Preparation method and application of epitope imprinting bionic receptor for self-recruitment of endogenous osteogenic growth peptide

Abstract

The invention belongs to the field of biomedical polymer materials, and discloses a preparation method and application of an epitope imprinting bionic receptor for self-recruitment of endogenous osteogenic growth peptide. According to the invention, from the angle of collection of endogenous growth factors, the OGP is taken as a target molecule, the epitope imprinting polymer nanoparticles MIP with specific recognition capability are prepared by adopting an epitope imprinting technology, and specific capture of the endogenous OGP is realized. Further, according to the invention, the MIP is introduced into a polyethylene glycol PEG hydrogel system, and the epitope imprinting bionic receptor MIP (at) PEG capable of collecting endogenous osteogenic factors is prepared. Directional capture and slow release of OGP and activation of local osteogenesis signals are realized in vivo, and formation of new bones in a bone defect area is remarkably promoted. The invention provides a new thought and experimental basis for developing a novel tissue engineering scaffold based on endogenous growth factor recruitment, and has important clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials and relates to the preparation method and application of a biomimetic receptor for self-recruiting endogenous osteogenic factors epitope imprinting. Background Technology

[0002] Bone defects are a common and challenging clinical problem with diverse causes, including comminuted fractures, bone tumors, and congenital malformations. The incidence of these conditions is increasing annually, leading to a continuous rise in the number of patients with bone defects worldwide. Bone tissue has limited self-repair capabilities, and large defects often fail to heal spontaneously. Currently, bone grafting is the primary clinical treatment for bone defects; however, this method faces numerous challenges, such as limited donor availability, immune rejection, and the risk of disease transmission, severely limiting its widespread application.

[0003] With the continuous development of tissue engineering technology, tissue engineering strategies based on the three elements of "seed cells-growth factors-scaffold materials" have brought new hope to bone defect repair. However, traditional bone tissue engineering methods mostly rely on exogenous growth factors or drugs to promote bone tissue regeneration. Although these exogenous growth factors can promote bone formation to a certain extent, they are also prone to causing adverse reactions (such as inflammation, immune responses, etc.) and even promoting carcinogenesis, requiring precise control, thus limiting their application in clinical treatment. On the other hand, recruiting endogenous osteogenic growth peptides (OGPs) from the body to the bone defect site can effectively avoid the various problems caused by exogenous growth factors, while efficiently promoting tissue healing and achieving in situ treatment of bone defects.

[0004] To achieve efficient capture and delivery of endogenous OGPs among numerous biomolecules in vivo, highly selective recognition of OGPs is crucial. Epitope imprinting technology (EIT) provides the design basis for achieving this goal. It is an advanced technology based on the molecular recognition principle of epitope-active peptide chains to prepare molecularly imprinted polymers (MIPs) capable of specifically recognizing biomacromolecules. Summary of the Invention

[0005] This invention provides a method for preparing and applying an epitope-imprinted biomimetic receptor capable of recruiting endogenous osteogenic factors. The epitope imprinting is achieved using OGP epitope peptide chains as template molecules via solid-phase imprinting, resulting in epitope-imprinted nanoparticles exhibiting excellent recognition and selection properties for OGP. The epitope-imprinted biomimetic receptor is prepared by encapsulating epitope-imprinted nanoparticles in a PEG-crosslinked hydrogel. The PEG-crosslinked hydrogel undergoes chemical modification to achieve a stable crosslinked structure. The epitope-imprinted biomimetic receptor can bind to endogenous OGP in vivo and present it to stem cells, inducing stem cells to differentiate into osteoblasts, accelerating the regeneration of damaged bone tissue, and achieving therapeutic repair of bone defects.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing an epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides includes the following steps: (1) Glass bead surface fixation template Surface-activated glass beads (GBs) were poured into a mixture of 3-(isobutenoyloxy)propyltrimethoxysilane and anhydrous toluene and shaken in a constant temperature shaker. After shaking, the GBs were rinsed with anhydrous ethanol, dried in an oven, and then sieved for later use. The sieved GBs were poured into a deoxygenated PBS solution containing template molecules, and a solution of sodium dithionite and photoinitiator were added. The GBs were then bound using thiol-ene click chemistry and reacted under UV irradiation. The GBs were washed with deionized water to obtain GBs with fixed templates. The GBs were then loaded into a constant temperature chromatography column and connected to a peristaltic pump. Pure water was pumped in to rinse the glass beads. (2) Solid-phase synthesis of epitope-imprinted polymers Weigh the temperature-responsive monomer, functional monomer, and crosslinking agent and dissolve them in PBS solution. Sonicate the solution until completely dissolved, place it in an ice-water bath and purge with nitrogen to form a prepolymer solution. Then, under anaerobic conditions, add the redox initiator potassium persulfate and tetramethylethylenediamine to the prepolymer solution and pump the prepolymer solution into the chromatographic column obtained in step (1). Turn on the circulating water bath switch and circulate in the ice-water bath for 1-2 h. Then, set the temperature to 37 ℃ and react at a constant temperature for 12-18 h. At 37 ℃, pump in PBS to wash out the unreacted monomers and oligomers of the non-imprinted polymer NIP. Next, lower the temperature of the circulating water bath to 6 ℃, pump in PBS to wash out the molecularly imprinted polymer MIP, filter, dialyze, freeze dry, and place in a vacuum drying oven for later use. (3) Polyethylene glycol double bond functionalization Weigh out polyethylene glycol (PEG) and dissolve it in dichloromethane. Add triethylamine to dissolve the PEG and sonicate it. Purge the solution with nitrogen gas and add acryloyl chloride dropwise. After stirring, filter to remove the solid and wash with potassium bicarbonate solution. Dry the solution with anhydrous sodium sulfate. Precipitate the double-bond functionalized PEG in cold diethyl ether, filter it, and dry it under vacuum overnight. (4) Preparation of epitope-imprinted biomimetic receptors At room temperature, prepare a PBS solution of photoinitiator, weigh the MIP obtained in step (2) and the double-bond functionalized PEG obtained in step (3), mix and sonicate in a centrifuge tube, place in a well plate, and irradiate under ultraviolet light to prepare an epitope-imprinted biomimetic receptor MIP@PEG that recruits endogenous osteogenic factors.

[0007] In step (1), the glass beads GBs have a particle size of 90~110 μm; the surface activation process is as follows: GBs are boiled in 4 M NaOH solution for 15 min, washed with pure water until neutral, and then dried in an oven at 40~50 ℃ for later use.

[0008] In step (1), the volume ratio of 3-(isobutyryloxy)propyltrimethoxysilane to anhydrous toluene in the mixture of 3-(isobutyryloxy)propyltrimethoxysilane and anhydrous toluene is 1:50; the temperature of the constant temperature oscillator is 25 ℃, the oscillation time is 12-24 h, and the oscillation rate is 45 r / min.

[0009] In step (1), the template molecule is ALKRQGRTLC, which is composed of the inactive terminal epitope polypeptide chain segment ALKRQGRTL of the endogenous osteogenic growth peptide OGP and a cysteine ​​C. The concentration of the template molecule in the PBS solution containing the template molecule is 25 mM.

[0010] In step (1), the concentration of sodium dithionite solution is 50 mM, the concentration of photoinitiator solution is 0.5% (w / v), and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP).

[0011] In step (1), the ratio of the amount of GBs after sieving, PBS solution containing template molecules, sodium dithionite solution and LAP solution is 50 g: 40~50 mL: 0.1-0.2 mL: 0.5-1 mL; the reaction time under ultraviolet irradiation is 6-12 h.

[0012] In step (2), the temperature-responsive monomer is a thermosensitive monomer. N -Isopropylacrylamide NIPAAm; the functional monomer is 2-(trifluoromethyl)acrylate TFMAA and N A mixture of -tert-butylacrylamide (TBAm); the crosslinking agent is...N,N - Methylenebisacrylamide (BIS); temperature-responsive monomers, functional monomers, and crosslinking agents are referred to as the total prepolymer; the molar proportions of NIPAAm, TFMAA, TBAm, and BIS in the total prepolymer are 85-87%, 5%, 3-5%, and 5%, respectively.

[0013] In step (2), the ratio of the initiator potassium persulfate (KPS), tetramethylethylenediamine (TEMED), and total prepolymer is 15-20 mg: 1-2 μL: 2 mmol.

[0014] The concentration of PBS solution was 25 mM; the dialysis time was 5-7 days. In step (3), the ratio of polyethylene glycol (PEG), dichloromethane, and acryloyl chloride is 10 g: 50 mL: 2 mL; wherein, the molecular weight of PEG is 5000 Da; In step (3), the stirring time is 12-24 h; the concentration of the potassium bicarbonate solution is 2 M.

[0015] In step (4), the ratio of the amount of MIP, double-bond functionalized PEG, and photoinitiator in PBS solution is 100~120 μg: 9~10 mg: 300 μL, wherein the concentration of photoinitiator in PBS solution is 0.2-0.5% (w / v); the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid lithium LAP.

[0016] In step (4), the irradiation time under ultraviolet light is 1-3 min.

[0017] The epitope-imprinted biomimetic receptor MIP@PEG of the self-recruiting endogenous osteogenic growth peptide obtained in this invention can be used as a tissue engineering scaffold to induce osteogenic differentiation of stem cells or promote bone defect repair.

[0018] The specific plan is as follows: (A) In vitro cell experiments to evaluate the effect of epitope-imprinted biomimetic receptor MIP@PEG on promoting osteogenic differentiation of BMSCs The above solution was incubated in 1 mL of 10 nM OGP solution for 12-16 h, followed by rinsing 2-3 times with PBS solution. Rat bone marrow mesenchymal stem cells (BMSCs) were seeded in 24-well plates (2 × 10⁻⁶). 4Cells per well (1 mL), then place MIP@PEG in a Transwell chamber and culture for 14 days in medium at 37°C. Fix cells with 4% paraformaldehyde solution, wash with pure water, and then stain with an ALP kit. After 21 days of culture, fix cells with 4% paraformaldehyde solution, wash with pure water, and then stain with an ARS staining kit.

[0019] (B) In vivo rat experiments to evaluate the effect of epitope-imprinted biomimetic receptor MIP@PEG self-recruiting OGP on promoting bone repair. MIP@PEG was implanted into a femoral condyle defect model in SD rats, and the experiment was conducted at 4 and 8 weeks, with bone tissue samples collected at specified time points. The bone tissue samples were treated with 4% paraformaldehyde solution for 2 days. After fixation, the bone tissue samples were scanned using Micro-CT, followed by 3D reconstruction, and the reconstructed model was quantitatively evaluated. The bone tissue samples underwent decalcification using EDTA decalcification solution, with the solution changed every 3 days until decalcification was complete. Finally, the bone tissue samples were embedded in paraffin and sectioned for HE and Masson staining to assess the bone repair effect.

[0020] The recruitable endogenous osteogenic factor epitope imprinted biomimetic receptor can recruit endogenous OGPs at bone defects; recruit BMSCs to migrate to damaged tissue; induce BMSCs to differentiate into osteoblasts, accelerate the bone defect repair process, and realize its application in bone tissue regeneration.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention proposes an innovative concept of "preserving overall function through local epitope recognition." It successfully screened non-bioactive specific epitopes of osteoglobulin (OGP) for the first time, prepared epitope-imprinted nanoparticles (MIPs), and based on this, constructed a MIP@PEG biomimetic receptor preparation strategy for self-recruiting endogenous OGP. The core of this strategy lies in achieving the overall specific capture of OGP and the preservation and utilization of its biological functions through the recognition of local epitopes. This epitope-imprinted biomimetic receptor system not only possesses good biocompatibility and mechanical properties but also enables the specific directional capture, sustained release, and activation of local osteogenic signals of OGP. This specific capture capability allows MIPs to accurately recognize and bind to OGP in complex biological environments, thereby providing crucial endogenous growth factor support for subsequent bone defect repair. Compared with the application of exogenous growth factors, this invention not only avoids the immune reactions and adverse reactions that may be caused by exogenous substances but also reduces treatment costs and improves the safety and effectiveness of treatment.

[0023] The method for preparing and applying epitope-imprinted biomimetic receptors proposed in this invention provides a safe and effective novel material for bone defect repair, and is expected to become one of the future technologies in the field of bone tissue engineering. This innovative material design and application not only solves many problems existing in traditional bone defect repair methods, but also provides new ideas and experimental basis for developing novel bone tissue engineering materials based on the recruitment of endogenous growth factors. Attached Figure Description

[0024] Figure 1 XPS spectra of surface-activated GBs (a) and grafted epitope templates GBs (b); Figure 2 The particle size distribution of the prepared MIP and NIP was measured by DLS; Figure 3 Images of the MIP@PEG biomimetic receptor before (a) and after (b) photocuring; Figure 4 The frequency (Δf) of different concentrations of MIP and NIP introduced into the QCM sensor at 37℃ changes over time (a); the isotherm of MIP binding with epitope template molecules (b); Figure 5 The frequency (Δf) of different molecular chips grafted onto the MIP after the QCM sensor was introduced at 37℃ varies with time; (a)-template peptide, (b)-GRGDS, (c)-VQDID; Figure 6 The isothermal titration binding curves of MIP with epitope template molecules (a) and OGP (b) at 37℃ are shown. Figure 7 Images of MIP@PEG biomimetic receptors promoting osteogenic differentiation of BMSCs, including alkaline phosphatase staining (a) and alizarin red staining (b); Figure 8 Micro-CT and 3D reconstruction images of MIP@PEG biomimetic receptors promoting bone tissue healing; Figure 9 HE staining (a) and Masson staining (b) of bone tissue in SD rats to promote the repair of femoral condyle defects by MIP@PEG biomimetic receptor. Detailed Implementation

[0025] The present invention can be further described through the following embodiments; however, the scope of the present invention is not limited to the following embodiments. The present invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Those skilled in the art will understand that, unless otherwise specified below, the materials and operating methods used in the present invention are well-known in the art, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from chemical reagent companies.

[0026] Example 1

[0027] (1) Molecular imprint template molecules are fixed on the surface of glass beads. Activation of glass bead surface: Heat 4 M NaOH solution to boiling, add GBs with a diameter of about 100 μm, stir and continue heating for 15 min, cool and wash with pure water until the pH value of the GBs filtrate reaches 7.2~7.4, then take out the GBs, place them in a glass petri dish, transfer them to an oven, dry at 40~50℃ and then sieve for later use.

[0028] The surface-activated GBs were poured into a mixture of 3-(isobutenoyloxy)propyltrimethoxysilane and anhydrous toluene in a volume ratio of 1:50 and shaken in a constant temperature shaker for 12 h. The GBs were then rinsed with anhydrous ethanol, dried in an oven, and sieved for later use. The sieved GBs were poured into a deoxygenated PBS solution containing 50 mg of the template molecule ALKRQGRTLC, and 0.1 mL of sodium dithionite and 1 mL of the UV photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphine sulfate (LAP) were added. The mixture was reacted under UV irradiation for 6 h. After washing the GBs with deionized water, the GBs with the fixed template were obtained, packed into an isothermal chromatographic column, and connected to a peristaltic pump. Pure water was pumped in to rinse the glass beads.

[0029] like Figure 1 To activate the XPS spectra of GBs and grafted epitope templates GBs, after fixing the template molecules, the characteristic peaks of N and S appeared in the XPS spectra, indicating that the template modification was successful.

[0030] (2) Solid-phase synthesis of molecularly imprinted polymers Weigh out 2 mmol of NIPAAm, TFMAA, TBAm, and BIS in a molar ratio of 86:5:4:5 and dissolve them in PBS solution. Then, sonicate to completely dissolve the monomers. Place the prepolymer solution in an ice-water bath for 30 min to remove oxygen. Next, add 0.07 mmol of KPS to the prepolymer solution and continue purging with nitrogen for 30 min. Finally, add 1.5 μL of TEMED to the prepolymer solution. Pump the prepolymer solution into the chromatographic column at a rate of 3 mL / min. After the monomers are introduced into the column, turn on the circulating water bath and circulate in an ice-water bath for 1 h to allow the monomers to self-assemble around the template molecule ALKRQGRTLC. Then, heat the water bath to 37℃ and polymerize for 15 h. After the reaction was completed, 250 mL of PBS was bubbled into the chromatography column to wash away the unreacted monomers and low-affinity NIPs in the column. Then, the temperature of the circulating water bath was lowered to 6°C, and PBS was bubbled through the column to elute the high-affinity MIP nanoparticles. The collected eluent was filtered and dialyzed for 5 days. After lyophilization, it was placed in a vacuum drying oven for later use to obtain the molecularly imprinted polymer.

[0031] (3) Preparation of double bond functionalized PEG 10 g of PEG was weighed and dissolved in 50 mL of dichloromethane. Triethylamine was added to dissolve the PEG, and the mixture was sonicated. Nitrogen gas was introduced, and 2 mL of acryloyl chloride was added dropwise to the solution. After stirring for 24 h, the solution was washed with 2 M potassium bicarbonate solution and dried with anhydrous sodium sulfate. The product was precipitated in cold diethyl ether, filtered, and dried under vacuum overnight.

[0032] (4) Preparation of epitope-imprinted biomimetic receptors A 0.2% (w / v) LAP photoinitiator was prepared. The MIP prepared in step (2), the above-mentioned PEG, and the LAP photoinitiator were mixed in a centrifuge tube at a ratio of 100 μg: 10 mg: 300 μL. The mixture was ultrasonically mixed, and then the mixed solution was placed in a 24-well plate or glass bottle and irradiated under ultraviolet light for 1 min. The epitope-imprinted biomimetic receptor MIP@PEG was obtained.

[0033] In step (4), MIP is replaced with NIP collected in step (2), and the product is NIP@PEG.

[0034] Example 2

[0035] (1) Molecular imprint template molecules are fixed on the surface of glass beads, the steps are the same as step (1) in Example 1; (2) Solid-phase synthesis of molecularly imprinted polymers Weigh out 2 mmol of NIPAAm, TFMAA, TBAm, and BIS in a molar ratio of 85:5:5:5 and dissolve them in PBS. Then, sonicate to completely dissolve the monomers. Place the prepolymer solution in an ice-water bath for 30 min to remove oxygen. Next, add 0.07 mmol of KPS to the prepolymer solution and continue purging with nitrogen for 30 min. Finally, add 1.5 μL of TEMED to the prepolymer solution. Pump the prepolymer solution into the chromatographic column at a rate of 3 mL / min. After the monomers are introduced into the column, turn on the circulating water bath and circulate in an ice-water bath for 1 h to allow the monomers to self-assemble around the template molecule ALKRQGRTLC. Then, heat the water bath to 37°C and polymerize for 15 h. After the reaction was completed, 250 mL of PBS was bubbled into the chromatography column to wash away the unreacted monomers and low-affinity NIPs. Then, the temperature of the circulating water bath was lowered to 6°C, and PBS was bubbled through the column to elute the high-affinity MIPs. The collected eluent was filtered and dialyzed for 5 days. After lyophilization, it was placed in a vacuum drying oven for later use to obtain the molecularly imprinted polymer.

[0036] A 0.1% (w / v) polymer solution was prepared using deionized water, filtered through a 0.22 μm filter membrane, and placed in an ice-water bath. The solution was sonicated for 5 min to disperse the MIP and NIP particles. After sonication, 1 mL of the solution was transferred to a quartz cuvette. At 25°C, dynamic light scattering analysis revealed that the MIP nanoparticles had a diameter of approximately 43 nm, and the NIP nanoparticles had a diameter of approximately 30 nm. (See attached image) Figure 2 As shown.

[0037] (3) Preparation of double bond functionalized PEG 10 g of PEG was weighed and dissolved in 50 mL of dichloromethane. Triethylamine was added to dissolve the PEG, and the solution was sonicated. Nitrogen gas was then introduced, and 2 mL of acryloyl chloride was added dropwise to the solution. After stirring for 24 h, the solution was washed with 2 M potassium bicarbonate solution and dried over anhydrous sodium sulfate. The product was precipitated in cold diethyl ether, filtered, and dried under vacuum overnight.

[0038] (4) Preparation of epitope-imprinted biomimetic receptors Prepare a 0.2% (w / v) LAP photoinitiator solution. Mix the MIP prepared in step (2), the above-mentioned PEG, and the LAP photoinitiator in a centrifuge tube at a ratio of 100 μg: 10 mg: 300 μL. Sonicate the mixture until homogeneous, then place the solution in a 24-well plate or glass bottle and irradiate it under ultraviolet light for 1 min. See attached... Figure 3 As shown, the solution was cured under ultraviolet irradiation for 1 minute to prepare the epitope-imprinted biomimetic receptor MIP@PEG.

[0039] In step (4), MIP is replaced with NIP collected in step (2), and the product is NIP@PEG.

[0040] Experimental Example 1

[0041] This experimental example investigates the performance of the molecularly imprinted polymer prepared in step (2) of Example 2 in specifically recognizing the template molecule ALKRQGRTLC using the following method: The specific steps are as follows: Specificity and selectivity were tested using a quartz microbalance QCM. First, template molecules were immobilized on the chip surface. Then, the chip was placed into the QCM system, and a peristaltic pump was turned on with a flow rate of 3 mL / h. After the air baseline leveled out, 25 mM PBS was pumped into the QCM sensor until the baseline leveled out again. A series of concentration gradients (5–250 μg / mL) were prepared from the lyophilized MIP and NIP prepared in Example 2 for later use. Subsequently, MIP or NIP solutions were passed through at 15-minute intervals, increasing in concentration, and the frequency signal changes were observed. The dissociation constant of the MIP or NIP to the template molecule was calculated by analyzing the Langmuir equation and fitting the curve based on the concentration gradient and the detected frequency change, thus calculating the equilibrium dissociation constant. K D ) value.

[0042] The results are attached. Figure 4 (a) The chip frequency (Δf) changes over time after different concentrations of MIP and NIP are introduced into the QCM sensor at 37℃. The frequency drop of the MIP group is significantly greater than that of the NIP group, indicating that the former has a higher affinity for the template molecule; (b) The binding isotherm of MIP and template molecule shows that the MIP... K D The value was 214.70 μg / mL (433.12 nM).

[0043] Experimental Example 2

[0044] This experimental example investigates the selective recognition performance of the molecularly imprinted polymer prepared in step (2) of Example 2 using the following method: Specificity and selective recognition performance were tested using a quartz microbalance-based quartz microbalance-based quantization (QCM). First, different polypeptide segments (GRGDS, VQDID, and the template polypeptide segment ALKRQGRTLC) were immobilized on the chip surface. The chips were then placed into the QCM system, and the temperature was set to 37°C. The peristaltic pump speed was 3 mL / h. After the air baseline leveled out, 25 mM PBS was pumped into the QCM sensor until the baseline leveled out again. A series of concentration gradients (5–250 μg / mL) were prepared from the lyophilized MIP prepared in Example 2 for later use. Subsequently, the MIP solution was bubbled through at 15-minute intervals, increasing in concentration, and the frequency signal changes were observed.

[0045] The results are attached. Figure 5 The frequency (Δf) of grafting different molecular chips onto the QCM sensor at different concentrations of MIP at 37℃ changed over time. Since MIP and the corresponding multi-template peptide are spatially complementary, the binding frequency of MIP to other molecules is much lower than that of the corresponding polypeptide chain; this indicates that nanoparticles have excellent selective recognition ability.

[0046] Experimental Example 3

[0047] This experimental example investigates the specific recognition ability of the molecularly imprinted polymer prepared in step (2) of Example 2 for OGP protein using the following method: The thermodynamic parameters between the MIP-epitope template and OGP prepared in Example 2 were tested using an isothermal titration calorimeter (ITC). First, 40 μL of a 10.6 μM MIP and NIP solution was prepared and injected into a syringe; 300 μL of a 1 μM template molecule solution was prepared and injected into the sample cell. The solutions were degassed before testing. The reaction temperature was set at 37 °C, the stirring speed at 750 r / min, and 19 injections were performed, each injecting 2 μL at 120 s intervals. The Gibbs free energy formula was used to calculate the free energy, enthalpy change, and entropy change.

[0048] The results are attached. Figure 6 In (a), the MIP binds to the epitope template, has a free energy less than 0, and the reaction proceeds spontaneously. The heat change generated by the binding is the main driving force of the reaction. K D The value is 205.76 nM; while NIP shows no obvious binding to the epitope template. In (b), MIP binds to OGP, and has a smaller free energy. K D The value was 81.30 nM; while NIP showed no significant binding to OGP. The experimental results indicate that MIP can recognize not only epitope templates but also OGP, and exhibits excellent binding performance.

[0049] Test Example 4

[0050] This experimental example was used to evaluate the ability of the epitope-imprinted biomimetic receptor MIP@PEG, the final product prepared in Epitope Example 2, to promote osteogenic differentiation as a tissue engineering scaffold. The specific steps were as follows: The epitope-imprinted biomimetic receptor MIP@PEG described in Example 2 was incubated in 10 nM OGP (1 mL) solution. BMSCs cells were seeded in 24-well plates (2 × 10⁻⁶ cells / well). 4Cells per well (1 mL), MIP@PEG was placed in a Transwell chamber and cultured for 14 days at 37°C. Cells were then fixed with 4% paraformaldehyde solution, washed with pure water, and stained with an ALP kit. Cells cultured for 21 days were fixed with 4% paraformaldehyde solution, washed with pure water, and stained with an ARS staining kit.

[0051] To compare the osteogenic ability of MIP@PEG, pure PEG hydrogel (PEG) and non-imprinted hydrogel (NIP@PEG) were prepared. The preparation method was the same as step (4) in Example 2, except that MIP nanoparticles were not added when preparing pure PEG hydrogel, and MIP was replaced with NIP in Example 2 when preparing NIP@PEG hydrogel.

[0052] The in vitro cell experiments were grouped as follows: (a) Control, (b) PEG, (c) PEG+OGP, (d) NIP@PEG+OGP, (e) MIP@PEG+OGP.

[0053] As attached Figure 7 As shown, BMSCs co-cultured with MIP@PEG+OGP exhibited the most significant osteogenic differentiation trend, the deepest alkaline phosphatase staining (a), and the highest number of alizarin red calcium nodules (b). This indicates that the OGP-loaded MIP@PEG biomimetic receptor effectively released OGP during long-term osteogenic differentiation induction, promoting the differentiation process of BMSCs.

[0054] Experimental Example 5

[0055] This study established a rat femoral condyle defect model to evaluate the performance of the epitope-imprinted biomimetic receptor MIP@PEG as a tissue engineering scaffold in promoting the repair of rat femoral condyle defects. The specific steps are as follows: Healthy male SD rats aged 6 weeks were selected and housed in the SPF experimental animal facility. The rats were divided into four groups: (a) Control, (b) PEG (pure PEG hydrogel as described in Example 4), (c) NIP@PEG (non-imprinted hydrogel as described in Example 4), and (d) MIP@PEG. Each group contained 4 rats, and the experimental period was 4 weeks and 8 weeks. After random selection, the rats were generally anesthetized by intraperitoneal injection of 0.3 ml / 100 g of 2.5% sodium pentobarbital. Hair in the intercondylar fossa of the femur was removed using an animal electric razor and a non-irritating depilatory cream to fully expose the surgical area. After disinfection of the surgical site, a 3 mm diameter hole was drilled near the intercondylar fossa of the femur. The defect was rinsed with physiological saline, and the above-mentioned hydrogel precursor solution was injected, followed by curing under ultraviolet light. The rats were kept in normal housing, and bone tissue samples were collected by periodic sacrifice. After fixing and decalcifying the tissue, it was sectioned in paraffin and then stained with hematoxylin and eosin (HE) and Masson stain.

[0056] As attached Figure 8 As shown in the Micro-CT and 3D reconstructed images, bone tissue treated with MIP@PEG biomimetic receptors was effectively treated, with a significant reduction in bone defect volume in rats, and an increase in the number and density of trabeculae within the bone tissue. These changes indicate that the MIP@PEG biomimetic receptor can effectively recognize and capture free OGP in vivo, and then present it to surrounding cells, thereby promoting bone tissue repair and regeneration.

[0057] As attached Figure 9 As shown, at 4 weeks, HE staining clearly revealed round bone defects. The bone tissue in the control group was discontinuous and internally porous, while the MIP@PEG biomimetic receptor group showed newly formed trabecular bone structures. This improvement was even more pronounced at 8 weeks, with reduced cavity volume and enhanced bone tissue continuity, indicating a trend towards improved repair. Masson staining also assessed the capabilities of the MIP@PEG biomimetic receptor, which exhibited greater collagen deposition and osteoid formation, suggesting that a self-recruitment strategy can accelerate bone tissue repair and reduce the recovery period.

[0058] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an epitope imprinted biomimetic receptor that self-recruits endogenous osteogenic growth peptides, characterized by, Comprising the following steps: (1) Epitope imprinting template is fixed on the surface of glass beads Pour the surface-activated glass beads GBs into a mixture of 3-(isobutyryloxy) propyl trimethoxysilane and anhydrous toluene, and place it in a constant temperature oscillator; after the vibration is completed, the GBs are washed with anhydrous ethanol, dried in an oven and sieved for use; Pour the sieved GBs into the template molecule-containing PBS solution after oxygen removal, add sodium dithionite solution and light initiator solution, combine using thiol-alkene click chemistry, and react under ultraviolet irradiation; the GBs are washed with deionized water to obtain GBs with fixed templates; the GBs are loaded into a constant temperature chromatographic column and connected to a peristaltic pump, and pure water is pumped to wash the glass beads; (2) Solid-phase synthesis of epitope imprinting polymer Weigh the temperature-responsive monomer, functional monomer and crosslinking agent into a PBS solution, ultrasonically dissolve them, place them in an ice water bath and introduce nitrogen to form a pre-polymer solution; Then, under anaerobic conditions, add the redox initiator potassium persulfate and tetramethyl ethylenediamine to the pre-polymer solution, and pump the pre-polymer solution into the chromatographic column obtained in step (1); Turn on the circulating water bath switch, circulate the ice water bath for a certain period of time, set the temperature to T1, and react for t1; at T1, pump in PBS to elute out the non-imprinted polymer NIP of unreacted monomers and oligomers; then, reduce the temperature of the circulating water bath to T2, pump in PBS to elute out the molecularly imprinted polymer MIP, filter, dialyze, freeze-dry and place in a vacuum drying oven for standby; (3) Double bond functionalization of polyethylene glycol Weigh the polyethylene glycol PEG into dichloromethane, add triethylamine to dissolve, ultrasonically, introduce nitrogen, and add acryloyl chloride dropwise to the solution; after stirring, filter out the solids, wash with potassium bicarbonate solution, and dry with anhydrous sodium sulfate; sediment the double bond functionalized PEG in cold ether, filter, and dry under vacuum overnight; (4) Preparation of epitope imprinting biomimetic receptor At room temperature, prepare a PBS solution of a light initiator, weigh the MIP obtained in step (2) and the double bond functionalized PEG obtained in step (3), mix in a centrifuge tube, ultrasonically, place in a well plate, and irradiate under ultraviolet light to prepare an epitope imprinting biomimetic receptor MIP@PEG that can recruit endogenous osteogenic factors.

2. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (1), The particle size of the glass beads GBs is 90-110 μm; The surface activation process is as follows: after boiling the GBs in 4 M NaOH solution for 15 min, wash with pure water to neutralize, and place in a 40-50 ℃ oven to dry for standby; In the mixture of 3-(isobutyryloxy) propyl trimethoxysilane and anhydrous toluene, the volume ratio of 3-(isobutyryloxy) propyl trimethoxysilane to anhydrous toluene is 1:50; the temperature of the constant temperature oscillator is 25 ℃, the vibration time is 12-24 h, and the vibration rate is 45 r / min.

3. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (1), the template molecule is ALK RQGRTLC, which is composed of ALK RQGRTLC, a non-active epitope polypeptide segment of endogenous osteogenic growth peptide OGP, and a cysteine C; the concentration of the template molecule in the PBS solution containing the template molecule is 25 mM; the concentration of the sodium hydrosulfite solution is 50 mM; the concentration of the solution of the photoinitiator is 0.5% (w / v), and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP); the ratio of the use amounts of the screened GBs, the PBS solution containing the template molecule, the sodium hydrosulfite solution, and the LAP solution is 50 g: 40-50 mL: 0.1-0.2 mL: 0.5-1 mL; and the reaction time under ultraviolet irradiation is 6-12 h.

4. The method of claim 1, wherein the self-recruiting, epitope-imprinted, biomimetic receptor for endogenous osteogenic growth peptides is prepared by the steps of: In step (2), the temperature-responsive monomer is a temperature-sensitive monomer N - isopropyl acrylamide NIPAAm; the functional monomer is a mixture of 2- (trifluoromethyl) acrylate TFMAA and N - tert-butyl acrylamide TBAm; the crosslinker is N,N - methylene bisacrylamide BIS; the temperature-responsive monomer, the functional monomer and the crosslinker are referred to as the total prepolymer; The molar use amounts of the NIPAAm, TFMAA, TBAm, and BIS account for 85-87%, 5%, 3-5%, and 5% of the total prepolymer, respectively.

5. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (2), the ratio of the use amounts of the initiator potassium persulfate (KPS), tetramethyl ethylenediamine (TEMED), and the total prepolymer is 15-20 mg: 1-2 μL: 2 mmol.

6. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (2), the ice-water bath circulation time is 1-2 h; T1 is 37 ℃, t1 is 12-18 h; T2 is 6 ℃; the concentration of the PBS solution is 25 mM; and the dialysis time is 5-7 days.

7. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (3), the ratio of the use amounts of the polyethylene glycol (PEG), dichloromethane, and acryloyl chloride is 10 g: 50 mL: 2 mL; and the molecular weight of the PEG is 5000 Da.

8. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (3), the stirring time is 12-24 h; and the concentration of the potassium bicarbonate solution is 2 M.

9. The method for preparing the epitope-imprinted biomimetic receptor for self-recruiting endogenous osteogenic growth peptides as described in claim 1, characterized in that, In step (4), the ratio of the use amounts of the MIP, the double-bond functionalized PEG, and the PBS solution of the photoinitiator is 100-120 μg: 9-10 mg: 300 μL, and the concentration of the PBS solution of the photoinitiator is 0.2-0.5% (w / v); the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP); The ultraviolet irradiation time is 1-3 min.

10. Use of the epitope imprinting biomimetic receptor MIP@PEG of self-recruited endogenous osteogenic growth peptide prepared by the preparation method in any one of claims 1-9 as a tissue engineering scaffold for inducing osteogenic differentiation of stem cells or promoting repair of bone defects.

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