Extracellular matrix hydrogel microspheres and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biological materials, and discloses extracellular matrix hydrogel microspheres and a preparation method therefor. The preparation method comprises: constructing an extracellular matrix derived from newborn rat bone marrow mesenchymal stem cells; preparing hydrogel microspheres by means of microfluidic technology; and using a condensing agent DMTMM to activate carboxyl groups on the surface of the hydrogel microspheres, and reacting the activated hydrogel microspheres with amino groups on the extracellular matrix to prepare extracellular matrix hydrogel microspheres. In the present invention, the extracellular matrix hydrogel microspheres are constructed, so that the advantages of the extracellular matrix hydrogel microspheres and the stem cell derived extracellular matrix are complemented, and a hydrogel precursor material has mechanical properties while the biocompatibility of the hydrogel precursor material is ensured. In addition, the extracellular matrix is combined, so that the in-vitro osteogenesis of the bone marrow mesenchymal stem cells can be remarkably promoted, the differentiation of osteoclasts is inhibited, and more possibilities are provided for skull defect repair.

Description

Extracellular matrix hydrogel microspheres and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biomaterials, in particular to a preparation method of extracellular matrix hydrogel microspheres and application thereof. BACKGROUND

[0002] Bone tissue, as a hard tissue of the human body, can support various activities of people and protect various organs in daily life. In addition, bone tissue has the function of maintaining calcium and phosphorus balance in the body, and according to the classic definition, a critical size defect is the smallest size of tissue defect that will not completely heal within the natural life span of an animal, and a critical size bone defect is still a major health challenge for elderly patients. When bone defects occur, bone marrow mesenchymal stem cells (BMSCs) are one of the main endogenous repair cells. However, the tissue repair ability of bone marrow mesenchymal stem cells (BMSCs) derived from elderly patients is significantly reduced, and it is usually difficult to rely on self-repair, and the repair of bone defects must be promoted by treatment. In the current clinical treatment of bone defects, autologous bone and allogeneic bone transplantation is the most common method of bone repair. However, this bone transplantation method has some limitations, such as limited supply source, self-occurring rejection and limited immune response, and autologous bone transplantation cannot effectively improve the decreased bone regeneration ability of elderly patients. These shortcomings limit the development of bone defect repair process and bring great trouble to the life of elderly patients. Therefore, improving the bone repair ability of elderly patients through tissue engineering technology will become an effective means to cure aged bone defects.

[0003] Extracellular matrix (ECM) derived from stem cells is a promising candidate biomaterial for bone tissue engineering. On the one hand, compared with tissue-derived ECM, stem cell-derived ECM has more controllable quality and can avoid the risk of disease or pathogen transmission at the donor site of tissue-derived ECM. On the other hand, stem cell-derived ECM is mainly composed of collagen and various matrix components, such as collagen, fibroin, and elastin, which are similar to the organic phase of bone tissue. Bone homeostasis is maintained by the balance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Stem cell-derived ECM not only can significantly promote the in vitro osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but also can effectively inhibit the differentiation of osteoclasts. However, by culturing mesenchymal stem cells (MSCs) on tissue culture polystyrene (TCPS), the obtained extracellular matrix is often difficult to be directly applied to in vivo bone defect repair due to poor mechanical properties.

[0004] Hydrogel is a kind of polymer material with three-dimensional network structure, which shows great potential in the field of tissue engineering due to its good biocompatibility, biodegradability and adjustable mechanical properties. Hyaluronic acid (HA) is a natural extracellular matrix component with good biocompatibility and adjustable mechanical properties. Hyaluronic acid methacrylate (HAMA) can be prepared by introducing methacryl groups into the molecular chain of hyaluronic acid. Hyaluronic acid methacrylate not only retains the excellent biological properties of hyaluronic acid, but also can be rapidly cross-linked and cured under the irradiation of ultraviolet light due to its photo-curing property, forming a hydrogel. The carboxyl and hydroxyl groups on the hyaluronic acid methacrylate molecule enable it to be chemically modified in various ways.

[0005] In addition, gelatin methacrylate (GelMA), sodium alginate methacrylate (AlgMA), silk fibroin methacrylate (SilMA), chondroitin sulfate methacrylate (ChsMA) and collagen methacrylate (COMA) also have good biocompatibility and can be chemically modified. However, traditional bulk hydrogel has the problems of too large external size (millimeter level and above), too long cell culture period, need for surgical implantation, and too large postoperative trauma. Compared with traditional bulk hydrogel, hydrogel microspheres not only retain most of the original properties of hydrogel, but also show some unique advantages, such as larger specific surface area, more excellent injectability, more significant structural porosity and more convenient modular design. Therefore, compared with traditional bulk hydrogel, hydrogel microspheres are more suitable for bone tissue engineering. The above hydrogels can be prepared into injectable size hydrogel microspheres by microfluidic technology. However, hyaluronic acid, gelatin and silk fibroin lack certain biological activity, which limits the therapeutic effect of hydrogel microspheres in bone defect repair. SUMMARY

[0006] The present application provides an extracellular matrix hydrogel microsphere which can overcome the defects of poor mechanical properties of extracellular matrix and insufficient biological activity of hydrogel microspheres in repairing bone defects.

[0007] The present application provides a preparation method of an extracellular matrix hydrogel microsphere, which comprises: constructing neonatal rat bone marrow mesenchymal stem cell-derived extracellular matrix; preparing hydrogel microspheres by microfluidic technology; activating the carboxyl groups on the surface of the hydrogel microspheres by using condensing agent DMTMM and reacting the activated hydrogel microspheres with the amino groups on the extracellular matrix to obtain the extracellular matrix hydrogel microspheres.

[0008] As a priority, the carboxyl groups on the surface of the hydrogel microspheres are activated by using a condensing agent DMTMM, and the activated hydrogel microspheres are reacted with the amino groups on the extracellular matrix, specifically including the following steps: Step S1, soaking the hydrogel microspheres in a DMTMM solution, centrifugal stirring at room temperature, and freeze-drying the extracellular matrix, and grinding the freeze-dried extracellular matrix, dissolving it with sterile deionized water to obtain an extracellular matrix suspension; Step S2, adding the hydrogel microspheres to the extracellular matrix suspension and reacting on a shaking table.

[0009] As a priority, the construction of neonatal rat bone marrow mesenchymal stem cell-derived extracellular matrix specifically includes the following steps: Step S11, seeding neonatal rat-derived BMSCs into a cell culture dish, and culturing with complete medium until the cell density reaches 90%; Step S12, adding ascorbic acid with a concentration of 80-200 μM to the complete medium for 3-10 days; Step S13, discarding the old medium and adding a decellularization solution for incubation; Step S14, removing the decellularization solution and adding deoxyribonuclease for further incubation; Step S15, discarding the deoxyribonuclease and washing with PBS buffer; Step S16, collecting the extracellular matrix using a cell scraper.

[0010] As a priority, the composition of the decellularization solution in step S13 is 99.5% PBS buffer, 0.5% Triton X-100, and 20-35 mM NH4OH, and the incubation time is 5-10 min; the concentration of deoxyribonuclease in step S14 is 80-150 U / ml, and the action time is 1-3 h.

[0011] As a priority, the preparation of hydrogel microspheres by microfluidic technology specifically includes the following steps: Step S21, preparing a hydrogel precursor solution containing a photoinitiator with deionized water as the water phase; Step S22, mixing Span 80 and isopropyl myristate uniformly as the oil phase; Step S23, loading the water phase and the oil phase into syringes, respectively, using a syringe pump to control different flow rates, pushing the water phase and the oil phase out at a constant speed, and solidifying the water phase into stable spherical shape by a UV lamp; Step S24, washing the surface of the hydrogel microspheres with ethanol; Step S25, collecting the washed hydrogel microspheres and freeze-drying.

[0012] As a priority, the hydrogel precursor solution in step S21 includes one or more of hyaluronic acid methacrylate, methacrylated gelatin, methacrylated silk fibroin, and methacrylated collagen; the concentration of the hyaluronic acid methacrylate solution is 1%-5%, the concentration of the methacrylated gelatin solution is 5%-20%, the concentration of the methacrylated silk fibroin solution is 7.5%-20%, and the concentration of the methacrylated collagen solution is 1%-20%.

[0013] As a priority, the DMTMM solution in step S1 is configured by sterile deionized water, the concentration is 5-15 mg / mL, the stirring time at room temperature is 30-60 min, the centrifugation condition is 4 DEG C, 1200 r / min, 5 min, and the centrifugation times are 3 times.

[0014] As a priority, the reaction temperature of the hydrogel microspheres and the extracellular matrix suspension in step S2 is 4-25 DEG C, and the reaction time is 3-8 h.

[0015] An extracellular matrix hydrogel microsphere is prepared by the preparation method of the extracellular matrix hydrogel microsphere.

[0016] The application of the extracellular matrix hydrogel microsphere in bone defect repair products.

[0017] The application has the following technical effects: the extracellular matrix hydrogel microsphere is constructed, the advantages of the stem cell-derived extracellular matrix are successfully complementary, the mechanical performance is also possessed while the biocompatibility of the hydrogel precursor material is ensured, the combination of the extracellular matrix can significantly promote the in-vitro osteogenesis of the bone marrow mesenchymal stem cells and inhibit the differentiation of the osteoclasts, and more possibilities are provided for the skull defect repair. BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is a construction of the young-state extracellular matrix in embodiment 1 of the application; FIG. 2 is a coomassie brilliant blue staining of the extracellular matrix hydrogel microsphere in embodiment 1 of the application; FIG. 3 is a light microscope and particle size distribution diagram of the extracellular matrix hydrogel microsphere in embodiment 1 of the application; FIG. 4 is a diagram of the treatment effect evaluated by Micro-CT analysis in embodiment 1 of the application; and FIG. 5 is a diagram of the relative content of collagen type I in the hydrogel microspheres in embodiments 1-8 and comparative examples 1-2 of the application. DETAILED DESCRIPTION

[0019] In order to further understand the content of the application, the application is described in detail in combination with the drawings and embodiments. It should be understood that the embodiments are only used to explain the application but not to limit the application. Embodiment 1

[0020] As shown in FIG. 1, a preparation method of an extracellular matrix hydrogel microsphere comprises the following steps: constructing a new-born rat bone marrow mesenchymal stem cell-derived extracellular matrix; preparing a hydrogel microsphere by using a microfluidic technology; and activating the carboxyl on the surface of the hydrogel microsphere by using a condensing agent DMTMM and reacting the activated hydrogel microsphere with the amino on the extracellular matrix to obtain the extracellular matrix hydrogel microsphere.

[0021] Specifically, the following steps are included: step 101, culturing the new-born rat bone marrow mesenchymal stem cells in a culture dish, and the inoculation density is 3000 / cm2 ; when the cells reached 90% confluence, 100 μM ascorbic acid was added to induce for 8 days; then, a decellularization solution was added, wherein the decellularization solution contained 99.5% PBS, 0.5% Triton X-100, and 20 mM NH4OH; the cells were incubated in the incubator for 5 min; the decellularization solution was discarded, and 100 U / mL DNase was added to incubate for 1 h; the cells were washed with PBS, and the extracellular matrix was collected by scraping; step 102, 5% hyaluronic acid methacrylate hydrogel solution was prepared by using a LAP solution containing a photoinitiator, and hyaluronic acid methacrylate hydrogel microspheres were prepared by microfluidic technology, specifically including: step S21, deionized water containing a photoinitiator was used to prepare a hydrogel precursor solution as an aqueous phase; step S22, Span 80 and isopropyl myristate were mixed uniformly as an oil phase; step S23, the aqueous phase and the oil phase were loaded into syringes, respectively, and the different flow rates were controlled by using a syringe pump, and the aqueous phase and the oil phase were pushed out at a constant speed, and the aqueous phase was solidified into stable spheres by a UV lamp; step S24, the surface of the hydrogel microspheres was washed with ethanol; step S25, the washed hydrogel microspheres were collected and freeze-dried.

[0022] Step 103, the freeze-dried extracellular matrix was ground thoroughly and dissolved in sterile deionized water to obtain an extracellular matrix suspension; step 104, the hyaluronic acid methacrylate hydrogel microspheres were soaked in a DMTMM solution, stirred at room temperature and centrifuged for 3 times to obtain a precipitate; the DMTMM solution was prepared by using sterile deionized water, and the concentration was 5-15 mg / mL; the stirring time was 30-60 min at room temperature; the centrifugation condition was 4℃, 1200 r / min, and 5 min; step 105, the precipitate was added to the extracellular matrix suspension, and the reaction temperature was 4-25℃, and the reaction was performed on a shaking table for 4 h; step 106, the hyaluronic acid methacrylate extracellular matrix hydrogel microspheres were recovered and washed with sterile deionized water; step 107, freeze-drying was performed to obtain the extracellular matrix hydrogel microspheres.

[0023] In the embodiment, the extracellular matrix hydrogel microspheres prepared were photographed by light microscopy, stained by Coomassie brilliant blue, and subjected to type I collagen immunofluorescence staining, and the results showed that the stem cell-derived extracellular matrix was successfully combined with the hyaluronic acid methacrylate hydrogel microspheres (FIG. 2), and the extracellular matrix hydrogel microspheres obtained by the method had uniform particle size and good dispersibility (FIG. 3); the Micro-CT results showed that the young-state extracellular matrix hydrogel microspheres could effectively promote the repair of the skull defects of old rats in vivo (FIG. 4).

[0024] The extracellular matrix hydrogel microspheres prepared in the embodiment successfully realize the effective combination of stem cell-derived extracellular matrix and hydrogel microspheres. The young-state extracellular matrix hydrogel microspheres prepared by the method can effectively improve the bone regeneration ability of old rats and promote the repair of critical skull defects. The application provides a new idea for using stem cell-derived extracellular matrix for cell and tissue engineering research and expands the application range of hydrogel microspheres. Example 2

[0025] The same as example 1, except that the concentration of NH4OH in the decellularization solution in step 101 of the embodiment is 10 mM, and the incubation time in the incubator is 5 min. Example 3

[0026] The same as example 1, except that the concentration of NH4OH in the decellularization solution in step 101 of the embodiment is 35 mM, and the incubation time in the incubator is 5 min. Example 4

[0027] The same as example 1, except that the concentration of NH4OH in the decellularization solution in step 101 of the embodiment is 20 mM, and the incubation time in the incubator is 10 min. Example 5

[0028] The same as example 1, except that the concentration of DNase in step 101 of the embodiment is 80 U / mL, and the incubation time is 1 h. Example 6

[0029] The same as example 1, except that the concentration of DNase in step 101 of the embodiment is 100 U / mL, and the incubation time is 30 min. Example 7

[0030] The same as example 1, except that the hydrogel material in step 102 of the embodiment is 10% methacrylated gelatin. Example 8

[0031] The same as example 1, except that the hydrogel material in step 102 of the embodiment is 12% methacrylated silk fibroin solution. Example 9

[0032] The same as example 1, except that the hydrogel material in step 102 of the embodiment is 2% methacrylated collagen. Comparative Example 1

[0033] The same as example 1, except that the embodiment specifically includes the following steps: step 101, preparing a 5% hyaluronic acid methacrylate hydrogel solution containing a photoinitiator LAP solution, and preparing hyaluronic acid methacrylate hydrogel microspheres by microfluidic technology; step 102, washing the hyaluronic acid methacrylate hydrogel microspheres with sterile deionized water; step 103, freeze-drying to obtain hyaluronic acid methacrylate hydrogel microspheres. Comparative Example 2

[0034] The same as example 1, the difference is that this example specifically includes the following steps: Step 101, place 18-month-old rat bone marrow mesenchymal stem cells in a culture dish for culture, with a seeding density of 3000 / cm 2 ; when the cells reach 90% confluence, add 100 μM ascorbic acid for induction for 8 days; then add the decellularization solution, wherein the content of each component of the decellularization solution is 99.5% PBS, 0.5% Triton X-100, and 20 mM NH4OH, and incubate in an incubator for 5 min; discard the decellularization solution, add 100 U / mL deoxyribonuclease for incubation for 1 h; wash with PBS, and collect the extracellular matrix with a cell scraper; Step 102, prepare a 5% hyaluronic acid methacrylate hydrogel solution with a LAP solution containing a photoinitiator, and prepare hyaluronic acid methacrylate hydrogel microspheres through microfluidic technology; Step 103, thoroughly grind the freeze-dried extracellular matrix, and dissolve it with sterile deionized water; Step 104, soak the hyaluronic acid methacrylate hydrogel microspheres in a DMTMM solution, stir at room temperature, and centrifuge three times to retain the precipitate; Step 105, add the hyaluronic acid methacrylate hydrogel microspheres to the extracellular matrix suspension, and react on a shaking bed for 4 h; Step 106, recover the hyaluronic acid methacrylate extracellular matrix hydrogel microspheres, and wash them with sterile deionized water; Step 107, freeze-dry to obtain the hyaluronic acid methacrylate extracellular matrix hydrogel microspheres.

[0035] As shown in FIG. 5, by comparing the content of collagen type I in the extracellular matrix hydrogel microspheres prepared by example 1 and examples 2-4, the optimal concentration of NH4OH in the decellularization solution is 20 mM, and the optimal incubation time is 5 min.

[0036] By comparing the content of collagen type I in the extracellular matrix hydrogel microspheres prepared by example 1 and examples 5 and 6, the optimal concentration of deoxyribonuclease is 100 U / mL, and the optimal incubation time is 1 h.

[0037] By comparing the content of collagen type I in the extracellular matrix hydrogel microspheres prepared by example 1 and examples 7-8, whether hyaluronic acid methacrylate, methacrylated gelatin, or methacrylated collagen is used as the main material of the hydrogel microspheres, all can achieve loading of extracellular matrix, and can effectively promote bone repair.

[0038] By comparing the content of collagen type I in the hydrogel microspheres prepared by examples 1-8 and comparative examples 1 and 2, the hydrogel microspheres without extracellular matrix, or the hydrogel microspheres loaded with extracellular matrix obtained from old mice, have a significantly weakened effect on bone defect repair, which further illustrates the important role of extracellular matrix in maintaining bone homeostasis.

[0039] An extracellular matrix hydrogel microsphere as shown in Fig. 2 is obtained by the method for preparing the extracellular matrix hydrogel microsphere of any one of embodiments 1-9, successfully combines stem cell-derived extracellular matrix and microspheres, and achieves uniform particle size and good dispersibility. The prepared microspheres can promote the repair of bone defects while ensuring good biocompatibility. Embodiment 11

[0040] An application of the extracellular matrix hydrogel microsphere of embodiment 10 in bone defect repair products, such as repair of senile skull defects, which can promote the repair of bone defects.

[0041] It is easily understood that, based on one or more embodiments provided in the present application, a person skilled in the art can combine, split, recombine, etc. the embodiments of the present application to obtain other embodiments, and these embodiments do not exceed the protection scope of the present application.

[0042] The above describes the present application and its embodiments in a schematic manner, which is not limited, and the embodiments shown are only part of the embodiments of the present application, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired thereby, without departing from the spirit of the present application, similar structural modes and embodiments can be designed without creativity, which shall belong to the protection scope of the present application.

Claims

1. A method of preparing extracellular matrix hydrogel microspheres, characterized by, include: Constructing an extracellular matrix derived from bone marrow mesenchymal stem cells of newborn rats; Hydrogel microspheres were prepared using microfluidic technology. Extracellular matrix hydrogel microspheres were prepared by activating the carboxyl groups on the surface of hydrogel microspheres with the condensing agent DMTMM and then reacting the activated hydrogel microspheres with amino groups on the extracellular matrix.

2. The method for preparing extracellular matrix hydrogel microspheres according to claim 1, characterized in that, The carboxyl groups on the surface of hydrogel microspheres are activated using the condensing agent DMTMM, and the activated hydrogel microspheres react with amino groups on the extracellular matrix. The specific steps include: Step S1: Soak the hydrogel microspheres in DMTMM solution, centrifuge at room temperature with stirring, freeze-dry the extracellular matrix, grind the freeze-dried extracellular matrix thoroughly, dissolve it in sterile deionized water to obtain an extracellular matrix suspension. Step S2: Add the hydrogel microspheres to the extracellular matrix suspension and react on a shaker.

3. The method for preparing extracellular matrix hydrogel microspheres according to claim 1, characterized in that, The construction of extracellular matrix derived from newborn rat bone marrow mesenchymal stem cells specifically includes the following steps: Step S11: Inoculate newborn rat-derived BMSCs into cell culture dishes and culture them in complete culture medium until the cell density reaches 90%. Step S12: Add ascorbic acid at a concentration of 80-200 μM to the complete culture medium and continue for 3-10 days; Step S13: Discard the old culture medium and add decellularization solution for incubation; Step S14: Remove the decellularization solution and add deoxyribonuclease for further incubation; Step S15: Discard the deoxyribonuclease and wash with PBS buffer; Step S16: Collect extracellular matrix using a cell scraper.

4. The method of claim 3, wherein the cell extracellular matrix hydrogel microspheres are prepared by the method of claim 1 or 2. In step S13, the decellularization solution consists of 99.5% PBS buffer, 0.5% Triton X-100, and 20-35mM NH4OH, and the incubation time is 5-10 min; in step S14, the concentration of deoxyribonuclease is 80-150 U / ml, and the reaction time is 1-3 h.

5. The method for preparing extracellular matrix hydrogel microspheres according to claim 1, characterized in that, The preparation of hydrogel microspheres using microfluidic technology includes the following steps: Step S21: Prepare a hydrogel precursor solution as the aqueous phase using deionized water containing a photoinitiator; Step S22: Mix Span 80 and isopropyl myristate evenly to form the oil phase; Step S23: Load the aqueous phase and oil phase into the syringe respectively, use the syringe pump to control different flow rates, push out the aqueous phase and oil phase at a constant speed, and solidify the aqueous phase into a stable sphere by ultraviolet light, etc. Step S24: Clean the surface of the hydrogel microspheres with ethanol; Step S25: Collect the cleaned hydrogel microspheres and freeze-dry them.

6. The method for preparing extracellular matrix hydrogel microspheres according to claim 5, characterized in that, The hydrogel precursor solution in step S21 includes one or more of hyaluronic acid methacrylate, methacrylamide gelatin, methacrylamide silk fibroin, and methacrylamide collagen; the concentration of the hyaluronic acid methacrylate solution is 1%-5%, the concentration of the methacrylamide gelatin solution is 5%-20%, the concentration of the methacrylamide silk fibroin solution is 7.5%-20%, and the concentration of the methacrylamide collagen solution is 1%-20%.

7. The method for preparing extracellular matrix hydrogel microspheres according to claim 2, characterized in that: The DMTMM solution in step S1 is configured by sterile deionized water with a concentration of 5-15 mg / mL, stirring at room temperature for 30-60 min, centrifugation at 4°C, 1200 r / min for 5 min, and centrifugation for 3 times.

8. The method for preparing extracellular matrix hydrogel microspheres according to claim 2, characterized in that: The reaction temperature of the hydrogel microspheres and the extracellular matrix suspension in step S2 is 4-25°C, and the reaction time is 3-8 h.

9. An extracellular matrix hydrogel microsphere, characterized in that: The preparation method of the extracellular matrix hydrogel microspheres is prepared by the method of any one of claims 1-8.

10. The extracellular matrix hydrogel microspheres of claim 9 are used in bone defect repair products.

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