Multi-particulate drug delivery hydrogels and their use in promoting stem cell regeneration of cartilage

Abstract

A kind of composition containing microspheres, including the first kind of microspheres releasing the first kind of active substance to the environment, to promote chondrogenesis in early mesenchymal stem cell differentiation;And the second kind of microspheres releasing the second kind of active substance to the environment, to inhibit angiogenesis or invade cartilage, promote cartilage growth, form cartilage pit and cartilage-specific ECM deposition.It has been verified that the composition of the application not only realizes the regulation of early chondrogenesis and the inhibition of late angiogenesis, thereby meeting the dynamic requirements of bone marrow mesenchymal stem cell chondrogenesis, but also realizes the stable regeneration of cartilage tissue in ectopic environment, without the operation of first implementing pre-induction cartilage tissue in vitro and then implanting in vivo, and the convenience of tissue repair is significantly improved.

Description

Technical Field

[0001] This invention relates to a biomaterial, and more particularly to a composition carrying chondrogenic active molecules for promoting chondrogenic differentiation and regeneration of stem cells, and its application in medical devices. Background Technology

[0002] Engineered cartilage using autologous chondrocytes as seed cells has been preliminarily applied in the clinical use of auricle, nose, trachea, meniscus, and joints. However, chondrocyte-based cartilage regeneration strategies are limited in clinical application due to limited cell sources, unavoidable trauma to the donor site, and difficulty in maintaining the phenotype after repeated expansion. Bone marrow mesenchymal stem cells (BMSCs) offer unique advantages as ideal seed cells for engineered cartilage, including minimal trauma during cell acquisition, high proliferative capacity, and well-defined chondrogenic potential. In ectopic environments, the cartilage phenotype is difficult to maintain because vascular infiltration inevitably leads to endochondral ossification during chondrogenesis, differentiation, and development. Despite these advances, BMSC-based cartilage regeneration still requires significant in vitro pre-induction time to differentiate into chondrocytes before in vivo transplantation, limiting its clinical application. Therefore, an ideal stem cell-based cartilage regeneration technology needs to meet the time-dependent requirements of early in vivo cartilage formation and late anti-angiogenic microenvironment without in vitro pre-induction.

[0003] Drug delivery systems based on microparticles (MPs) and nanoparticles (NPs), including liposomes and polymeric and inorganic particles, are considered viable carriers for modulating stem cell fate and simultaneously promoting tissue-specific regeneration. For example, FDA-approved poly(lactic-co-glycolic acid) microparticles have been widely used to precisely deliver multifunctional bioactive substances such as growth factors (e.g., transforming growth factor β and bone morphogenetic proteins). Recent studies have shown that transforming growth factor β3 generates potent chondrogenic function by activating the TGFβ / Smad signaling pathway.

[0004] Another factor synergistically regulating stem cell cartilage regeneration is the use of biocompatible scaffolds capable of carrying bioactive substances. Unlike scaffoldless cell sheet culture modalities, hydrogel-based three-dimensional (3D) culture modalities possess a microenvironment that mimics the extracellular matrix (ECM), facilitating the encapsulation of microparticles for multi-drug delivery. In our previous study, photocrosslinked matrix hydrogels mimicked key components of the ECM by incorporating proteoglycans and glycosaminoglycans. Decellularized cartilage or umbilical cord matrix has also been used as a bioactive component to provide a satisfactory cartilage microenvironment. Therefore, it is reasonable to develop an ideal stem cell culture platform by combining cartilage-specific matrix hydrogels with MPs-based drug delivery systems. To date, breakthroughs in dynamically regulating in vivo stem cell cartilage regeneration using 3D hydrogel scaffolds have been very limited. Summary of the Invention

[0005] One object of the present invention is to provide a composition containing microspheres that regulates the process of cell differentiation into chondrocytes and promotes cartilage growth and repair.

[0006] Another objective of this invention is to provide a composition containing a variety of microspheres, wherein the release cycles of the active substances provided by the various microspheres are different, and the different active substances are released in a programmed manner at different stages of stem cell differentiation, which is beneficial to regulating the process of cell differentiation into chondrocytes and promoting cartilage growth and repair.

[0007] Another objective of this invention is to provide a hydrogel containing a variety of microspheres, wherein the microspheres are loaded within the hydrogel to provide a growth environment for cells, and to facilitate the release of active substances from the microspheres to regulate the process of cell differentiation into chondrocytes, thereby promoting chondrocyte growth and repair.

[0008] Another objective of this invention is to provide a hydrogel containing a variety of microspheres that regulates the differentiation process of stem cells (such as mesenchymal stem cells) into chondrocytes, thereby promoting chondrocyte growth and repair.

[0009] The fifth objective of this invention is to provide a hydrogel containing a variety of microspheres for use as a medical device in bone repair.

[0010] A composition containing microspheres, comprising:

[0011] The first type of microspheres releases first-type active substances into the environment to promote chondrogenesis in the early stages of mesenchymal stem cell differentiation; and

[0012] The second type of microspheres releases second-type active substances into the environment, which inhibit angiogenesis or matrix degradation after cartilage formation, promote cartilage growth, and form cartilage lacunae and cartilage-specific ECM deposits.

[0013] Type I microspheres typically have the characteristic of rapidly releasing type I active substances into the environment. Materials suitable for preparing type I microspheres include, but are not limited to, PLGA with a molecular weight of 5kDa to 10kDa.

[0014] Type II microspheres typically possess the characteristic of delayed release of Type II active substances into the environment. Materials suitable for preparing Type I microspheres include, but are not limited to, PLGA with a molecular weight of 50 kDa or higher, PLGA with a molecular weight of 100 kDa or higher, PLL with a molecular weight of 10 kDa or higher, as well as materials such as mesoporous silica (MSNs) and metal-organic frameworks (MOFs).

[0015] The first type of active substances are growth factors such as transforming growth factor β3 and Kartogenin (KGN), which are small molecules that help promote the chondrogenic differentiation of stem cells in the early stages of stem cell differentiation, forming white cartilage-like tissue.

[0016] The second type of active substances are anti-angiogenic molecules, such as, but not limited to, small molecules like lenvatinib and bevacizumab. These substances are controlled by second-type microspheres. Most of these substances (e.g., more than 40%, 50%, or 60% of the amount contained in the microspheres) are released into the environment late in chondrogenesis, thus achieving anti-angiogenesis. They provide an avascular microenvironment for chondrogenic tissue, inhibit vascular invasion of chondrogenic tissue and inhibit the degradation of the cartilage matrix, and promote the growth of chondrogenic tissue to form cartilage lacunae and cartilage-specific deposition, among other cartilage properties.

[0017] To facilitate cartilage formation and growth, the composition of the present invention further includes a hydrogel, in which both the first type of microspheres and the second type of microspheres are loaded.

[0018] A composition containing multiple microspheres, comprising:

[0019] The first type of microspheres releases the first type of active substances they contain into the environment to promote cartilage formation in the early stages of chondrogenesis.

[0020] The second type of microspheres releases second-type active substances into the environment, which, after cartilage formation, inhibit angiogenesis or cartilage invasion, forming cartilage lacunae and cartilage-specific ECM deposition, maintaining a stable regenerated cartilage phenotype; and

[0021] The hydrogel, in which first-class and second-class microspheres are loaded, provides a microenvironment for cartilage formation and growth.

[0022] To accelerate cartilage formation and growth, cells, such as bone marrow mesenchymal stem cells and adipose-derived mesenchymal stem cells, are loaded into the hydrogel.

[0023] Another composition containing multiple microspheres includes:

[0024] The first type of microspheres releases the first type of active substances they contain into the environment to promote cartilage formation in the early stages of chondrogenesis.

[0025] The second type of microspheres releases the second type of active substances into the environment, which inhibit angiogenesis or cartilage invasion after cartilage formation, forming cartilage lacunae and cartilage-specific ECM deposition, and maintaining the stability of regenerated cartilage.

[0026] Mesenchymal cells, used for cartilage repair; and

[0027] The hydrogel contains type I microspheres, type II microspheres, and mesenchymal stem cells.

[0028] In this invention, the hydrogel is typically a medical hydrogel, such as hyaluronic acid, gelatin, chondroitin sulfate, and their cross-linked modified derivatives, such as gels GelMA and HAMA suitable for photocrosslinking. GelMA concentrations are typically 5% w / v to 10% w / v, particularly 5%, 6%, 7%, 8%, 9%, and 10% w / v. HAMA concentrations are typically 0.3% w / v to 2% w / v, particularly 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, and 1.2% w / v.

[0029] To facilitate cartilage formation and growth, the hydrogel of this invention is a cartilage-specific matrix (ACM), a viscous solution obtained by decellularizing and digesting cartilage with proteases. After modification with methacrylic anhydride, it becomes a gel suitable for photocrosslinking, denoted as ACMMA, with concentrations such as 0.3% w / v to 2% w / v, especially 0.7% w / v, 0.8% w / v, 0.9% w / v, 1.0% w / v, 1.1% w / v, and 1.2% w / v.

[0030] Another composition containing multiple microspheres includes:

[0031] The first type of microspheres releases the first type of active substances contained therein into the environment to promote chondrogenesis in the early stages of bone marrow mesenchymal stem cell differentiation.

[0032] The second type of microspheres releases second-type active substances into the environment, which, after cartilage formation, inhibit angiogenesis or cartilage invasion, forming cartilage lacunae and cartilage-specific ECM deposition, thus maintaining the stability of regenerated cartilage; and

[0033] Hydrogels, including one or more of GelMA and HAMA, as well as ACMMA;

[0034] Type I and Type II microspheres are loaded in a hydrogel.

[0035] Another composition containing multiple microspheres includes:

[0036] The first type of microspheres releases the first type of active substances they contain into the environment to promote cartilage formation in the early stages of chondrogenesis.

[0037] The second type of microspheres releases the second type of active substances they contain into the environment. After cartilage formation, these substances inhibit angiogenesis or invade cartilage, promote cartilage growth, and form cartilage lacunae and cartilage-specific ECM deposits.

[0038] Mesenchymal cells, used for cartilage repair; and

[0039] Hydrogels, including one or more of GelMA and HAMA, as well as ACMMA;

[0040] Type I microspheres, Type II microspheres, and mesenchymal stem cells are loaded into a hydrogel.

[0041] The composition provided by this invention has different release cycles of active substances provided by various microspheres, exhibiting a programmed sequential release of active substances. At different stages of stem cell differentiation, various active substances play different regulatory roles, which is beneficial to the process of cell differentiation into chondrocytes, promotes cartilage generation and growth, and realizes the repair of cartilage tissue.

[0042] Using this composition as a medical device, after loading cells, especially mesenchymal stem cells, can further accelerate the repair of cartilage tissue.

[0043] In vitro experiments have shown that the composition of the present invention rapidly releases first-type active substances (such as transforming growth factor β3) through first-type microspheres (P... T -MPs) have good cell compatibility, regulate chondrogenesis, and slowly release second-type active substances (P-MPs) from second-type microspheres. L -MPs provide anti-angiogenic effects, inhibiting the degradation of regenerated cartilage matrix and promoting the stability of regenerated cartilage. In vivo experiments have confirmed that effective anti-angiogenic regulation through the VEGF / TIMP signaling pathway plays a crucial role in inhibiting endochondral ossification and maintaining the phenotype of regenerated cartilage.

[0044] The technical solution provided by this invention not only achieves the regulation of early cartilage formation and the inhibition of late-stage angiogenesis, thereby meeting the dynamic requirements of bone marrow stromal cell cartilage regeneration, but also achieves stable regeneration of cartilage tissue in an ectopic environment, eliminating the need for pre-induction of cartilage tissue in vitro before implantation in vivo, thus significantly improving the convenience of tissue repair. Attached Figure Description

[0045] Figure 1 Figure 1 shows the characterization results of various PLGA-loaded drug-eluting MPs; where A represents the in vitro release curves of various TGFβ3-loaded MPs, B represents the in vitro release curves of various Levatinib-loaded MPs, and C represents the P... T -MPs and P L -MPs SEM image, D represents P T -MPs and P L Particle size distribution diagram of -MPs;

[0046] Figure 2 Figure 1 shows the hydrogel characterization results; where A is the 1H NMR spectrum of ACM and ACMMA, B is the 1H NMR tracer spectrum of the GHA gel precursor before and after light exposure, and C is the NMR spectrum of GH, GHA, and P. T @GHA and PT -P L The time-scan rheological analysis results of the GHA hydrogel are shown in the figure, where D represents GH, GHA, and P. T @GHA and P T -P L The results of quantitative analysis of the shear modulus of the GHA hydrogel are shown in the figure. E represents GH, GHA, and P. T @GHA and P T -P L The swelling ratio of the GHA hydrogel is shown in the figure, where F represents GH, GHA, and P. T @GHA and P T -P L Degradation rate results of @GHA hydrogel;

[0047] Figure 3 Bright field results of gel precursor (i.e., solution state) solidifying into a solidified hydrogel;

[0048] Figure 4 The results of the in vitro chondrogenic differentiation function verification of PT-MPs are shown in Figure A. Among them, A is a statistical graph of the expression results of SOX9 gene mRNA in each experimental group at 7 days and 14 days, B is a statistical graph of the expression results of COLIIA1 gene mRNA in each experimental group at 7 days and 14 days, and C is a graph of the tissue staining results of each experimental group at 7 days.

[0049] Figure 5 For P L -MPs in vitro anti-angiogenic function verification results; where A represents the control group, 0μM, 20μM lenvatinib drug solutions, and P. L -MPs extract cultured human umbilical vein endothelial cells wound healing, B is a typical image of tubule formation under the field of view, C is the quantitative analysis result of the total tube length of HUVECs, and D is the quantitative analysis result of tube branching.

[0050] Figure 6 The images show the tissue changes in nude mice 4 weeks after subcutaneous gel implantation in each experimental group.

[0051] Figure 7 Histological staining results of nude mice in each experimental group 8 weeks after subcutaneous gel implantation;

[0052] Figure 8 Figure 1 shows the results of subcutaneous gel implantation in nude mice in each experimental group 8 weeks later; where A represents BMSCs loaded with P T -P L @GHA hydrogel diagram (i) and subcutaneous implantation diagram (ii), B represents subcutaneous implantation of various BMSCs-containing hydrogels (GHA, P... T @GHA、P T -P LGross image 8 weeks after implantation of various BMSCs-containing hydrogels (GHA, P) under the skin, C represents subcutaneous implantation of various BMSCs-containing hydrogels (GHA, P). T @GHA、P T -P L @GHA) Micro-CT images after 8 weeks, D is the quantitative analysis result of BV / TV in each group, and E is the quantitative analysis result of BDM in each group;

[0053] Figure 9 The results show the immunofluorescence staining of angiogenesis-related indicators and the expression of cartilage-related genes in nude mice after subcutaneous gel implantation in each experimental group. Among them, A is the statistical graph of CD31 fluorescence staining area at 4 and 8 weeks after gel implantation in each group; B is the statistical graph of vWF fluorescence staining area at 4 and 8 weeks after gel implantation in each group; C is the statistical graph of α-SMA fluorescence staining area at 4 and 8 weeks after gel implantation in each group; and D is the relative mRNA expression level of regenerated cartilage at 8 weeks after gel implantation in each group. Detailed Implementation

[0054] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0055] The specific experimental methods used in the following embodiments of the present invention are described below:

[0056] 1)P T -MPs and P L Synthesis of -MPs

[0057] Transforming growth factor 3 (TGFβ3) was dissolved in sterile deionized water as the aqueous phase, and polylactic acid-glycolic acid was dissolved in 4 mL of dichloromethane (hereinafter: oil phase). The aqueous and oil phases were mixed and stirred at high speed (e.g., 12,000 rpm / min) to form a homogeneous dispersion. This dispersion was then slowly added to a 1% polyvinyl alcohol solution, and the mixture was stirred at low speed (e.g., 400 rpm) for 5 hours using a magnetic stirrer to allow complete evaporation of the dichloromethane. The mixture was then collected by centrifugation to obtain P. T -MPs.

[0058] Following a similar method, an anti-angiogenic compound (e.g., levatinib) was dissolved in 4 mL of oil to form a homogeneous dispersion. This dispersion was then slowly added to a 1% polyvinyl alcohol solution. The mixture was stirred at a low speed (e.g., 400 rpm) with a magnetic stirrer for 5 hours to allow the dichloromethane to completely evaporate. The solution was then collected by centrifugation to obtain P. L -MPs.

[0059] 2) Morphological and structural characterization of MPs

[0060] P was observed using a scanning electron microscope. T -MPs and P L The morphology of -MPs was determined. After drying, the sample was fixed to the sample stub with double-sided carbon ribbon and sputtered with gold under vacuum conditions, and then imaged at an accelerating voltage of 10 kV.

[0061] 3) Encapsulation efficiency (EE)

[0062] Determination of P by solvent extraction method T -MPs and P L Encapsulation efficiency of -MPs in microspheres. The concentrations of extracted TGFβ3 and lenvatinib were determined using ELISA kits and UV-Vis spectrophotometry (n = 3 / group). Drug loading (DL) and EE were calculated using the following formulas:

[0063] DL(%) = W1 / W2, Equation (1);

[0064] EE(%) = Actual drug load / Theoretical drug load, Equation (2); where,

[0065] W1 and W2 represent the weight of the drug within the microparticle and the total weight of the microparticle, respectively, and were analyzed separately. Results are expressed as mean ± standard deviation.

[0066] 4) Sustained-release curve

[0067] Add an appropriate amount of MPs to a volumetric flask containing 20 ml of phosphate buffer (pH 7.4). Collect samples at different time points, and take 1 ml of supernatant each time, then add 1 ml of PBS. Quantitatively detect the samples using an ELISA kit or UV-Vis spectrophotometry (n = 3 / group).

[0068] 5) Preparation of cartilage-specific matrix (ACM) hydrogel

[0069] Auricular cartilage was isolated from Bama miniature pigs under aseptic conditions. After removing the skin, the auricular cartilage tissue was cut into small pieces. For decellularization, the cartilage was successively immersed in 0.5% trypsin / phosphate-buffered saline (PBS), 10 mM Tris-HCl, a nuclease solution consisting of 50 U / mL deoxyribonuclease and 1 U / mL ribonuclease A, 10 mM Tris-HCl (pH = 7.4), and 1% Triton X-100 / Tris-HCl solution (pH = 7.4). The decellularized cartilage was continuously stirred in 1.5 mg / mL collagenase solution at room temperature for 36 h to form a flowing viscous solution. The viscous solution was dialyzed against deionized water in a 3500D dialysis membrane for 72 h, and the resulting ACM was freeze-dried and stored at -20°C until use.

[0070] 6) ACM modification

[0071] ACM was modified with methacrylic anhydride (MA). 0.5 g of water-soluble ACM was dissolved in deionized water, and then 0.5 mL of MA was added in an ice bath at a rate of 0.5 mL / min. The pH was maintained at approximately 8 using 5 M sodium hydroxide. At the end of the reaction, the solution was neutralized with 1 M hydrochloric acid, dialyzed against distilled water for one week in a 3500 D membrane, frozen, and then freeze-dried.

[0072] 7) Rheological analysis

[0073] The rheological behavior of the gel precursor was tested at 25°C using a Hackmarck rotational rheometer with a parallel plate structure (P20TIL, 20 mm diameter). Time-scan oscillation experiments were conducted with 10% strain (CD mode), a frequency of 1 Hz, a 0.5 mm gap, and a duration of 60 s (illuminance: 365 nm, 20 mW / cm²). 2 The gel point refers to the time at which the storage modulus (G') exceeds the loss modulus (G").

[0074] 8) Mechanics Experiment

[0075] The hydrogel was prepared into cylinders with a diameter of 10 mm and a height of 3 mm, with 3 cylinders per group. The mechanical properties of the hydrogel were tested using a dynamic mechanical analyzer (Instron-5542). Compression was performed at 1 mm / min until the compression depth reached 60% of the initial height. The elastic modulus was calculated from the strain-stress curve of the initial 10%–20% compression.

[0076] 9) In vitro swelling rate and enzyme degradation

[0077] The initial wet weight of the hydrogel is denoted as W0, and the wet weight after 48 hours is denoted as Ws. The swelling rate is defined as Ws / W0 × 100%. Furthermore, the initial dry weight of the hydrogel is denoted as Wd, and the dry weight of the hydrogel after soaking in enzyme solution (1 U / mL collagenase) for different times is denoted as Wt. The degradation rate is defined as Wt / Wd × 100%.

[0078] 10) Isolation and culture of rabbit bone marrow mesenchymal stem cells

[0079] Bone marrow mesenchymal stem cells (BMSCs) were isolated from adult rabbit bone marrow, suspended in mesenchymal stem cell culture medium (7501, Science Cell), transferred to culture dishes, and cultured at 37°C with 5% CO2. Experiments were conducted when the BMSCs expanded to the second generation.

[0080] 11) Cytotoxicity test: Bone marrow mesenchymal stem cells were injected at a concentration of 2 × 10⁻⁶. 4Cells were seeded at a concentration of [number] cells / mL in the supernatant of a lyophilized hydrogel soaked in DMEM containing 10% fetal bovine serum for 72 hours, and their cytotoxicity was determined. To determine the amount of drug to be used in subsequent experiments, stem cells and endothelial cells were treated with different concentrations of Leva solution for 72 hours, and cell proliferation was measured using a cell counting kit (CCK-8; Dojindo) according to the manufacturer's instructions.

[0081] 12) Live / dead cell staining, spreading, and chondrogenic function: Bone marrow mesenchymal stem cells were mixed with various hydrogels (G, GH, GHA, P... T @GHA, P T -P L @GHA, 4×10 7 The mixture was prepared at a concentration of (cell / mL) and then injected into a cylindrical mold (10 mm in diameter and 2 mm in height). The mixture was then exposed to light at 365 nm and 20 mW / cm². 2 Rapid polymerization was performed for approximately 5 seconds followed by 60 seconds of incubation at 37°C with 5% CO2 for 7 days. Following the manufacturer's instructions, the activity of BMSCs encapsulated in the hydrogel was assessed using live and dead cell viability analysis (Invitgen, USA) and confocal microscopy (TCS SP8 STED 3X). To observe cell spread in the hydrogel, F-actin and cell nuclei were stained with podophyllotoxin and DAPI, respectively, on day 7. Cells in the hydrogel were also subjected to ColIIA1 immunofluorescence staining on day 7, and the chondrogenic differentiation capacity of BMSCs in the hydrogel was observed under a confocal microscope.

[0082] 13) Wound healing experiment

[0083] The content of microparticles encapsulated in the hydrogel was determined according to the cytotoxicity assay, and P was used. L The following experiments were performed using HUVECs extract (1 × 10⁻⁶ MPs per well). 5 Cells were seeded in 6-well plates and cultured until a monolayer formed. Cells were then divided into 4 groups, scored with the tip of a medium-sized pipette, washed with PBS, and treated with 20 ng / mL VEGFA and culture medium (control group), two concentrations of Levatinib solution, or P... L Cells were co-treated with MPs extract solution. Cell migration patterns were recorded under an optical microscope at 0, 6, and 24 hours.

[0084] 14) Tube formation experiment: Add 50 μL of matrix gel to each well of a 96-well plate and incubate for 30 min to solidify. HUVECs are then suspended in DMEM and subjected to 2 × 10⁻⁶ cycles. 4 / wells were seeded into 96-well plates. Cultured human umbilical vein endothelial cells were divided into 4 groups, and seeded with 20 ng / mL VEGFA and culture medium (control group), two concentrations of rifatinib solution, or P, respectively.L Incubate with MPs solution for 4 hours and observe angiogenesis.

[0085] 15) Animal surgical methods

[0086] Male nude mice (approximately 4 weeks old) were anesthetized via intraperitoneal injection of pentobarbital, placed in a prone position, and draped after disinfection. A 0.2 cm incision was made on the back of the mouse, and the skin around the incision was bluntly dissected. With the assistance of toothless forceps, the cell material complex was implanted subcutaneously into the nude mouse. The incision was sutured with 5-0 sutures to ensure the skin adhered tightly to the regenerating tissue. Subcutaneous cultures were collected after 4 and 8 weeks for further analysis.

[0087] 16) Micro-CT Analysis: Samples were fixed in 4% (w / v) paraformaldehyde and analyzed using a Micro-CT μ80 scanner (Sanco Medical, Switzerland). Scanning parameters were set as follows: voltage 70 kV, current 114 μA, pixel resolution 1024 × 1024. Data were analyzed using evaluation software (Sanco Medical, Switzerland). Two-dimensional cross-sectional images were obtained using Micro-CT, and relative bone volume fraction (BV / TV) and bone mineral density (BMD) were measured.

[0088] 17) Histological and immunohistochemical analysis

[0089] Collected specimens were fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and cut into 5-micrometer-thick sections. Hematoxylin-eosin (H&E), saffron O / Strong Green (SO / FG), and Masson staining were performed. Immunohistochemical staining for COLIIA1, OCN, and COLX was used to analyze cartilage and bone-specific proteins. Immunofluorescence staining for CD31, vWF, and αSMA was performed according to the methods described above. Images were analyzed using ImageJ software to quantify the area of ​​vascular endothelial cells.

[0090] 18) Quantitative Real-Time PCR

[0091] Total RNA was extracted from cells using a Trizol kit (Omega), and genes were obtained by reverse transcription using a gene synthesis kit (Takara). Real-time quantitative PCR was performed using a SYBR Green PCR Master Mix (Takara) and a CFX96 real-time PCR detection system (Bio-Rad).

[0092] 19) Statistical methods

[0093] Data were analyzed using SPSS software. One-way ANOVA was used for comparisons between groups. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference. All values ​​are expressed as mean ± standard deviation.

[0094] Example 1: Characterization of drug-loaded microspheres

[0095] MPs were prepared using polylactic acid-glycolic acid copolymers (L / G = 85 / 15) with average relative molecular masses of 10 kDa, 50 kDa, and 100 kDa, and the effects of relative molecular mass and drug properties on the release rate were investigated. Figure 1 A and Figure 1 As can be seen from B, the relative molecular mass of PLGA polymers is the main factor affecting the sustained-release time. In vitro cumulative release experiments show that low molecular weight polylactic acid microspheres can sustain release (~96%) within 4 weeks, while high molecular weight polylactic acid microspheres have a longer release period, reaching ~40% after 56 days. Meanwhile, the solubility of factors or drugs indirectly affects the release rate. Water-soluble TGFβ3 is more conducive to the release rate from MPs, while the lipid-soluble angiogenesis inhibitor Levatinib is difficult to penetrate before MPs are destroyed, thus achieving a long-term sustained-release effect.

[0096] 10kDa PLGA used to prepare PLGA loaded with TGFβ 1w Microspheres (P) T -MP S A 100kDa PLGA was used to fabricate a PLGA loaded with LEVA. 10w Microspheres (P) L -MP S ).like Figure 1 As shown in Figure C, scanning electron microscopy (SEM) reveals P T -MP S and P L -MP S They are evenly distributed and spherical. Figure 1 D displays, P T -MP S The average volumetric particle size is approximately 90 μm, slightly smaller than P. L -MP S The average volumetric particle size is ~100 μm.

[0097] Therefore, this embodiment successfully established a rapid-release PLGA using transforming growth factor β3 as a carrier. 1w -MP S PLGA with slow-release Levatinib 10w -MP SA dual programmed drug delivery system is used to regulate early chondrogenesis and inhibit late angiogenesis, thereby meeting the dynamic requirements of bone marrow stromal cell chondrogenesis.

[0098] Example 2: Cartilage-Specific Matrix Hydrogel

[0099] In this embodiment, methacryloyl gelatin (GelMA) was selected as the protein component, and methacryloyl hyaluronic acid (HAMA) was selected as the glycosaminoglycan component, serving as a hybrid carrier to simulate the ECM microenvironment. To further provide a cartilage-specific microenvironment for BMSCs to differentiate into chondrocytes, methacryloyl decellularized chondrocyte matrix (ACMMA) was introduced into GelMA / HAMA to prepare a cartilage-specific matrix hydrogel (GelMA / HAMA / ACMMA, GHA) composed of 5% w / v GelMA, 1% w / v HAMA, and 1% w / v ACMMA. ACMMA was synthesized by modifying the decellularized chondrocyte matrix with methacrylate, as confirmed by its 1H NMR spectrum. Figure 2 A). For example Figure 3 As shown, the GHA gel precursor (i.e., in its fluid state) under light irradiation (365nm LED, 20mW / cm²). 2 Rapid cross-linking under ( ) conditions. 1 H NMR spectroscopy further confirmed the photopolymerization mechanism of GHA hydrogel, which is due to the reduction of the signal of methacrylate groups by 5.4–7.8 ppm under light irradiation. Figure 2 B). P T -MPS and P L -MPS was added to GHA hydrogels to prepare P T -P L @GHA hydrogel, under light conditions, P T -P L The GHA hydrogel also underwent a rapid sol-gel transition. For example... Figure 2 C and Figure 2 D. Time-scan rheological and storage modulus experiments showed that the addition of ACMMA increased the shear modulus of the GH hydrogel from 1175±102 Pa to 1878±63 Pa. P T @GHA and P T P L The mechanical properties of @GHA were not significantly different from those of GHA, further confirming the good crosslinking properties of these gel precursors. Furthermore, P... T -P L @GHA hydrogel has suitable swelling and degradation rates. Figure 2 E and Figure 2 F)

[0100] Example 3 Animal Experiment

[0101] To evaluate the gradient modulation function of programmed delivery systems in early chondrogenesis and late avascular microenvironment, BMSCs were directly encapsulated into GHA and P without in vitro pre-induction. T @GHA and P T -P L The gels were then embedded in various hydrogels such as GHA and transplanted into nude mice in each experimental group. Figure 8 A). Four weeks after implantation, the GHA group showed significant osteogenic tendency and significant angiogenesis, while the P group... T @GHA and P T -P L In the GHA group, white cartilage-like tissue had already formed due to the early cartilage microenvironment resulting from the formation of cartilage-specific matrix components and the early release of transforming growth factor β-3. With the subcutaneous implantation time extended to 8 weeks, the GHA group and the P group... T @GHA group has formed osteoid-like tissue with widespread neovascularization, while at this time P T -P L In the GHA group, the sustained release of lenvatinib inhibited angiogenesis, providing an avascular microenvironment for late-stage cartilage growth, resulting in the presence of white cartilage-like tissue without vascular invasion. Figure 8 B). Micro-CT images show GHA group and P T The GHA group showed significant osteoid calcification, a result of direct contact with the subcutaneous vascular environment. However, P T -P L @GHA group maintained cartilage condition despite continuous release of lenvatinib ( Figure 8 C). Bone volume fraction (BV / TV) of the samples collected in each group. Figure 8 D) and bone mineral density (BMD) Figure 8 The statistical data from E) confirms the above observations.

[0102] Histological staining showed that at 4 weeks, the GHA group exhibited a mixed structure of cartilage and osteoid tissue, with positive immunohistochemical staining for COLIIA1 and osteocalcin (OCN). Conversely, the group containing P... T The -MPS group regenerated homogeneous cartilage-like tissue at 4 weeks, exhibiting typical cartilage lacunae, cartilage-specific deposition, and positive COLIIA1 immunostaining. Figure 6 At 8 weeks, histological staining showed that the GHA group had developed typical osteoid structures. T The @GHA group also began ossification, with mature bone-specific ECM deposition and strong positive OCN immunohistochemical staining. However, P T- P L The @GHA group still had stable chondroid tissue, with typical lacunae and cartilage-specific ECM deposits. Figure 7These results demonstrate that the programmed release system of this embodiment possesses in vivo chondrogenesis and anti-angiogenesis capabilities, thereby regenerating stable cartilage tissue in an ectopic environment without the need for in vitro pre-induction procedures.

[0103] To further investigate its anti-angiogenic effect in vivo, immunofluorescence staining was used to identify vascular endothelial cells. CD31 immunofluorescence staining showed vascular-specific expression in the GHA group at 4 weeks, while no significant expression was observed in the other two groups. Von von Willebrand factor (vWF) and smooth muscle actin (SMA) staining showed that vascular endothelial cells were only found in the ossified area of ​​the GHA group, and in P... T @GHA group and P T -P L In the GHA group, almost no endothelial cells were detected in the newly formed cartilage area. Eight weeks after implantation, the number of endothelial cells in the ossification area of ​​the GHA group increased significantly. T In the GHA group, partial vascular invasion was observed. Conversely, in the P group... T -P L @GHA group due to P L -MPS slowly releases angiogenesis inhibitors while the observed number of endothelial cells is negligible, findings supported by semi-quantitative analysis of the fluorescent regions. Figure 9 A, Figure 9 B and Figure 9 C). Quantitative reverse transcription polymerase chain reaction results also showed that the expression levels of chondrogenesis-related genes (SOX9, TGFβ3, Smad, COLIIA1, and CHM1) were significantly upregulated at 8 weeks, confirming the early chondrogenic effect of the transforming growth factor β / Smad signaling pathway in vivo. Figure 9 D). The expression of angiogenesis-related genes (VEGFA, CD31, and ANGPT1) and matrix deposition-related genes (TIMP3, MMP13, and SDC) was significantly downregulated at 8 weeks, thereby effectively suppressing the expression levels of osteogenic-related genes (BMP2, RUNX2, OPN, OCN, and ALP).

[0104] As can be seen, the strategy provided in this embodiment has advantages in maintaining the stability of cartilage phenotype. It successfully achieves dynamic regulation of in vivo stem cell cartilage regeneration without the need for an in vitro pre-induction process, providing a new stem cell cartilage regeneration method for the repair of multiple cartilage defects.

Claims

1. The use of a microsphere-containing composition in the manufacture of a medical device for promoting cartilage growth and repair, characterized in that... include: The first type of microspheres rapidly releases the first type of active substances they contain into the environment to promote chondrogenesis in the early stages of mesenchymal stem cell differentiation. and The second type of microspheres slowly releases their second type of active substances into the environment. After cartilage formation, these substances inhibit angiogenesis or matrix degradation, promote cartilage growth, and form cartilage lacunae and cartilage-specific ECM deposits; and The first type of microspheres uses PLGA with a material selected from 5kDa to 10kDa; The second type of microspheres uses PLGA with a strength of 50 kDa or higher. The first type of active substance is TGFβ3; The second type of active substance is selected from lenvatinib or bevacizumab; The first type of microspheres and the second type of microspheres are loaded in a hydrogel, wherein the hydrogel is composed of... It is a mixture of 5% w / v to 10% w / v methacryloyl gelatin (GelMA), 0.3% w / v to 2% w / v methacryloyl decellularized cartilage matrix (ACMMA), and 0.3% w / v to 2% w / v methacryloyl hyaluronic acid (HAMA).

2. The application according to claim 1, characterized in that... The second type of microspheres uses PLGA with a strength of 100 kDa or higher.

3. The application according to claim 1, characterized in that... Cells are also loaded into the hydrogel.

4. A medical device, characterized in that... The composition containing microspheres as described in claim 1.

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