Injectable hydrogel based on modification of platelet lysate as well as preparation method and application of injectable hydrogel

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses injectable hydrogel based on platelet lysate modification, a preparation method of the injectable hydrogel and application of the injectable hydrogel in osteochondral defect repair. The platelet lysate modified injectable hydrogel is photosensitive dual-network hydrogel with a uniform structure, which is constructed by taking platelet lysate (PL), methacrylic anhydride (MA), E7 peptide and polyethylene glycol diacrylate (PEGDA) as raw materials through a photocuring technology, and the hydrogel is cured after in-situ injection at a defect position to promote osteochondral regeneration. The dual-network hydrogel can simultaneously meet the extracellular matrix bionic requirements of cartilage and bone due to the characteristics of rigidity and softness; the combination of the E7 peptide and the PL can effectively utilize a local microenvironment and promote migration and directional differentiation of surrounding stem cells. Therefore, the invention provides a self-adaptive hydrogel scheme which is simple and convenient in preparation process and can promote layered repair of osteochondral defects by using a surrounding matrix environment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an injectable hydrogel based on platelet lysate modification, its preparation method, and its application in the repair of osteochondral defects. Background Technology

[0002] Repairing osteochondral defects in joints caused by trauma, degeneration, and tumor resection remains a major clinical challenge. Joint tissue is a complex structure composed of cartilage and subchondral bone, a typical multi-layered, progressively changing structure. Achieving effective layered repair remains a significant challenge. Current techniques include microfracture surgery, autologous osteochondral transplantation, and bio-scaffold material filling techniques.

[0003] An ideal osteochondral scaffold should provide a suitable microenvironment for natural cell growth and tissue regeneration, while also considering the layered structural differences of articular osteochondral material. Most existing osteochondral repair materials remain in the preclinical stage, often employing biomimetic gradient structure designs to mimic the layered characteristics of natural osteochondral. For example, Rongtai Sun et al. prepared a continuous gradient mineralized hydrogel using gravity infiltration, with the compressive modulus of the surface and subchondral layers simulating the mechanical properties of cartilage and subchondral bone, respectively (Advanced Functional Materials, 2024, 34, 2408249). However, the preparation of continuous gradient mineralized hydrogels relies on a complex gravity infiltration process, and the gradient interface may pose a risk of abrupt changes in mechanical properties. Another magnesium alloy composite hydrogel scaffold (CN116236623A) exhibits controllable regulation of antibacterial and osteochondral regeneration-promoting capabilities, but its preparation process is complex, and stress concentration points easily form at the interface of the three layers, potentially leading to microcrack propagation after long-term implantation. Furthermore, its three-layer scaffold needs to be prepared separately, making the process cumbersome and difficult to scale up. Summary of the Invention

[0004] To address the issues of unstable properties in existing biomimetic gradient structure scaffolds, as well as the complexity of multilayer structure fabrication, this invention provides a homogeneous hydrogel that is low-cost, simple to prepare, easy to use, and utilizes the microenvironment driving the stratification of autologous defects and stem cell recruitment to achieve layered repair of osteochondral tissue.

[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing an injectable hydrogel based on platelet lysis buffer modification, comprising the following steps:

[0007] Step 1. Synthesis of PEGDA:

[0008] Polyethylene glycol (PEG) was mixed with triethylamine and acryloyl chloride and reacted. The reaction mixture was then precipitated, purified and dialyzed, and finally lyophilized to obtain PEGDA powder.

[0009] Step 2. Synthesis of PLMA:

[0010] Platelet lysis buffer (PL) was mixed with methacrylic anhydride (MA) and reacted. The reaction mixture was then dialyzed to remove excess methacrylic acid, and finally lyophilized to obtain PLMA powder.

[0011] Step 3. Preparation of PP7 hydrogel:

[0012] PLMA powder, SH-E7 powder, PEGDA powder and photoinitiator LAP powder were dissolved in PBS solution and cured by UV irradiation to obtain PEGDA@PLMA-E7(PP7) hydrogel.

[0013] Preferably, the preparation method includes the following steps:

[0014] Step 1. Synthesis of PEGDA:

[0015] PEG was dissolved in anhydrous toluene at 40°C under N2 conditions. After cooling to room temperature, triethylamine was added and stirred until homogeneous. Acryloyl chloride was then added dropwise and stirred at room temperature for 2 hours. The resulting polymer was precipitated in n-hexane. The precipitate was further purified three times by toluene and n-hexane. The purified PEGDA was then dissolved in deionized water and dialyzed for three days. After lyophilization, PEGDA was obtained.

[0016] Step 2. Synthesis of PLMA:

[0017] The PL lyophilized powder was completely dissolved in PBS solution, and then methacrylic anhydride (MA) was added dropwise to the PL solution to adjust the pH to 9.0. The mixture was stirred at room temperature for 24 hours. The reaction mixture was then dialyzed in pure water for 3-5 days to remove excess methacrylic acid. Finally, the PLMA powder was obtained by lyophilization.

[0018] Step 3. Preparation of PP7 hydrogel:

[0019] PLMA powder was dissolved in PBS solution, NaOH solution was added, then SH-E7 powder was added and sonicated to dissolve completely; then PEGDA powder was added and sonicated to dissolve completely; finally, photoinitiator LAP powder was added and sonicated to dissolve completely. After curing by UV curing lamp, PEGDA@PLMA-E7 (PP7) hydrogel was obtained.

[0020] Further, in step 1, the concentration of PEG dissolved in anhydrous toluene is 10% w / v; the volume ratio of triethylamine to anhydrous toluene is 3:200; and the volume ratio of acryloyl chloride to anhydrous toluene is 1:100.

[0021] Furthermore, in step 1, the molecular weight cutoff of the dialysis bag used for dialyzing the reaction mixture in pure water is 1000 Da.

[0022] Furthermore, in step 2, the concentration of PL dissolved in PBS is 1% w / v; the volume ratio of MA to PBS is 1:100.

[0023] Furthermore, in step 2, the molecular weight cutoff of the dialysis bag used for dialysis of the reaction mixture in pure water is 3500 Da.

[0024] Furthermore, in step 3, the concentration of PLMA dissolved in PBS is 10% w / v; the volume ratio of NaOH solution to PBS is 3:100; the concentration of SH-E7 dissolved in PBS is 1% w / v; and the concentration of LAP dissolved in PBS is 0.3% w / v.

[0025] Furthermore, in step 3, the concentration of PEGDA dissolved in PBS is 5-15% w / v. When the concentration of PEGDA is 10% w / v, the resulting hydrogel has better mechanical properties.

[0026] Furthermore, in step 3, the ultrasonic time after adding SH-E7 powder and PEGDA powder is 5 minutes.

[0027] Furthermore, in step 3, the ultrasonic time after adding LAP powder is 2 minutes.

[0028] Furthermore, in step 3, the UV lamp curing time is 0.5-3 minutes.

[0029] Secondly, the present invention provides an injectable hydrogel based on platelet lysis fluid modified by the above method. This hydrogel uses platelet lysis fluid (PL), methacrylic anhydride (MA), E7 peptide, and polyethylene glycol diacrylate (PEGDA) as raw materials. A uniform photosensitive dual-network hydrogel is constructed using photocuring technology. After in-situ injection at the defect site, it solidifies, promoting osteochondral regeneration.

[0030] Thirdly, the present invention provides the application of the above-mentioned injectable hydrogel modified with platelet lysate in the preparation of products for the repair of osteochondral defects.

[0031] Furthermore, the application method is as follows: inject the hydrogel solution into the defect until it is filled, and use ultraviolet light to cure it for 0.5-3 minutes, so that the hydrogel forms a gel state that adheres tightly to the wound surface.

[0032] Because the cartilage component in joint tissue requires a hydrated viscoelastic matrix with relatively low compressive modulus for compensation, hydrogels can meet the mechanical requirements of this component. Conversely, the bone component requires a rigid framework with relatively high modulus. This invention provides a solution for this: a dual-network hydrogel. The dual-network hydrogel consists of two interpenetrating elastic networks, primarily formed through a two-step polymerization reaction. The first network is rigid and brittle, acting as a sacrificial network to effectively dissipate energy. Simultaneously, the second network is flexible, possessing a certain degree of extensibility to ensure the integrity of the hydrogel during deformation. The dynamic adaptability of the dual-network hydrogel can meet the scaffold material requirements of different tissue repair processes. In this invention, methacrylated platelet lysate (PLMA) combined with polyethylene glycol diacrylate (PEGDA) dual-network hydrogel serves as flexible and rigid components, respectively, and its porous structure provides ample space for cell and tissue growth. On the other hand, we grafted the stem cell homing peptide E7 onto PLMA, which can effectively drive synovial fluid mesenchymal stem cells (SFMSCs) in the joint cavity and bone marrow mesenchymal stem cells (BMSCs) in the bone marrow to migrate to the defect area. The abundant growth factors contained in platelet lysate (PL) can promote the directed differentiation of stem cells in different microenvironments (bone or cartilage).

[0033] In summary, the dual-network hydrogel's combination of rigidity and flexibility can simultaneously meet the biomimetic requirements of the extracellular matrix for both cartilage and bone; the E7 peptide combined with PL can effectively utilize the local microenvironment to promote the migration and directed differentiation of surrounding stem cells. This invention provides an adaptive hydrogel solution with a simple preparation process that can utilize the surrounding matrix environment to promote the layered repair of osteochondral defects.

[0034] The present invention has the following beneficial effects:

[0035] 1. The resulting hydrogel can be used for complex osteochondral defects, conforming to defects of any shape and precisely inducing their regeneration;

[0036] 2. The resulting hydrogel adheres firmly to the defect, effectively preventing changes in the support structure caused by joint weight-bearing and movement;

[0037] 3. The resulting hydrogel has a uniform structure, and there is no need to adjust the hydrogel composition according to the actual defect layer;

[0038] 4. The resulting hydrogel is rich in porosity and bioactive factors, has good biocompatibility, and can promote the proliferation, migration and directional differentiation of mesenchymal cells at the defect site, which is beneficial to osteochondral regeneration;

[0039] 5. The materials required for the hydrogel of this invention are widely available, inexpensive, easy to prepare, easy to store and transport, and simple to use. Attached Figure Description

[0040] Figure 1 The images show the 1H NMR spectra of PL and PLMA.

[0041] Figure 2 The NMR spectra of PEG and PEGDA are shown in the form of 1H NMR.

[0042] Figure 3 Infrared spectra of PL and PLMA.

[0043] Figure 4 Infrared spectra of PEG and PEGDA.

[0044] Figure 5 Infrared spectra of PLMA / PEGDA (hydrogel prepared without SH-E7) and PLMA / SH-E7 / PEGDA (PP7 hydrogel).

[0045] Figure 6 These are scanning electron microscope images of four groups of hydrogels.

[0046] Figure 7 The diagrams show the time-rheological and frequency-rheological analyses of the four hydrogels.

[0047] Figure 8 The swelling curves of the four hydrogels are shown.

[0048] Figure 9 These are the compressive stress-strain curves for four groups of hydrogels.

[0049] Figure 10 The degradation curves of 15% PP7 hydrogel in the presence and absence of lysozyme are shown.

[0050] Figure 11 Statistical analysis of CCK8 cells co-cultured with 15% PP7 hydrogel and bone marrow mesenchymal stem cells (BMSC) and synovial fluid-derived mesenchymal stem cells (SFMSC).

[0051] Figure 12 This study presents qPCR results of osteogenic markers of mRNA from BMSCs co-cultured in a bone microenvironment with 15% PP7 hydrogel and ordinary BMSCs, as well as qPCR results of chondrogenic markers of mRNA from SFMSCs co-cultured in a cartilage microenvironment with 15% PP7 hydrogel and ordinary SFMSCs.

[0052] Figure 13 ICRS histological scoring and microCT bone volume quantification for PP7-treated rats with osteochondral defects. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0055] Example 1: Preparation of 5% PP7 hydrogel

[0056] Step 1. Synthesis of PEGDA:

[0057] 8 g of PEG (4000 Da) was dissolved in 80 mL of anhydrous toluene at 40 °C under N2 atmosphere. After cooling to room temperature, 1.2 mL of triethylamine was added, and the mixture was stirred until homogeneous. Then, 0.8 mL of acryloyl chloride was added dropwise, and the mixture was stirred at room temperature for 2 h. The resulting polymer was precipitated in n-hexane. The precipitate was further purified three times by passing it through toluene and n-hexane (i.e., the precipitate was dissolved in anhydrous toluene and then precipitated in n-hexane, repeated three times to wash away impurities). The purified PEGDA was then dissolved in deionized water and dialyzed (MW: 1000 Da) for three days. After lyophilization, PEGDA was obtained and stored at 4 °C for later use.

[0058] Step 2. Synthesis of PLMA:

[0059] 1 g of lyophilized PL powder (purchased from Fujian Sany Hematopoietic Co., Ltd., catalog number RC-002-500) was completely dissolved in 100 mL of PBS solution. Then, 1 mL of methacrylic anhydride was added dropwise to the PL solution to adjust the pH to 9.0, and the mixture was stirred at room temperature for 24 h. The reaction mixture was then dialyzed against pure water (3500 Da dialysis bag) for 3-5 days to remove excess methacrylic acid. Finally, the mixture was lyophilized to obtain PLMA powder.

[0060] Step 3. Preparation of PEGDA@PLMA-E7 hydrogel:

[0061] Weigh 0.1g of PLMA powder and dissolve it in 1mL of PBS solution. Add 30μL of 1mol / L NaOH solution, then add 10mg of SH-E7 powder (purchased from Xi'an Ruixi Biotechnology Co., Ltd., batch number RM0241241, a thiolized polypeptide derivative with the sequence EPLQLKM-Cys). Sonicate for 5min to completely dissolve the powder. Then add 0.05g of PEGDA powder and sonicate for 5min. After dissolving the PEGDA powder, add 0.003g of LAP powder and sonicate for 2min. After irradiation with a UV curing lamp for 3min, a 5% PP7 hydrogel is obtained.

[0062] Example 2: Preparation of 10% PP7 hydrogel

[0063] Step 1. Synthesis of PEGDA:

[0064] 8 g of PEG (4000 Da) was dissolved in 80 mL of anhydrous toluene at 40 °C under N2 atmosphere. After cooling to room temperature, 1.2 mL of triethylamine was added, and the mixture was stirred until homogeneous. Then, 0.8 mL of acryloyl chloride was added dropwise, and the mixture was stirred at room temperature for 2 h. The resulting polymer was precipitated in n-hexane, and the precipitate was further purified three times by passing it through toluene and n-hexane. The purified PEGDA was then dissolved in deionized water and dialyzed (MW: 1000 Da) for three days. After lyophilization, PEGDA was obtained and stored at 4 °C for later use.

[0065] Step 2. Synthesis of PLMA:

[0066] 1 g of PL lyophilized powder was completely dissolved in 100 mL of PBS solution. Then, 1 mL of methacrylic anhydride was added dropwise to the PL solution to adjust the pH to 9.0, and the mixture was stirred at room temperature for 24 h. The reaction mixture was then dialyzed against pure water (3500 Da dialysis bag) for 3-5 days to remove excess methacrylic acid, and finally lyophilized to obtain PLMA powder.

[0067] Step 3. Preparation of PP7 hydrogel:

[0068] Weigh 0.1g of PLMA powder and dissolve it in 1mL of PBS solution. Add 30μL of 1mol / L NaOH solution, then add 10mg of SH-E7 powder and sonicate for 5min to dissolve it completely. Add 0.1g of PEGDA powder and sonicate for 5min. After it dissolves, add 0.003g of LAP powder and sonicate for 2min. After irradiation with a UV curing lamp for 3min, 10% PP7 hydrogel is obtained.

[0069] Example 3: Preparation of 15% PP7 hydrogel

[0070] Step 1. Synthesis of PEGDA:

[0071] 8 g of PEG (4000 Da) was dissolved in 80 mL of anhydrous toluene at 40 °C under N2 atmosphere. After cooling to room temperature, 1.2 mL of triethylamine was added, and the mixture was stirred until homogeneous. Then, 0.8 mL of acryloyl chloride was added dropwise, and the mixture was stirred at room temperature for 2 h. The resulting polymer was precipitated in n-hexane, and the precipitate was further purified three times by passing it through toluene and n-hexane. The purified PEGDA was then dissolved in deionized water and dialyzed (MW: 1000 Da) for three days. After lyophilization, PEGDA was obtained and stored at 4 °C for later use.

[0072] Step 2. Synthesis of PLMA:

[0073] 1 g of PL lyophilized powder was completely dissolved in 100 mL of PBS solution. Then, 1 mL of methacrylic anhydride was added dropwise to the PL solution to adjust the pH to 9.0, and the mixture was stirred at room temperature for 24 h. The reaction mixture was then dialyzed against pure water (3500 Da dialysis bag) for 3-5 days to remove excess methacrylic acid, and finally lyophilized to obtain PLMA powder.

[0074] Step 3. Preparation of PEGDA@PLMA-E7 hydrogel:

[0075] Weigh 0.1g of PLMA powder and dissolve it in 1mL of PBS solution. Add 30μL of 1mol / L NaOH solution, then add 10mg of SH-E7 powder and sonicate for 5min to dissolve it completely. Add 0.15g of PEGDA powder and sonicate for 5min. After it dissolves, add 0.003g of LAP powder and sonicate for 2min. After irradiation with a UV curing lamp for 3min, 15% PP7 hydrogel is obtained.

[0076] Comparative Example 1: Preparation of hydrogel without PEGDA crosslinking (PLMA-E7)

[0077] Step 1. Synthesis of PLMA:

[0078] 1 g of PL lyophilized powder was completely dissolved in 100 mL of PBS solution. Then, 1 mL of methacrylic anhydride was added dropwise to the PL solution to adjust the pH to 9.0, and the mixture was stirred at room temperature for 24 h. The reaction mixture was then dialyzed against pure water (3500 Da dialysis bag) for 3-5 days to remove excess methacrylic acid, and finally lyophilized to obtain PLMA powder.

[0079] Step 2. Preparation of PLMA-E7 hydrogel:

[0080] Weigh 0.1g of PLMA powder and dissolve it in 1mL of PBS solution. Add 30μL of 1mol / L NaOH solution, then add 10mg of SH-E7 powder. Sonicate for 5min to dissolve completely. After dissolving, add 0.003g of LAP powder and sonicate for 2min. After irradiation with a UV curing lamp for 3min, PLMA-E7 hydrogel is obtained.

[0081] Characterization performance testing:

[0082] (1) Nuclear magnetic resonance test

[0083] Test method: Weigh 20 mg of PL and PLMA, PEG, and PEGDA samples and dissolve them in heavy water or deuterated chloroform. Transfer the solution to an NMR tube and perform structural determination using an NMR spectrometer at room temperature. Then, perform spectral analysis using MestReNova software.

[0084] Test Results: The chemical structure of the substances before and after modification can be investigated by using 1H NMR spectroscopy, thereby determining the effect of the modification. In this experiment, PL was grafted with MA to double-bond PL, and the chemical structures of PL and PLMA were analyzed by 1H NMR spectroscopy. Figure 1 The 1H NMR spectra of PL and PLMA show that the absorption peaks at 5.16 ppm and 5.48 ppm are due to the C=C bond vibrations after methacrylation, which proves that MA successfully modified PL.

[0085] pass 1 HMNR spectral analysis was used to further confirm the synthesis results of PEGDA. Figure 2 The NMR spectra of PEG and PEGDA are shown. Compared with the spectrum of PEG, PEGDA showed three different proton peaks in the chemical shift range of 5.8-6.5 ppm. These are attributed to the three H proton peaks at the -CH=CH2 ends of the double bond after acrylate esterification of PEG. The appearance of these three peaks confirms that the terminal hydroxyl group of PEG has been replaced by acrylate group. Another newly generated H proton peak appeared near 4.3 ppm, which corresponds to the -CH2O-CO- hydrogen signal on the PEGDA skeleton. This proves that the modification of PEG by DA (diacrylate) was successful.

[0086] (2) Infrared testing

[0087] Test method: Weigh 20 mg of the sample and an appropriate amount of dry potassium bromide powder (approximately 5% by mass) into an agate mortar, grind them thoroughly to ensure uniform mixing, and then compress an appropriate amount of the ground sample powder into a thin sample film. Set the scanning range to 4000-500 cm⁻¹. -1 The detection was performed using a Fourier transform infrared spectrometer.

[0088] Test results: Infrared characterization can be used to assist in the analysis of the synthesis results of chemical reactions before and after modification. Figure 3 The figures show the infrared spectra of PL and PLMA. It can be seen from the figures that, compared to PL, PLMA has a lower infrared spectrum at 1635 cm⁻¹. -1 The appearance of a vibrational absorption peak of the C=C double bond indicates that MA was successfully grafted onto PL.

[0089] FTIR spectra of PEG and PEGDA are as follows Figure 4 As shown, PEG and PEGDA at 2892 cm⁻¹ -1 A strong absorption peak, generated by the stretching vibration of the CH bond, was detected nearby. Compared to PEG, the absorption peak of PEGDA was at 1718 cm⁻¹. -1An absorption peak generated by the C=O double bond vibration can be found at the point. This vibrational absorption peak is a characteristic peak generated after the PEG end group is esterified by acyl chloride, which can preliminarily prove the successful synthesis of PEGDA.

[0090] According to literature, the IR range of CSC bonds is between 900 and 1300 cm⁻¹. -1 Between. By Figure 5 It can be seen that after the thiol group of SH-E7 binds to PLMA, the thiol group disappears. Therefore, the infrared spectrum of PLMA / SH-E7 / PEGDA does not have a thiol peak, but it does have a CSC bond peak. It can be clearly seen in the figure that, compared to PLMA / PEGDA, PLMA / SH-E7 / PEGDA has a peak at 1204.81 cm⁻¹. -1 There is a distinct CSC bond absorption peak. This proves that SH-E7 was successfully grafted with PLMA.

[0091] (3) Electron microscopy tests were performed on the hydrogel composite materials prepared in Examples 1-3 and Comparative Example 1.

[0092] Test method: To observe the internal microstructure of the hydrogel, 400 μL of the prepared hydrogel was refrigerated in a freezer at -20°C and freeze-dried in a freeze dryer. The freeze-dried sample was cut with a scalpel to expose its cross-section. The sample was fixed onto the electron microscope stage with conductive adhesive and sputtered with gold for 90 seconds. The surface morphology was observed using a scanning electron microscope.

[0093] Test results: Figure 6 The images show the microstructure of hydrogels with different PEGDA contents under a scanning electron microscope. As can be seen from the images, all four groups of hydrogels have a dense porous structure. The smaller pore structure helps to avoid stress concentration and crack propagation, thereby enhancing the mechanical strength of the hydrogel. Furthermore, as the PEGDA content changes from 0 to 15%, the number of pores tends to decrease. When the PEGDA concentration of the hydrogel increases, the number of cross-linking points in the gel increases, and the network structure becomes more compact and dense, thus reducing the number of pores in the gel.

[0094] (4) Rheological testing of the hydrogel composite materials prepared in Examples 1-3 and Comparative Example 1: The mixed hydrogel was subjected to time scanning and frequency scanning in sequence using a rheometer.

[0095] Time scan: The prepared hydrogel was transferred to the rheometer sample stage, and the scan time was set to 0-5 min. The time change was "linear law change". The changes of sample modulus G' and G" over time were observed.

[0096] Frequency scan: After time scan, set the temperature to 37℃, select a constant strain value, shear strain γ = 0.5%, frequency change to "logarithmic change", angular frequency ω = 0.01~100Hz, set the sampling time control to be set by the equipment, and observe the changes of sample modulus G' and G” with frequency.

[0097] Test results: such as Figure 7 As shown, G' is consistently greater than G" over time, indicating that the hydrogel exhibits a stable gel state and further revealing its rapid gelation ability. Furthermore, during the 0.1-1 Hz scanning process, the G' value showed no significant change with increasing frequency, and G' > G" indicates that the hydrogel has a well-developed crosslinking density and a stable crosslinked network structure.

[0098] (5) The swelling properties of the hydrogel composite materials prepared in Examples 1-3 and Comparative Example 1 were tested.

[0099] Test method: The swelling rate of different groups of hydrogels in PBS solution was determined by gravimetric method. The weight before swelling was m0. Then, the hydrogels were transferred to PBS solution at 37°C. After the predetermined time, they were removed, the surface moisture was wiped off with filter paper, and the weight was m. t The swelling ratio (S) of the hydrogel is calculated using the following formula:

[0100]

[0101] Test results: Swelling performance is one of the important evaluation indicators of hydrogels, reflecting the ability of hydrogel materials to transport substances. In tissue engineering, swelling performance affects whether the scaffold is conducive to the transport of nutrients and the excretion of metabolic waste; in drug release, it affects the rate at which drugs are released into the target tissue. Figure 8 The swelling rate curves of the four groups of hydrogels show that the four groups of hydrogels with different PEGDA contents reached swelling equilibrium within 4 hours, and the swelling rate increased with the increase of PEGDA content. The maximum swelling ratios were 16.2%, 36.0%, 59.5%, and 79.8%, respectively. A PEGDA concentration of more than 10% can bring good water absorption performance.

[0102] (6) The compressibility of the hydrogel composite materials prepared in Examples 1-3 and Comparative Example 1 was tested.

[0103] Test method: The compression test was carried out in a dynamic universal testing machine at 25°C. The hydrogel was made into a cylindrical frustum sample with a diameter of 10 mm and a height of 8 mm, and compressed at a compression rate of 1 mm / min until the sample ruptured.

[0104] Test results: Hydrogels not only require good water absorption, but also good mechanical properties to meet practical needs. For biological hydrogels, high mechanical strength hydrogels are not easily broken under stress and have low friction. Figure 9 The effect of different PEGDA contents on the mechanical properties of PLMA / PEGDA composite hydrogels was shown. As the PEGDA concentration increased from 0% to 10%, the strength and modulus showed an increasing trend. At this point, the compressive strength of the hydrogel system with a PEGDA concentration of 10% reached 167.7 kPa, a significant increase compared to pure PLMA hydrogel (PLMA-E7). When the PEGDA concentration was further increased to 15%, the compressive strength decreased to 38.9 kPa. The fracture strain of the hydrogel decreased with increasing concentration. This is because the increased PEGDA content leads to an increase in the number of double bonds undergoing free radical polymerization, resulting in a more densely crosslinked and less flexible network structure. In comparison, it can be concluded that the hydrogels of Examples 1-3 all showed significantly improved compressive properties compared to Comparative Example 1, and the hydrogel of Example 2 (i.e., when the PEGDA concentration was 10%) exhibited the best compressive mechanical properties.

[0105] (7) Degradation performance test of the hydrogel composite material prepared in Example 3.

[0106] Test Method: A PBS solution with pH 7.4 was used to simulate human body fluids, and the in vitro degradation performance of the hydrogel at 37°C was measured. The degradation status of the hydrogel was evaluated by the percentage of weight retention. The specific procedures were as follows: The hydrogel was first swollen to equilibrium, then freeze-dried and weighed, recorded as w0. One group was transferred to an equal volume of PBS solution and placed in a 37°C water bath for slow degradation, while the other group was transferred to an equal volume of PBS + Lysozyme (1000 U / mL) solution and placed in a 37°C water bath for slow degradation. The hydrogel was removed from the solution at predetermined times (0, 7, 14, 21, 28 days), freeze-dried, and accurately weighed, recorded as w0. t The mass retention rate (W) of the hydrogel is then calculated using the following formula:

[0107]

[0108] Test Results: The biodegradability of polymer materials is of great significance for their application in the biomedical field. This invention tested the degradation performance of 10% PP7 hydrogel in PBS solution and PBS+Lysozyme solution, such as... Figure 10 As shown, the hydrogel degrades faster under enzymatic conditions, indicating that enzymes can accelerate the degradation rate of hydrogels. After 28 days, both hydrogels were almost completely degraded, demonstrating their good biodegradability.

[0109] (8) In vitro experiments were conducted on the hydrogel composite material prepared in Example 3.

[0110] Experimental Methods: Primary rat BMSCs (bone marrow-derived mesenchymal stem cells, naturally possessing osteogenic differentiation potential) and SFMSCs (synovial mesenchymal stem cells, naturally possessing chondrogenic differentiation potential) were used. A hydroxyapatite-coated 24-well plate (CSR-BRA-24P) from CosmoBio (Japan) was used to simulate the bone microenvironment, in which BMSCs were co-cultured with PP7 hydrogel. Ten layers of collagen 2 / chondroitin sulfate coating were deposited on 14nm cell slides using a layer-by-layer self-assembly technique and placed in 24-well plates to simulate the cartilage microenvironment, where SFMSCs were co-cultured with PP7 hydrogel. The absorbance of the culture medium was measured at 450nm after 1, 3, and 7 days of co-culture to reflect cell proliferation capacity. After 14 days of co-culture, mRNA from both types of stem cells was extracted and analyzed by qPCR.

[0111] Experimental results: Cell proliferation capacity test results are as follows Figure 11 As shown, compared with the control group, the proliferation rate of BMSCs and SFMSCs in the PP7 hydrogel co-culture group in the corresponding microenvironment was significantly increased, demonstrating its good cell proliferation-promoting ability. The results of qPCR assays for osteogenic and chondrogenic abilities are shown below. Figure 12 As shown, compared with the control group, the osteogenic marker genes (Col1a1, Runx2, Bglap, AlpI) of BMSCs and the chondrogenic marker genes (Col2a1, Sox9, Acan) of SFMSCs were significantly increased in the PP7 hydrogel co-culture group. This demonstrates that PP7 promotes osteogenic differentiation of mesenchymal stem cells in the bone microenvironment and chondrogenic differentiation of mesenchymal stem cells in the cartilage microenvironment, and has the ability to induce directed differentiation of mesenchymal stem cells in an adaptive manner to the intra-articular osteochondral microenvironment.

[0112] (9) In vivo experiments were conducted on the hydrogel composite material prepared in Example 3.

[0113] Experimental Methods: First, a rat model of osteochondral defect was established using microfracture surgery. Specifically, after disinfecting and exposing the rat knee joint cavity, a 1.5-2 cm longitudinal incision was made along the midline of the right knee joint to expose the quadriceps tendon. The joint capsule was incised, and the patella was pulled laterally to expose the medial femoral condyle. Then, the osteochondral defect model was created: a cylindrical osteochondral defect with a diameter of 1.5 mm and a depth of 1.5 mm was formed in the center of the medial femoral condyle cartilage surface using a dental drill. Next, microfracture treatment was performed: a 0.5 mm diameter Kirschner wire connected to a low-speed motor was used to drill holes in the cancellous bone region at the base of the defect, each hole 1.0-1.2 mm deep. A small amount of bone marrow blood was observed after drilling. Finally, the wound was closed, and the model was completed. Postoperatively, antibiotics were administered subcutaneously for 3 consecutive days for infection control. PP7 hydrogel solution was injected into the knee joint defect of the rats with osteochondral defect and photocured with ultraviolet light for 30 seconds. Normal rats injected with PBS served as negative controls, while rats subjected to sham surgery (the procedure was the same as for microfracture surgery, but without osteochondral defect modeling and microfracture treatment) served as positive controls. Two months after treatment, biological evaluations were performed on rat samples, including ICRS histological scoring and microCT bone volume quantification.

[0114] Experimental results: such as Figure 13 As shown, the quantitative results of bone volume by micro-CT indicate that the PP7 hydrogel group has a significant ability to promote bone tissue regeneration; the ICRS histological score results indicate that the PP7 hydrogel group has a significant ability to promote cartilage and bone tissue regeneration.

[0115] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A method for preparing an injectable hydrogel based on platelet lysis buffer modification, characterized in that, Includes the following steps: Step 1. Synthesis of PEGDA: Polyethylene glycol was mixed with triethylamine and acryloyl chloride and reacted. The reaction mixture was then precipitated, purified and dialyzed, and finally lyophilized to obtain PEGDA powder. Step 2. Synthesis of PLMA: Platelet lysis buffer (PL) was mixed with methacrylic anhydride (MA) and reacted. The reaction mixture was then dialyzed to remove excess methacrylic acid, and finally lyophilized to obtain PLMA powder. Step 3. Preparation of PP7 hydrogel: PLMA powder, SH-E7 powder, PEGDA powder and photoinitiator LAP powder were dissolved in PBS solution and cured by UV irradiation to obtain PEGDA@PLMA-E7(PP7) hydrogel.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: Step 1. Synthesis of PEGDA: PEG was dissolved in anhydrous toluene at 40°C under N2 atmosphere, and after cooling to room temperature, triethylamine was added. After stirring evenly, acryloyl chloride was added dropwise and stirred at room temperature for 2 hours. The resulting polymer was precipitated in n-hexane, and the precipitate was further purified three times by toluene and n-hexane. The purified PEGDA was then dissolved in deionized water and dialyzed for three days. After lyophilization, PEGDA was obtained. Step 2. Synthesis of PLMA: The PL lyophilized powder was completely dissolved in PBS solution, and then MA was added dropwise to the PL solution. The pH was adjusted to 9.0, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was then dialyzed in pure water for 3-5 days, and finally lyophilized to obtain PLMA powder. Step 3. Preparation of PP7 hydrogel: PLMA powder was dissolved in PBS solution, NaOH solution was added, then SH-E7 powder was added and sonicated to dissolve completely; then PEGDA powder was added and sonicated to dissolve completely; finally, photoinitiator LAP powder was added and sonicated to dissolve completely. After curing by UV curing lamp, PP7 hydrogel was obtained.

3. The preparation method according to claim 2, characterized in that, In step 1, the concentration of PEG dissolved in anhydrous toluene was 10% w / v, the volume ratio of triethylamine to anhydrous toluene was 3:200, and the volume ratio of acryloyl chloride to anhydrous toluene was 1:100; the molecular weight cutoff of the reaction mixture in the dialysis bag used for dialysis in pure water was 1000 Da.

4. The preparation method according to claim 2, characterized in that, In step 2, the concentration of PL dissolved in PBS is 1% w / v; the volume ratio of MA to PBS is 1:100; and the molecular weight cutoff of the reaction mixture in the dialysis bag used for pure water dialysis is 3500 Da.

5. The preparation method according to claim 2, characterized in that, In step 3, the concentration of PLMA dissolved in PBS is 10% w / v; the volume ratio of NaOH solution to PBS is 3:100; the concentration of SH-E7 dissolved in PBS is 1% w / v; the concentration of LAP dissolved in PBS is 0.3% w / v; and the concentration of PEGDA dissolved in PBS is 5-15% w / v.

6. The preparation method according to claim 2, characterized in that, In step 3, the ultrasonic time after adding SH-E7 powder and PEGDA powder is 5 minutes; the ultrasonic time after adding LAP powder is 2 minutes.

7. The preparation method according to claim 2, characterized in that, In step 3, the UV lamp curing time is 0.5-3 minutes.

8. The injectable hydrogel based on platelet lysis buffer modified by the preparation method according to any one of claims 1-7, characterized in that, It is a photosensitive dual-network hydrogel with a uniform structure.

9. The use of the platelet lysate-modified injectable hydrogel of claim 8 in the preparation of products for the repair of osteochondral defects.

10. The application according to claim 9, characterized in that, The application method is as follows: Inject the hydrogel solution into the defect until it is filled, and use ultraviolet light to cure it for 0.5-3 minutes, so that the hydrogel forms a gel state that adheres tightly to the wound surface.

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