CN117717655A - Multiparticulate drug delivery hydrogel and application thereof in promoting stem cell regeneration cartilage - Google Patents
Multiparticulate drug delivery hydrogel and application thereof in promoting stem cell regeneration cartilage Download PDFInfo
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- CN117717655A CN117717655A CN202311194935.3A CN202311194935A CN117717655A CN 117717655 A CN117717655 A CN 117717655A CN 202311194935 A CN202311194935 A CN 202311194935A CN 117717655 A CN117717655 A CN 117717655A
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Abstract
A microsphere-containing composition comprising a first type of microspheres that release a first type of active agent to the environment to promote chondrogenesis at an early stage of mesenchymal stem cell differentiation; and a second class of microspheres that release a second class of active substances to the environment to inhibit angiogenesis or invasion of cartilage, promote cartilage growth, form cartilage pits, and cartilage-specific ECM deposition. Proved by verification, the composition not only realizes the regulation of early cartilage formation and the inhibition of late angiogenesis, thereby meeting the dynamic requirement of bone marrow mesenchymal stem cell cartilage regeneration, but also realizes the stable regeneration of cartilage tissues in an ectopic environment, and the convenience of tissue repair is obviously improved without the operation of firstly carrying out pre-induced cartilage tissues in vitro and then implanting the cartilage tissues into a body.
Description
Technical Field
The invention relates to a biological material, in particular to a composition loaded with cartilage-forming active molecules, which is used for promoting differentiation and regeneration of stem cells into cartilage and application in medical devices.
Background
Engineered cartilage using autologous chondrocytes as seed cells has been primarily used in clinical applications in auricles, nose, trachea, meniscus and joints. However, chondrocyte-based cartilage regeneration strategies have limited clinical application due to limited cell sources, unavoidable trauma to the donor site, and difficult maintenance of phenotype after multiple expansion. As ideal engineering cartilage seed cells, bone marrow mesenchymal stem cells (BMSCs) have the unique advantages of small trauma, good proliferation capacity, definite cartilage differentiation potential and the like when obtaining cells. In an ectopic environment, the cartilage phenotype is difficult to maintain because vascular penetration inevitably leads to endochondral bone formation during the chondrogenic differentiation and development stages. Despite these advances, BMSCs-based cartilage regeneration still requires a significant amount of in vitro pre-induction time to differentiate into chondrocytes prior to in vivo transplantation, which limits its clinical use. Therefore, an ideal stem cell cartilage regeneration technique would need to meet the time-dependent requirements of early cartilage formation and late anti-angiogenic microenvironment in vivo without in vitro pre-induction.
Drug delivery systems based on Microparticles (MPs) and Nanoparticles (NPs), including liposomes and polymers and inorganic particles, are considered to be viable carriers for modulating stem cell fate and for simultaneously promoting tissue-specific regeneration. For example, U.S. food and drug administration approved poly (lactic-co-glycolic acid) microparticles have been widely used for delivery of multifunctional bioactive substances by precision delivery, such as: growth factors (e.g., transforming growth factor beta and bone morphogenic proteins). Recent studies have shown that transforming growth factor beta 3 produces potent cartilage-inducing functions by activating tgfβ/Smad signaling pathways.
Another factor that synergistically regulates stem cell cartilage regeneration is the use of biocompatible scaffolds capable of carrying biologically active substances. Unlike the stentless cell sheet culture mode, the hydrogel-based three-dimensional (3D) culture mode has a microenvironment that mimics the extracellular matrix (ECM), which facilitates encapsulation of microparticles for multi-drug delivery. In our previous studies, photocrosslinked matrix hydrogels mimic the major components of ECM by binding proteoglycans and glycosaminoglycans. Cartilage or umbilical cord decellularized matrix is also used as a bioactive ingredient 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, the breakthroughs for dynamically regulating stem cell cartilage regeneration in vivo using 3D hydrogel scaffolds are very limited.
Disclosure of Invention
It is an object of the present invention to provide a microsphere-containing composition that regulates the progression of cell chondrogenic differentiation, and promotes cartilage growth and repair.
Another object of the present invention is to provide a composition comprising a plurality of microspheres, wherein the release cycles of the active substances provided by the microspheres are different, and the active substances are released in a programmed manner at different stages of stem cell differentiation, thereby being beneficial to regulating the differentiation process of cells into cartilage and promoting cartilage growth and repair.
It is still another object of the present invention to provide a hydrogel containing a plurality of microspheres, wherein the microspheres are loaded in the hydrogel to provide a growth environment for cells, and the active substances released from the microspheres are beneficial to regulating the chondrogenic differentiation process of cells, and promoting cartilage growth and repair.
It is still another object of the present invention to provide a hydrogel containing a plurality of microspheres, which regulates the progress of differentiation of stem cells (e.g., mesenchymal stem cells) into cartilage, and promotes cartilage growth and repair.
A fifth object of the present invention is to provide a hydrogel containing a plurality of microspheres for use as a medical device in bone repair.
A microsphere-containing composition comprising:
a first type of microspheres that release the contained first type of active substance to the environment to promote chondrogenesis at an early stage of mesenchymal stem cell differentiation; and
and a second class of microspheres that release the contained second class of active substances to the environment, inhibit angiogenesis or matrix degradation after cartilage production, promote cartilage growth, form cartilage pits, and cartilage-specific ECM deposition.
The first type of microspheres generally have the property of rapidly releasing the first type of active substance into the environment, and materials suitable for use in the first type of microspheres are: but are not limited to PLGA having a molecular weight of 5kDa to 10 kDa.
The second type of microspheres generally have the property of delayed release of the second type of active substance into the environment, and materials suitable for the first type of microspheres are as follows: but are not limited to, PLGA having a molecular weight of 50kDa or more, PLGA having a molecular weight of 100kDa or more, PLL having a molecular weight of 10kDa or more, and Mesoporous Silica (MSNs) and organometallic framework (MOF).
The first class of active agents are growth factors such as: transforming small molecular substances such as growth factors beta 3 and Kartogenin (KGN) and the like are beneficial to promoting the chondrogenic induction differentiation of stem cells in the early stage of stem cell differentiation, so as to form white cartilage-like tissues.
The second class of active substances are anti-angiogenic molecules, known as: but not limited to, lenvatinib, bevacizumab and other small molecular substances, controlled by the second type of microspheres, most of the substances (such as more than 40%, more than 50% or more than 60% of the amount of substances carried in the microspheres) are released into the environment after the cartilage is formed, so that anti-angiogenesis is realized, a vascular-free microenvironment is provided for cartilage-like tissues, blood vessels are inhibited from invading the cartilage-like tissues and degradation of cartilage matrixes is inhibited, and growth of the cartilage-like tissues is promoted to form cartilage characteristics such as cartilage pits and cartilage-specific deposition.
To facilitate cartilage formation and growth, the composition of the present invention further comprises a hydrogel, wherein the first type of microspheres and the second type of microspheres are both loaded in the hydrogel.
A composition comprising a plurality of microspheres, comprising:
a first type of microspheres that release the contained first type of active substance to the environment to promote cartilage formation at an early stage of cartilage formation;
a second class of microspheres that release the contained second class of active substances to the environment, inhibit angiogenesis or invasion of cartilage after cartilage production, form cartilage pits and cartilage-specific ECM deposition, maintain a stable regenerated cartilage phenotype; and
hydrogels, in which a first type of microsphere and a second type of microsphere are loaded, provide a microenvironment for cartilage production and growth.
To accelerate cartilage production and growth, cells are also contained in hydrogels, such as: bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and the like.
Another composition comprising a plurality of microspheres, comprising:
a first type of microspheres that release the contained first type of active substance to the environment to promote cartilage formation at an early stage of cartilage formation;
a second type of microspheres that release the contained second type of active substance to the environment, inhibit angiogenesis or invasion of cartilage after cartilage formation, form cartilage pits and cartilage-specific ECM deposition, and maintain stability of regenerated cartilage;
mesenchymal cells for repair of cartilage; and
hydrogel, first class microsphere, second class microsphere and mesenchymal stem cell are loaded therein.
In the present invention, the hydrogel is generally a medical hydrogel, such as: hyaluronic acid, gelatin, chondroitin sulfate and crosslinked modified derivatives thereof, such as: gel GelMA and HAMA suitable for photocrosslinking, and the like. GelMA concentrations are as follows: 5% w/v to 10% w/v, especially 5% w/v, 6% w/v, 7% w/v, 8% w/v, 9% w/v and 10% w/v etc. HAMA concentrations were as follows: 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 etc.
In order to facilitate the generation and growth of cartilage, the hydrogel is a cartilage specific matrix ACM, and is a viscous solution prepared by decellularizing cartilage and digesting with protease. After modification with methacrylic anhydride, a gel suitable for photocrosslinking was obtained, noted: ACMMA, the concentration of which is as follows: 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 etc.
Another composition comprising a plurality of microspheres, comprising:
a first type of microspheres that release the contained first type of active substance to the environment to promote chondrogenesis at an early stage of differentiation of bone marrow mesenchymal stem cells;
a second type of microspheres that release the contained second type of active substance to the environment, inhibit angiogenesis or invasion of cartilage after cartilage formation, form cartilage pits and cartilage-specific ECM deposition, and maintain regenerated cartilage stability; and
hydrogels, including one or more of GelMA and HAMA, and ACMMA;
the first type of microspheres and the second type of microspheres are loaded in a hydrogel.
Another composition comprising a plurality of microspheres, comprising:
a first type of microspheres that release the contained first type of active substance to the environment to promote cartilage formation at an early stage of cartilage formation;
a second class of microspheres that release the contained second class of active substances to the environment, inhibit angiogenesis or invasion of cartilage after cartilage formation, promote cartilage growth, form cartilage pits, and cartilage-specific ECM deposition;
mesenchymal cells for repair of cartilage; and
hydrogels, including one or more of GelMA and HAMA, and ACMMA;
the first type of microspheres, the second type of microspheres and the mesenchymal stem cells are loaded on the hydrogel.
The composition provided by the invention has different release periods of active substances provided by various microspheres, shows the characteristic of procedural sequential release of the active substances, and has different regulation effects on various active substances at different stages of stem cell differentiation, thereby being beneficial to the differentiation process of cells into cartilage, promoting cartilage generation and growth and realizing the repair of cartilage tissues.
The composition can be used as medical equipment, and after being loaded with cells, especially mesenchymal stem cells, the composition can further accelerate the repair of cartilage tissues.
In vitro experiments show that the composition of the invention rapidly releases the first type of microspheres (P) of the first type of active substances (such as transforming growth factor beta 3) T -MPs) haveGood cytocompatibility, and has cartilage formation regulating effect, and can slowly release second type of active substance (P L -MPs) provide an anti-angiogenic effect, inhibit degradation of the regenerative cartilage matrix, and facilitate stabilization of the regenerative cartilage. In vivo experiments prove that the VEGF/TIMP signal channel is used for effectively resisting angiogenesis regulation, and plays a vital role in inhibiting the phenotype maintenance of cartilage internal bone formation and regenerated cartilage.
The technical scheme provided by the invention not only realizes the regulation of early cartilage formation and the inhibition of late angiogenesis, thereby meeting the dynamic requirement of bone marrow stromal cell cartilage regeneration, but also realizes the stable regeneration of cartilage tissue in an ectopic environment, and the operation of pre-inducing the cartilage tissue from outside the body and then implanting the cartilage tissue into the body is not needed, so that the convenience of tissue repair is obviously improved.
Drawings
FIG. 1 is a graph of characterization results of MPs of various PLGA drug carriers prepared; wherein A is the in vitro release curve of various MPs carrying TGF beta 3, B is the in vitro release curve of various MPs carrying Levatinib, and C is P T -MPs and P L SEM electron microscopy of MPs, D is P T -MPs and P L -a particle size distribution profile of MPs;
FIG. 2 is a graph of hydrogel characterization results; wherein A is nuclear magnetic resonance hydrogen spectrogram of ACM and ACMMA, B is nuclear magnetic resonance hydrogen spectrogram trace of GHA gel precursor before and after illumination, and C is GH, GHA and P T @GHA and P T -P L Time scanning rheological analysis result graph of @ GHA hydrogel, wherein D is GH, GHA and P T @GHA and P T -P L Shear modulus quantitative analysis result graph of @ GHA hydrogel, wherein E is GH, GHA and P T @GHA and P T -P L Swelling ratio results of @ GHA hydrogels, F is GH, GHA, P T @GHA and P T -P L A degradation rate result graph of the @ GHA hydrogel;
FIG. 3 is a graph of the bright field results of coagulation of a gel precursor (i.e., in solution) into a set-like hydrogel;
FIG. 4 is a graph showing the results of in vitro chondrogenic differentiation function verification of PT-MPs; wherein A is a SOX9 gene mRNA expression result statistical graph of each test group at 7 days and 14 days, B is a COLIIA1 gene mRNA expression result statistical graph of each test group at 7 days and 14 days, and C is a tissue staining result graph of each test group at 7 days;
FIG. 5 is P L -MPs in vitro anti-vascular function verification result map; wherein A is a control group, 0 mu M,20 mu M of lenvatinib drug solution and P L -map of wound healing of human umbilical vein endothelial cells cultured with MPs extract, B being typical images of tubule formation in visual field, C being a graph of quantitative analysis of total tube length of HUVEC, D being a graph of quantitative analysis of tube branching;
FIG. 6 is a graph showing the results of tissue changes 4 weeks after the subcutaneous implantation of gel in nude mice of each test group;
FIG. 7 is a graph showing histological staining results of each experimental group of nude mice 8 weeks after subcutaneously implanting the gel;
FIG. 8 is a graph showing the results of subcutaneously implanting gel for 8 weeks in nude mice of each experimental group; wherein A is BMSCs load P T -P L Graph (i) of GHA hydrogel and graph (ii) of subcutaneous implant, B is the subcutaneous implant of various BMSCs-containing hydrogels (GHA, P T @GHA、P T -P L Rough image after 8 weeks @ GHA), C was the subcutaneous implantation of various BMSCs-containing hydrogels (GHA, P T @GHA、P T -P L Microscopic CT images after 8 weeks @ GHA), D is a BV/TV quantitative analysis result graph of each group, and E is a BDM quantitative analysis result graph of each group;
FIG. 9 shows the results of immunofluorescence staining of the angiogenesis-related index and expression of cartilage-related genes after subcutaneous implantation of gel in nude mice of each experimental group; wherein, A is the CD31 fluorescent staining area statistical graph at 4 weeks and 8 weeks of each group of gel implantation, B is the vWF fluorescent staining area statistical graph at 4 weeks and 8 weeks of each group of gel implantation, C is the alpha-SMA fluorescent staining area statistical graph at 4 weeks and 8 weeks of each group of gel implantation, and D is the relative mRNA expression level of regenerated cartilage at 8 weeks of each group of gel implantation.
Detailed Description
The technical scheme of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only for illustrating the technical scheme of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical scheme of the present invention, which is intended to be covered by the scope of the claims of the present invention.
The test methods used in the following examples of the present invention are specifically described below:
1)P T -MPs and P L Synthesis of MPs
Transforming growth factor 3 (TGF-beta 3) was dissolved in sterile deionized water as an aqueous phase, and polylactic acid-glycolic acid was dissolved in 4mL of methylene chloride (hereinafter: oil phase). Mixing water phase and oil phase, stirring at high speed (such as 12,000 rpm/min) to form uniform dispersion, slowly adding into 1% polyvinyl alcohol solution, stirring at low speed (such as 400 rpm) with magnetic stirrer for 5 hr to volatilize dichloromethane completely, centrifuging, and collecting to obtain P T -MPs。
Dissolving anti-angiogenic compound (such as Levalatinib, levatinib) in 4mL of oil phase to form uniform dispersion, slowly adding into 1% polyvinyl alcohol solution, stirring with magnetic stirrer at low speed (such as 400 rpm) for 5 hr to volatilize dichloromethane completely, centrifuging, and collecting to obtain P L -MPs。
2) MPs morphological structure characterization
Observation of P with a scanning electron microscope T -MPs and P L -morphology of MPs. The dried sample was fixed on a sample stub with a double-sided carbon tape and sputtered to gold plate under vacuum and imaged at an accelerating voltage of 10 kv.
3) Encapsulation Efficiency (EE)
Determination of P by solvent extraction T -MPs and P L Encapsulation efficiency of MPs in microspheres. The concentrations of extracted tgfβ3 and lenvatinib were measured (n=3/group) using ELISA kit and uv-vis spectrophotometry, respectively. Drug Loading (DL) and EE were calculated from the following formulas:
DL (%) =w1/W2, formula (1);
EE (%) = actual drug loading/theoretical drug loading, formula (2); wherein,
w1 and W2 represent the weight of the drug in the microparticles and the weight of the entire microparticles, respectively, and were analyzed. Results are expressed as mean ± standard deviation.
4) Sustained release profile
A suitable amount of MPs was added to a volumetric flask (pH 7.4) containing 20ml of phosphate buffer. Samples were collected at different time points, 1ml of supernatant was taken each time, and 1ml of pbs was added, and assayed using ELISA kit or uv-vis spectrophotometry (n=3/group).
5) Preparation of cartilage-specific matrix (ACM) hydrogels
Separating the small-sized pig auricle cartilage of Bama under aseptic condition, removing skin, and cutting auricular soft bone tissue into small pieces. For decellularization, cartilage was immersed in a nuclease solution consisting of 50U/mL deoxyribonuclease and 1U/mL ribonuclease A, 10mM Tris-HCl (pH=7.4), and 1% Triton X-100/Tris-HCl solution (pH=7.4) in sequence in 0.5% pancreatin/Phosphate Buffered Saline (PBS), 10mM Tris-HCl. The decellularized cartilage was stirred continuously in 1.5mg/mL collagenase solution at room temperature for 36h to form a flowing viscous solution. The viscous solution was dialyzed against deionized water in 3500D dialysis membrane for 72h, and the resulting ACM was lyophilized and stored at-20 ℃ until use.
6) ACM modification
ACM was modified with Methacrylic Anhydride (MA). 0.5g of water-soluble ACM was dissolved in deionized water, then 0.5mL of MA was added to the ice bath at a rate of 0.5mL/min, and the pH was maintained at about 8 with 5M sodium hydroxide. At the end of the reaction, the solution was neutralized with 1M hydrochloric acid, dialyzed against distilled water in 3500D membrane for 1 week, frozen, and then freeze-dried.
7) Rheology analysis
The rheological behaviour of the gel precursor was tested at 25℃using a Ha Kema-sided rotary rheometer of parallel plate construction (P20 TIL, diameter 20 mm). Time-scanning oscillation test using 10% strain (CD mode), 1 Hz frequency, 0.5mm gap of 60s (light: 365nm,20mW/cm 2 ). Gel point refers to the time that the storage modulus (G') exceeds the loss modulus (G ").
8) Mechanical experiment
Hydrogels were prepared in cylinders 10mm in diameter and 3mm in height, 3 per set. The mechanical properties of the hydrogels were tested on a dynamic mechanical analyzer (Instron-5542). Compression was performed at 1mm/min until the compression depth reached 60% of the initial height. The modulus of elasticity was calculated from the initial 10% -20% strain-stress curve.
9) In vitro swelling ratio and enzymatic degradation
The initial wet mass of the hydrogel is noted as W 0 Wet weights after 48h were noted as Ws. The swelling ratio is defined as Ws/W 0 X 100%. In addition, the initial dry weight of the hydrogel was noted Wd, and the dry weight of the hydrogel after being immersed in the enzyme solution (1U/mL collagenase) for various times was noted Wt. Degradation rate was defined as Wt/Wd.times.100%.
10 Isolation and culture of rabbit bone marrow mesenchymal stem cells
Taking adult rabbit bone marrow to separate bone marrow mesenchymal stem cells, suspending in mesenchymal stem cell culture solution (7501,Science cell), transferring into a culture dish, and transferring into a culture dish at 37deg.C and 5% CO 2 Culturing. Experiments were performed when BMSCs were amplified to generation 2.
11 Cytotoxicity test): mesenchymal stem cells of bone marrow are used for 2×10 4 The cytotoxicity was measured by inoculating the serum into a supernatant of lyophilized hydrogel soaked in DMEM containing 10% fetal bovine serum for 72 hours at a concentration of one/mL. To determine the amount of drug used in subsequent experiments, stem cells and endothelial cells were treated with Leva solutions at different concentrations for 72 hours, and cell proliferation was measured using a cell count kit (CCK-8; dojindo) according to the manufacturer's instructions.
12 Live/dead cell staining, spreading, and chondrogenic functions): combining bone marrow mesenchymal stem cells with hydrogel of each group (G, GH, GHA, P) T @GHA，P T -P L @GHA，4×10 7 cell/mL) and then injected into a cylindrical mold (diameter 10mm, height 2 mm), under light (365 nm,20 mW/cm) 2 ) Rapid polymerization 60S at 37℃with about 5S, 5% CO 2 Culturing for 7d. The activity of BMSCs encapsulated in hydrogels was evaluated using living and dead cell activity assays (invitrogen, USA) and confocal microscopy (TCS SP8 STED 3X) according to the manufacturer's instructions. To observe the spreading of cells in hydrogels, on day 7, F-actin and nuclei were used separatelyPodophylline and DAPI were stained. Cells in the hydrogel were subjected to Col IIA1 immunofluorescent staining on day 7, and the chondrogenic differentiation ability of BMSCs in the hydrogel was observed under confocal microscopy.
13 Wound healing experiment
The content of the particles encapsulated in the hydrogel was determined according to cytotoxicity test and used with P L The following experiments were carried out on the MPs extract. HUVECs at 1X 10 per well 5 Inoculated in 6-well plate and cultured until monolayer is formed. Cells were divided into 4 groups, scored with medium sized pipette tips, rinsed with PBS, treated with 20ng/mL VEGFA and culture medium (control), and either Levatinib solution or P at two concentrations L -co-processing of the MPs extract solution. Migration patterns of cells were recorded under an optical microscope at 0, 6, 24 hours, respectively.
14 Tube forming experiment): 50 mu L of matrigel is added to each well of a 96-well plate, and the mixture is incubated in an incubator for 30min for solidification. HUVECs were suspended in DMEM and then mixed at a rate of 2X 10 4 Wells were seeded in 96-well plates. The cultured human umbilical vein endothelial cells were divided into 4 groups, and 20ng/mL VEGFA and culture solution (control group), li Fan tinib solution or P at two concentrations were used respectively L Incubation of the MPs solution for 4 hours, and observation of the vascularization.
15 Animal surgery method
Male nude mice (about 4 weeks) were used, and pentobarbital was anesthetized by intraperitoneal injection, in prone position, sterilized and toweletted. Taking back incision with length of about 0.2cm, incising skin, blunt separating skin around the incision, embedding cell material compound under nude mice skin with assistance of toothless forceps, and suturing the incision with 5-0 suture to make skin cling to regenerated tissue. The nude mice were cultured subcutaneously for 4 weeks and 8 weeks, respectively, and then subjected to correlation analysis.
16 Micro-CT analysis: samples were fixed in 4% (w/v) paraformaldehyde and subjected to Micro-CT analysis using a Micro-CT μ80 scanner (Sanco Medical, switzerland). The scan parameters are set as: the voltage is 70kV, the current is 114 mu A, and the pixel resolution is 1024 multiplied by 1024. The 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 density (BMD) were measured.
17 Histological and immunohistochemical analysis
The collected specimens were fixed with 4% paraformaldehyde, decalcified, paraffin embedded and cut into 5 μm thick sections. Hematoxylin-eosin (H & E), saffron O/solid green (SO/FG) and Masson staining. Immunohistochemical staining of colia 1, OCN and COLX was used to analyze specific proteins of cartilage and bone. Immunofluorescent staining of CD31, vWF and αSMA was performed as described previously. The images were analyzed with ImageJ software to quantify the area of vascular endothelial cells.
18 Fluorescent quantitative PCR
Total RNA of cells was extracted with Trizol kit (Omega), and the gene was obtained by reverse transcription with gene synthesis kit (Takara). Real-time quantitative PCR detection was performed using SYBR Green PCR Master Mix (Takara) and CFX96 real-time PCR detection system (Bio-Rad).
19 Statistical method
The data were analyzed using SPSS software. The comparison between groups used One-Way ANOVA, where P <0.05, P <0.01, P <0.001, ns, no significant difference. All values are expressed as mean ± standard deviation.
Example 1 characterization of drug-loaded microspheres
MPs were prepared using polylactic acid-glycolic acid copolymers (L/g=85/15) with average relative molecular masses of 10kDa, 50kDa and 100kDa, respectively, and the effect of the relative molecular mass and drug properties on release rate was examined. As can be seen from fig. 1A and 1B, the relative molecular mass of the PLGA polymer is a main factor affecting the slow release time, and in vitro cumulative release experiments show that the low molecular polylactic acid microsphere can be released continuously (96%) within 4 weeks, while the release period of the macromolecular polylactic acid microsphere is longer, reaching 40% after 56 days. Meanwhile, the solubility of factors or medicines indirectly influences the drug release rate, the water-soluble TGF beta 3 is more beneficial to the release rate diffused from MPs, and the fat-soluble angiogenesis inhibitor Leretinib is difficult to permeate before the MPs are damaged, so that the long-term slow release effect can be achieved.
Preparation of TGF-beta-loaded PLGA from 10kDa PLGA 1w Microsphere (P) T -MP S ) 100kDa PLGA for preparationLEVA-loaded PLGA 10w Microsphere (P) L -MP S ). As shown in FIG. 1C, a Scanning Electron Microscope (SEM) displays P T -MP S And P L -MP S Evenly distributed and spherical. FIG. 1D shows, P T -MP S The average volume particle diameter of (3) is about 90 μm, which is slightly smaller than P L -MP S Average volume particle diameter (100 μm).
Thus, this example successfully established rapid release PLGA using transforming growth factor beta 3 as a carrier 1w -MP S PLGA with slow release of Leretinib 10w -MP S The dual programmed drug delivery system is used for regulating early cartilage formation and inhibiting late angiogenesis, thereby meeting the dynamic requirement of bone marrow stromal cell cartilage regeneration.
EXAMPLE 2 cartilage specific matrix hydrogels
In this example, methacryloyl gelatin (GelMA) was chosen as the protein component and methacryloyl hyaluronic acid (HAMA) as the glycosaminoglycan-containing component as the hybrid carrier that mimics the ECM microenvironment. To further provide a cartilage specific microenvironment for BMSCs to chondrocyte differentiation, methacryloyl decellularized cartilage matrix (ACMMA) was introduced into GelMA/HAMA, and a hydrogel for cartilage specific matrix (GelMA/HAMA/ACMMA, GHA) was prepared from 5% w/v GelMA, 1% w/v HAMA and 1% w/v ACMMA mixed. ACMMA was synthesized by modification of acellular cartilage matrix with methacrylate, as demonstrated by its nmr hydrogen spectrum (fig. 2A). As shown in FIG. 3, the GHA gel precursor (i.e., in a flowable state) was exposed to light (365 nm LED,20mW/cm 2 ) And rapidly cross-linking. 1 H nuclear magnetic resonance spectroscopy further confirmed the photopolymerization mechanism of GHA hydrogels due to the 5.4 to 7.8ppm reduction in the signal of methacrylate groups under light irradiation (fig. 2B). Will P T -MPS and P L MPS was added to GHA hydrogel separately to prepare P T -P L The @ GHA hydrogel, P under the illumination condition T -P L Rapid sol-gel transition also occurred with the @ GHA hydrogel. As shown in FIGS. 2C and 2D, time-swept rheology and storage modulus experiments indicate that the addition of ACMMA results in GH hydrogelsThe shear modulus of (2) is increased from 1175.+ -.102 Pa to 1878.+ -.63 Pa. P (P) T @GHA and P T P L The mechanical properties of @ GHA are not significantly different from those of GHA, and it is further proved that the gel precursors have good crosslinking property. In addition, P T -P L The @ GHA hydrogel has a suitable swelling and degradation rate. (FIGS. 2E and 2F)
Example 3 animal experiments
To assess gradient regulatory function of programmed delivery systems in early-stage chondria and late-stage avascular microenvironment, BMSCs were directly encapsulated into GHA, P without in vitro pre-induction T @GHA and P T -P L Various hydrogels such as @ GHA were transplanted into nude mice of each test group (fig. 8A). After 4 weeks of implantation, the GHA group had a pronounced osteogenic tendency and a pronounced angiogenesis, whereas P T @GHA and P T -P L The group @ GHA has formed white cartilage-like tissue due to the early cartilage microenvironment formed by the cartilage-specific matrix components and the early release of transforming growth factor β -3. GHA group and P as subcutaneous implantation time extended to 8 weeks T The group @ GHA had formed hard bone-like tissue with extensive distribution of new blood vessels, at which point P T -P L The group @ GHA provided a non-vascular microenvironment in the late growth phase of cartilage due to the slow release of lenvatinib inhibiting angiogenesis, with white cartilage-like tissue, without vascular invasion (fig. 8B). Micro-CT image display GHA group and P T The group @ GHA had significant osteoid calcification, a result of direct contact with the subcutaneous vascular environment. However, P T -P L The @ GHA group maintained the cartilage state with sustained release of lenvatinib (fig. 8C). The statistics of bone volume fraction (BV/TV, fig. 8D) and bone density (BMD, fig. 8E) of each set of collected samples confirm the observations.
Histological staining showed that, at 4 weeks, the GHA group had presented mixed structures of cartilage and bone-like tissue, with colia 1 and Osteocalcin (OCN) immunohistochemical staining positive. In contrast, contain P T The group of MPS regenerated uniform cartilage-like tissue at 4 weeks, with typical cartilage dimpling, cartilage-specific deposition and COLIIA1 immunostaining positivity (fig. 6). At 8 weeks, histological staining showed that the GHA group had formed typical patternsBone-like structures. P (P) T The @ GHA group also began ossifying, mature bone-specific ECM deposition, and OCN immunohistochemical staining was strongly positive. However, P T- P L The @ GHA group still had stable cartilage-like tissue with typical cartilage pits and cartilage-specific ECM deposition (fig. 7). These results demonstrate that the programmed release system of this example has both in vivo chondrogenic and anti-angiogenic functions, thereby regenerating stable cartilage tissue in an ectopic environment without the need for in vitro pre-induction procedures.
To further study its anti-angiogenic effect in vivo, vascular endothelial cells were identified using immunofluorescent staining. CD31 immunofluorescent staining showed vascular specific expression in the GHA group at 4 weeks, while no apparent expression was seen in the other two groups. Von willebrand factor (vWF) and Smooth Muscle Actin (SMA) showed vascular endothelial cells to be found only in the ossified region of the GHA group, at P T Group @ GHA and P T -P L Almost no endothelial cells were detected in the nascent cartilage region of group @ GHA. After 8 weeks of implantation, the endothelial cell number of the GHA group ossified region is obviously increased, and P T Part of the vascular invasion was seen in group @ GHA. In contrast, P T -P L Group @ GHA due to P L The slow release of angiogenesis inhibitor by MPS and the negligible number of endothelial cells observed were supported by semi-quantitative analysis of the fluorescence region (fig. 9A, 9B and 9C). Quantitative reverse transcription polymerase chain reaction results also showed that the expression levels of cartilage formation related genes (SOX 9, tgfβ3, smad, COLIIA1 and CHM 1) were significantly up-regulated at 8 weeks, confirming early cartilage induction of transforming growth factor β/Smad signaling pathways in vivo (fig. 9D). Expression of angiogenesis-related genes (VEGFA, CD31 and ANGPT 1) and matrix deposition-related genes (TIMP 3, MMP13 and SDC) was significantly down-regulated at 8 weeks, thereby effectively inhibiting the expression levels of osteogenesis-related genes (BMP 2, RUNX2, OPN, OCN and ALP).
Therefore, the strategy provided by the embodiment has the application advantage of maintaining the stability of the cartilage phenotype, successfully realizes the dynamic regulation of the cartilage regeneration of the stem cells in vivo, does not need an in vitro pre-induction process, and provides a novel stem cell cartilage regeneration method for repairing the multiple cartilage defects.
Claims (10)
1. A microsphere-containing composition comprising:
a first type of microspheres that release the contained first type of active substance to the environment to promote chondrogenesis at an early stage of mesenchymal stem cell differentiation; and
and a second class of microspheres that release the contained second class of active substances to the environment, inhibit angiogenesis or matrix degradation after cartilage production, promote cartilage growth, form cartilage pits, and cartilage-specific ECM deposition.
2. The microsphere-containing composition of claim 1, wherein the first type of microspheres comprises a material selected from the group consisting of PLGA of 5kDa to 10 kDa.
3. The microsphere-containing composition of claim 1, wherein the second type of microspheres comprises a material selected from the group consisting of PLGA above 50kDa, PLGA above 100kDa, PLL above 10kDa, and one of mesoporous silica and organometallic frameworks.
4. The microsphere-containing composition of claim 1, wherein the second substance is selected from the group consisting of Le Fa tinib and bevacizumab.
5. The microsphere-containing composition of claim 1, further comprising a hydrogel, wherein the first type of microspheres and the second type of microspheres are loaded therein to provide a cartilage producing and growing microenvironment.
6. The microsphere-containing composition of claim 5, further comprising loading cells into the hydrogel.
7. The microsphere-containing composition of claim 5, wherein the hydrogel is selected from one or more of hyaluronic acid, gelatin, gelMA, HAMA and ACMMA.
8. The microsphere-containing composition of claim 5, wherein the hydrogel is selected from one or more of the following:
GelMA at a concentration of 0.5% w/v to 7% w/v;
HAMA at a concentration of 0.3% w/v to 2% w/v;
ACMMA at a concentration of 0.3% w/v to 2% w/v.
9. The microsphere-containing composition of claim 5, wherein the hydrogel is selected from one or more of the following:
GelMA at a concentration of 0.7% w/v, 0.8% w/v, 0.9% w/v, 1% w/v, 2% w/v, 3% w/v, 4% w/v, 5% w/v or 6% w/v;
HAMA at a concentration of 0.7% w/v, 0.8% w/v, 0.9% w/v, 1.0% w/v, 1.1% w/v, or 1.2% w/v; and
ACMMA at a concentration of 0.7% w/v, 0.8% w/v, 0.9% w/v, 1.0% w/v, 1.1% w/v or 1.2% w/v.
10. A medical device comprising a microsphere-containing composition according to any one of claims 1 to 9.
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