Multifunctional hydrogel composite scaffold as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and particularly provides a multifunctional hydrogel composite scaffold capable of regulating and controlling drug release in a spatio-temporal sequence. The invention creatively provides a multi-stage linkage synergistic treatment strategy based on cascade reaction to enhance osteochondral regeneration, according to the method, a ROS response liposome technology, a 3D printing technology, a gelatin space-occupying method technology, a sound control space-time regulation technology and other technologies are combined, a hydrogel composite double-layer stent with responsiveness is prepared through a gelatin space-occupying method, and the hydrogel composite double-layer stent with responsiveness is used for treating osteochondral regeneration. Then through ultrasonic control, the loaded anti-inflammatory medicine, cartilage forming medicine and osteogenesis medicine are sequentially released, and meanwhile, a slow release function is achieved. According to the technical scheme, an innovative strategy is provided for application of novel osteochondral integrated repair cooperating with inflammation management.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly to a multifunctional hydrogel composite scaffold capable of spatiotemporally sequential regulation of drugs, and a preparation method and application thereof. Background Art

[0002] In the late stage of osteoarthritis, osteochondral defects will occur, including lesions of articular hyaline cartilage and subchondral bone. At present, various clinical treatment strategies, such as microfracture and autologous cell implantation, etc., are used to treat osteochondral defects. However, due to the complex composition and structure of cartilage and subchondral bone, there is still a lack of treatment strategies that simulate the integrated osteochondral structure. Hydrogel composite multiphase scaffolds can ideally meet the physical structure of osteochondral and meet the needs of the regeneration of these two lineages in the defect.

[0003] Currently, the recognized injury repair process is multiple different but consecutive physiological stages, including hemostasis, inflammation, proliferation, and maturation stages, which work together to improve tissue regeneration. In addition to the osteochondral regeneration stage, the inflammation stage is crucial in the process of osteochondral defect repair. Hydrogels can be used to delay and accelerate the healing process by adjusting the inflammatory microenvironment. In addition, in the late stage of inflammation, the osteoinductive signal in the bone repair stage can be improved, which can match the treatment biological window to optimize the delivery system of osteogenic biomolecules. Therefore, in order to obtain the desired osteochondral regeneration effect, the orderly regulation of the inflammatory response is necessary. However, most current osteochondral defect repair strategies only consider the regulation of a single regeneration stage, ignoring the integrity and continuity of different stages, and cannot provide the optimal strategy for osteochondral regeneration through multi-stage collaborative treatment. Although sequential drug delivery strategies based on the idea of treatment cascades have been used to improve tissue regeneration. However, there are few reports on enhancing osteochondral regeneration through responsive sequential treatment strategies.

[0004] In addition, as a load-bearing unit, bone has specific requirements for the mechanical properties of its repair materials to maintain the necessary spatial structure. 3D-printed biological scaffolds are used in the field of accelerating osteochondral defect regeneration because they can simulate the osteochondral structure and provide the necessary mechanical support. So far, many biomimetic scaffolds have been designed and applied to the repair of osteochondral, and great research results have been achieved. However, these research results only focus on the cartilage and subchondral bone stages through simulating the structure, but ignore the collaborative treatment of different stages in osteochondral regeneration. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a multifunctional hydrogel composite scaffold capable of spatiotemporal sequential regulation of drugs. The present invention creatively provides a multi-stage linkage and synergistic treatment strategy based on cascade reactions to enhance osteochondral regeneration. This method combines various technologies such as ROS-responsive liposome technology, 3D printing technology, gelatin placeholder method technology, and acoustic-controlled spatiotemporal regulation technology. A responsive hydrogel composite bilayer scaffold is prepared by the gelatin placeholder method, and then through ultrasonic control, it sequentially releases the loaded anti-inflammatory drugs, chondrogenic drugs, and osteogenic drugs, and at the same time has a slow-release function. The technical solution of the present invention provides an innovative strategy in the application of novel synergistic inflammation management for osteochondral integration repair.

[0006] The object of the present invention can be specifically achieved through the following technical solutions: In the first aspect of the present invention, there is provided a PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold capable of spatiotemporal sequential regulation of drugs, and the composite scaffold has a bilayer structure.

[0007] Further, the composite scaffold may be loaded with one or more of anti-inflammatory drugs, chondrogenic drugs, and osteogenic drugs.

[0008] Further, the composite scaffold can sequentially release the loaded anti-inflammatory drugs, chondrogenic drugs, and osteogenic drugs.

[0009] Further, the composite scaffold comprises a bilayer PLGA scaffold, a fibrin-thrombin hydrogel, liposomes responsive to reactive oxygen species (ROS), and anti-inflammatory, chondrogenic, and osteogenic drugs.

[0010] In the second aspect of the present invention, there is provided a preparation method of the multifunctional hydrogel composite scaffold capable of spatiotemporal sequential regulation of drugs according to the present invention. Specifically, it includes the following steps: Step 1: Prepare nano-liposomes LPKD or nano-liposomes LPB that are responsive to reactive oxygen species and loaded with drugs; Step 2: Mix the LPKD nano-carrier or LPB nano-carrier containing a photosensitizer obtained in Step 1 with thrombin, and then crosslink with fibrin to obtain a composite hydrogel FT-LPKD or FT-LPB with controllable release of reactive oxygen species-responsive drugs; Step 3: Prepare a PLGA bilayer scaffold using 3D printing technology; Step 4: Coat the responsive hydrogel prepared in Step 2 on the inside of the PLGA bilayer scaffold by the gelatin placeholder method to obtain a PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold.

[0011] Further, the preparation steps of the LPKD described in Step 1 include: Dissolve HSPC, cholesterol, and DSPE-TK-mPEG in chloroform. Dissolve the photosensitizer (PpIX) and KGN in DMF and DMSO respectively, and add them to the above chloroform solution at a mass of 0.4% of the total liposomes. Rotate evaporate under reduced pressure in the dark to remove the solvent to obtain a phospholipid film. Dry the film in a vacuum oven overnight. Then, the water-soluble non-steroidal anti-inflammatory drug diclofenac sodium can be actively loaded into the hydrophilic core of the liposome in the form of a high loading rate through the pH gradient method. Then, add the calcium acetate solution to the above flask and hydrate it in a water bath at 65 °C. Reciprocally extrude the activated liposomes under a polycarbonate membrane using a liposome extruder to adjust the size of the synthesized liposomes. Subsequently, dialyze the synthesized nanocarriers in physiological saline through a dialysis membrane (MWCO = 7000 Da) in the dark. Finally, add diclofenac to the above liposome solution and incubate it in a constant temperature shaker at 37 °C. Use the gel column method (G50) to remove free diclofenac sodium to prepare LPKD. Store the obtained nanoparticles at -80 °C. In addition, synthesize liposomes Liposome@PpIX-KGN (LPK) without loading DS drugs using the same preparation steps.

[0012] Furthermore, in the preparation process of LPKD described in step 1, the molar ratio of HSPC, cholesterol, and DSPE-TK-mPEG is 85:10:5.

[0013] Furthermore, the preparation steps of LPB described in step 1 include: Dissolve HSPC, cholesterol, and DSPE-TK-mPEG in chloroform. Dissolve the photosensitizer PpIX in DMF and add it to the above chloroform solution at a mass of 0.4% of the total liposomes. Use a rotary evaporator to rotate evaporate under reduced pressure in the dark to remove the solvent to obtain a phospholipid film. Dry the film in a vacuum oven overnight to ensure complete removal of the solvent. Then, add the DPBS solution to the above flask and hydrate it in a water bath at 65 °C. Cool the activated liposomes to room temperature and add BMP-2 to the above solution at a concentration of 2 μg / mL, and stir in an ice bath. Reciprocally extrude the synthesized liposomes under a polycarbonate membrane using a liposome extruder to adjust the size. Wash the nanoparticles with a 100 KWD ultrafiltration tube under the conditions of 30 min and 4500 rpm to obtain Liposome@PpIX@BMP-2 (LPB).

[0014] Furthermore, in the preparation process of LPKD described in step 1, the molar ratio of HSPC, cholesterol, and DSPE-TK-mPEG is 85:10:5.

[0015] Furthermore, the drugs described in step 1 include: diclofenac sodium, Kartogenin, and BMP-2.

[0016] Further, the preparation method of the composite hydrogel described in step 2 specifically includes: fibrinogen is completely dissolved in physiological saline at a concentration of 100 mg / mL at 37 °C. The thrombin concentration is 100 UI / mL and the KGN concentration in the LPKD nanosolution is adjusted to 200 μg / mL. The fibrinogen solution and thrombin are injected using a double-barrel syringe at a volume ratio of 1:1, and the mixture undergoes a liquid-solid transformation (FT-LPKD) within 1-2 min at 37 °C. Similarly, an FT-LPB hydrogel containing 200 ng / mL BMP-2 is prepared according to the above method.

[0017] Further, the double-layer scaffold described in step 3 is a double-layer PLGA scaffold obtained by 3D cryoprinting technology. PLGA is completely dissolved in 1,4-dioxane to prepare a 40% PLGA solution. At 25 °C, it is printed onto a cold plate at -20 °C using a 21G dispensing needle with a moving rate of 2 mm / s. The parameters for printing the double-layer scaffold are as follows: the fiber spacing of the upper-layer PLGA scaffold is 300 μm and the height is 1 mm; the fiber spacing of the lower-layer PLGA scaffold is 500 μm and the height is 2 mm. The printed scaffold is dried in a freeze-dryer for 3 days. The double-layer PLGA scaffold is printed into a cuboid (35×35×3 mm) and stored in a desiccator.

[0018] Further, the preparation method of the PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold described in step 4 includes: first, the lower-layer PLGA scaffold is immersed in 5% gelatin at 37 °C for 30 min and stored at 4 °C for 10 min. The voids of the lower-layer scaffold are filled with gelatin. Then, the LPKD fibrinogen hydrogel precursor solution and thrombin solution are dropped into the upper-layer PLGA scaffold and left to stand for 2 min to completely solidify into a gel. The scaffold is then placed in an incubator at 37 °C for 5 min to melt the gelatin. After taking out the scaffold, it is washed 3 times with DPBS at 37 °C. The lower-layer PLGA scaffold can be filled with FT-LPB hydrogel in the same way to obtain the PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold.

[0019] In the third aspect of the present invention, there is provided an application of the PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold capable of spatiotemporally regulating drugs in drug preparation and a controlled-release drug delivery system. Further, the composite gel scaffold can be applied to the treatment of osteochondral regeneration.

[0020] In the fourth aspect of the present invention, there is provided an application of the ROS-responsive LPKD or LPB nanoliposomes in drug preparation and a controlled-release drug delivery system. Further, the nanoliposomes can be applied to the treatment of osteochondral regeneration.

[0021] In the fifth aspect of the present invention, there is provided an application of the above-mentioned FT-LPKD or FT-LPB composite hydrogel in drug preparation and a controlled-release drug delivery system. Further, the composite hydrogel can be applied to the treatment of osteochondral regeneration.

[0022] Compared with the prior art, the present invention has the following effects: First, for the first time, a gelatin placeholder method is used to construct a responsive soft-hard combined composite scaffold system by combining a hydrogel loaded with responsive liposomes with a 3D bilayer scaffold and applying it to the integrated repair of osteochondral.

[0023] Second, the composite scaffold can achieve sequential release and spatio-temporal control of drugs under ultrasonic stimulation.

[0024] Third, the composite scaffold system has multiple functions such as inflammation management, cartilage regeneration, and bone regeneration. Description of the Drawings

[0025] Figure 1 . Synthesis and physicochemical properties of ROS-responsive nanoliposomes with multi-factor loading. (a) Schematic diagram of the preparation process of LPKD and LPB nanoparticles. (b) UV spectra of Lip, LPK, LPKD, and LPB nanoparticles at 200-800 nm. (c) Zeta potential diagrams of Lip, LPK, LPKD, and LPB nanoparticles. (d, e) Representative TEM and size distribution diagrams of LPKD nanoparticles with or without ultrasonic treatment. (f, g) Representative TEM and size distribution diagrams of LPB nanoparticles with or without ultrasonic treatment.

[0026] Figure 2 . Preparation and characterization of in-situ nano-composite hydrogels. (a) Schematic diagram of the preparation of in-situ nano-composite FT-FLPKD / LPB hydrogels. (b) Photos of FT-FLPKD hydrogels before and after gelation. (c) SEM diagrams of FT-LPB and FT-LPKD nano-composite hydrogels. (d) Rheological property diagrams of FT, FT-LPB, and FT-LPKD nano-composite hydrogels. (e) Swelling rate maps of FT, FT-LPB, and FT-LPKD nano-composite hydrogels. (f) Degradation property diagrams of FT, FT-LPB, and FT-LPKD hydrogels.

[0027] Figure 3. Preparation and performance diagram of multi-factor loaded FT-LPKD, FT-LPB nano-hydrogel composite double-layer PLGA scaffolds. (a) Schematic diagram of the preparation of PLGA-FT-LPKD / LPB nano-hydrogel composite scaffolds. (b) SEM images of the upper, lower and cross-sections of PLGA-FT-LPKD / LPB nano-hydrogel composite scaffolds. (c, d) Compressive strength and compressive modulus of PLGA double-layer scaffolds and PLGA-FT-LPKD / LPB nano-hydrogel composite double-layer scaffolds. (e) Cumulative release of DS at 0.5, 2, 4, 8, 12, 24, 48 and 72 hours. (f) Cumulative release of KGN and BMP-2 at 1, 3, 5, 7, 9, 11, 13, 15, 21, 28 days with or without ultrasound treatment.

[0028] Figure 4 . 3D culture of BMSCs and biocompatibility of multi-factor loaded nano-hydrogel composite double-layer scaffolds. (a) Live / dead staining images inside the nano-hydrogel composite double-layer scaffolds after 3 days of culture. (b) Cytoskeleton morphology of BMSCs in the hydrogel composite 3D scaffolds (scale bar: 50 μm). (c) Cytotoxicity detection of multi-factor loaded nano-hydrogel composite double-layer scaffolds. (d) Cell proliferation of BMSCs in the nano-hydrogel composite 3D scaffolds at 1, 3 and 7 days. (e) Expression of anti-inflammatory factor IL-1β in the nano-hydrogel composite 3D scaffolds detected by ELISA kit. (*p<0.05, **p<0.01, ***p<0.001) Figure 5 . Evaluation of in vitro osteochondrogenic differentiation using multi-factor loaded nano-composite hydrogel composite double-layer scaffolds. (a) Alcian blue staining was used to evaluate the differentiation of BMSCs into cartilage in the upper layer of PLGA-FT-LPKD at 7 days. (b) ALP and ARS staining were used to evaluate the differentiation of BMSCs into osteoblasts in the lower layer of PLGA-FT-LPB at 14 days. (c) Anti-inflammatory gene expression of IL-1β and IL-6 at 3 days. (d) Cartilage gene expression of ACAN, Col2al and Sox9 at 14 days. (e) Osteogenic gene expression of RUNX2, BMP-2, OCN and Col-1 at 14 days. (*p<0.05, **p<0.01, ***p<0.001) Figure 6. Effects of Multifactor-Loaded Nanocomposite Hydrogel Composite Double-Layer Scaffold on Osteochondral Repair in SD Rats. (a) Treatment protocol for osteochondral defect model in SD rats. (b) Image of PLGA-FT-LPKD / LPB scaffold implanted in osteochondral defect area. (c) Micro-CT images of scaffold bone defect after implantation for 4 W and 8 W. (d) Quantitative data of micro-CT were further confirmed by BV / TV and Tb. Th. (n = 5, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001).

[0029] Figure 7 . Preparation of Multifactor-Loaded Nanocomposite Hydrogel Composite Double-Layer Scaffold and Schematic Diagram of Its Osteochondral Repair Process Detailed Embodiments

[0030] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0031] Example 1 Preparation of Multifactor-Loaded Nanocomposite Hydrogel Composite Double-Layer Scaffold 1.1 Preparation of LPKD-Responsive Nanoliposomes Dissolve HSPC, cholesterol, and DSPE-TK-mPEG in 5 mL of chloroform at a molar ratio of 85:10:5. Dissolve the photosensitizer (PpIX) and KGN in DMF and DMSO respectively, and add them to the above chloroform solution at a mass of 0.4% of the total liposome. Use a rotary evaporator to rotary evaporate under reduced pressure for 30 min at 50 °C in the dark to remove the solvent and obtain a phospholipid film. Dry the film in a vacuum oven overnight to ensure complete removal of the solvent. The water-soluble non-steroidal anti-inflammatory drug diclofenac sodium can be actively loaded into the hydrophilic core of the liposome in the form of a high loading rate by the pH gradient method. Then add 5 mL of calcium acetate solution (120 mM) to the above flask and hydrate it in a water bath at 65 °C for 1 h. Adjust the size of the synthesized liposome by reciprocally extruding the activated liposome 15 times through a 200 nm polycarbonate membrane using a liposome extruder. Subsequently, dialyze the synthesized nanocarrier against 4 °C physiological saline through a dialysis membrane (MWCO = 7000 Da) in the dark for 48 h to remove excess calcium acetate anhydrous, free HSPC, cholesterol, DSPE-TK-PEG, PpIX, and KGN. Finally, add 2 mg of diclofenac to the above liposome solution and incubate it in a constant temperature shaker at 37 °C for 10 min. Remove free diclofenac sodium using the gel column method (G50). Store the obtained nanoparticles at -80 °C. In addition, liposomes Liposome@PpIX-KGN (LPK) without loading DS drugs were synthesized using the same preparation steps.

[0032] 1.2 Preparation of LPB-responsive nanoliposomes Dissolve HSPC, cholesterol, and DSPE-TK-mPEG in 5 mL of chloroform at a molar ratio of 85:10:5. Dissolve the photosensitizer PpIX in DMF and add it to the above chloroform solution at a mass of 0.4% of the total liposome. Use a rotary evaporator to rotary evaporate under reduced pressure for 30 min at 50 °C in the dark to remove the solvent and obtain a phospholipid film. Dry the film in a vacuum oven overnight to ensure complete removal of the solvent. Then add 5 mL of DPBS solution to the above flask and hydrate it in a water bath at 65 °C for 1 h. Cool the activated liposome to room temperature and add BMP-2 to the above solution at a concentration of 2 μg / mL, and stir it in an ice bath for 30 min. Adjust the size of the synthesized liposome by reciprocally extruding it 15 times through a 200 nm polycarbonate membrane using a liposome extruder. Wash the nanoparticles 3 times with a 100 KWD ultrafiltration tube under the conditions of 30 min and 4500 rpm to obtain Liposome@PpIX@BMP-2 (LPB).

[0033] 1.3 Preparation of ROS-responsive fibrin-based nanohydrogels Fibrinogen was completely dissolved in physiological saline at a concentration of 100 mg / mL at 37 °C. The thrombin concentration was 100 UI / mL and the KGN concentration in the LPKD nanosolution was adjusted to 200 μg / mL. The fibrinogen solution and thrombin were injected using a double-syringe at a volume ratio of 1:1, and the mixture achieved liquid-solid transformation (FT-LPKD) within 1-2 min at 37 °C. Similarly, the FT-LPB hydrogel containing 200 ng / mL BMP-2 was prepared according to the above method.

[0034] 1.4 Preparation of double-layer PLGA scaffolds Double-layer PLGA scaffolds were obtained by 3D cryoprinting technology. PLGA was completely dissolved in 1,4-dioxane to prepare a 40% PLGA solution. At 25 °C, it was printed onto a -20 °C cold plate using a 21G dispensing needle with a moving rate of 2 mm / s. The parameters for printing the double-layer scaffold were as follows: the fiber spacing of the upper-layer PLGA scaffold was 300 μm and the height was 1 mm; the fiber spacing of the lower-layer PLGA scaffold was 500 μm and the height was 2 mm. The printed scaffolds were dried in a freeze-dryer for 3 days. The double-layer PLGA scaffolds were printed into a cuboid (35×35×3 mm) and stored in a desiccator.

[0035] 1.5 Construction of multifunctional ROS-responsive hydrogel composite double-layer scaffolds PLGA-FT-LPKD / LPB double-layer scaffolds were prepared by the gelatin placeholder method. First, the lower-layer PLGA scaffold was immersed in 5% gelatin at 37 °C for 30 min and stored at 4 °C for 10 min. The precursor solution of the LPKD fibrinogen hydrogel and the thrombin solution were dropped into the upper-layer PLGA scaffold and allowed to stand for 2 min to completely solidify into a gel. Then the scaffold was placed in an incubator at 37 °C for 5 min to melt the gelatin. After taking out the scaffold, it was washed 3 times with DPBS at 37 °C. The lower-layer PLGA scaffold was filled with the FT-LPB hydrogel in the same way.

[0036] Example 2 Synthesis and characterization of ROS-responsive nanosystems with multi-factor loading ROS-responsive LPKD / LPB liposomal NPs loaded with anti-inflammatory drug DS / chondrogenic factor KGN and osteogenic protein BMP-2 were synthesized by the thin-film rehydration and pH gradient methods ( Figure 1 a). To confirm the successful loading of the LPKD / LPB nanoparticles, ultraviolet spectroscopy was applied for qualitative analysis. The results ( Figure 1b) The absorbance peak of KGN in LPK at 278 nm was shown. The KGN and DS absorbance peaks of LPKD were at 278 nm and 270 nm respectively, indicating successful drug loading. Meanwhile, an ELISA kit was used to measure the BMP-2 loading of LPB because LPB had no specific absorbance peak. The Zeta potential values were also measured, showing that Lip was -24.91 ± 0.781 mV, LPK was -25.01 ± 0.22, LPKD was -26.33 ± 0.65 mV, and LPB was -33.16 ± 3.01 mV ( Figure 1 c). It should be noted that the zeta potential value of LPB was lower than those of the Lip, LKP, and LPKD groups, indicating that the negatively charged BMP-2 protein could reduce the zeta charge of the liposomes. In addition, the TEM images of LPB / LPKD nanoparticles (Figure 1d, 1f) showed a uniform spherical structure. Moreover, DLS measurements showed that the average diameters of the synthesized LPB / LPKD nanoparticles were 125.7 ± 0.7 nm (PDI = 0.185 ± 0.015) and 201 ± 2.7 nm (PDI = 0.242 ± 0.062). More importantly, to demonstrate the ROS-responsive rupture of LPB / LPKD nanoparticles, TEM images and DLS measurements were performed on LPB / LPKD nanoparticles after 3 min of ultrasonic (US) treatment. The results ( Figure 1 e, 1g) showed that with the increase in DLS size, the destruction of the spherical structure and the aggregation of NPs ±4.8 nm (PDI = 0.514 ± 0.014) and 776.8 ± 12.8 nm (PDI = 0.769 ± 0.04) after US treatment, which indicated that ultrasound triggered the generation of ROS, further leading to the cleavage of the TK bond and the rupture of LPB / LPKD nanoparticles.

[0037] Example 3 Preparation and Characterization of In-Situ Nano-Composite Hydrogels Doped with ROS-Responsive Nanoliposomes In-situ nano-composite FT-LPKD or FT-LPB hydrogels were prepared using the enzymatic reaction of fibrinogen and thrombin. Among them, pre-synthesized LPKD and LPB nanoliposomes were incorporated into the hydrogel precursors to construct a ROS-responsive drug delivery platform ( Figure 2 a). As shown in Figure 2 b, the in-situ gelatin of the FT-LPKD nano-composite hydrogel formed within 2 min. The SEM images ( Figure 2 c) showed that the FT-LPKD and FT-LPB hydrogels had an interconnected and uniformly distributed porous structure, which was beneficial for cell adhesion and migration. In addition, the rheological behaviors of the FT-LPKD and FT-LPB hydrogels were also evaluated to determine the influence of LPB / LPKD nanoparticles on the gelation process. The results ( Figure 2d) It was shown that when fibrinogen was connected to the two precursor aqueous solutions of thrombin-LPB / LPKD for 70 - 75 seconds, the storage modulus (G′) began to exceed the loss modulus (G″), indicating the start of the gelation process from liquid to gel. After mixing fibrinogen and thrombin-LPB / LPKD, the values of G′ and G″ continued to increase. The G″ values of the FT-LPKD and FT-LPB hydrogels (2061.13 Pa and 1843.73 Pa respectively) were significantly higher than that of the FT hydrogel (1218.19 Pa), indicating that the FT-LPKD and FT-LPB hydrogels had superior viscoelasticity. The swelling ratios of the FT-LPKD and FT-LPB hydrogels were measured by the weighing method ( Figure 2 e). The results showed that after DPBS acted at 37 °C for 24 h, the swelling ratios reached 664.95% and 672.67% respectively, achieving swelling equilibrium. In addition, the appropriate biodegradation of hydrogels plays an important role in drug delivery and tissue regeneration. To evaluate the biodegradation performance of the hydrogels, they were incubated in DPBS at 37 °C for 28 days. At each time point, the remaining weights of the FT and FT-LPKD, FT-LPB hydrogels were measured after freeze-drying. The results showed that after 28 days in DPBS, the FT-LPKD and FT-LPB hydrogels retained 42.76% and 46.13% of their original mass respectively, while the mass ratio of the FT hydrogel only retained 24.72% ( Figure 2 f). The results indicated that the degradation rate of the FT-LPKD and FT-LPB nanocomposite hydrogels was slower than that of the pure fibrin hydrogel, which might be because in the nanocomposite hydrogels, in addition to the enzymatic reaction of fibrinogen and thrombin, hydrogen bonds were formed between the NH2-residues of fibrin and the OH-groups of liposomes (DSPE-TK-mPEG / HSPC). The formation of hydrogen bonds could enhance the intermolecular force between the hydrogel and liposome molecules, further improving the mechanical strength of the nanocomposite FT-LPKD and FT-LPB hydrogels and reducing their degradation rate.

[0038] Example 4 Preparation and Characterization of a ROS-Controlled Release Multifactor-Loaded Nanohydrogel Composite Double-Layer Scaffold A double-porous double-layer PLGA 3D scaffold mimicking the natural osteochondral structure was prepared by the low-temperature deposition method. Then, the thrombin-LPKD / LPB precursor and fibrinogen were respectively injected into the upper layer pores (PLGA-FT-LPKD) and lower layer pores (PLGA-FT-LPB) of the double-layer PLGA scaffold, and a multifactor-loaded nanohydrogel composite double-layer scaffold was prepared by the enzymatic reaction using the gelatin placeholder method ( Figure 3 a). SEM images showed the microstructure of the nanohydrogel composite double-layer scaffold from different angles. As Figure 3As shown in Fig. b, the composite hydrogel scaffold consists of two layers, with a regular interconnected porous structure, which can provide spatial mechanical support and a three-dimensional cell culture microenvironment for tissue growth. Among them, the upper layer is composed of a PLGA hard scaffold with a fiber spacing of 300 μm and an FT-LPKD hybrid soft hydrogel, allowing the cascading release of small molecule water-soluble DS and inhibiting the early inflammation of OA development. The FT-LPKD hybrid hydrogel, as a ROS-controlled KGN drug delivery system, induces cartilage regeneration through external ultrasound stimulation. The lower layer is composed of a PLGA hard scaffold with a fiber spacing of 500 μm and an FT-LPB hybrid hydrogel, promoting the controlled release of BMP-2 protein and subchondral bone regeneration. In addition, we also evaluated the mechanical properties of the PLGA-FT-LPKD / LPB scaffold ( Figure 3 Figs. c, 3d). The average compressive strength and compressive modulus of the PLGA-FT-LPKD / LPB nanogel composite scaffold were 8.3 MPa and 15.3 MPa, respectively. The compressive strength and compressive modulus of the PLGA double-layer scaffold were slightly lower, 7.2 MPa and 13.2 MPa, respectively. These results indicate that the coating of FT-LPKD and FT-LPB hydrogels may slightly improve the mechanical properties of the PLGA scaffold, but will make a great contribution to the three-dimensional microenvironment culture and dual-lineage differentiation of future MSCs.

[0039] Example 5 Sequential Release of Multiple Factors Loaded in Nanohydrogel Composite Double-Layer Scaffold Drug release experiment of the hydrogel composite double-layer scaffold: To evaluate the release patterns of the loaded anti-inflammatory drug DS and chondrogenic and osteogenic growth factors KGN and BMP-2. To achieve accurate measurement of the drugs, the total drug loading was increased in this experiment, and a 6×6×3 mm hydrogel composite scaffold was used, with the contents of DS, KGN, and BMP-2 being 667.3 μg, 100 μg, and 300 ng, respectively. These samples were immersed in a 50 mL centrifuge tube (5 mL DPBS) and placed in a 37 °C constant temperature shaker (Bluepard) and shaken at a speed of 120 rpm. At the required time points, 200 μL of DPBS was taken from the centrifuge tube to measure the corresponding drug concentration, and then 200 μL of fresh DPBS was replenished.

[0040] To evaluate the release pattern of DS, the samples were sampled at 0.5, 2, 4, 8, 12, 24, 48, 72 h and determined by high performance liquid chromatography. To evaluate the release behavior of KGN and BMP-2, the samples were sampled at 1, 2, 3, 5, 11, 14, 21, 28 days, and at 3, 5, 11 days, with 1 W / cm 2Treat with an ultrasonic instrument with a 50% duty cycle for 3 min. Then, measure the drug concentration of KGN by HPLC at each sampling point, and detect the corresponding concentration of BMP-2 at each of the above sampling points by ELISA.

[0041] The spatio-temporal sequential drug delivery strategy plays a crucial role in the multi-stage collaborative treatment of osteochondral defects. To trigger stepwise anti-inflammatory, chondrogenic, and osteogenic responses during the multi-stage continuous osteochondral regeneration process, an anti-inflammatory drug (DS) was effectively loaded into LPKD nanoparticles by the pH gradient method to achieve rapid release and inhibit the early inflammatory response. The chondrogenic factor (KGN) and osteogenic protein (BMP-2) were loaded into LPKD / LPB nanoparticles by the membrane-rehydration method, and spatio-temporal controlled release was achieved through ultrasonic stimulation to promote the dual-lineage differentiation of MSCs and promote osteochondral regeneration. As Figure 3 shown in e, the PLGA-FT-LPKD nanohydrogel composite scaffold exhibited a burst release of water-soluble DS, and the cumulative release amount of DS reached 79.48% within 24 h. After 48 h, the DS cumulative release curve of the PLGA-FT-LPKD nanohydrogel composite scaffold gradually reached a plateau of 80.87%. The rapid release of DS from the nanohydrogel composite scaffold would be an excellent delivery strategy for the inflammatory phase of osteochondral defects without any additional stimulation. The encapsulation efficiencies of KGN and BMP-2 in LPKD and LPB were determined by high-performance liquid chromatography and enzyme-linked immunosorbent assay, and the encapsulation efficiencies were 86.23% and 36.16%, respectively. In addition, the spatio-temporal sequential release of KGN and BMP-2 in PLGA-FT-LPK and PLGA-FT-LPB was controlled by ultrasonic treatment (1 W / cm 2 , 3 min). Figure 3 Figure f shows the release curves of KGN and BMP-2 from the PLGA-FT-LPK and PLGA-FT-LPB nanohydrogel composite scaffolds within 28 days. A small amount of lipophilic KGN was released in the first three days, approximately 8.33%. The release amount of KGN increased significantly on the 3rd, 7th, and 11th days after ultrasonic stimulation, and the cumulative release amount reached 91.30% by the 15th day. In contrast, the sustained release of KGN from the PLGA-FT-LPKD nanohydrogel composite scaffold without ultrasonic stimulation was slower, and the cumulative release amount within 28 days was 38.74%. Similarly, under ultrasonic stimulation, the release kinetics of BMP-2 in the PLGA-FT-LPB nanohydrogel composite scaffold also followed a similar trend, and the cumulative release amount on the 13th day was 72.18%.

[0042] Example 6 Cellular and Zoological Analyses of the Multifactor-Loaded Nanohydrogel Composite Double-Layer Scaffold 6.1 Cell Seeding in the Hydrogel Composite Double-Layer Scaffold The BMSCs used in this experiment were from SD rats. The cell culture methods involved in this experiment were to first resuspend the cells in the fibrin precursor solution and then composite them into the 3D scaffold of PLGA-FT-LPKD / LPB. Specifically, first, fibrinogen was sterilized by ultraviolet for 1 h. The third-generation BMSCs from the femoral bone marrow of SD rats (4 days old) were resuspended in a 100 mg / mL fibrinogen solution, and the cell concentration was adjusted to 2 × 10 7 cells per milliliter. The PLGA scaffold was soaked in 75% alcohol for 3 h, irradiated under ultraviolet for 1 h, and dried in a fume hood. The manufacturing method of PLGA-FT-LPKD / LPB was as described above. Then, PLGA-FT-LPKD / LPB was co-cultured with a complete medium containing α-MEM, 10% fetal bovine serum, and 1% double antibody at 37 °C and 5% CO2.

[0043] 6.2 Cell proliferation experiment of the hydrogel composite double-layer scaffold As per the above operation steps, the FT, PLGA-FT, PLGA-FT-PLKD / PLB, and PLGA-FT-PLKD / PLB+US groups were immersed in the complete medium and cultured for 1, 3, and 7 days. And on the 3rd and 5th days, the cells in PLGA-FT-LPKD / LPB were exposed to ultrasound at a duty cycle of 50% and 1 W / cm 2 for 3 min. Similarly, the absorbance of the cells was measured using the CCK-8 method to calculate the proliferation of the cells in the double-layer hydrogel composite porous scaffold system.

[0044] The CCK-8 kit was used to detect the viability of BMSCs in the PLGA-FT-LPKD / LPB+US nanohydrogel composite scaffold ( Figure 4 c). The results showed that under both ultrasonic stimulation and non-ultrasonic stimulation, mesenchymal stem cells of the bone marrow had relatively high cell viability (93.04% - 95.61%) and no obvious toxicity, indicating that the PLGA-FT-LPKD / LPB nanohydrogel composite scaffold had good biocompatibility. The proliferation of mesenchymal stem cells of the bone marrow in the PLGA-FT-LPKD / LPB nanohydrogel composite scaffold was detected by CCK-8 on the 1st, 3rd, and 7th days of culture. As Figure 4 shown in d, the proliferation ability of BMSCs in the nanohydrogel composite scaffold was better than that of the FT group, which might be due to the successful construction of a soft and hard matrix composite, enhancing the cell proliferation ability by simulating the ECM microenvironment.

[0045] 6.3 Cell AM / PI staining experiment of the hydrogel composite double-layer scaffold Visual assessment of the viability of BMSCs in PLGA-FT-LPKD / LPB was performed using a live / dead cell staining kit. The FT, PLGA-FT, and PLGA-FT-PLKD / PLB groups were immersed in complete medium and cultured for 24 h. The PLGA-FT-PLKD / PLB+US group was immersed in complete medium and cultured for 20 h, and then exposed to ultrasound at 50% duty cycle and 1 W / cm 2 for 3 min. BMSCs were labeled with propidium iodide (PI, 4 μM) and calcein-AM (2 μM), and stored in the incubator in the dark for 30 min. Then, after fixation with 4% paraformaldehyde for 30 min, the samples were rinsed three times with DPBS. 3D cell status was imaged using CLSM (FV3000-ILSW, Olympus, Japan) at an emission wavelength of 494 nm and an excitation wavelength of 528 nm.

[0046] To evaluate the three-dimensional culture effect of the PLGA-FT-LPKD / LPB nanohydrogel composite bilayer scaffold on bone marrow mesenchymal stem cells, live / dead staining images and FITC / DAPI-stained cytoskeleton morphology were observed after 3 days of culture. As Figure 4 shown in a, almost all bone marrow mesenchymal stem cells remained viable and were well-distributed in the three-dimensional hydrogel-like ECM microenvironment of the PLGA-FT-LPKD / LPB nanohydrogel composite bilayer scaffold. In addition, the cytoskeleton morphology of mesenchymal stem cells in different groups ( Figure 4 b) showed that bone marrow mesenchymal stem cells were well-distributed in the PLGA-FT-LPKD / LPB nanohydrogel composite bilayer scaffold, with many cell pseudopodia.

[0047] 6.4 Cytotoxicity experiment of the hydrogel composite bilayer scaffold The effects of the PLGA-FT-LPKD / LPB scaffold system on cell viability were studied in vitro using a CCK-8 kit. BMSCs cultured in a pure fibrin hydrogel 3D were used as the control group. To verify the effects of the upper and lower double-layer scaffolds and their loaded responsive hydrogel systems on cells, this experiment was divided into FT, PLGA-FT, PLGA-FT-PLKD (Upper), PLGA-FT-PLB (Lower), PLGA-FT-PLKD (Upper)+US, and PLGA-FT-PLB (Lower)+US groups, and cultured in complete medium for 24 h. Among them, the PLGA-FT-PLKD (Upper)+US and PLGA-FT-PLB (Lower)+US groups were ultrasonically treated after co-culturing for 20 h. The medium was replaced with fresh medium containing 10% CCK-8. After co-incubating in a cell incubator for 2 h, the absorbance of the supernatant was analyzed at 450 nm using a multifunctional microplate reader, and cell viability was calculated.

[0048] 6.5 In vitro chondrogenic differentiation gene expression experiment of hydrogel composite double-layer scaffold To explore the ability of the double-layer hydrogel composite porous scaffold to induce BMSCs to differentiate into chondrocytes in vitro, qRT-PCR was used to analyze the relative gene expression levels of ACAN, Col II, and Sox9 in BMSCs after culturing in the scaffold for 14 days. After incubating the 24-well plates of the FT, PLGA-FT, PLGA-FT-LPK (Upper), PLGA-FT-LPK+US (Upper), PLGA-FT-LPKD (Upper), and PLGA-FT-LPKD+US (Upper) groups for 24 h, the medium was replaced with chondrogenic induction medium containing DMEM high glucose, 10% fetal bovine serum, 1% double antibody, 50 μg / mL ascorbic acid, 100 mM dexamethasone, and 1% ITS and cultured for 14 days. The PLGA-FT-LPB+US (Upper) and PLGA-FT-LPKD+US (Upper) scaffold groups were ultrasonically treated on the 3rd, 7th, and 11th days. The induced scaffolds were frozen in liquid nitrogen for 1 min and treated with a homogenizer for 30 s, and then mRNA was extracted using an RNAiso kit (TaKaRa, Japan). A reverse transcription kit (TaKaRa, Japan) was used for reverse transcription (RT) to obtain cDNA. qRT-PCR was performed using TBGreen qRT-PCR (TaKaRa, Japan) on a detection system. The qRT-PCR cycling conditions were 95 °C for 3 min, then 95 °C for 3 s, 60 °C for 30 s, for 42 cycles. The mRNA expression levels were measured by GAPDH (housekeeping gene) and detected by the threshold cycle (Ct) method.

[0049] The double-layer PLGA scaffold has multiple functions in the comprehensive repair of osteochondral defects. Specifically, the upper composite scaffold (PLGA-FT-LPKD) is designed to induce cartilage regeneration, while the lower composite scaffold (PLGA-FT-LPB) is designed to promote bone regeneration. Therefore, we separately evaluated the chondrogenic differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells in the upper and lower layers of the PLGA-FT-LPKD / LPB scaffold. To evaluate the bone marrow mesenchymal stem cells in the PLGA-FT-LPKD scaffold, after 7 days of induction culture in the 3D scaffold, Alcian blue staining was used to evaluate its chondrogenic differentiation ability ( Figure 5 a). The results showed that glycosaminoglycan was most expressed in the PLGA-FT-LPKD scaffold group, suggesting its good chondrogenic differentiation potential. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the chondrogenic differentiation of bone marrow mesenchymal stem cells on the PLGA-FT-LPKD scaffold, and the expression levels of typical chondrogenic gene markers (ACAN, Sox9, and Col-II) were detected. The results ( Figure 5 d) showed that compared with the non-ultrasound group, the expression levels of ACAN, Sox9, and Col-II increased by 2.03-fold, 1.92-fold, and 1.72-fold, respectively. In addition, we evaluated the PLGA-FT-LPK group without the DS scaffold by the same method, and the results showed that the expression levels of ACAN, Sox9, and Col-II genes in PLGA-FT-LPK + US were higher than those in the FT group, indicating that PLGA-FT-LPK + US had good cartilage regeneration ability.

[0050] 6.6 In vitro osteogenic differentiation gene expression experiment of hydrogel composite double-layer scaffold According to the above steps, osteogenic induction medium DMEM high glucose, 10% fetal bovine serum, 1% double antibody, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 0.2 mM ascorbic acid were prepared. The FT, PLGA-FT, PLGA-FT-LPB (Lower), and PLGA-FT-LPB+US (Lower) groups were cultured with osteogenic induction medium for 14 days, and the lower layer PLGA-FT-LPB+US scaffold group was examined by ultrasound on the 3rd, 5th, and 11th days. As in the above chondrogenic gene operation steps, the expression of osteogenesis-related genes (RUNX2, ColII, BMP-2, OCN) was detected.

[0051] After co-culture for 7 d and 14 d, alkaline phosphatase (ALP) and alizarin red staining were used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in the PLGA-FT-LPB scaffold. The results of ALP staining ( Figure 5 b) showed that compared with the PLGA-FT-LPB group, the staining of the PLGA-FT-LPB +US scaffold was enhanced after co-culture. The results ( Figure 5e) showed that compared with the non-ultrasound group, the expression levels of ALP, BMP-2, OCN, and Col I genes in the PLGA-FT-LPB + US group were significantly increased by 1.37-fold, 1.22-fold, 1.13-fold, and 1.27-fold, respectively. This indicates that under ultrasound similar to ALP and ARS staining, the PLGA-FT-LPB scaffold can significantly promote bone regeneration. Thus, it can be seen that the PLGA-FT-LPB + US hydrogel composite scaffold has good bioactivity for inducing osteogenic differentiation of BMSCs and can promote the reconstruction of subchondral bone after in vivo implantation.

[0052] 6.7 In vitro anti-inflammatory performance study of multi-factor loaded nano-hydrogel composite bilayer scaffold To evaluate the in vitro anti-inflammatory effects of 3D bioprinted PLGA bilayer composite scaffolds coated with FT-LPK and FT-LPKD, we cultured bone marrow mesenchymal stem cells in TNF-α-induced inflammatory mediators for 3 days and detected them using an ELISA Kit. The results ( Figure 4 e) showed that the FT-LPKD nano-hydrogel composite scaffold significantly reduced the level of IL-1β compared with the FT group. In addition, there was no significant difference in the level of IL-1β after ultrasound (1 W / cm 2 , 3 min) treatment, indicating that the PLGA-FT-LPKD / LPB scaffold under ultrasound stimulation does not promote the inflammatory response. Interestingly, the expression of IL-1β in bone marrow mesenchymal stem cells in the PLGA-FT-LPKD / LPB and PLGA-FT-LPKD / LPB + US nano-hydrogel composite scaffolds was similar to that in the DS group. The expression levels of related genes IL-1β and IL-6 were detected by qRT-PCR. The results ( Figure 5 c) showed that the expressions of IL-1β and IL-6 genes were significantly downregulated in both the PLGA-FT-LPKD / LPB and PLGA-FT-LPKD / LPB+US nano-hydrogel composite scaffolds. There was no significant difference between the PLGA-FT-LPKD / LPB, PLGA-FT-LPKD / LPB+US nano-hydrogel composite scaffolds and the DS group.

[0053] 6.8 Alcian blue staining for chondrogenesis experiment of hydrogel composite bilayer scaffold To further detect the effect of inducing chondrogenesis in vitro for 7 days in the hydrogel composite porous scaffold system, Alcian blue staining was used to evaluate the potential of BMSCs to differentiate into chondrocytes in the upper layer FT, PLGA-FT, PLGA-FT-LPK (Upper), PLGA-FT-LPK+US (Upper), PLGA-FT-LPKD (Upper), and PLGA-FT-LPKD+US (Upper) groups. The scaffolds were placed in 24-well plates and ultrasonically treated for PLGA-FT-LPK (Upper) and PLGA-FT-LPKD+US (Upper) on the 3rd and 5th days of induction in chondrogenic induction medium. All scaffold samples were fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS. Then, the scaffolds were stained with Alcian blue for 1 h, and the scaffolds were washed with DPBS until no color was released in the DPBS, and then soaked overnight. Alcian blue staining images of the scaffolds and hydrogels were obtained through a stereomicroscope.

[0054] 6.9 Osteogenic differentiation experiment of hydrogel composite bilayer scaffold by alkaline phosphatase (ALP) staining in vitro Similarly, according to the above culture process, the potential of BMSCs to differentiate into osteoblasts in the lower layer FT, PLGA-FT, PLGA-FT-LPB (Lower), and PLGA-FT-LPB+US (Lower) was evaluated using alkaline phosphatase. The above scaffolds were induced with osteogenic induction medium for 14 days and ultrasonically treated on the 3rd, 5th, and 11th days. After fixation, an alkaline phosphatase chromogenic kit was used to evaluate the osteogenic differentiation of BMSCs in the scaffolds. After staining for 1 h, it was washed 3 times with DPBS and photographed. To quantify alkaline phosphatase staining, the same procedure was used with an alkaline phosphatase detection kit to quantitatively analyze the osteogenic differentiation ability of BMSCs in the scaffolds. Similarly, the scaffolds applied to the osteogenic induction medium on the 3rd, 5th, and 11th days were ultrasonically treated.

[0055] 6.10 Osteogenic differentiation experiment of hydrogel composite bilayer scaffold by alizarin red (ARS) staining in vitro The potential of BMSCs to differentiate into osteoblasts in the lower layer FT, PLGA-FT, PLGA-FT-LPB (Lower), and PLGA-FT-LPB+US (Lower) was evaluated using alizarin red (ARS). The above scaffolds were induced with osteogenic induction medium for 14 days and ultrasonically treated on the 3rd, 5th, and 11th days. After fixation, it was stained with alizarin red for 1 h, washed, and photographed. To quantitatively analyze ARS, a 5% SDS-HCl solution was prepared, 500 μL was added to each well, and it was dissolved at room temperature for 1 h. The liquid was taken out, centrifuged at 1000 rpm / min, and the supernatant was taken. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader at 405 / 415 nm.

[0056] 6.11 Establishment of osteochondral injury model Establishment of cartilage-bone defect model: All animal experiments followed the "Principles of Laboratory Animal Care" of the Animal Experiment Ethics Committee (SCXK2018SCXK2018-0004). Sprague-Dawley (SD) rats (6 w, male, body weight 180-200 g) were selected as the model construction objects, and 10 rat models were constructed in each group. The SD rats were anesthetized by intraperitoneal injection of pentobarbital (35 mg / kg). The right leg joint of the rats was opened, and a cartilage-bone defect model (Ф3.2×3 mm) was constructed in the center of the patellar groove of the SD rats using a dental drill. Thus, the model was successfully established. After implanting the above different scaffolds at the cartilage-bone defect model site, the wound was sutured, and antibiotics were used continuously for 3 days after the operation to prevent wound infection. The operation steps of the ultrasound group were as follows: The rats were exposed to ultrasound with a 50% duty cycle and 1 W / cm 2 for 3 min on the 3rd, 5th, and 11th days using an ultrasound instrument. After 4 W and 8 W of treatment, 5 SD rats' femurs were collected from each group.

[0057] 6.12 In vivo Micro-CT scanning The right leg femurs were collected and scanned by a micro-computed tomography scanner (Inveon PET / CT, Siemens, Germany). The cylindrical region of interest (ROI, diameter: 3.2 mm, height: 3 mm) was concentrically located in the modeling area. Then, 3D reconstruction was performed using computer-aided design software (Midivi, Kings Medical, Changzhou, China). Bone volume fraction (BV / TV) and trabecular thickness (Tb.Th) data were also calculated based on the ROI.

[0058] To evaluate the ability of the PLGA-FT-LPKD / LPB+US scaffold to promote osteochondral regeneration in vivo, an SD rat osteochondral defect model with a diameter of 3.2 mm and a height of 3 mm was used. The treatment protocol for the scaffold to treat the osteochondral defects in SD rats was as Figure 6 shown in a. The PLGA, PLGA+FT, PLGA-FT-LPK / LPB, PLGA-FT-LPKD / LPB, and PLGA-FT-LPKD / LPB+US scaffolds were transplanted ( Figure 6 b) and were respectively called the PLGA, PLGA+FT, PLGA-FT-LPK / LPB, PLGA-FT-LPKD / LPB, and PLGA-FT-LPKD / LPB+US groups. The rats were sacrificed at 4 W and 8 W after the operation to collect joint specimens, and it was observed that the defect areas in the PLGA-FT-LPK / LPB+US group and the PLGA-FT-LPKD / LPB+US group were filled with regenerated tissue and were tightly combined with the surrounding cartilage.

[0059] Using micro-CT ( Figure 6c) and related quantitative data ( Figure 6 d) The subchondral bone and bone were evaluated after 4 W and 8 W of treatment, and it was found that the new bone mass of the PLGA-FT-LPKD / LPB+US scaffold was higher than that of other groups. Compared with other groups, the bone volume fraction (BV / TV) value of the PLGA-FT-LPKD / LPB+US scaffold group showed an increasing trend. Among them, the BV / TV value (16.92%) of the PLGA-FT-LPKD / LPB+US scaffold group at 4 w was significantly higher than that of the blank group (5.6%), the PLGA-FT-LPK / LPB+US group (14.81%) and the PLGA-FT-LPK / LPB group (10.8%). Compared with the blank group (12.75%), the PLGA-FT-LPK / LPB+US group (29.97%) and the PLGA-FT-LPK / LPB group (24.96%), the BV / TV value of the PLGA-FT-LPKD / LPB+US scaffold group (31.76%) further increased at 8 W. In addition, the trabecular number (Tb.Th) of the PLGA-FT-LPKD / LPB+US group was significantly higher than that of other groups at 4 W and 8 W.

Claims

1. A PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold for spatiotemporal sequential regulation of drugs, characterized in that, The composite scaffold has a bilayer structure.

2. The multifunctional hydrogel composite scaffold according to claim 1, wherein The composite scaffold can be loaded with one or more of anti-inflammatory drugs, chondrogenic drugs, and osteogenic drugs.

3. The multifunctional hydrogel composite scaffold according to claim 1, wherein The composite scaffold can sequentially release the loaded anti-inflammatory drugs, chondrogenic drugs, and osteogenic drugs.

4. The multifunctional hydrogel composite scaffold according to claim 1, wherein The composite scaffold includes a bilayer PLGA scaffold, a fibrin-thrombin hydrogel, ROS-responsive liposomes, and anti-inflammatory, chondrogenic, and osteogenic drugs.

5. A method for preparing the multifunctional hydrogel composite scaffold capable of spatiotemporally regulating drugs according to claims 1 to 4, characterized in that, Specifically, it includes the following steps: Step 1: Prepare nano-liposomes LPKD or nano-liposomes LPB with reactive oxygen species response and loaded with drugs. Step 2: Mix the LPKD nano-carrier or LPB nano-carrier containing the photosensitizer obtained in Step 1 with thrombin, and then crosslink with fibrin to obtain a composite hydrogel FT-LPKD or FT-LPB with controllable release of reactive oxygen species-responsive drugs. Step 3: Prepare a PLGA bilayer scaffold using 3D printing technology. Step 4: Coat the responsive hydrogel prepared in Step 2 on the inside of the PLGA bilayer scaffold using the gelatin placeholder method to obtain a PLGA-FT-LPKD / LPB multifunctional hydrogel composite scaffold.

6. The preparation method according to claim 5, characterized in that, The drugs described in Step 1 include one or more of anti-inflammatory drugs, chondrogenic drugs, and osteogenic drugs.

7. The preparation method according to claim 5, characterized in that, The drugs described in Step 1 include diclofenac sodium, Kartogenin, and BMP-2.

8. Application of the multifunctional hydrogel composite scaffold capable of spatiotemporally sequential regulation of drugs described in Claims 1 to 4 in drug preparation and controllable drug delivery systems.

9. Application of the reactive oxygen species-responsive LPKD or LPB nano-liposomes described in Claim 5 in drug preparation and controllable drug delivery systems.

10. Application of the FT-LPKD or FT-LPB composite hydrogel described in Claim 5 in drug preparation and controllable drug delivery systems.

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