Hydrogel microspheres loaded with genetically engineered BMSCs (bone marrow mesenchymal stem cells) as well as preparation method and application of hydrogel microspheres

Abstract

The invention discloses hydrogel microspheres loaded with genetically engineered BMSCs (bone mesenchymal stem cells) as well as a preparation method and application thereof, phenylboronic acid modified polyamide-amine dendrimer PPBA is used for loading plasmid pRUNX2, then the loaded plasmid pRUNX2 is delivered into BMSCs to induce osteogenic differentiation transfection of the BMSCs, the transfected BMSCspRUNX2 is mixed with hydrogel, and the hydrogel microspheres are prepared by a hydrothermal method. A water-in-oil type solution is obtained in a micro-fluidic mode, and finally, the water-in-oil type polymer is obtained after curing. The directional osteogenic differentiation capability of the BMSCs engineered based on pRUNX2 is superior to that of a control group; the used hydrogel material has good biocompatibility, so that the viability and the treatment effect of cells are greatly improved; meanwhile, the hydrogel microspheres can prolong the residence time of cells at the defect part, improve the treatment effect and promote bone defect repair.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and specifically relates to a hydrogel microsphere loaded with genetically engineered BMSCs and a preparation method and application thereof. Background Art

[0002] Bone defects caused by traffic accidents, bone diseases, trauma, and surgical resection have put tremendous pressure on public healthcare systems worldwide. The self-repair of defects is a slow process accompanied by cell migration, chondrocyte differentiation, and calcium deposition. To accelerate repair, traditional therapies have developed autologous transplantation, allogeneic transplantation, and xenotransplantation, but these methods face the limitations of insufficient donor sources, donor site morbidity, infection risks, and host immune responses. Stem cell therapy has been studied as an alternative for tissue transplantation. Its efficacy mainly depends on the survival rate and density of the delivered cells in the target local tissue. Mesenchymal stem cell (MSCs) injection therapy has the advantages of easy administration and minimal invasiveness. It is one of the most promising stem cell therapies for bone defect repair, but it is still unsatisfactory in some aspects. Direct injection of cells usually leads to rapid cell loss, while encapsulating cells in hydrogel blocks hinders effective nutrient absorption. In addition, the differentiation of MSCs into osteoblasts is usually silenced and needs further induction. Therefore, there is still an urgent need to improve innovative MSCs therapies.

[0003] Genetic engineering uses innovative gene editing methods to control the expression of specific genes, which enables precise and targeted manipulation of the biological behavior of cells. RUNX2 is an osteogenic differentiation-specific transcription factor that regulates the transduction of many genes that affect osteoblast differentiation and chondrocyte maturation of BMSCs. Specifically, when BMSCs are genetically engineered with pRUNX2, they show a tendency to differentiate directly into osteoblasts, which is beneficial for bone defect repair. In contrast, hydrogel microcarriers are an effective platform for delivering cells due to their high permeability, large surface area, good biocompatibility, and 3D microenvironment similar to the extracellular matrix. Microfluidics is the most effective method to prepare cell-friendly hydrogel microcarriers with customized size and local cell density. Because these microcarriers shorten the distance between cells and the environment, they allow more efficient exchange of cell nutrients and metabolites. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a microcarrier composed of BMSCs transfected with pRUNX2 and hydrogel for improving the therapeutic effect of bone defects in view of the deficiencies in the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows: A method for preparing hydrogel microspheres loaded with genetically engineered BMSCs, using phenylboronic acid-modified polyamidoamine dendrimer PPBA to load plasmid pRUNX2, and then delivering the loaded pRUNX2 plasmid to BMSCs to induce osteogenic differentiation transfection, and then transfecting the transfected BMSCs pRUNX2 The mixture is mixed with hydrogel, and an oil-in-water solution is obtained by microfluidics, and finally solidified.

[0006] Furthermore, the preparation method of the hydrogel microspheres loaded with genetically engineered BMSCs of the present invention specifically comprises the following steps: S1. Prepare phenylboronic acid-grafted polyamide-amine dendrimer PPBA, then mix PPBA with plasmid pRUNX2 to prepare nanomaterial PPBA@pRUNX2, and co-incubate PPBA@pRUNX2 with BMSCs to obtain transfected BMSCs pRUNX2 ; S2. BMSCs after transfection pRUNX2 , a photoinitiator and a hydrogel solution are mixed, and the mixed hydrogel solution is used as an inner phase, and liquid paraffin and a surfactant are used as an outer phase, and a water-in-oil solution is prepared by a microfluidic device; S3. The water-in-oil solution prepared in step S2 is cured by ultraviolet irradiation, and then washed with sterile PBS.

[0007] Specifically, in step S1, the phenylboronic acid grafted polyamide-amine dendrimer PPBA is prepared by the following method: 4-bromomethylphenylboronic acid and the fifth generation polyamide-amine dendrimer are added to methanol at a molar ratio of 50-150:1 and reacted for 10-12 hours to obtain the obtained product.

[0008] Specifically, in step S1, the nanomaterial PPBA@pRUNX2 is prepared by mixing the polyamide-amine dendrimer PPBA grafted with phenylboronic acid with the plasmid pRUNX2 in a mass ratio of 0.5-2:1-4 and letting it stand for more than half an hour.

[0009] Specifically, in step S1, transfection of BMSCs was performed by adding the prepared nanomaterial PPBA@pRUNX2 to BMSCs at a final concentration of 1 mg / mL and incubating for 12 to 48 hours. The number of BMSCs was 1 to 10 × 10 6 .

[0010] Specifically, in step S2, the hydrogel used refers to a 5%-10% by mass fraction of methyl benzoate acylated gelatin or methyl methacrylate acylated hyaluronic acid hydrogel PBS solution; in the internal phase mixed hydrogel solution, the transfected BMSCs are used pRUNX2 The number is 1~10×10 5The concentration of the added photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone is 0.1~0.5% v / v.

[0011] Specifically, in step S2, the external phase is obtained by mixing liquid paraffin and a surfactant in a volume ratio of 90~95:10~5; the parameters for preparing the water-in-oil solution using a microfluidic device are: the flow rate of the internal phase is 2 μL / min~10 μL / min, and the external phase is 20~100 μL / min.

[0012] Specifically, in step S3, the ultraviolet irradiation curing time is 30 to 90 seconds.

[0013] Furthermore, the genetically engineered BMSCs-loaded hydrogel microspheres prepared by the above preparation method are also within the protection scope of the present invention.

[0014] Furthermore, the present invention also claims to protect the use of the above-mentioned genetically engineered BMSCs-loaded hydrogel microspheres in the preparation of drugs for treating bone defects. Beneficial Effects

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The ability of BMSCs engineered with pRUNX2 to differentiate into directional osteoblasts is superior to that of the control; the hydrogel material used has good biocompatibility, which greatly improves the viability of cells and the therapeutic effect; at the same time, the hydrogel microspheres can prolong the retention time of cells in the defect site, improve the therapeutic effect, and promote bone defect repair.

[0016] (2) The preparation method of the genetically engineered BMSCs-loaded hydrogel microspheres of the present invention has the advantages of low cost, simple operation, controllable size, and large-scale mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0018] Figure 1 Schematic diagram of the preparation method of hydrogel microspheres loaded with genetically engineered BMSCs.

[0019] Figure 2 SEM image of hydrogel microspheres.

[0020] Figure 3 To express osteogenic-related genes in genetically engineered BMSCs.

[0021] Figure 4 Quantitative analysis of ALP and calcium nodule staining for hydrogel microspheres loaded with genetically engineered BMSCs.

[0022] Figure 5 To evaluate the therapeutic effect of hydrogel microspheres loaded with genetically engineered BMSCs in bone defects in rats. DETAILED DESCRIPTION

[0023] The present invention can be better understood with reference to the following examples.

[0024] Combination Figure 1 The preparation method of the genetically engineered BMSCs-loaded hydrogel microspheres of the present invention specifically comprises the following steps: S1. Prepare phenylboronic acid-grafted polyamide-amine dendrimer PPBA, then mix PPBA with plasmid pRUNX2 to prepare nanomaterial PPBA@pRUNX2, and co-incubate PPBA@pRUNX2 with BMSCs to obtain transfected BMSCs pRUNX2 ; S2. BMSCs after transfection pRUNX2 , a photoinitiator and a hydrogel solution are mixed, and the mixed hydrogel solution is used as an inner phase, and liquid paraffin and a surfactant are used as an outer phase, and a water-in-oil solution is prepared by a microfluidic device; S3. The water-in-oil solution prepared in step S2 is cured by ultraviolet irradiation, and then washed with sterile PBS. Example 1

[0025] Plasmid pRUNX2 was transfected into BMSCs.

[0026] Preparation of nanomaterial PPBA@pRUNX2: 4-bromomethylphenylboronic acid and the fifth-generation polyamide-amine dendrimer were added to a methanol solution in a molar ratio of 128:1, heated at 70°C for 24 hours, transferred to a dialysis bag, and dialyzed in methanol and deionized water respectively. The product PPBA was separated and purified. The PBS solution of PPBA (1 mg / mL) was mixed with the plasmid pRUNX2 in a mass ratio of 1:1 and allowed to stand for thirty minutes to obtain PPBA@pRUNX2.

[0027] Transfect BMSCs: When the number of BMSCs cells reaches 10 6 PPBA@pRUNX2 was added to BMSCs at a final concentration of 1 mg / mL and cultured for 24 h, then washed three times with PBS, digested with trypsin, and centrifuged for later use. Example 2

[0028] Preparation of hydrogel microspheres loaded with genetically engineered BMSCs.

[0029] 10 5 BMSCs pRUNX2, methacrylate gelatin (GelMA, 5wt%) and photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP, 0.1% v / v) were mixed evenly as the internal phase of the microfluidic system, with a total volume of 1 mL. The external phase of the microfluidic system was liquid paraffin and surfactant (Span-80), with a volume ratio of 95:5, which were mixed evenly. The internal and external phase liquids flowed into the microfluidic device through the inner and outer capillaries respectively. Under the action of shear force and surface tension, the internal phase liquid was pinched off into monodisperse droplets to prepare an oil-in-water solution.

[0030] The water-in-oil solution was collected and photocured under 365 nm UV light for 60 seconds, and the microspheres were washed with a large amount of sterile PBS. Figure 2 As shown, it can be seen that the hydrogel microspheres after freeze-drying are 240 μm in size and have a reticular porous hydrogel structure on the surface, which can release substances that are beneficial to the transfer of cells in the microspheres and the outside world. Example 3

[0031] Evaluation of the in vitro osteogenic differentiation properties of hydrogel microspheres loaded with genetically engineered BMSCs.

[0032] BMSCs pRUNX2 The hydrogel microspheres were placed in stem cell culture medium containing 0.1 μM dexamethasone, ascorbic acid (50 μg / mL) and sodium β-glycerophosphate (10 nM), and the culture medium was changed every four days. On the seventh day of culture, the expression of osteogenesis-related genes (ALP, OCN, RUNX2 and COL-1) was detected by qPCR technology, and the amount of ALP was detected by alkaline phosphatase kit. On the 14th day of culture, the amount of microsphere calcium nodules was identified by using Alizarin red staining. The above results are shown in Figure 3 and Figure 4 As shown. Figure 3 It can be seen that BMSCs pRUNX2 The expression of bone-related genes in the hydrogel microsphere group was significantly higher than that in the control group, among which the expression of RUNX2 gene was 22 times higher than that in the control group. Figure 4 It can be seen that BMSCs pRUNX2 The ALP expression and calcium nodule number of hydrogel microspheres were significantly higher than those of the control group. pRUNX2 The hydrogel microsphere group can improve the osteogenic differentiation ability of BMSCs in vitro. Example 4

[0033] Application of hydrogel microspheres loaded with genetically engineered BMSCs in bone defect repair.

[0034] Male SD rats (weighing approximately 200-250 g) were adaptively fed for 1 week and then randomly divided into 4 groups, with 3 rats in each group. After the SD rats were rinsed with sterile saline, two circular skull defects with a diameter of 5 mm were made using a bone drill. After the skull defects were rinsed with saline, they were filled with sterilized experimental materials. All rats were free to drink water containing ceftriaxone sodium during the first 4 days to avoid infection. The rats were divided into the following four groups: 1) blank defect (control group); 2) blank microsphere group; 3) BMSCs microsphere-loaded group; 4) BMSCs microsphere-loaded group. After 8 weeks, skull tissues were collected for macroscopic observation and Micro CT scanning, and 3D software was used to reconstruct tomographic images based on the reconstructed micro CT images. Bone defect healing was evaluated for each group of rats, as shown in the following table. Figure 5 As shown, genetically engineered BMSCs microspheres have a significant effect on the treatment of bone defects in rats. This strategy can be widely used in the field of cell therapy and tissue regeneration.

[0035] The present invention provides a method and idea of ​​loading genetically engineered BMSCs hydrogel microspheres and a preparation method and application thereof. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing hydrogel microspheres loaded with genetically engineered BMSCs, characterized in that: The plasmid pRUNX2 was loaded with polyamidoamine dendrimer PPBA modified with phenylboronic acid, and then the loaded pRUNX2 plasmid was delivered to BMSCs to induce their osteogenic differentiation transfection. pRUNX2 The mixture is mixed with hydrogel, and an oil-in-water solution is obtained by microfluidics, and finally solidified.

2. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 1, characterized in that: The steps include: S1. Prepare phenylboronic acid-grafted polyamide-amine dendrimer PPBA, then mix PPBA with plasmid pRUNX2 to prepare nanomaterial PPBA@pRUNX2, and co-incubate PPBA@pRUNX2 with BMSCs to obtain transfected BMSCs pRUNX2 ; S2. BMSCs after transfection pRUNX2 , a photoinitiator and a hydrogel solution are mixed, and the mixed hydrogel solution is used as an inner phase, and liquid paraffin and a surfactant are used as an outer phase, and a water-in-oil solution is prepared by a microfluidic device; S3. The water-in-oil solution prepared in step S2 is cured by ultraviolet irradiation, and then washed with sterile PBS.

3. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 2, characterized in that: In step S1, the phenylboronic acid grafted polyamide-amine dendrimer PPBA is prepared by the following method: 4-bromomethylphenylboronic acid and the fifth generation polyamide-amine dendrimer are added to methanol at a molar ratio of 50-150:1 and reacted for 10-12 hours to obtain the obtained product.

4. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 2, characterized in that: In step S1, the nanomaterial PPBA@pRUNX2 is prepared by mixing the polyamide-amine dendrimer PPBA grafted with phenylboronic acid with the plasmid pRUNX2 at a mass ratio of 0.5-2:1-4 and letting it stand for more than half an hour.

5. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 2, characterized in that: In step S1, transfection of BMSCs was performed by adding the prepared nanomaterial PPBA@pRUNX2 to BMSCs at a final concentration of 1 mg / mL and incubating for 12 to 48 hours. The number of BMSCs was 1 to 10 × 10 6 .

6. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 2, characterized in that: In step S2, the hydrogel used refers to a 5%-10% by mass fraction of methyl benzoate acylated gelatin or methyl methacrylate acylated hyaluronic acid hydrogel PBS solution; in the internal phase mixed hydrogel solution, the transfected BMSCs are used pRUNX2 The number is 1~10×10 5 The concentration of the added photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone is 0.1~0.5% v / v.

7. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 2, characterized in that: In step S2, the external phase is obtained by mixing liquid paraffin and a surfactant in a volume ratio of 90-95:10-5; the parameters for preparing the water-in-oil solution using a microfluidic device are: the flow rate of the internal phase is 2 μL / min-10 μL / min, and the flow rate of the external phase is 20-100 μL / min.

8. The method for preparing hydrogel microspheres loaded with genetically engineered BMSCs according to claim 2, characterized in that: In step S3, the ultraviolet irradiation curing time is 30 to 90 seconds.

9. Hydrogel microspheres loaded with genetically engineered BMSCs prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the genetically engineered BMSCs-loaded hydrogel microspheres according to claim 1 in the preparation of a drug for treating bone defects.

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