula is C0. 36 H 48N6O7, with a precise molecular weight of 676.8. This peptide can promote fibroblast regeneration and functional activation by activating the Nrf2 signaling pathway, accelerate the repair of soft tissues surrounding bone, maintain cellular protein homeostasis, reduce oxidative stress-induced cell damage, and create a favorable local microenvironment for bone repair. 3. VGVAPG short peptide: The amino acid sequence is valine-glycine-valine-alanine-proline-glycine, and the chemical formula is C0. 22 H 38 N6O7, with a precise molecular weight of 498.57, is a peptide that can bind to EBP and αvβ3 integrin on the cell surface, transmitting osteogenic induction signals, promoting osteoblast migration and differentiation, while regulating MMP expression and participating in extracellular matrix remodeling to match the matrix renewal rate with the bone tissue growth rate.

[0010] Table 1 compares the core parameters and functions of the three short peptides:

[0011] To achieve optimal synergistic effects, the mass ratio of the three short peptides, KTTKS, FVAPFP, and VGVAPG, was set at 1:1:1. This ratio has been verified through multiple cell and animal experiments, ensuring that each peptide can fully exert its function while avoiding cytotoxicity or functional antagonism caused by excessive amounts of a single peptide, thus maximizing bone repair efficiency.

[0012] This invention utilizes the Fmoc solid-phase synthesis method to prepare three short peptides: KTTKS, FVAPFP, and VGVAPG. This method significantly improves amino acid coupling efficiency and product purity through precise control of reaction parameters and optimization of washing and detection steps. The specific steps are as follows: Preparation of raw materials and reagents Resin: Rink Amide MBHA resin was selected as the carrier for solid-phase synthesis, which has good chemical stability and amino acid loading capacity. Amino acids: Fmoc-Lys(Boc)-OH (lysine), Fmoc-Thr(tBu)-OH (threonine), Fmoc-Ser(tBu)-OH (serine), Fmoc-Phe-OH (phenylalanine), Fmoc-Val-OH (valine), Fmoc-Ala-OH (alanine), Fmoc-Pro-OH (proline), Fmoc-Gly-OH (glycine); Solvents and reagents: Dichloromethane (DCM, analytical grade, used for amino acid activation and cleaning), N,N-dimethylformamide (DMF, analytical grade, reaction solvent and cleaning agent), 1-hydroxybenzotriazole (HOBT, analytical grade, carboxyl activator, to improve coupling efficiency), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, analytical grade, coupling activator), N,N-diisopropylethylamine (DIEPA, analytical grade, to adjust the pH of the reaction system and inhibit amino acid racemization), piperidine (analytical grade, to remove Fmoc protecting groups), trifluoroacetic acid (TFA, analytical grade, to cleave peptides and resin), icy diethyl ether (analytical grade, for product precipitation), deionized water (ultrapure water grade, for impurity removal).

[0013] Specific preparation steps Activate the resin and couple the first amino acid (AA1). Accurately weigh Rink Amide MBHA resin and add it to a solid-phase synthesis tube, ensuring uniform resin spreading. Based on the target short peptide sequence, weigh the first amino acid (AA1), with a molar amount 1.2-1.5 times the resin loading, preferably 1.5 times; add it to the solid-phase synthesis tube. Add DCM to the tube and gently shake to fully disperse the resin and AA1. Then add DIEPA and adjust the pH of the reaction system to 8.0-8.5 (this pH range maximizes coupling efficiency and reduces amino acid racemization). Cap the solid-phase synthesis tube and place it on a shaker at a speed of 120-150 r / min, preferably 130 r / min; shake the reaction to ensure sufficient reaction between AA1 and the amino groups on the resin surface.

[0014] Cleaning residual amino acids After the reaction is complete, the reaction solution in the solid-phase synthesis tube is removed by vacuum pump to avoid residual liquid affecting subsequent steps. DCM is added to the tube and it is shaken on a shaker to fully dissolve the uncoupled AA1 in the DCM. The above washing operation is repeated 5 times. After each washing, the final washing solution is collected and detected by thin-layer chromatography (TLC): the developing solvent is chloroform-methanol. Under ultraviolet light, if there are no characteristic spots of AA1, it indicates that the residual amino acids have been cleaned. If there are still spots, it is necessary to continue washing 1-2 more times.

[0015] Blocked resin A mixed blocking solution was prepared according to a volume ratio of DCM:MeOH:DIEPA = 8:1.5:0.5. This blocking solution can bind to unreacted active sites (amino groups) on the resin to form stable amide bonds, preventing subsequent non-specific coupling of amino acids. After filtering the liquid in the solid-phase synthesis tube, the blocking solution was added first, and the tube was placed on a shaker to ensure full contact between the blocking solution and the resin. The blocking solution was then removed by filtration, and the remaining blocking solution was added. Shaking was continued to ensure complete blocking of the active sites on the resin surface. After blocking, a small amount of resin was taken and tested using the ninhydrin colorimetric method: if the resin did not show color, the blocking efficiency was ≥98%; if color was shown, the blocking solution needed to be added and the shaking time extended.

[0016] Cleaning resin After removing the liquid from the blocking reaction, add DMF to the solid-phase synthesis tube and shake it on a shaker to fully dissolve the remaining blocking solution on the resin surface in the DMF. Filter to remove the DMF, and repeat the above cleaning operation 5 times. Collect the cleaning solution after each cleaning and detect it using high performance liquid chromatography (HPLC) (column: C18 column, mobile phase: acetonitrile-water = 30:70 v / v, detection wavelength 254 nm). If there are no characteristic peaks of the blocking solution (MeOH, DIEPA) in the cleaning solution, it indicates that the resin has been cleaned. If there are characteristic peaks, it is necessary to continue cleaning 2-3 times.

[0017] Deprotection of Fmoc Preparation of a 20% (w / w) piperidine solution: Mix pure piperidine with DMF, stir thoroughly, and use immediately (piperidine is volatile, so the concentration must be accurate); add the 20% (w / w) piperidine solution to the solid-phase synthesis tube, place it on a shaker and oscillate to initially remove the Fmoc protecting group on the AA1 amino group (the Fmoc protecting group is an amino protecting group and must be removed before the next coupling step); filter to remove the piperidine solution, add another 20% (w / w) piperidine solution, and continue oscillation to ensure complete removal of the Fmoc protecting group (Fm... The Fmoc protecting group removal reaction is a two-step reaction. In the first step, the protecting group is rapidly removed to generate dibenzo-fullen. In the second step, dibenzo-fullen reacts with piperidine to generate a stable product. After the reaction, the piperidine solution is removed by filtration. DMF is added to the tube, and the mixture is shaken and washed five times. The last washing solution is collected and detected by ultraviolet spectrophotometry (detection wavelength 290 nm, the characteristic absorption wavelength of the Fmoc protecting group). If the absorbance value is ≤0.02, it indicates that the Fmoc protecting group has been completely washed away. If the absorbance value is >0.02, it is necessary to continue washing 2-3 times.

[0018] Coupled with the second amino acid (AA2) According to the target short peptide sequence, weigh the second amino acid (AA2) in a molar amount that is 1.2-1.5 times the resin loading, preferably 1.5 times; at the same time, weigh 0.15 mmol of HOBT and 0.15 mmol of AA2 and place them in an EP tube. Add DMF to the EP tube and place it on a vortex mixer to shake until AA2, HOBT, and TBTU are completely dissolved to form a homogeneous reaction solution (HOBT and TBTU can synergistically activate the carboxyl group of AA2, improve coupling efficiency, and reduce racemization). After the deprotected reaction solution in the solid-phase synthesis tube is filtered clean, the above-dissolved reaction solution is added, and DIEPA is added at the same time to adjust the pH of the reaction system to 8.0-8.5. Cover the solid phase synthesis tube and place it on a shaker to allow the activated carboxyl group of AA2 to fully react with the amino group of AA1 on the resin. After the reaction was completed, the coupling efficiency was detected by HPLC: a small amount of resin was taken, cut with TFA and detected. If the coupling efficiency was ≥95%, the next step was carried out; if the coupling efficiency was <95%, AA2, HOBT, TBTU and DIEPA were added, and the mixture was shaken and detected again until the coupling efficiency was ≥95%.

[0019] (7) Couple subsequent amino acids (until the last amino acid is linked) Repeat the operation process of steps (4)-(6), namely "cleaning resin → removing Fmoc protection → coupling the next amino acid", and couple the next amino acid in sequence according to the target short peptide sequence (KTTKS: Ser→Lys→Thr→Thr→Lys; FVAPFP: Phe→Pro→Ala→Val→Phe→Pro; VGVAPG: Gly→Pro→Ala→Val→Gly→Val); After each amino acid is coupled, the coupling efficiency must be detected by HPLC to ensure that the coupling efficiency of each step is ≥95% (if the coupling efficiency of a certain step is not up to standard, it must be coupled again according to the addition method in step (6)). For the last amino acid, no Fmoc deprotection is required after coupling (if the last amino acid is a drug molecule, deprotection is also not required), and proceed directly to the next washing step.

[0020] Cleaning of target peptides After the last amino acid is coupled, the target peptide is subjected to a multi-step washing process to remove residual coupling reagents, unreacted amino acids, and protecting group fragments: Step 1: Clean with DMF 5 times, shaking each time to remove residual AA, HOBT, and TBTU; Step 2: If the last linker is an amino acid (non-drug molecule), add a 20% piperidine solution and shake. After filtration, add another 20% piperidine solution and shake to remove the Fmoc protecting group of the last amino acid. Then wash with DMF 5 times. If the last linker is a drug molecule, piperidine washing is not required. Step 3: Wash with DCM 5 times, shaking each time to remove residual DMF and piperidine; after washing, use HPLC to detect the washing solution (chromatographic conditions are the same as in step (4)). If there are no impurity peaks in the washing solution, it indicates that the target peptide has been cleaned.

[0021] Resin cleavage (separation of peptides from resin) Remove the resin (with the target peptide bound to its surface) from the solid-phase synthesis tube, scrape it clean with a clean scraper to avoid resin residue, and transfer it to a round-bottom flask. Add TFA (TFA can break the amide bond between the peptide and the resin, and at the same time remove the protecting group of the amino acid side chain, such as Boc, tBu) to the flask, place it on a magnetic stirrer, and set the speed to 300-350 r / min, preferably 320 r / min; stir at room temperature for 1.5 hours (the stirring speed should be moderate, too fast will easily cause the resin to agglomerate, and too slow will result in insufficient cutting). After stirring, the filtrate (containing the target peptide) was collected into another clean round-bottom flask by vacuum filtration. The resin was washed three times with a small amount of TFA until the washing solution obtained by vacuum filtration was colorless and transparent (indicating that the peptide had been completely cut off from the resin). The washing solution was combined with the main filtrate to ensure that the peptide was completely collected.

[0022] Rotary evaporation to concentrate and cut products The combined filtrate was transferred to a flask in a rotary evaporator. The rotary evaporator was connected, and the water bath heating temperature was set to 65°C (this temperature allows TFA to evaporate rapidly without damaging the peptide structure). The rotation speed was 60-80 r / min, preferably 70 r / min. Turn on the rotary evaporator and continue rotary evaporation until a uniform thin film without obvious liquid droplets is observed to form on the inner wall of the flask (at this point, most of the TFA has been removed, and the residual TFA content is ≤5%). Turn off the rotary evaporator and wait for the flask to cool to room temperature before removing it. The flask will then contain the concentrated peptide product.

[0023] Product precipitation and centrifugal purification Add ice-cold ether (ice-cold ether temperature is -20℃, which can reduce the solubility of peptides and promote precipitation) slowly, in small amounts, to the concentrated product after rotary evaporation. The dropping rate is controlled at 1-2 mL / min, preferably 1.5 mL / min; the total amount is 5-8 times the volume of the concentrated product. Gently shake the flask during the dropwise addition process to ensure thorough mixing of the icy ether with the concentrated product until a white flocculent precipitate (target peptide) appears at the bottom of the flask. Use a clean scraper to scrape off the precipitate and transfer it to an EP tube; add an equal weight of deionized water to the EP tube, gently shake to disperse the precipitate, and balance the tube (ensure that the EP tube is subjected to uniform force during centrifugation). Place the EP tube into a centrifuge. After centrifugation, carefully discard the supernatant (containing residual TFA, protective group fragments, and other impurities) and collect the precipitate at the bottom of the centrifuge tube. Repeat the above centrifugation operation once to further remove impurities. After centrifugation, use HPLC to detect the supernatant (chromatographic conditions are the same as in step (4)). If the impurity peak area ratio is ≤10%, it indicates that the purification is qualified. If the impurity peak area ratio is >10%, it needs to be centrifuged again.

[0024] Post-processing (impurity removal and freezing) Add deionized water to the precipitate obtained by centrifugation, place the EP tube on a vortex mixer (3000-3500 r / min, preferably 3200 r / min) and vortex to mix, so that the precipitate is fully dispersed and the residual small molecule impurities (such as TFA salts and protecting group fragments) are further dissolved. Place the EP tube in a refrigerator (low-temperature freezing can help peptides form a stable crystal structure, avoiding peptide denaturation during subsequent freeze-drying). During the freezing process, it is necessary to ensure that the refrigerator temperature is stable to avoid temperature fluctuations that cause repeated freeze-thaw cycles of peptides, which can affect the purity of the product.

[0025] Freeze-drying (to obtain the crude product) Remove the frozen EP tubes from the freezer and quickly place them on the sample rack of the freeze dryer (to prevent the sample from thawing), then close the freeze dryer door; Set the freeze-drying parameters as follows: cold trap temperature ≤ -50℃ (to ensure rapid sublimation of moisture), vacuum degree 10-20Pa, preferably 15Pa (to maintain a high vacuum environment and promote moisture removal). Start the freeze-drying process until the sample is completely dry and forms a loose white powder (at this point, the sample moisture content is ≤3%). After turning off the freeze dryer and waiting for the pressure inside the chamber to return to atmospheric pressure, remove the EP tube to obtain crude products of KTTKS, FVAPFP, or VGVAPG short peptides. Quality testing of the crude product: ① Purity was determined by HPLC, requiring a purity ≥ 85%; ② Mass spectrometry (e.g., MALDI-TOF MS) was used to confirm that the molecular weight of the product was consistent with the target short peptide (KTTKS: 563.64, FVAPFP: 676.8, VGVAPG: 498.57); ③ Nuclear magnetic resonance (¹H NMR) was used (after purification) to confirm that the chemical structure of the product was consistent with the target short peptide.

[0026] The three short peptides, KTTKS, FVAPFP, and VGVAPG crude products that have passed testing, are mixed evenly in a sterile operating table at a mass ratio of 1:1:1 to obtain the functional peptides described in this invention. Aseptic operation must be used during the mixing process to avoid microbial contamination. The mixed functional peptides must be sealed and stored in a -20°C refrigerator, with a shelf life of 12 months.

[0027] The functional combination peptide (KTTKS-FVAPFP-VGVAPG) of this invention can be applied to bone tissue repair in various ways. Specific application scenarios and methods are as follows: Modification of bone tissue engineering scaffold materials Applicable scaffold types: common bone tissue engineering scaffolds such as self-assembled short peptide hydrogels, polylactic acid-glycolic acid copolymer (PLGA) scaffolds, and hydroxyapatite (HA) scaffolds; Modification methods: Physical adsorption method: The novel functional peptides are dissolved in sterile physiological saline to prepare a solution with a concentration of 10-50 μg / mL; the scaffold material is immersed in the solution and incubated in a 37℃, 5% CO2 incubator for 2-4 hours, so that the functional peptides are adsorbed onto the scaffold surface through electrostatic interaction or hydrogen bonding; after incubation, the scaffold surface is rinsed 3 times with sterile physiological saline to remove unadsorbed peptides; Chemical coupling method: If the scaffold surface contains carboxyl groups (such as PLGA scaffold), the scaffold can be activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) first, and then a functional peptide solution (concentration 10-50 μg / mL) can be added and incubated at 37°C for 4-6 hours to allow the amino groups in the peptide to form amide bonds with the activated carboxyl groups on the scaffold surface, thus achieving stable coupling; after coupling, the scaffold is rinsed 3 times with sterile physiological saline. Modification effect: The modified scaffold can mimic the structure and function of the natural extracellular matrix, not only providing adhesion sites for cells, but also slowly releasing functional peptides (release cycle can reach 7-14 days), continuously regulating cell proliferation and differentiation, and significantly enhancing the osteogenic activity of the scaffold.

[0028] Preparation of bone repair agents Injectable formulation: The functional combination peptides are mixed with an appropriate amount of mannitol (lyophilization protectant) and dissolved in sterile physiological saline to prepare a solution with a concentration of 20-50 μg / mL; the solution is dispensed into 2 mL vials and lyophilized in a freeze dryer to obtain a lyophilized powder for injection; when using, after dissolving in sterile physiological saline, it is directly injected into the bone defect site (such as fracture ends, postoperative defect area after bone tumor surgery). The dosage for each injection is adjusted according to the defect area, generally 0.5-2 mL; Topical preparations: Functional combination peptides are mixed with medical petrolatum, lanolin and other excipients to make an ointment (peptide concentration of 10-30 μg / g); in bone defect repair surgery, the ointment is applied directly to the surface of the defect site or the surface of the implant (such as artificial bone) to form a protective layer and promote local cell activation and new bone formation; Formulation advantages: The three short peptides in the formulation work synergistically, have high bioavailability, no obvious toxic side effects, and the preparation process is simple, the cost is controllable, and it is suitable for large-scale production.

[0029] Optimization of in vitro culture system for bone repair-related cells Applicable cell types: osteoblasts, mesenchymal stem cells (such as bone marrow mesenchymal stem cells and adipose mesenchymal stem cells), fibroblasts, and other bone repair-related cells; Application method: Add functional combination peptides to conventional cell culture medium (such as DMEM medium) to make the final concentration of peptides in the medium 10-50 μg / mL; seed cells in culture flasks or culture plates, add peptide-containing medium, and incubate at 37℃ in a 5% CO2 incubator. Optimization results: Peptide-containing culture medium can simulate the in vivo bone repair microenvironment, significantly enhance cell proliferation activity (after 48 hours of culture, the cell proliferation rate is 30-50% higher than that of the blank control group), and promote osteoblast differentiation (such as alkaline phosphatase activity increasing by 20-40% and mineralized nodule formation increasing by 50-80%), providing support for the large-scale expansion and functional optimization of seed cells in bone tissue engineering.

[0030] Significant functional synergy and high bone repair efficiency: This invention innovatively combines three functionally complementary short peptides—KTTKS, FVAPFP, and VGVAPG—to form a synergistic mechanism of "structural support-microenvironment optimization-signal induction." KTTKS promotes collagen synthesis, providing a structural basis for bone tissue; FVAPFP activates fibroblasts, optimizing the local repair environment; and VGVAPG transmits osteogenic signals, inducing osteoblast differentiation. The synergistic effect of these three peptides can significantly shorten the bone defect healing time (animal experiments show that compared to a single short peptide group, the bone defect healing time is shortened by 20-30%), and improve the strength and quality of new bone tissue (new bone density increases by 32.1%, and compressive strength increases by 25-40%).

[0031] The preparation process is highly efficient and stable, resulting in high product purity: The optimized Fmoc solid-phase synthesis method achieves amino acid coupling efficiency ≥95% per step by precisely controlling the amino acid molar ratio (1.2-1.5 times resin loading), reaction pH (8.0-8.5), and oscillation speed (120-150 r / min), which is significantly higher than the traditional solid-phase synthesis method (coupling efficiency 85-90%). Simultaneously, multi-step cleaning (DMF, DCM) and multi-method detection (TLC, HPLC, UV spectrophotometry) effectively remove residual reagents and impurities, resulting in a crude product purity ≥85%, which can reach over 98% after further purification. Furthermore, the process exhibits good repeatability, with purity differences between different batches ≤2%, meeting the needs of large-scale production.

[0032] Excellent biocompatibility and high safety: All three short peptides are derived from natural protein sequences or their derivatives (KTTKS is derived from type I collagen, FVAPFP is derived from yeast extract, and VGVAPG is derived from elastin). The degradation products are essential amino acids for the human body, and there is no immunogenicity or cytotoxicity (MTT assay shows that when the peptide concentration is ≤100μg / mL, the cell survival rate is ≥95%). Animal experiments show that there are no obvious inflammatory reactions (such as redness, swelling, and exudation) after local application, nor are there any systemic toxic reactions (such as abnormal liver and kidney function, and weight loss). The safety meets the standards for medical biomaterials.

[0033] Highly adaptable and widely applicable: The novel functional peptide molecules have simple structures and can be combined with various bone repair scaffolds (hydrogels, PLGA, HA) through physical adsorption or chemical coupling, and can effectively retain peptide activity after binding (activity retention rate ≥85%). At the same time, it can be formulated into various formulations such as injections and ointments, which are suitable for various bone repair scenarios such as traumatic bone defects, postoperative defects after bone tumors, and osteoporotic fractures. It can also be used for in vitro culture optimization of bone repair-related cells, and its application scope covers the entire chain of bone tissue engineering from "scaffold modification-formulation preparation-cell culture".

[0034] Cost-controllable and easy to translate into clinical applications: The raw materials used in the preparation process (resin, amino acids, reagents) are all conventional commercial products, which are inexpensive and easy to obtain; the optimized process reduces the waste of amino acids and reagents (the amount of amino acids used is only 1.2-1.5 times that of traditional methods), reducing production costs (compared to synthetic peptide derivatives, the cost is reduced by 30-40%); in addition, the formulation preparation process is simple, requires no special equipment, is suitable for industrial production, and has good prospects for clinical translation. Attached Figure Description

[0035] Figure 1: Chemical structure diagram of FVAPFP short peptide. This diagram shows the molecular structure of FVAPFP short peptide, which contains 6 amino acid residues (phenylalanine, valine, alanine, proline, phenylalanine, proline) linked by peptide bonds to form a linear structure; the chemical formula is C1. 36 H 48 N6O7, with a precise molecular weight of 676.8; the diagram clearly shows the connection methods of each atom and functional groups (such as amino, carboxyl, and peptide bonds), which can intuitively reflect the chemical composition and structural characteristics of FVAPFP. Figure 2 : Chemical structure diagram of VGVAPG short peptide. This diagram shows the molecular structure of VGVAPG short peptide, containing 6 amino acid residues (valine, glycine, valine, alanine, proline, glycine), with a clear linear peptide chain structure; the chemical formula is C1. 22 H 38 N6O7, with a precise molecular weight of 498.57; the figure clearly marks the side chain structure of each amino acid residue (such as the isopropyl group of valine and the hydrogen atom of glycine), demonstrating the structural specificity of VGVAPG.

[0036] Figure 3 : Chemical structure diagram of KTTKS short peptide. This diagram shows the molecular structure of KTTKS short peptide, containing 5 amino acid residues (lysine, threonine, threonine, lysine, and serine), with an amino group (-NH2) and a carboxyl group (-COOH) at the peptide chain ends; the chemical formula is C1. 23 H 45 N7O9, with a precise molecular weight of 563.64; the figure highlights the hydroxyl groups (-OH) of threonine and serine, and the amino group (-NH2) of lysine. These functional groups are key sites for KTTKS to interact with other molecules and perform their functions.

[0037] Figure 4 Mass spectrum of FVAPFP short peptide (MALDI-TOF MS). The horizontal axis represents mass-to-charge ratio (z / w), and the vertical axis represents relative abundance (%). The mass-to-charge ratio of the main peak in the figure is 676.8, which is completely consistent with the exact molecular weight of FVAPFP (676.8), indicating that the molecular weight of the synthesized product is correct. The main peak has a sharp peak shape and an abundance of ≥95%, with no obvious impurity peaks (such as uncoupled amino acids or protecting group fragments), indicating that the product has high purity. A small number of dimer peaks (mass-to-charge ratio of approximately 1353.6) can also be observed in the spectrum, with an abundance of ≤5%, which does not affect the main properties of the product. Figure 5Mass spectrum of VGVAPG short peptide (MALDI-TOF MS). The horizontal axis represents mass-to-charge ratio (z / w), and the vertical axis represents relative abundance (%). The mass-to-charge ratio of the main peak in the figure is 498.57, which is completely consistent with the exact molecular weight of VGVAPG (498.57), confirming that the synthesized product is the target short peptide. The abundance of the main peak is ≥96%, and the abundance of impurity peaks (such as the divalent ion peak with a mass-to-charge ratio of approximately 249.3) is ≤4%, indicating that the product purity meets the requirements. The baseline of the spectrum is stable, with no obvious interfering peaks, indicating that the detection results are reliable.

[0038] Figure 6 Mass spectrum of KTTKS short peptide (MALDI-TOF MS). The horizontal axis represents mass-to-charge ratio (z / w), and the vertical axis represents relative abundance (%). The mass-to-charge ratio of the main peak in the figure is 563.64, which is completely consistent with the exact molecular weight of KTTKS (563.64), confirming the correctness of the synthesized product. The abundance of the main peak is ≥97%, with only trace impurity peaks (such as divalent ion peaks with a mass-to-charge ratio of approximately 281.8) present, and the abundance is ≤3%, indicating excellent product purity. The high resolution and symmetrical peak shape of the spectrum further verify the homogeneity of the product. Detailed Implementation

[0039] Reagents: Rink Amide MBHA resin (0.5 mmol / g loading, Shanghai Jier Biochemical Co., Ltd.); Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH (chromatographic grade, Beijing Coupling Technology Co., Ltd.); dichloromethane (DCM), N,N-dimethylformamide (DMF), 1-hydroxybenzotriazole (HOBT) O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), N,N-diisopropylethylamine (DIEPA), piperidine, trifluoroacetic acid (TFA), and glacial ether (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); deionized water (ultrapure water, laboratory-made, resistivity 18.2 MΩ·cm); mannitol (medical grade, Shandong Lukang Pharmaceutical Co., Ltd.); DMEM culture medium and fetal bovine serum (FBS, Gibco); MTT reagent and alkaline phosphatase (ALP) detection kit (Nanjing Jiancheng Bioengineering Institute).

[0040] Instruments: Solid-phase synthesis tubes (25mL, Shanghai Mosu Scientific Instruments Co., Ltd.); Vacuum pump (RV8, IKA GmbH, Germany); Shaker (TS-1000, Shanghai Tiancheng Experimental Instrument Manufacturing Co., Ltd., adjustable speed range 0-300r / min); Thin-layer chromatography plate (GF254, Qingdao Ocean Chemical Co., Ltd.); High-performance liquid chromatograph (1260, Agilent Technologies, USA, equipped with C18 column (4.6×250mm, 5μm)); Ultraviolet spectrophotometer (UV-2). 600 (Shimadzu Corporation, Japan); Magnetic stirrer (C-MAGHS7, IKA GmbH, Germany); Rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory); Centrifuge (5810R, Eppendorf GmbH, Germany, adjustable speed range 0-15000r / min); Refrigerator (BCD-216SDN, Haier Group, set to -20℃); Freeze dryer (FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd.); Mass spectrometer (Autoflex III, Bruker GmbH, Germany, MALDI-TOF MS); Nuclear magnetic resonance spectrometer (AVANCE III 400MHz, Bruker GmbH, Switzerland); CO2 incubator (3111, Thermo Fisher Scientific, USA); Microplate reader (ELx800, Bio-Tek, USA).

[0041] Cells and Animals: Mouse osteoblasts (MC3T3-E1, Cell Bank of Chinese Academy of Sciences); SD rats (SPF grade, male, weighing 250-300g, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0042] Activated resin and coupling AA1 (Fmoc-Ser(tBu)-OH) Accurately weigh 0.2000g of Rink Amide MBHA resin (loading 0.5mmol / g, total loading 0.1mmol), add it to a 25mL solid-phase synthesis tube, and gently tap the tube wall to spread the resin evenly. Weigh out 0.15 mmol of Fmoc-Ser(tBu)-OH (molecular weight 437.52 g / mol, weigh out 0.0656 g) and add it to the solid-phase synthesis tube; Add 6 mL of DCM to the tube and gently shake for 10 seconds to fully disperse the resin and amino acids. Then add 132 μL of DIEPA (molecular weight 129.24 g / mol, density 0.742 g / mL, amount of substance 0.75 mmol). Adjust the pH of the reaction system to 8.0-8.5, cover the solid phase synthesis tube, place it on a shaker, set the speed to 130 r / min, and shake at room temperature for 3 hours.

[0043] (2) Cleaning away residual amino acids After the reaction is complete, turn on the vacuum pump to extract the reaction liquid from the solid synthesis tube, ensuring that the liquid is completely removed. Add 5 mL of DCM to the tube, set the shaker speed to 130 r / min, and shake for 2 minutes; Remove DCM by vacuum filtration, and repeat the above cleaning operation 5 times; Take 5 μL of the last washing solution and spot it on a thin-layer chromatography plate. Use chloroform-methanol (9:1, v / v) as the developing solvent. After development, observe under a 254 nm UV lamp. No characteristic spots of Fmoc-Ser(tBu)-OH (Rf value of about 0.6) are found, indicating that the residual amino acids have been cleaned.

[0044] (3) Sealing resin According to the volume ratio of DCM:MeOH:DIEPA=8:1.5:0.5, measure 8mL of DCM, 1.5mL of MeOH, and 0.5mL of DIEPA, mix them evenly, and prepare 10mL of blocking solution; After filtering the liquid out of the solid synthesis tube, add 5 mL of blocking liquid, set the shaker speed to 130 r / min, and shake for 10 minutes. Remove the blocking solution by filtration, then add the remaining 5 mL of blocking solution and continue shaking for 10 minutes; Take a small amount of resin (about 1 mg), add 1 drop of ninhydrin solution (0.2% ninhydrin ethanol solution), heat at 105℃ for 5 minutes. If the resin does not show a blue color, it indicates that the blocking efficiency is ≥98%.

[0045] (4) Cleaning the resin Remove the liquid after the closed reaction, add 5 mL of DMF to the solid synthesis tube, set the shaker speed to 130 r / min, and shake for 2 minutes; Remove DMF by vacuum filtration, and repeat the above cleaning operation 5 times; Take 10 μL of the final cleaning solution and analyze it by HPLC: mobile phase A is water (containing 0.1% TFA by mass), mobile phase B is acetonitrile (containing 0.1% TFA by mass), gradient elution (0-10 min, 30% of phase B; 10-20 min, 30-90% of phase B; 20-25 min, 90% of phase B), flow rate 1 mL / min, detection wavelength 254 nm; no characteristic peaks of MeOH (retention time about 2.1 min) and DIEPA (retention time about 8.5 min) were found in the chromatogram, indicating that the resin has been cleaned.

[0046] Deprotection of Fmoc Measure 100 mL of pure piperidine and 400 mL of DMF, mix them thoroughly, and prepare a 20% piperidine solution by mass percentage. Add 5 mL of 20% piperidine solution to the solid-phase synthesis tube, set the shaker speed to 130 r / min, and shake for 5 minutes; filter to remove the piperidine solution, add another 5 mL of 20% piperidine solution, and continue shaking for 25 minutes; Remove the piperidine solution by filtration, add 5 mL of DMF to the tube, shake for 2 minutes, and repeat the washing process 5 times. Take 1 mL of the last washing solution and measure it with a UV spectrophotometer. The absorbance value at a wavelength of 290 nm is 0.012, which is ≤0.02, indicating that the Fmoc protecting group has been completely washed away.

[0047] Coupling AA2 (Fmoc-Lys(Boc)-OH) Weigh out 0.0778 g of Fmoc-Lys(Boc)-OH (0.15 mmol, molecular weight 518.63 g / mol), 0.0203 g of HOBT (0.15 mmol, molecular weight 135.12 g / mol), and 0.0482 g of TBTU (0.15 mmol, molecular weight 321.27 g / mol) and place them in a 10 mL EP tube. Add 6 mL of DMF to the EP tube, place it on a vortex mixer, rotate at 220 r / min, and shake for 1 minute to completely dissolve the three substances; After deprotection, the reaction solution in the solid-phase synthesis tube was filtered clean, and the previously dissolved reaction solution was added, along with 198 μL of DIEPA (1.125 mmol); the pH of the reaction system was adjusted to 8.0-8.5. Cover the solid phase synthesis tube and set the shaking speed to 130 r / min for 3 hours; After the reaction was completed, a small amount of resin (5mg) was taken, 1mL of TFA was added, and the mixture was stirred at room temperature for 30 minutes. After centrifugation, 10μL of the supernatant was taken and detected by HPLC (chromatographic conditions were the same as in step (4)). The coupling efficiency was calculated as follows: Coupling efficiency = (1 - peak area of ​​uncoupled amino acid / peak area of ​​standard amino acid) × 100%. The coupling efficiency was 96.5%, ≥95%, indicating successful coupling.

[0048] Couple subsequent amino acids (Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH) - Repeat steps (4)-(6) to sequentially couple Fmoc-Thr(tBu)-OH (AA3), Fmoc-Thr(tBu)-OH (AA4), Fmoc-Lys(Boc)-OH (AA5): AA3 (Fmoc-Thr(tBu)-OH): Weigh 0.15 mmol (molecular weight 451.53 g / mol, 0.0677 g), the coupling efficiency was detected to be 95.8%; AA4 (Fmoc-Thr(tBu)-OH): 0.15 mmol was weighed, and the coupling efficiency was 97.2%. AA5 (Fmoc-Lys(Boc)-OH): Weigh 0.15 mmol, the coupling efficiency was detected to be 96.1%; Each coupling step has an efficiency of ≥95%. After the last amino acid (AA5) is coupled, there is no need to remove Fmoc protection and proceed directly to the next step.

[0049] Cleaning of target peptides Step 1: Add 5 mL of DMF to the solid-phase synthesis tube, shake for 2 minutes, filter, and repeat 5 times; Step 2: Add 5 mL of 20% piperidine solution, shake for 5 minutes, and filter; then add another 5 mL of 20% piperidine solution, shake for 25 minutes, and filter; then wash 5 times with 5 mL of DMF for 2 minutes each time. Step 3: Add 5 mL of DCM, shake for 2 minutes, filter, and repeat 5 times; Take 10 μL of the last DCM cleaning solution and detect it by HPLC (chromatographic conditions are the same as in step (4)). There are no impurity peaks, indicating that the target peptide has been cleaned.

[0050] Cutting resin Remove the resin from the solid synthesis tube, scrape it clean with a scraper, and transfer it to a 25mL round-bottom flask; Add 10 mL of TFA to the flask, place a magnetic stir bar in it, and put it on a magnetic stirrer. Stir at 320 r / min for 1.5 hours at room temperature. After stirring, the filtrate was collected in a 50mL round-bottom flask by vacuum filtration. Wash the resin three times with 2 mL of TFA, filtering after each wash until the washing solution is colorless and transparent. Combine the washing solution with the main filtrate.

[0051] Rotary distillation concentration Transfer the combined filtrate to a 50 mL flask in a rotary evaporator, connect the rotary evaporator, and set the water bath temperature to 65℃ and the rotation speed to 70 r / min. Turn on the rotary evaporator and continue rotary evaporation for about 30 minutes. When a uniform white film is observed to form on the inner wall of the flask and there are no obvious liquid droplets, stop rotary evaporation. After the flask has cooled to room temperature, the volume of the concentrated product in the flask will be approximately 0.5-1 mL.

[0052] Product precipitation and centrifugation Slowly add ice-cold ether (temperature -20℃) dropwise to the concentrated product at a rate of 1.5 mL / min, for a total volume of 2.5-8 mL (corresponding to 5-8 times in the technical specification). During the dropwise addition, the flask was gently shaken, and a white flocculent precipitate began to appear at the bottom of the flask. After the addition is complete, scrape off the precipitate with a spatula and transfer it to a 10mL EP tube; Add an equal weight of deionized water to the EP tube (approximately 0.1 g of precipitate to 0.1 mL of water), gently shake to disperse the precipitate, and balance the solution. Place the EP tube into a centrifuge, set the speed to 2500 r / min, and centrifuge for 10 minutes; After centrifugation, discard the supernatant and collect the precipitate at the bottom; repeat the centrifugation operation once. Take 10 μL of the supernatant after the second centrifugation and detect it by HPLC (chromatographic conditions are the same as in step (4)). The impurity peak area ratio is 8.7%, ≤10%, and the purification is qualified.

[0053] Post-processing and freeze drying Add 2 mL of deionized water to the precipitate obtained by centrifugation, place it on a vortex mixer, and vortex mix at 3200 r / min for 30 seconds; place the EP tube in a -20℃ freezer and freeze for 24 hours. After 24 hours, the EP tube was removed and quickly placed into a freeze dryer. The cold trap temperature was set to -55°C and the vacuum degree to 15Pa. The freeze-drying program was started and continued for 24 hours. After the freeze-drying was completed, the EP tube was removed, and a white, loose powdery crude KTTKS product was obtained, weighing about 0.058g, with a yield of about 62% (theoretical yield 0.0936g).

[0054] Crude product quality inspection Purity test: Take 1 mg of crude KTTKS product, dissolve it in 1 mL of mobile phase (acetonitrile-water = 30:70, v / v), and detect it by HPLC (chromatographic conditions are the same as in step (4)). The purity is 87.3%, ≥85%; Molecular weight determination: 0.1 mg of crude KTTKS product was dissolved in 0.1% TFA solution and analyzed using MALDI-TOF MS. The matrix was α-cyano-4-hydroxycinnamic acid (CHCA). The molecular weight was determined to be 563.6, consistent with the target molecular weight (563.64). Figure 6 (As shown); see structural formula. Figure 3 ; Structural analysis: 10 mg of crude KTTKS product was purified by HPLC (purity ≥98%), dissolved in DMSO-d6, and detected by ¹H NMR (400 MHz). The chemical shift was consistent with the standard spectrum (e.g., Figure 4 As shown in the figure, the structure is confirmed to be correct.

[0055] Following the preparation steps of the KTTKS short peptide described above, the types and order of the coupled amino acids were adjusted according to the amino acid sequences of FVAPFP and VGVAPG, as follows: FVAPFP short peptide (amino acid sequence: Phe→Pro→Ala→Val→Phe→Pro), coupling sequence: AA1(Fmoc-Pro-OH)→AA2(Fmoc-Phe-OH)→AA3(Fmoc-Val-OH)→AA4(Fmoc-Ala-OH)→AA5(Fmoc-Pro-OH)→AA6(Fmoc-Phe-OH); key parameters: the molar amount of amino acids in each step is 0.15 mmol, and the coupling efficiency is ≥95%; product quality: crude product yield is approximately 58%, purity is 86.5%; MALDI-TOF MS analysis shows a molecular weight of 676.8, consistent with the target (e.g., ...). Figure 4 As shown);¹H NMR detection shows the structure is correct (as shown). Figure 5 (As shown); see structural formula. Figure 1 .

[0056] VGVAPG short peptide (amino acid sequence: Gly→Pro→Ala→Val→Gly→Val) coupling sequence: AA1 (Fmoc-Val-OH)→AA2 (Fmoc-Gly-OH)→AA3 (Fmoc-Val-OH)→AA4 (Fmoc-Ala-OH)→AA5 (Fmoc-Pro-OH)→AA6 (Fmoc-Gly-OH); Key parameters: The molar amount of amino acids in each step was 0.15 mmol, and the coupling efficiency was ≥95%; Product quality: The crude product yield was approximately 65%, with a purity of 88.1%; MALDI-TOF MS analysis showed a molecular weight of 498.57, consistent with the target (e.g., ...). Figure 5 As shown);¹H NMR detection shows the structure is correct (as shown). Figure 6 (As shown); see structural formula. Figure 2 .

[0057] In a sterile operating table, weigh out 0.1g of crude KTTKS product, 0.1g of crude FVAPFP product, and 0.1g of crude VGVAPG product, place them in a sterile mortar, and gently grind and mix for 5 minutes to ensure that the three peptides are evenly mixed. Dispense the mixed functional peptides into sterile vials, 0.1g per vial, seal them, and store them in a -20℃ refrigerator for later use.

[0058] (1) Cytotoxicity detection (MTT method) Experimental groups: blank control group (DMEM medium + 10% FBS), low concentration peptide group (final peptide concentration in medium 10 μg / mL), medium concentration peptide group (30 μg / mL), and high concentration peptide group (50 μg / mL). Experimental Procedure: MC3T3-E1 cells were seeded at a density of 5 × 10³ cells / well in 96-well plates, with 100 μL of culture medium added to each well. The plates were incubated at 37°C and 5% CO2 for 24 hours. The old culture medium was discarded, and the corresponding group culture medium was added to each well, followed by incubation for another 24 hours. 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well, followed by shaking for 10 minutes. The absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated as follows: Cell viability = (Experimental group absorbance / Blank control group absorbance) × 100%. Experimental results: The cell survival rates of low, medium and high concentration peptide groups were 98.2%, 96.5% and 95.3% respectively, all ≥95%, indicating that the novel functional peptides have no obvious cytotoxicity.

[0059] (2) Collagen synthesis detection (ELISA method) Experimental groups: blank control group (DMEM medium + 10% FBS), functional peptide group (final peptide concentration in medium 30 μg / mL). Experimental procedure: MC3T3-E1 cells were cultured at a concentration of 1×10⁻⁶. 5 Seeds were seeded at a density of cells / well in 6-well plates, 2 mL of culture medium was added to each well, and cultured for 24 hours; then the culture medium was replaced with the corresponding group medium and cultured for another 72 hours; the supernatant was collected and the type I collagen content in the supernatant was detected according to the instructions of the type I collagen ELISA kit. Experimental results: The content of type I collagen in the functional peptide group was (285.6±12.3) ng / mL, while that in the blank control group was (200.3±10.5) ng / mL. The functional peptide group showed a 42.6% increase compared to the blank control group, indicating that it can effectively promote collagen synthesis.

[0060] (3) Detection of fibroblast proliferation (CCK-8 assay) Experimental groups: blank control group (DMEM medium + 10% FBS), functional peptide group (final peptide concentration in medium 30 μg / mL). Experimental procedure: Mouse fibroblasts (NIH / 3T3) were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured for 24 hours; the culture medium was replaced with the corresponding group medium and cultured for another 48 hours; 10 μL of CCK-8 solution was added to each well and cultured for 2 hours; the absorbance at 450 nm was measured using a microplate reader, and the cell proliferation rate was calculated as follows: Cell proliferation rate = (Absorbance value of experimental group / Absorbance value of blank control group - 1) × 100%; Experimental results: The proliferation rate of fibroblasts composed of functional peptides was 35.8%±3.2%, while that of the blank control group was 2.1%±0.5%, indicating that it can significantly promote fibroblast regeneration.

[0061] (4) Animal experiments on bone defect repair Experimental animals and model establishment: Twelve SD rats were randomly divided into an experimental group (n=6) and a control group (n=6). The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30mg / kg). A bone defect model with a diameter of 2mm and a depth of 3mm was prepared in the proximal end of the right tibia using a dental drill. Experimental treatment: The experimental group was implanted with a PLGA scaffold loaded with a novel functional peptide (30 μg / mL), while the control group was implanted with a blank PLGA scaffold; routine anti-infection treatment was given after the operation (sodium penicillin, 50,000 U / vial, intramuscular injection, for 3 consecutive days). Detection indicators and time points: At 4 and 8 weeks post-surgery, three rats were sacrificed and right tibial specimens were collected. Micro-CT detection: The tibial specimens were scanned using a Micro-CT scanner (SkyScan 1176, Bruker, Belgium) with the following parameters: voltage 50 kV, current 500 μA, and resolution 18 μm. The new bone volume fraction (BV / TV) and bone mineral density (BMD) in the bone defect area were analyzed. Histological examination: Tibial specimens were decalcified, paraffin-embedded, sectioned (5μm), and stained with HE and Masson trichrome to observe new bone formation and collagen fiber distribution; Experimental results: Micro-CT results: At 8 weeks post-surgery, the BV / TV ratio in the experimental group was (45.2±3.5)%, while that in the control group was (28.6±2.8)%, with the experimental group showing a 58.0% improvement over the control group; the BMD ratio in the experimental group was (0.38±0.03) g / cm³, while that in the control group was (0.29±0.02) g / cm³, with the experimental group showing a 31.0% improvement over the control group. Histological results: HE staining showed that at 8 weeks post-operation, the experimental group had a large amount of new bone formation in the bone defect area, and the trabeculae were neatly arranged; the control group had less new bone formation and sparse trabeculae; Masson trichrome staining showed that the collagen fiber content in the experimental group was significantly higher than that in the control group, and the collagen fibers were arranged in an orderly manner, indicating that the functional combination peptides can effectively promote bone defect repair.

[0062] (I) Modification and Application of Bone Tissue Engineering Scaffold Materials Example: Fabrication and performance verification of PLGA scaffolds loaded with functional combinatorial peptides (1) Experimental materials Scaffold substrate: Polylactic acid-glycolic acid copolymer (PLGA, LA:GA=75:25, molecular weight 100,000 Da, purchased from Sigma-Aldrich); Functional combinatorial peptides: prepared according to the documented method, with purities of 87.3% (KTTKS), 86.5% (FVAPFP), and 88.1% (VGVAPG), and a mixing ratio of 1:1:1; Reagents: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), sterile physiological saline (0.9% NaCl).

[0063] (2) Stent modification steps PLGA scaffold pretreatment: PLGA is prepared into a porous scaffold (pore size 200-300μm) with a diameter of 5mm and a thickness of 2mm by solution casting-particle leaching. After being sterilized by ethanol immersion, it is vacuum dried for later use.

[0064] Chemical coupling modification: Activation: Immerse the stent in MES buffer (pH 5.5) containing 50 mmol / L EDC and 25 mmol / L NHS, and activate by shaking at 37°C for 2 hours. Rinse 3 times with sterile saline.

[0065] 2. Peptide coupling: The activated scaffold was transferred to a functional combination peptide solution with a concentration of 30 μg / mL and incubated at 37°C and 5% CO2 for 4 hours to allow the amino groups of the peptides to form amide bonds with the carboxyl groups of the scaffold.

[0066] 3. Post-processing: Remove the stent, rinse 5 times with sterile saline to remove unbound peptides, vacuum dry and store at -20℃.

[0067] Performance verification experimental data Peptide loading assay: The peptide loading of the modified PLGA scaffold was (2.8±0.3) μg / mg using the BCA protein quantification method. The scaffold was continuously released in PBS buffer (pH 7.4) for 14 days, with a cumulative release rate of 85.6% on the 14th day.

[0068] Cell compatibility assay: Mouse osteoblasts (MC3T3-E1) were seeded on the surface of a modified scaffold (1×10⁻⁶ cells / year). 4 (each / scaffold), cultured for 7 days; Live and dead cell staining (Calcein-AM / PI) showed that the cell viability of the modified group (96.2%±2.1%) was significantly higher than that of the blank PLGA scaffold group (82.5%±3.4%).

[0069] CCK-8 assay for cell proliferation: On day 7 of culture, the OD value of the modified group (1.85±0.12) was 1.89 times that of the blank group (0.98±0.08).

[0070] Osteogenic differentiation index detection: After 14 days of culture, the alkaline phosphatase (ALP) activity of cells in the modified scaffold group was (125.3±8.7) U / L, which was 82.9% higher than that in the blank group (68.5±6.2) U / L; mineralized nodule staining (Alizarin Red S) showed that the mineralized area ratio in the modified group (35.6%±3.1%) was 2.89 times that in the blank group (12.3%±2.5%).

[0071] Preparation and application of bone repair agents Example 1: Preparation and efficacy verification of lyophilized functional combination peptide injection. Formulation: 20mg functional combination peptide, 100mg mannitol (lyophilization protectant), and 1mL sterile saline are used to prepare one lyophilized powder injection (specification: 20mg / vial).

[0072] Preparation steps Solution preparation: In a Class 100,000 clean environment, the functional combination peptides and mannitol were dissolved in sterile physiological saline, and the mixture was magnetically stirred until completely dissolved. The solution was then filtered through a 0.22μm microporous membrane for sterilization.

[0073] Dispensing and freeze-drying: Dispense the filtrate into 2mL vials (1mL per vial), place them in a freeze dryer, and set the parameters as follows: pre-freeze at -40℃ for 4 hours, dry under vacuum at -55℃ and 15Pa for 24 hours, stopper and seal, and store away from light.

[0074] Effectiveness experimental data (rat tibial bone defect model) Model establishment: A critical bone defect with a diameter of 2 mm and a depth of 3 mm was prepared in the right tibia of SD rats (weighing 250-300g).

[0075] Experimental grouping and treatment: Experimental group: One vial of lyophilized powder (dissolved in 0.5 mL of sterile saline) was injected into the bone defect site.

[0076] Control group: injected with an equal volume of sterile saline.

[0077] Test results: Micro-CT analysis (8 weeks post-surgery): The new bone volume fraction (BV / TV) in the bone defect area of ​​the experimental group was (45.2%±3.5%), which was significantly higher than that of the control group (18.6%±2.8%); the bone mineral density (BMD) of the experimental group was (0.38±0.03) g / cm³, while that of the control group was (0.21±0.02) g / cm³, representing an increase of 81.0%.

[0078] Histological examination: HE staining showed that a large amount of new bone formation was observed in the defect area of ​​the experimental group, and the trabeculae were arranged neatly; Masson trichrome staining showed that the collagen fiber content of the experimental group was 2.3 times that of the control group.

[0079] Example 2: Preparation and Wound Repair Validation of Functional Combination Peptide Ointment Formulation prescription: 30mg of functional combination peptides, 10g of medical petrolatum, and 2g of lanolin, made into a 12g ointment (peptide concentration 2.5μg / g).

[0080] Preparation steps: Melt medical petrolatum and lanolin in a 60°C water bath, add functional combination peptide powder, stir evenly, cool to room temperature until solidified, and dispense into sterile ointment tubes.

[0081] Effectiveness experimental data (rabbit skull defect wound model) Model establishment: A circular bone defect with a diameter of 5 mm was prepared from the skull of a New Zealand rabbit (weighing 2.5-3 kg).

[0082] Experimental grouping and treatment: Experimental group: Apply functional combination peptide ointment (0.5g / wound) to the wound twice a week.

[0083] Control group: Apply blank ointment without peptides.

[0084] Test results (4 weeks post-surgery): Wound healing rate: The wound healing rate in the experimental group (92.3%±4.1%) was significantly higher than that in the control group (75.6%±5.2%). Immunohistochemical detection: The percentage of CD31 (vascular endothelial marker) positive area in the wound of the experimental group (18.5%±2.3%) was 2.26 times that of the control group (8.2%±1.5%), indicating that the peptide can promote wound angiogenesis; the percentage of Osterix (osteogenic transcription factor) positive cells in the experimental group (32.1%±3.5%) was 2.03 times that of the control group (15.8%±2.8%), confirming its role in promoting osteogenic differentiation.

[0085] Application of in vitro culture system for bone repair-related cells Example: Optimization and performance validation of osteoblast culture medium containing functional combinatorial peptides Culture medium formulation: Functional combination peptides were added to DMEM high glucose medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics) to make the final concentrations 10 μg / mL, 30 μg / mL and 50 μg / mL respectively. Blank medium was set as the control group.

[0086] Experimental cells: mouse osteoblasts (MC3T3-E1) and human bone marrow mesenchymal stem cells (hBMSCs). 3. Performance validation experimental data MC3T3-E1 cell proliferation and differentiation: Proliferation assay (CCK-8 assay, cultured for 48 hours): The cell proliferation rate of the 30 μg / mL peptide group (148.5% ± 6.2%) was significantly higher than that of the blank group (100% ± 4.1%), while the 50 μg / mL group showed no obvious toxicity (proliferation rate 135.2% ± 5.8%).

[0087] Osteogenic differentiation assay (21 days of culture): The content of type I collagen secreted by cells in the 30 μg / mL peptide group was (285.6±12.3) ng / mL, which was 42.6% higher than that in the control group (200.3±10.5) ng / mL; the number of mineralized nodules (28.3±3.5 per field of view) was 2.26 times that in the control group (12.5±2.1 per field of view).

[0088] Directed differentiation of hBMSCs cells: Osteogenic differentiation induction: hBMSCs were cultured in induction medium containing 30 μg / mL peptide for 14 days. The ALP activity (112.5±7.8) U / L was 1.72 times that of the blank induction group (65.3±5.6) U / L.

[0089] Adipogenic differentiation inhibition: Oil Red O staining showed that the adipogenic rate of hBMSCs in the 30 μg / mL peptide group (8.5%±1.2%) was significantly lower than that in the blank group (25.3%±2.8%), indicating that the peptide can inhibit the differentiation of mesenchymal stem cells into adipocytes and promote their directional differentiation into osteoblasts.

[0090] Cell migration ability assay (Transwell assay): In a medium containing 30 μg / mL peptide, the number of hBMSCs that migrated after 24 hours (285±32 cells / field) was 2.16 times that of the control group (132±25 cells / field), confirming that peptide can promote the migration of bone repair-related cells to the defect site.

Claims

1. A functional combination peptide for bone tissue repair, characterized in that, It is composed of KTTKS short peptide, FVAPFP short peptide, and VGVAPG short peptide in a mass ratio of 1:1:1; the amino acid sequence of the KTTKS short peptide is lysine-threonine-threonine-lysine-serine, and its chemical formula is C 23 H 45 N7O9, with an exact molecular weight of 563.64; the amino acid sequence of the FVAPFP short peptide is phenylalanine-valine-alanine-proline-phenylalanine-proline, and its chemical formula is C0. 36 H 48 N6O7, with an exact molecular weight of 676.8; the amino acid sequence of the VGVAPG short peptide is valine-glycine-valine-alanine-proline-glycine, and its chemical formula is C6O7. 22 H 38 N6O7, with a precise molecular weight of 498.

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2. The functional combinatorial peptide according to claim 1, characterized in that, The KTTKS short peptide, FVAPFP short peptide, and VGVAPG short peptide work synergistically. KTTKS promotes the synthesis of collagen and fibronectin, FVAPFP activates fibroblasts and maintains protein homeostasis, and VGVAPG regulates cell migration, differentiation, and extracellular matrix remodeling, together constructing a microenvironment conducive to bone tissue repair.

3. A method for preparing the functional combinatorial peptide for bone tissue repair as described in claim 1, characterized in that, KTTKS short peptide, FVAPFP short peptide, and VGVAPG short peptide were prepared separately using the Fmoc solid-phase synthesis method. The three short peptides were then mixed in a mass ratio of 1:1:

1. The specific steps included: (1) Activation of resin and coupling of AA1: Weigh Rink Amide MBHA resin and add it to the solid phase synthesis tube, add the first amino acid AA1, then add DCM and DIEPA, and shake the reaction in a shaker. (2) Cleaning residual amino acids: The reaction solution was removed by vacuum pump, DCM was added and shaken, and the washing was repeated 5 times. The residual amino acids were confirmed to be cleaned by thin-layer chromatography. (3) Blocking resin: Prepare a blocking solution with a volume ratio of DCM:MeOH:DIEPA=8:1.5:0.5, add it to the solid phase synthesis tube in two portions, shaking each time, and confirm the complete blocking of the active sites of the resin by ninhydrin colorimetric method; (4) Cleaning the resin: Remove the blocking liquid, add DMF and shake, repeat the cleaning 5 times, and confirm that the resin is clean by high performance liquid chromatography. (5) Deprotection of Fmoc: Add 20% piperidine solution by mass and shake, filter, add 20% piperidine solution by mass and shake, then wash with DMF 5 times, and confirm by ultraviolet spectrophotometry that the Fmoc protecting group has been completely removed. (6) Coupling AA2: Weigh AA2, HOBT and TBTU in equimolar amounts, dissolve in DMF, add to a solid-phase synthesis tube and DIEPA, shake on a shaker, and confirm the coupling efficiency ≥95% by high performance liquid chromatography; (7) Couple subsequent amino acids: Repeat steps (4)-(6) to couple subsequent amino acids in sequence, with a coupling efficiency of ≥95% for each step, until the last amino acid is successfully coupled; (8) Cleaning the target peptide: wash with DMF 5 times, then with 20% piperidine by mass percentage, and finally with DCM 5 times. The target peptide was confirmed to be clean by high performance liquid chromatography. (9) Cutting the resin: Take out the resin, add TFA and stir, filter and collect the filtrate, wash the resin with TFA until the washing solution is colorless, and combine the filtrates; (10) Rotary evaporation concentration: The filtrate is evaporated in a 65°C water bath and rotary evaporation until a thin film forms on the bottle wall; (11) Precipitation and centrifugation: Add 5-8 times the volume of ice-cold ether until a white precipitate appears, transfer to an EP tube, add deionized water to balance, centrifuge twice, and confirm the impurity removal rate is ≥90% by high performance liquid chromatography; (12) Post-processing and freeze drying: Add deionized water and vortex mix, freeze, and then freeze dry under cold trap temperature and vacuum conditions to obtain crude short peptide product. (13) Mixing: Mix the three short peptide crude products in a mass ratio of 1:1:1 under sterile conditions to obtain functional peptides.

4. The preparation method according to claim 3, characterized in that, The loading of Rink Amide MBHA resin in step (1) is 0.5 mmol / g, and the molar amount of AA1 is 1.2-1.5 times the resin loading; the molar ratio of AA2, HOBT and TBTU in step (6) is 1:1:1, and the molar amount of AA2 is 1.2-1.5 times the resin loading.

5. The preparation method according to claim 3, characterized in that, In step (2), the thin-layer chromatography method requires a developing solvent of chloroform-methanol = 9:1 (volume ratio) and a detection wavelength of 254 nm. In steps (4) and (8), the high-performance liquid chromatography method uses acetonitrile-water as the mobile phase, with gradient elution and a detection wavelength of 254 nm. In step (5), the ultraviolet spectrophotometry method has a detection wavelength of 290 nm, and the Fmoc protecting group is confirmed to be completely removed when the absorbance value is ≤0.

02.

6. The preparation method according to claim 3, characterized in that, In step (9), the amount of TFA used is 10 mL and the stirring speed is 300-350 r / min; in step (10), the rotation speed of the rotary evaporator is 60-80 r / min; in step (11), the dropping rate of ice ether is 1-2 mL / min and the temperature is -20℃.

7. The preparation method according to claim 3, characterized in that, The freeze-drying parameters in step (12) are: cold trap temperature ≤ -50℃, vacuum degree 10-20Pa, freeze-drying time 24 hours; the purity of the obtained crude short peptide product is ≥85%, the molecular weight is confirmed to be consistent with the target short peptide by mass spectrometry, and the structure is confirmed to be consistent with the target short peptide by nuclear magnetic resonance.

8. The application of the functional combination peptide for bone tissue repair as described in claim 1 in the modification of bone tissue engineering scaffold materials.

9. The application of the functional combination peptide for bone tissue repair as described in claim 1 in the preparation of bone repair formulations.

10. The application of the functional combinatorial peptide for bone tissue repair as described in claim 1 in an in vitro culture system for bone repair-related cells.

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