CN111195370A - High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof - Google Patents

Abstract

The invention provides a high-magnesium microenvironment bone marrow stem cell microsphere carrier, a preparation method and an application thereof, wherein magnesium ions are compounded into the microsphere carrier to form a local high-magnesium environment, the size of the microsphere carrier is within 500 mu m, and the concentration of the magnesium ions is 2.5mM-10 mM. The invention provides a concept and a preparation method for constructing a local high-magnesium microenvironment stem cell microsphere carrier, on one hand, when the concentration of screened magnesium ions is 2.5mM-5mM, the osteogenesis induction effect is optimal; secondly, the diameter of the prepared stem cell microsphere carrier is within 500 mu m, so that the loading rate and the survival rate of stem cells are effectively improved, the loaded stem cells are promoted to be osteogenic and differentiative by utilizing a local high-magnesium environment, and the defects of high cost, difficult slow release and the like caused by using protein factors are reduced; finally, when the prepared high-magnesium microenvironment stem cell microsphere carrier is used for bone defect regeneration repair, the number of new bones in the defect area is obviously increased, and the repair effect is obviously improved.

Description

High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof

Technical Field

The invention belongs to the field of tissue engineering and regenerative medicine, and particularly relates to a high-magnesium microenvironment bone marrow stem cell microsphere carrier, and a preparation method and application thereof.

Background

Stem cells have self-renewal ability and multipotential differentiation ability, and theoretically can be expanded in vitro to a sufficient number and induced to differentiate into specific adult cells for tissue regeneration treatment. Direct loading of cells onto scaffold materials is a common method, but due to the influence of material pore size and specific surface area, the number of transplanted cells is significantly limited: too small pore diameter of the porous scaffold material can limit the cells from permeating into the deep part of the scaffold, and the specific surface area is reduced along with the expansion of the pore diameter, thereby influencing the loading quantity of the cells. The microspheres are used as a stem cell carrier, compared with the traditional support, the microsphere has the advantages of small size, easy nutrition penetration and large specific surface area and large cell loading capacity, and meanwhile, themicrosphere 3D cultured stem cells effectively maintain the cell dryness, provide a stable local environment for the stem cells, effectively resist the immune rejection of a receptor and the like, and have more advantages in tissue regeneration application.

Magnesium is a major element of human body and is indispensable in bone growth and development, and the function of promoting osteogenesis by magnesium ions has been reported in many studies. In order to further improve the regeneration and repair effect, magnesium ions with osteogenic differentiation induction capability are compounded into microspheres, high-magnesium microenvironment microspheres are constructed, and stem cells are delivered for bone regeneration at the same time, and the research on the aspect is not reported in documents.

Disclosure of Invention

The invention aims to provide a bone marrow stem cell microsphere carrier with a high-magnesium microenvironment.

The second purpose of the invention is to provide a preparation method of the bone marrow stem cell microsphere carrier with the high magnesium microenvironment.

The third purpose of the invention is to provide the application of the bone marrow stem cell microsphere carrier with high magnesium microenvironment in bone regeneration.

In order to realize the first purpose of the invention, the invention discloses the following technical scheme: the high-magnesium microenvironment bone marrow stem cell microsphere carrier is characterized in that magnesium ions are compounded into the microsphere carrier to form a local high-magnesium environment, the size of the microsphere carrier is 100-500 mu m, and the concentration of the magnesium ions is 2.5-10 mM.

Preferably, the magnesium ion concentration is in the range of 2.5mM to 5mM, which is optimal for osteoinduction.

As a preferred scheme, the density of the stem cells loaded on the microsphere carrier is 5 x 10⁶-10⁷/ml。

In order to achieve the second purpose of the invention, the invention discloses the following technical scheme: a preparation method of a high-magnesium microenvironment bone marrow stem cell microsphere carrier is characterized by comprising the following steps:

(1) obtaining and culturing osteogenesis related stem cells;

(2) digesting and centrifuging the stem cells obtained in the step (1), then re-suspending the stem cells by using a high-magnesium culture solution, mixing the obtained cell suspension with gel, and dripping the gel into a cross-linking agent by using a syringe to obtain the cell-loaded microspheres, wherein the diameter of the cell-loaded microspheres is 100-500 microns, and the concentration of magnesium ions is 2.5-10 mM.

As a preferable embodiment, the magnesium ion concentration selected in step (2) is in the range of 2.5mM to 5mM, which is the most excellent for the osteogenesis inducing effect.

As a preferable scheme, the step (2) uses sodium alginate gel, and the final concentration of the sodium alginate gel after gelling is 1% -2%.

As a preferred embodiment, the crosslinking agent used is CaCl₂，SrCl₂Or BaCl₂One kind of (1).

Preferably, the inner diameter of the syringe needle is 150 μm to 180 μm, and the cell-gel mixture is dropped using a micro-applicator so that the drop volume is within 0.4. mu.L and the needle is within 1mm from the surface of the crosslinking agent.

In order to achieve the third purpose of the invention, the invention discloses the following technical scheme: the application of the high-magnesium microenvironment bone marrow stem cell microsphere carrier in bone regeneration. The hydrogel stem cell microsphere carrier can be injected alone or matched with a traditional bracket to repair bone defects, and meanwhile, the injection mode can reduce surgical wounds and avoid complications such as wound infection and the like.

The invention evaluates the influence of magnesium ions with different concentrations on the activity of the bone marrow stem cells, and screens out the concentration range of the magnesium ions suitable for the survival of the cells; on the basis, the influence of magnesium ions on the osteogenic differentiation of stem cells is detected, and the magnesium ion concentration range with the best osteoinduction effect is optimized; preparing a high-magnesium microenvironment bone marrow stem cell microsphere carrier, evaluating the survival rate of stem cells and the osteogenic differentiation effect, and screening the microsphere size with the best cell survival rate and the concentration of magnesium ions for promoting osteogenic differentiation of the stem cells; the prepared bone marrow stem cell microsphere carrier with high magnesium microenvironment is transplanted in vivo to observe the bone regeneration effect. The research result shows that: (1) the magnesium ion concentration is within 20mM, has no obvious cytotoxicity, and has certain effect of promoting cell proliferation (2) when the magnesium ion concentration is within 10mM, the osteogenesis induction effect is realized; when the concentration of magnesium ions is 2.5mM-5mM, the best effect of inducing the stem cell to osteogenesis and differentiate is achieved (3) the stem cell-loaded sodium alginate microspheres with the diameter within 1mM are successfully prepared, and the survival rate of cells is the highest when the diameter of the microspheres is 500 mu m; (4) the microsphere local magnesium ion concentration is 2.5mM-5mM, the osteogenic differentiation of the stem cells can be promoted, and the high-magnesium microenvironment stem cell microsphere has a cell proliferation promoting effect (5) the bone defect repair is obviously promoted.

The invention has the advantages that: the invention provides a concept and a preparation method for constructing a local high-magnesium microenvironment stem cell microsphere carrier, on one hand, screened magnesium ions with the concentration within 20mM have no toxicity to cells and have the effect of promoting cell proliferation, the magnesium ions have the osteogenesis induction effect when the concentration is 2.5mM-10mM, and the osteogenesis induction effect is optimal when the concentration is 2.5mM-5 mM; secondly, the diameter of the prepared stem cell microsphere carrier is 100-500 μm, so that the load rate and the survival rate of stem cells are effectively improved, and the defects of high cost, difficult slow release and the like caused by using protein factors are reduced by promoting osteogenic differentiation of the loaded stem cells in a local high-magnesium environment; finally, when the prepared high-magnesium microenvironment stem cell microsphere carrier is used for bone defect regeneration repair, the number of new bones in the defect area is obviously increased, and the repair effect is obviously improved. The invention does not need special, complex and expensive equipment, has simple operation flow and is beneficial to popularization and use.

Drawings

FIG. 1: effect of different concentrations of magnesium ions on stem cell activity. (a) Half maximal Inhibitory Concentration (IC) of magnesium ions₅₀) Curve, IC₅₀65.91mM and (b) MTT method for detecting the influence of different concentrations of magnesium ions on cell proliferation (★★, p)<0.01)。

FIG. 2 shows the induction of osteogenic differentiation of stem cells by magnesium ions at different concentrations, (a) ALP staining results, (b) ALP semiquantitative results (★★, p <0.01), (c) Weterblot for detecting the expression of Osteocalcin (OCN) in stem cells, and (d) stem cell OCN immunofluorescence staining results.

FIG. 3: the magnesium ions with different concentrations induce the expression change of a stem cell magnesiumion channel MagT 1. (a) The stem cell MagT1qPCR result after osteogenic induction; (b) performing immunofluorescence double staining on stem cells MagT1 and OCN after osteogenesis induction; (c) performing MagT1qPCR (quantitative polymerase chain reaction) on stem cells induced by magnesium ions with different concentrations; (d) stem cells were immunofluorescent stained with MagT1 following induction with different magnesium ions.

FIG. 4: the live and dead staining results of stem cells in microspheres of different sizes after 3 days of culture (green indicates live cells and red indicates dead cells).

FIG. 5: effect of high magnesium microenvironment on stem cell activity within microspheres. (a) Culturing for 1 day and 3 days to obtain live and dead staining results of stem cells in the microspheres; (b) the CCK8 method is used for detecting the survival condition of stem cells in the microspheres.

FIG. 6 shows that EdU staining detects the proliferation activity of stem cells in high-magnesium microenvironment microspheres, (a) the result of EdU staining after cryosectioning of the high-magnesium microenvironment stem cell microspheres (red is EdU positive cells, blue is cell nucleus, pink is EdU true positive cells), and (b) the proliferation rate of stem cells in microsphere stem cell carriers (★★, p < 0.01).

FIG. 7 shows the qPCR detection result of osteogenic differentiation of stem cells in high-magnesium microenvironment microspheres, (a) the expression of run-related transcription factor 2 (Runx 2) of stem cells in microspheres after 3 days of culture, and (b) the expression of OCN of stem cells in microspheres after 7 days of culture (★★, p < 0.01).

FIG. 8 shows the experimental results of repairing critical bone defects of rat skull by using high-magnesium microenvironment stem cell microsphere carrier, (a) X-ray and CT three-dimensional reconstruction images, (b) BV/TV results of each group analyzed by CT scanning, and (c) BMD results of each group analyzed by CT scanning (★★, p is less than 0.01).

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not specified, are generally conducted under conventional conditions or under conditions recommended by the manufacturers.

Example 1 detection of toxicity of magnesium ions on rat bone marrow Stem cells

Step one, isolated culture of rat bone marrow stem cells

Sprague Dawley rats of 3 to 4 weeks old were taken, and after death at the neck, the rat carcasses were soaked in 75% alcohol solution for 3 minutes for sterilization. And (4) after the disinfection, obtaining the mesenchymal stem cells in the ultra-clean bench. The skin was cut at the groin of the rat and the tissue was blunt dissected to expose the tibia and femur. Reducing the fracture of the knee joint by using scissors, taking out the tibia and the femur, removing soft tissues such as muscles on the surface of the bone, and the like, and cutting off epiphyses at two ends of the tibia; and (3) sucking a proper amount of DMEM complete culture solution by using an injector, inserting the needle of the injector into the marrow cavity, washing by using the DMEM culture solution, lifting up and down at the same time, ensuring that the content in the marrow cavity is flushed into a centrifugal tube for use, and washing by using the culture solution until the color of the long bone is white. Centrifuging the collected marrow cavity flushing fluid in a centrifuge at 1500-1800 rpm for 10 min; discarding the supernatant, resuspending the cell precipitate with culture solution, uniformly inoculating into a 10cm culture dish, and culturing in a constant-temperature incubator at 37 ℃; after 3-4 days, the cells were passaged when they reached 80% confluence, after observation and fluid exchange.Passage 2 through passage 4 bone marrow stem cells were used in the experiment.

Step two, magnesium ion IC₅₀Measurement of (2)

Taking rat bone marrowMesenchymal stem cells were seeded in a 96-well plate (n.6) at a density of 5000 cells/well, and 100. mu.L of DMEM complete medium containing magnesium ions at different concentrations was replaced when the cells were confluent (Mg ion concentration 1.6 mM; 2.5 mM; 5 mM; 10 mM; 20 mM; 40 mM; 60 mM; 80 mM; normal DMEM complete medium as a control). After 24h, the reaction solution was added to each well in 10. mu.L of MTT reaction solution in dark, and after careful mixing, the 96-well plate was returned to the cell culture chamber for further culture for 4h, at which time purple crystals appeared at the bottom of the well. Absorbing supernatant, adding 150 mu L of dimethyl sulfoxide (DMSO) into each well, shaking for 10min, detecting light absorption at 490nm after purple crystals are completely dissolved, comparing OD value of experimental group with that of control group, calculating cell survival rate, drawing magnesium ion IC50 curve by GraphPadprism 5.0 software, the experimental result is shown in figure 1(a), when magnesium ion concentration is within 20mM, cell activity is not obviously affected, after the concentration exceeds 20mM, cell activity is obviously reduced, IC is IC₅₀The concentration was 65.91 mM.

Example 2 Effect of magnesium ions on cellular bioactivity

Step one, influence of magnesium ions with different concentrations on cell proliferation

DMEM complete culture solutions with magnesium ion concentrations of 1.6, 2.5, 5, 10, and 20mM were prepared according to the magnesium ion concentration selected in example 1, rat bone marrow stem cells were seeded in a 96-well plate at a density of 1000 cells/well (n ═ 6), and after the cells were attached to the wall, 100 μ L of high-magnesium culture solution was replaced, and the normal DMEM complete culture solution was used as a control group, which was recorded asday 0. In 1, 4, 7 days, 10 mu l of MTT liquid is added into each hole, original liquid in the plate is sucked after incubation for 4 hours, 150 mu l of DMSO is added into each hole, and the vibration is carried out for 10min, so that crystals are fully dissolved. And detecting the absorbance value by using an enzyme-labeling instrument, and analyzing the cell proliferation condition.

The experimental results are shown in fig. 1(b), and the proliferation activity of the cells in the high magnesium ion concentration group is not significantly different from that in the control group atdays 1 and 4, and the proliferation activity of the cells in each high magnesium ion concentration group is stronger than that in the control group by day 7 (fig. 1 (b)). The above results indicate that the proliferation of mesenchymal stem cells is facilitated in an environment of high magnesium ion concentration.

Step two, alkaline phosphatase (ALP) staining and alkaline phosphatase semi-quantitative detection

Taking cells at5X 10⁴The cells were seeded in 24-well plates at a density of one/mL, and DMEM complete medium containing magnesium ions at different concentrations was replaced after the cells were confluent. After 7 days, the culture broth was aspirated and washed withPBS 3 times, fixed with 4% paraformaldehyde, washed withPBS 3 times, and stained with a staining solution prepared with BCIP/NBT alkaline phosphatase kit. Adding ALP activity semi-quantitative lysis solution into a pore plate, incubating at 37 ℃ until cell lysis is complete, mixing a sample and p-NPP working solution according to a ratio of 1:1, incubating at 37 ℃, and detecting absorbance at 405 nm. The protein concentration of each sample was measured by the BCA method, and the protein mass was calculated from the volume. Semi-quantitative Activity of ALP ═ OD₄₀₅(ii)/protein mass.

The results are shown in FIGS. 2(a), (b), and it was found by ALP staining that the concentration of magnesium ion was 2.5mM-10mM, and the number of ALP staining positive cells was increased as compared with the control group, with 2.5mM and 5mM being most significant. ALP activity semi-quantitative determination found that the ALP activity of the 2.5mM group and the 5mM group was significantly different from that of the control group, and the effect of 5mM was the best. This shows that magnesium ion concentration of 2.5mM-10mM has a certain osteogenic differentiation effect on the stem cells, wherein magnesium ion concentration of 2.5mM-5mM has the best osteogenic differentiation effect on the rat bone marrow mesenchymal stem cells.

Step three, Westernblot detection of expression condition of stem cell osteogenesis related protein under magnesium ion stimulation

Taking cells at 1 × 10⁶The cells were seeded at a density of one/mL in a 10cm dish and after cell fusion, 2.5mM and 5mM high magnesium medium were added according to the semiquantitative results for ALP. After 7 days, protein samples were collected, supernatant protein concentration was measured by BCA method, and sample protein was adjusted so that the amount was consistent. Performing SDS-PAGE electrophoresis, transferring the membrane to a PVDF membrane, and sealing with 5% skimmed milk for 1 h. OCN antibody was diluted 1:200, and glycerol phosphate dehydrogenase (GAPDH) antibody was diluted 1:5000, and incubated overnight at 4 ℃. PBST was washed 3 times, and after incubation of secondary antibody (dilution ratio) at room temperature for 1h, PBST was washed 3 times and developed.

The results are shown in FIG. 2(c), and compared with the normal culture medium, the OCN expression levels in the 2.5mM and 5mM groups are significantly increased, wherein the increase in the 5mM group is more significant and is consistent with ALP staining and semi-quantitative results.

Step four, immunofluorescence staining detection

Taking cells at5X 10⁵The cells are inoculated in a glass bottom dish at a density of one cell per mL, and the cells are replaced by DMEM complete culture solution with magnesium ions of different concentrations after being attached to the wall. After 5 days of culture, the old cell culture medium in the glass-bottom dish was blotted dry and rinsed 3 times with PBS buffer. After 4% paraformaldehyde fixation, immunofluorescence staining was performed to detect the expression of cellular OCN. The dilution ratio of the OCN antibody is 1: 50; the dilution ratio of the secondary antibody is 1: :200. After staining, the cells were stained with rhodamine-labeled phalloidin (dilution ratio 1:200) for 30min, washed with PBS buffer, and stained with DAPI (1:500) for 5 min. The photographs were observed and taken under a fluorescent microscope.

The results are shown in FIG. 2(d), and the OCN expression levels of the stem cells in the 2.5mM and 5mM groups are higher than those in the control group and even in the 5mM group. The above results all indicate that the high magnesium environment has the effect of promoting the osteogenic differentiation of stem cells.

Example 3 detection of changes in expression of magnesium ion channel MagT1 in Stem cells induced by magnesium ions at different concentrations

Step one, detecting expression change of stem cell MagT1 after osteogenesis induction

Taking rat bone marrow stem cells with the fusion degree of more than 90%, digesting, centrifuging, counting, inoculating into a 6-well plate and a confocal dish, replacing an osteogenesis inducing liquid (provided by Seisaku-Sho) when the cell fusion degree reaches 80%, taking a normal DMEM culture solution as a control (n is 3), carrying out osteogenesis induction, collecting samples after 3 days and 5 days of osteogenesis induction, carrying out qPCR detection and immunofluorescence detection, wherein the immunofluorescence staining method is the same as the above, adding trizol 1mL of lysed cells into a qPCR sample, collecting total RNA of each group of samples, measuring the concentration and purity of the extracted RNA under 260nm and 280nm of an ultraviolet spectrophotometer, synthesizing cDNA according to a reverse transcription kit, then carrying out PCR detection on the expression of MagT1, taking housekeeping gene GAPDH as an internal reference, representing a CT value for a Real-time PCR result, and calculating the relative expression of the genes according to a formula of 2- △△ CT.

The results of the experiment are shown in FIG. 3. As shown in fig. 3(a), MagT1 expression was significantly increased 5 days after osteogenic induction; FIG. 3(b) shows that both OCN and MagT1 expression were elevated in stem cells induced by osteogenesis for 5 days. This suggests that MagT1 is involved in osteogenic differentiation of stem cells and can be used as an indirect assessment indicator of osteogenic differentiation of stem cells.

Step two, the expression condition of stem cell MagT1 after the induction of magnesium ions with different concentrations

The preparation concentration is 1.6 mM; 2.5 mM; 5 mM; 10 mM; 20 mM; 40 mM; 60mM high magnesium medium, and normal DMEM complete medium as control. Taking rat bone marrow stem cells with good growth condition, digesting, centrifuging and inoculating the rat bone marrow stem cells in a culture dish; after the cells are full of cells, the high-magnesium culture solution (n is 3) is replaced, samples are collected after 5 days, and qPCR detection and immunofluorescence staining are carried out.

As shown in FIG. 3, it can be seen from FIG. 3(c) that the expression of dry cell MagT1 was significantly increased at magnesium ion concentrations of 2.5mM and 5mM, and that the immunofluorescence staining results of FIG. 3(d) were consistent with the qPCR results, indicating that magnesium ions were most effective in promoting bone differentiation at concentrations of 2.5mM to 5 mM.

Example 4 preparation of microsphere vectors for Stem cells of different sizes and evaluation of cell survival

Step one, preparation of stem cell microsphere carriers with different sizes

Taking rat bone marrow stem cells with fusion degree of more than 90%, digesting, centrifuging, re-suspending and counting the culture solution, mixing with 4% sodium alginate solution sterilized at high temperature and high pressure at a ratio of 1:1-3:1, adjusting cell density to 5 × 10⁶—10⁷After/ml, aspirate with a microsyringe with 0.1M CaCl₂Is a cross-linking agent, the inner diameter of the syringe needle is 150 μm, the needle and CaCl are controlled₂The liquid level distance is within 1mm, and the hydrogel-cell mixed solution is dripped into CaCl₂And (4) carrying out medium crosslinking for 3min, and controlling the volume of the liquid drop within 0.4 mu L. Abandon CaCl₂After washing with saline for 3 times, DMEM complete culture medium was added, and the microspheres were observed to have a diameter of about 500 μm under a microscope (see FIG. 4). Large-sized microspheres were prepared with a needle having an inner diameter of about 500 μm, and the microspheres were observed under a microscope to have a size of 1-2mm (see FIG. 4). The prepared stem cell microsphere carrier is placed in a 24-pore plate and cultured in a constant-temperature incubator at 37 ℃.

Step two, detecting the survival condition of the stem cells in the microsphere carrier by live-dead staining

And (3) after culturing the microspheres with different sizes prepared in the step one for 3 days, staining the microspheres by using a Calcein-AM/PI live-dead staining kit, detecting the microspheres by using a confocal microscope, and analyzing the survival condition of the cells after reconstruction.

The size of the microsphere is related to the inner diameter of a needle head (the composite size can be formed in an experimental selected range, if the microsphere is too large, the microsphere is large, if the microsphere is too small, the extrusion difficulty is large, cells are difficult to survive after being pressed), the distance between a liquid drop and the liquid surface (related to the shape of the microsphere, the shape of the microsphere is oval instead of round when the distance is higher), and the volume of the liquid drop.

The experimental results are shown in fig. 4, after live and dead staining, live cells emit green fluorescence under a mirror, dead cells show red fluorescence, and the graph shows that after 3 days of culture, the dead cells gradually increase along with the increase of the diameter, the cell death rate in microspheres with the diameter of 2mm is close to 50%, while fresh cells in microspheres with the diameter of 500 microns die, the cell survival rate is obviously improved compared with that in a large-size group, which indicates that the cell survival rate is 500 microns or less, and theoretically 100 microns-500 microns is the optimal size of the microsphere carrier under the current experimental conditions.

Example 5 preparation of microsphere vectors for high-magnesium Stem cells at various concentrations and evaluation of cell survival

Step one, preparation of high-magnesium stem cell microsphere carriers with different concentrations

According to the results of example 2, selecting 2.5mM and 5mM magnesium ion as experimental groups, taking rat bone marrow stem cells with fusion degree of more than 90%, digesting, centrifuging, re-suspending and counting with high magnesium culture solution with different concentrations (normal DMEM control), mixing with 4% sodium alginate solution sterilized at high temperature and high pressure at ratio of 1:1-3:1, adjusting cell density at 5 × 10⁶-10⁷In terms of magnesium concentration, the magnesium concentration is 2.5mM and 5 mM. Several microspheres of about 500 μm were prepared (same method as example 4), and cultured in normal DMEM medium at 37 ℃.

Step two, evaluating survival of cells in microspheres

And (3) taking different high-magnesium microenvironment microsphere carriers cultured for 1 day and 3 days, performing living and dead staining, and performing confocal microscope imaging. The results are shown in FIG. 5(a), and the high magnesium group showed less apoptosis at 1 day of culture, and slightly increased apoptosis at 3 days, but had no significant difference from the control group.

Taking 50 mu L (n is 3) of microspheres cultured for 0 day, 1 day and 3 days respectively, adding the microspheres into a physiological saline solution of 55mM sodium citrate, carrying out shaking table reaction at 37 ℃ for 5-10 min, centrifuging at 3000rpm for 5min after the sodium alginate microspheres are completely degraded, discarding supernatant, then resuspending the culture solution, and inoculating the suspension into a 96-well plate. After the cells are attached to the wall, 100 mu L of culture solution is changed, 10 mu L of CCK8 working solution is added into each hole, and the cells are incubated for 1.5h at 37 ℃ in a dark place. After the supernatant is absorbed, an enzyme-labeling instrument detects the OD value at 450nm, and GraphPad Prism 5.0 software analyzes and calculates the cell survival rate. The experimental result is shown in FIG. 5(b), the cell survival rate of the high magnesium group is not obviously different from that of the control group, and the survival rate is slightly reduced at 3 days, but still about 90%.

Example 6 Effect of high magnesium microenvironment on Stem cell bioactivity in microsphere Supports

Step one, preparation of stem cell microsphere carrier with high magnesium microenvironment

According to the results of examples 2 and 5, the final concentration of magnesium ions was 5 mM. The microspheres were prepared as described in example 4.

Step two, detecting the proliferation condition of stem cells in the microsphere carrier

Preparing an EdU solution with the concentration of 5mg/mL, diluting the EdU solution at a ratio of 1:1000, adding the diluted EdU solution into a culture solution, incubating for 24 hours, taking microspheres cultured for 1 day, 3 days and 7 days, freezing and slicing (n is 3), fixing the microspheres with the layer thickness of 5 microns by 95% ethanol for 30 minutes, washing for 5 minutes by running water, washing for 10 minutes by 2mg/mL glycine solution, permeating for 10 minutes by 0.5% triton solution, washing for 10 minutes by PBS buffer solution, incubating for 30 minutes at room temperature by the EdU staining solution in a dark place, washing for two times by 0.5% triton solution, 10 minutes each time, and washing for 10 minutes by the PBS buffer solution. DAPI stained nuclei for 5min, observed by fluorescence microscopy, EdU positive (pink cells) as proliferating cells. Image J software statistical analysis.

The experimental results are shown in fig. 6, as shown in (a), when cultured for 1 day, the cells in the microspheres proliferate less, the proportion of the proliferated cells increases gradually with time, the proportion of the proliferated cells increases obviously atday 7, and the proliferation of the cells is more active in the 5mM group compared with the control group, which indicates that the high-magnesium microenvironment can promote the proliferation of the stem cells in the microspheres.

Step three, detecting osteogenic differentiation condition of stem cells in microspheres

Taking 50uL (n is 3) of stem cell microspheres cultured for 3 days and 7 days respectively, dissolving the stem cell microspheres in a sodium citrate solution, centrifuging the solution, removing supernatant, adding trizol 1mL of lysed cells, collecting total RNA of samples of each group, detecting expression of related genes, measuring the concentration and purity of extracted RNA at 260nm and 280nm of an ultraviolet spectrophotometer, synthesizing cDNA according to a reverse transcription kit, then carrying out PCR (polymerase chain reaction) detection to obtain the expression of bone related genes Runx2 and OCN, taking housekeeping genes GAPDH as internal reference, taking CT values as Real-timePCR results, and calculating the relative expression of the genes according to a formula of 2- △△ CT.

The experimental result shows that the expression of Runx2 in the 5mM group is obviously higher than that in the control group (figure 7(a) ★★, p is less than 0.01) at 3 days, and the expression of OCN in the 5mM group is obviously higher than that in the control group (figure 7(b) ★★, p is less than 0.01) at 7 days.

Example 7 Effect of high-magnesium microenvironment stem cell microspheres in repairing Critical bone defect of rat skull

Step one, preparation of stem cell microsphere carrier with high magnesium microenvironment

According to the results of examples 2, 5 and 6, the final concentration of magnesium ions is 5 mM; according to the results of example 4, stem cell microspheres with a size of 500 μm were prepared according to example 4 and designated as SA-Mg/BMSCs group; suspending rat bone marrow stem cells by using a common DMEM culture solution to prepare microspheres with the same size, and marking as an SA-BMSCs group; and selecting 5mM of final magnesium ion concentration to prepare sodium alginate microspheres, and marking as an SA-Mg group, wherein the sodium alginate gel microspheres are a blank control group and are marked as an SA group.

Step two, preparation of critical bone defect of rat skull

Sprague Dawley rats of 6-8 weeks of age were weighed and anesthetized by injection of 10% chloral hydrate solution at a dose of 4. mu.L/g body weight. After leg-clamping reflection and corneal reflection of a rat disappear, shaving hair, disinfecting a skull top operation area by 75% alcohol, making a sagittal incision along a skull median suture to periosteum, separating the periosteum by a periosteum stripper, and preparing 5 mm-diameter circular bone defects along two sides of the skull median suture.

Step three, implanting the stem cell microsphere carrier

Stopping bleeding with gauze, sucking high magnesium stem cell microsphere carrier (SA-Mg/BMSCs group) with Pasteur tube, dripping to defect area, spreading microsphere uniformly with forceps, drawing periosteum, assisting microsphere retention, suturing periosteum skin in sequence, disinfecting operation area again, observing rat respiration and state, confirming rat state stability, marking, and returning to mouse cage. The SA-BMSCs, SA-Mg and SA groups were implanted in the same manner, and the microspheres in each group were 50. mu.L, and the sample size n in each group was 6.

Step four, evaluating the repairing effect of the critical bone defect of the skull of the rat

Four weeks after the operation, the rat was euthanized, and a skull specimen was fixed in formalin fixing solution; after fixation, X-ray imaging detection is carried out (the result is shown in figure 8); and then, detecting the formation condition of new bones by using a micro CT (micro computed tomography), and analyzing the bone formation effect of each group.

The results are shown in FIG. 7, and it can be seen from FIG. 8(a) that the SA group had the worst bone repair effect and remained a lot of defects, while the SA-Mg group and the SA-BMSCs group had partially formed new bones, and the difference between the two groups was small; compared with the other three groups, the new bone regeneration of the SA-Mg/BMSCs group is obviously increased, and the repair effect is most obvious. As can be seen from fig. 8 (b), BV/TV (%), SA-Mg/BMSCs 32.425 ± 4.337, SA-BMSCs 18.148 ± 3.911, SA-Mg 14.005 ± 2.864, and SA 3.492 ± 1.103; as can be seen from fig. 8(c), BMD (mgHA/cc), SA-Mg/BMSCs group is 344.690 ± 51.246; the SA-BMSCs group is 213.863 +/-35.839, the SA-Mg group is 180.706 +/-25.860, and the SA group is 85.622 +/-22.522; as can be seen, the new bone mass in the SA-Mg/BMSCs group was significantly higher than that in the other groups.

In summary, when the magnesium ion concentration is within 20mM, the cytotoxicity is low, and the osteogenesis inducing effect is achieved, and when the magnesium ion concentration is within 2.5mM-10mM, the cell proliferation promoting effect is achieved; when the concentration of magnesium ions is 2.5mM-5mM, the osteogenic differentiation effect of the induced stem cells is optimal; on the basis, the high-magnesium microenvironment stem cell microsphere carrier is constructed, when the microsphere size is 500 mu m, the cell survival rate is high, the proliferation activity is good, and the high-magnesium microenvironment stem cell microsphere carrier has stronger osteogenic differentiation capacity under the stimulation of local high magnesium. When the stem cell microsphere carrier is used for bone defect repair, the differentiation capacity of stem cells is improved under the stimulation of a local high-magnesium environment, the new bone formation amount is obviously improved compared with that of a control group, and the effect of promoting bone regeneration is achieved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, without departing from the principle of the present invention, a person skilled in the art may make several improvements and modifications, such as loading magnesium-containing particles for the purpose of slow release of magnesium ions and expanding the application range of labeled cells for binding magnesium ions or applying to other researches, and may also achieve the system for maintaining local high magnesium microenvironment by using other known hydrogels, such as common hydrogels of hyaluronic acid, silk protein, chitosan, gelatin, collagen, cyclodextrin, etc., and also for other tissue regeneration repair or in vitro micro tissue construction and related researches, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (9)

1. The high-magnesium microenvironment bone marrow stem cell microsphere carrier is characterized in that magnesium ions are compounded into the microsphere carrier to form a local high-magnesium environment, the size of the microsphere carrier is 100-500 mu m, and the concentration of the magnesium ions is 2.5-10 mM.

2. The bone marrow stem cell microsphere carrier with the high magnesium microenvironment according to claim 1, wherein the concentration of the magnesium ions is 2.5mM-5 mM.

3. The microsphere carrier of bone marrow stem cells with high magnesium microenvironment of claim 1, wherein the density of the stem cells loaded on the microsphere carrier is 5 x 10⁶-10⁷/ml。

4. A preparation method of a high-magnesium microenvironment bone marrow stem cell microsphere carrier is characterized by comprising the following steps:

(1) obtaining and culturing osteogenesis related stem cells;

(2) digesting and centrifuging the stem cells obtained in the step (1), then re-suspending the stem cells by using a high-magnesium culture solution, mixing the obtained cell suspension with gel, and dripping the gel into a cross-linking agent by using a syringe to obtain the cell-loaded microspheres, wherein the diameter of the cell-loaded microspheres is 100-500 microns, and the concentration of magnesium ions is 2.5-10 mM.

5. The method for preparing the bone marrow stem cell microsphere carrier with the high magnesium microenvironment according to the claim 4, wherein the magnesium ion concentration in the step (2) is 2.5mM to 5 mM.

6. The preparation method of the bone marrow stem cell microsphere carrier with the high magnesium microenvironment according to the claim 4, characterized in that the sodium alginate gel is used in the step (2), and the final concentration of the sodium alginate gel after the gelling is 1% -2%.

7. The method for preparing the bone marrow stem cell microsphere carrier with the high magnesium microenvironment according to the claim 4, wherein the cross-linking agent is CaCl₂，SrCl₂Or BaCl₂One kind of (1).

8. The method for preparing the microsphere carrier for the bone marrow stem cells in the high-magnesium microenvironment according to claim 4, wherein the inner diameter of the needle of the syringe is 150 μm to 180 μm, a microsyringe is used for dripping the cell-gel mixed solution, the volume of the liquid drop is within 0.4 μ L, and the distance between the needle and the liquid level of the cross-linking agent is within 1 mm.

9. The use of the high-magnesium microenvironment bone marrow stem cell microsphere carrier of claim 1 in bone regeneration.

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